Natural Products Targeting Liver X Receptors or Farnesoid X Receptor

Nuclear receptors (NRs) are a superfamily of transcription factors induced by ligands and also function as integrators of hormonal and nutritional signals. Among NRs, the liver X receptors (LXRs) and farnesoid X receptor (FXR) have been of significance as targets for the treatment of metabolic syndrome-related diseases. In recent years, natural products targeting LXRs and FXR have received remarkable interests as a valuable source of novel ligands encompassing diverse chemical structures and bioactive properties. This review aims to survey natural products, originating from terrestrial plants and microorganisms, marine organisms, and marine-derived microorganisms, which could influence LXRs and FXR. In the recent two decades (2000–2020), 261 natural products were discovered from natural resources such as LXRs/FXR modulators, 109 agonists and 38 antagonists targeting LXRs, and 72 agonists and 55 antagonists targeting FXR. The docking evaluation of desired natural products targeted LXRs/FXR is finally discussed. This comprehensive overview will provide a reference for future study of novel LXRs and FXR agonists and antagonists to target human diseases, and attract an increasing number of professional scholars majoring in pharmacy and biology with more in-depth discussion.


INTRODUCTION
Nuclear receptors (NRs), a superfamily of transcription factors incorporating a group of 48 members in humans and 49 in mice (Gronemeyer et al., 2004;Zhang et al., 2004), are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body (Calkin and Tontonoz, 2012). NRs comprise seven subfamilies, namely, thyroid hormone receptor-like (NR1), retinoid X receptor-like (NR2), estrogen receptor-like (NR3), nerve growth factor IB-like (NR4), steroidogenic factor-like (NR5), germ cell nuclear factor-like (NR6), miscellaneous (NR0), etc. (Zhang et al., 2020c). From N-terminus to C-terminus, NRs share common structure features and are composed of an activation function-1 (AF-1) domain, a DNA-binding domain (DBD), a ligand-binding domain (LBD), a ligand-dependent activation function-2 (AF-2) domain, as well as a hinge region linking the DBD and the LBD (Figure 1) (El-Gendy et al., 2018). NRs activate or repress genes expression required for virtually all aspects of development, reproduction, cell growth, metabolism, immunity, and inflammation by binding with regulatory regions of target genes and acting in concert with coactivators and corepressors. The activity of a large subgroup of NRs depends on small lipophilic, membranepermeable ligands binding to the LBD, such as fatty acids, oxysterols, and bile acids, functioning as important regulators and ideal drug targets in metabolic syndrome-related diseases for drug discovery and development (Lonard and O'Malley, 2007;McKenna et al., 2009;Hollman et al., 2012).
The farnesoid X receptor (FXR, NR1H4) identified in 1995 has been known as a member of NRs and a xenobiotic receptor (Niesor et al., 2001;Wang D. et al., 2018). FXR was originally named on the basis of the observation that farnesol and related derivatives possessed the potency of activating FXR (Calkin and Tontonoz, 2012;Zhang J. et al., 2020). In the subsequent years, a series of natural cholesterol metabolites including bile acids were identified as endogenous ligands, meaning that FXR is more appropriately classified as a nuclear bile acid (BA) receptor (Parks et al., 1999;Zhang T. et al., 2020). In accordance with its function as chief sensor of BA, FXR is most abundantly expressed in the liver, intestine, kidneys, and adrenal glands, thus, playing a pivotal role in the regulation of BA metabolism (Lefebvre et al., 2009;Chen et al., 2011). Furthermore, FXR has also been reported to exhibit the regulatory potential of several other physiological processes such as glucose and lipid metabolism (Forman et al., 1995;Parks et al., 1999). Similarly, FXR also forms obligate heterodimers with 9-cis retinoic acid receptor RXR like LXRs. FXR exists as two types of encoded genes, namely, FXRα and FXRβ, although the latter is a pseudogene. Hence, for the purpose of this review, FXRα will be simplified as FXR (Modica et al., 2010).
Natural products are a promising source for bioactive agents and lead compounds for new drug research (Hiebl et al., 2018;Wang D. et al., 2020), which not only include ethnopharmacologically used compounds from plants or herbals but also terrestrial or marine-derived microbial metabolites . There are plenty of bioactive components in traditional Chinese medicines (TCMs) that might serve as selective ligands for their respective receptors (Qiu, 2007;Cragg and Newman, 2013;Li et al., 2015). Furthermore, the oceans, with their unique environment and huge biodiversity, have the potential as a plentiful source of diverse natural products accompanying pharmacological activities (Carazo et al., 2019). In comparison with synthetic and combinatorial compounds, natural products display a great structural and chemical diversity (Jones et al., 2006;Shen 2015). Beyond that, they cover a large range of biodiversity and profuse functionality because of the capacity of interacting with multiple proteins or other biological targets (Wink, 2003;Jones et al., 2006;Hiebl et al., 2018).
However, there are two main drawbacks to the development of natural products, one of which is that natural products are often in limited supply. Another limitation is the difficulty of separation of trace active compounds and elucidation of unknown compounds . To date, several technological advances have helped to overcome the above drawbacks, particularly in the developments of high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) and high-resolution nuclear magnetic resonance (NMR) technologies, facilitating the identification and structure elucidation of unknown compounds (Strege, 1999;Koehn and Carter, 2005). Additionally, advances in other subjects, such as metabolic engineering, microbial cultivation, as well as genetic methods, might solve the supply of natural products and contribute to the development of natural product-derived drugs (Ling et al., 2015;Shen, 2015).
This review focuses on natural products targeting LXRs/FXR in the recent two decades . For a brief background of LXRs/FXR, we refer to several recent reviews and give comprehensive overviews regarding this topic, and the docking evaluation of desired natural products targeting LXRs/FXR is finally discussed.

PHYSIOLOGICAL FUNCTIONS OF LIVER X RECEPTORS AND FARNESOID X RECEPTOR
2.1 The role of liver X receptors and farnesoid X receptor in metabolic processes 2.1.1 Cholesterol and lipid metabolism A stringent control of systemic and cellular cholesterol levels is essential to physiological homeostasis (Bonamassa and Moschetta, 2013;Hong and Tontonoz, 2014). LXRs sense excess cholesterol and trigger various adaptive mechanisms protecting the cells from cholesterol overload. Activation of LXRs results in reverse cholesterol transport (RCT), inhibition of intestinal cholesterol absorption, and suppression of cholesterol synthesis and uptake by the cells (Beltowski, 2008).
ATP-binding cassette transporters A1 (ABCA1) is one of the earliest identified LXR target genes and one of the most highly regulated LXRs targets, required for the ability of LXRs agonists to stimulate cholesterol efflux to apolipoprotein AI (APOAI) (Ishibashi et al., 2013) acceptors. Another transporter ATPbinding cassette transporters G1 (ABCG1) that promotes cholesterol efflux from macrophages is also an LXR target gene (Tarling and Edwards, 2011). RCT is the stimulation of cholesterol removal from the cell, along with transport to the liver, and biliary excretion (Beltowski, 2008). In this process, LXRs upregulate the expression of transporters involved in cholesterol removal from plasma membrane to extracellular acceptors, namely the ABCA1 and ABCG1 , thus, induce cholesterol mobilization from the plasma membrane of nonhepatocyte cell types, cause the formation of high density lipoprotein (HDL) or apolipoproteins, reduce cholesterol expression in the cell membrane, and disrupt lipid rafts formation (Repa et al., 2000;Bradley et al., 2007). LXRs are also involved in the regulation of intracellular cholesterol traffic. LXRs agonists increase the expression of Niemann-Pick C1 (NPC1) and C2 (NPC2) proteins, two carriers mediating transportation from the endosomal compartment to the plasma membrane before efflux, which result in stimulating redistribution of cholesterol from the endosomal compartment to the plasma membrane where it becomes available for efflux to extracellular acceptors (Castrillo et al., 2003;Duval et al., 2006).
In terms of the indispensable interrelation between the intestinal absorption and the regulation of cholesterol levels within the body, LXRs agonists have been verified to stimulate cholesterol recycling from the enterocyte to the intestinal lumen by upregulating ATP-binding cassette transporters G5 (ABCG5) and ATP-binding cassette transporters G8 (ABCG8) (Yu et al., 2003). Additionally, Niemann-Pick C1-like 1 (NPC1L1) protein contained in the apical membranes of enterocytes and attenuated by LXR activation endows a paramount role in intestinal cholesterol absorption (Wang, 2007).
Collectively, cellular and systemic cholesterol homeostasis are maintained by the coordinated actions of sterol-regulatory element-binding proteins (SREBPs) and LXRs. SREBPs are activated in response to low cellular cholesterol levels, whereas LXRs are activated by elevated cholesterol levels (Calkin and Tontonoz, 2012).
FXR activation not only inhibits the uptake and conversion to bile acids of cholesterol, as well as impact the synthesis and excretion of cholesterol, but also promote the expression of hepatic scavenger receptors leading to the enhanced RCT (Neuschwander-Tetri et al., 2015). Growing lines of evidence indicate that FXR activation is an attractive approach for the regulation of lipid homeostasis. FXR-null mice are featured with hypertriglyceridemia, hypercholesterolemia, and growing intestinal cholesterol absorption, in close association with increase in HDL cholesterol and lipoprotein lipase activity, the generation and characterization of which are a breakthrough for uncovering and verifying the importance of FXR action for lipid metabolism (Sinal et al., 2000;Lambert et al., 2003). LXRs regulate the expression of proteins involved in lipid remodeling. The gene cluster of apolipoproteins E, C1, C2, and C4 (APOE, APOC1, APOC2, and APOC4), belonging to LXRs target genes, are implicated in lipid transport and catabolism (Hiebl et al., 2018). Also, FXR has been demonstrated to possess the ability of regulating LXRsmediated lipogenesis (Han et al., 2016).

Bile acid metabolism
BAs, the end products of cholesterol catabolism in the liver (Fiorucci et al., 2012a), are amphipathic molecules with a steroidal moiety derived from cholesterol (Han et al., 2016). FXR, widely expressing in nonclassical BA target tissues (Wang et al., 2018b), plays a critical role in maintaining BAs homeostasis by controlling their synthesis, transport, and metabolism (Sinal et al., 2000). Take for instance FXR. It protects the liver from the excess BAs by promoting excretion and preventing synthesis and uptake (Jian et al., 2014). Recent researches have demonstrated that BAsactivated FXR decreases BAs de novo synthesis in the liver, increases BAs secretion into the small intestine, promotes BA intestinal reabsorption, and inhibits hepatic basolateral BAs reuptake (Gadaleta et al., 2015).
The classic BA synthetic pathway, initiated by the first and rate-limiting enzyme converting cholesterol into BA, cholesterol 7α-hydroxylase (CYP7A1), is considered as the major BA biosynthetic pathway in humans (Russell, 2003). Likewise, FXR activation induces intestinal epithelial expression of fibroblast growth factor 19 (FGF19, also known as FGF15 in rodents), which suppresses BA synthesis by inhibiting CYP7A1 (Inagaki et al., 2005). FXR primarily controls BA synthesis via activating small heterodimer partner (SHP) to inhibit the expression of LXRs, which further suppresses the transcription of CYP7A1 (De Fabiani et al., 2001). As has been stated, CYP7A1 contains a responsive element for the LXRs/RXR heterodimer, proposing that LXRs also might regulate the BA synthetic pathways to a certain degree .

Glucose metabolism
Given the interdependence of lipid and carbohydrate metabolism, it is not surprising that the LXR signaling has affected glucose homeostasis and insulin sensitivity (Goodwin et al., 2008). Glucose transporter type 4 (GLUT4), an insulin-responsive glucose transporter expressed primarily in adipose tissues and skeletal muscle, is usually inhibited in diabetic patients and diabetic mice models. Several studies have illuminated that the expression of GLUT4 in some mice models could be regulated throughout direct interaction with LXR response elements (LXREs) (Mi et al., 2003;Goodwin et al., 2008). Indeed, phosphoenolpyruvate carboxy kinase (PEPCK) and glucose-6phosphatase (G6P), involved in hepatic gluconeogenesis, are dramatically inhibited in insulin-resistant rats administrated with LXR agonists, resulting in decreasing hepatic glucose, insulin sensitivity, and plasma glucose (Cao et al., 2003).
Another unanticipated area of intense study that recently arose is that FXR are currently under clinical investigation for the pleiotropic role to manage glucose homeostasis. Activation of hepatic FXR decreases plasma glucose levels, downregulates the gluconeogenic pathway, and alters the transcription, either indirectly or directly, of several genes that govern gluconeogenesis and glycolysis. Nevertheless, the underlying mechanisms involved in glycemic response remain controversial.

Liver X receptors and farnesoid X receptor as therapeutic target
The fact that LXRs and FXR play a pivotal role in metabolism homeostasis is of relevance for drug research, the functions of which are evidenced in many pathological conditions as illustrated in Figures 2 and 3.

Diabetic mellitus and obesity
It has been covered that human LXRs genes possess potential connections between obesity and diabetes with the advent of genome-wide association studies (Calkin and Tontonoz, 2012). LXR activation promotes triglyceride (TG) accumulation in skeletal muscle cells, probably through the induction of the expression of lipogenic enzymes. Furthermore, LXR agonist treatment improves glucose tolerance in diet-induced diabetic models, involved in glucose metabolism as a potential approach for the treatment of type 2 diabetes (T2D) characterized by highblood glucose and insulin resistance (Huang, 2014).
The observation that FXR activation results in insulin substrate receptor 1 (IRS-1) phosphorylation on the tyrosine residues in the liver and adipose tissue manifests a prospect application of FXR agonists for the management of patients with insulin resistance to improve insulin sensitivity, indicating that FXR agonists are suitable for T2D (Gadaleta et al., 2015;Han et al., 2016). Intestinal activation of FXR reduces diet-induced weight gain, hepatic glucose production, and steatosis. Meanwhile, it strongly stimulates FGF19 expression in the intestine, reverses high fat-induced diabetes, and enhances the metabolic rate while decreasing adiposity (Cave et al., 2016).

Cancer
LXRs inhibit the proliferation of multiple cancer cell lines from the liver, lung, skin melanoma, prostate, breast, cervical, epidermis, bone, and squamous carcinoma, as well as leukemia T cells (El-Gendy et al., 2018). LXR ligands have shown antiproliferative effects on different cancer cell types, altogether suggesting a ubiquitous and global effect of LXRs on proliferation and apoptosis, not only on cells with a tumor origin (Jakobsson et al., 2012;Boussac et al., 2013).
According to accumulating bibliographic data, FXR may prevent intestinal and hepatocellular tumorigenesis (Gadaleta et al., 2015;Masaoutis and Theocharis, 2018). Emerging evidence supports that FXR seems to possess various antioncogenic and less common prooncogenic attributes. In the case of the pancreas, elevated FXR expression seems to impart better prognosis to adenocarcinoma. In the case of breast cancer, immunohistochemical FXR positivity is also an independent favorable prognostic factor (Masaoutis and Theocharis, 2018).

Inflammation and immunity
Both LXRα and LXRβ are involved in regulating inflammatory responses since they are expressed in a variety of immune cells, and are involved in innate and adaptive immunity and inflammatory responses (Hong and Tontonoz 2008), the activation of which inhibit proinflammatory cytokine production in macrophages derived from wild-type mice rather than from LXRα-and LXRβ-knockout mice (Cave et al., 2016). Moreover, FXR is downregulated in a variety of disease states accompanying inflammation, such as fibrosis, cirrhosis, cardiovascular inflammatory, and cancer (Han et al., 2016). For the therapy of nonalcoholic steatohepatitis (NASH), chronic hepatitis B virus (HBV) infection (Wang et al., 2018c), and inflammatory bowel disease (IBD), FXR has provided a novel target.
Most of the studies conducted to find LXR modulators possessing therapeutic utility have been directed toward LXR agonists. However, the elevation of plasma TG and hepatic steatosis caused by LXRs in the liver, as a major drawback, has impeded its development into commercial drugs. To overcome this undesirable effect, new strategies have been put forward such as developing inverse agonists, LXRβ-selective agonists, and tissue-selective agonists (El-Gendy et al., 2018).
Based on emerging evidence that FXR activation by agonists treatment has a beneficial effect on the treatment of various diseases in animal models, some FXR agonists have been imported into clinical studies for its value as medicinal drugs (Han et al., 2016). At present, some FXR ligands impacted in cholestasis, T2D and metabolic syndrome, nonalcoholic fatty liver disease (NAFLD) or NASH, and primary BA diarrhea are measured in phases I and II clinical trials. Although there is therapeutic benefit of FXR ligands, the risks of various ligands, particularly on the intestines or kidneys, require profound investigation and cautious consideration (Hollman et al., 2012).

NATURAL PRODUCTS TARGETING LIVER X RECEPTORS
In this section, natural products regulating LXRs expression in different models are outlined. We categorized and summarized these natural products in Supplementary Tables S1 and S2.

Terpenes
Geraniol (1), a major constituent of essential oils from aromatic plants, including Cinnamomum tenuipilum Kosterm (Lauraceae), Valeriana officinalis L. (Caprifoliaceae), and Panax notoginseng (Burk.) F.H. Chen, leads to the activation of LXRα and FXR, as assessed in the hyperlipidemic rats, which induced significant and dose-dependent decrease of serum total cholesterol (TC), TG, and low-density lipoprotein cholesterol in hyperlipidemic rats (Ji and Gong, 2007;Galle et al., 2015;Lei et al., 2019). Cineole (2), also named cajeputol, is a monoterpene and a principal constituent of Mentha longifolia (L.) L., most eucalyptus oils, teas, rosemary, Psidium, and many other essential oils. Treatment with 2 induced remarkable increase in target genes associated with LXRs. It displayed decreased cellular lipid accumulation and reduced cholesterol levels, as evaluated via Oil Red O lipid staining and cholesterol quantification, suggesting practical implications for the development of hypercholesterolemia and atherosclerosis because it not only reduced cholesterol accumulation but also prevented the potential side-effect hepatic steatosis . Triterpene squalene (3), obtained from dried biomass of Schizochytrium mangrovei PQ6, was presented in nutrition, health care, cosmetics, and medicine. A recent study revealed that 3 significantly regulated target genes associated with RCT via stimulating the transactivation of LXRα/β, further confirmed as a potent pharmaceutical agent for the treatment of atherosclerosis and hypercholesterolemia, and the prevention of the potential side effect of hepatic steatosis as well (Hien et al., 2017).
Ganoderma capense has been documented to possess various pharmacological activities, including defending malignant tumors and hepatitis. Ganocalidin A (14), from the fruiting bodies of G. capense, could increase LXRα and reduced lipid accumulation as an LXRα agonist (Liao et al., 2019). Ganoboninone E (15) and ganoboninketals A-C (16-18), dominating compounds of the fruiting bodies of G. boninense, exhibited remarkable agonistic activity to the transactivation of LXRβ (Ma et al., 2015;Abdullah et al., 2020). Tripterygium wilfordii Hook. f. has been used as a traditional medicine for multiple pharmacological activities, including anticancer and anti-inflammatory effects. A major ingredient triptolide (19) from T. wilfordii Hook. f., influenced either LXRα or FXR through increasing the LXRα protein expression and suppressing FXR protein expression simultaneously, while the clinical application of 19 is greatly constrained by hepatotoxicity (Jiang et al., 2016;Zou et al., 2020).
Tirotundin (20) and tagitinin A (21) from Tithonia diversifolia (Hemsl.) A. Gray, which are used to treat diabetes and hepatoprotection, also act as LXRα/β and FXR agonists (Lin HR., 2013). Platycodin D (22), a triterpene saponin from Platycodon grandiflorum (Jacq.) A.DC., suppressed the generation of proinflammatory cytokine and subsequently inhibited LPS-induced inflammatory response, due to the upregulation of LXRα expression, which is considered as a potential therapeutic drug for mastitis. It exhibited that 22 was capable of attenuating NL-induced inflammatory response in osteoarthritis chondrocyte via LXRα activation (Qu et al., 2016;Wang L. et al., 2017). Saikosaponin A (23), a wellknown bioactive component of Radix bupleuri, induced cholesterol efflux from lipid rafts through LXRα-ABCA1 signaling pathway, and knockdown of LXRα led to the abrogation of the antiinflammatory effect of 23, suggesting a potency of attenuating oxidative and inflammatory responses (Fu et al., 2015). Viperidone (24), a major ingredient of Leptocereus quadricostatus (Bello) Britton & Rose, selectively activated LXRα, and its derivatives also owned the same activity Komati et al., 2017).

Flavonoids
Cyanidin (33), a very widely distributed flavonoid present in fruits and vegetables, possesses the function of regulating cellular lipid metabolism. It was reported that 33 accelerated LXRα and LXRβ transactivation and induced the recruitment of coactivator peptide with EC 50 3.5 and 125.2 μM, respectively. Meanwhile, surface plasmon resonance (SPR) assay demonstrated that 33 directly associated with both LXRα/β with the dissociation equilibrium constant (K D ) of 33 to LXRα at 2.16 μM and LXRβ at 73.2 μM, respectively . Another common flavonoid, hesperetin (34), increased ABCA1 promoter and LXRα enhancer activities, indicating the function of promoting cholesterol efflux (Iio et al., 2012a). Chrysin (35), present in honey, propolis, and plant extracts, significantly enhanced cholesterol efflux and promoted the mRNA level of a set of nuclear receptors including PPAR (peroxisome proliferator-activated receptor) γ, LXRα, ABCA1, and ABCG1 via upregulating the classical PPARγ-LXRα-ABCA1/ABCG1 pathway . Daidzein (36), widely existing in soybeans, is an indirect modifier of LXRs as it reduced the expression of SREBP-1c through suppressing LXRα, whereas it increased the expression of ABCG8 via activating LXRβ indirectly. Another bioactive flavonoid genistein (37) present in soybeans also was reported to inhibit LXRα activation induced by a synthetic agonist T0901317 while stimulating LXRβ activation. Besides, 37 might prevent NAFLD via the regulation of visceral adipocyte metabolism and adipocytokines (Kim et al., 2010;Gonzalez-Granillo et al., 2012). Naringenin (38), widespread in grapefruits, oranges, and tomatoes, acts as a modulator of LXRα activity. It activated and upregulated LXRα and associated target genes in an AMPK (adenosine 5′-monophosphateactivated protein kinase)-dependent manner, further preventing the deterioration of atherosclerosis and foam cell progression (Wolkow et al., 2010;Saenz et al., 2018). Persimmon tannin is isolated from Diospyros kaki L., also known as proanthocyanidins. In a recent study using highcholesterol diet mice model, persimmon tannin stimulated the expressions of LXRα, PPARα, and PPARγ, and simultaneously, it promoted their associated downstream gene expressions, further accelerating macrophage reverse cholesterol transport (Ge et al., 2017).
Quercetin (39), the pivotal aglycone in Medicago sativa L., elevated protein levels of LXRα and PPARγ in THP-1cells. Additionally, it increased cholesterol efflux from THP-1 macrophages and lowered the risk of atherosclerosis. A marked antihepatitis C virus (HCV) activity was exhibited by 39 in repliconcontaining cells when combined with interferon α, appearing to be an effective inhibitor of HCV replication Pisonero-Vaquero et al., 2014;Ren et al., 2018). Iristectorigenin B (40) derived from Belamcanda chinensis, used as a traditional medicine in East Asia for its inflammation property, increased ABCA1 and ABCG1 gene expression function as an agonist for both LXRα and LXRβ significantly. It also stimulated cholesterol efflux in macrophages without inducing hepatic steatosis, providing insights for treating hypercholesterolemia and atherosclerosis (Jun et al., 2012). A flavonoid that is very widespread in food plants like Phaseolus vulgaris L. and in medicinal plants like (41), as an LXRα/β dual agonist. It exhibited potent LXRα/β agonistic activity with EC 50 values of 1.8 μM (Dong et al., 2007;He et al., 2012). Interestingly, its aglycone kaempferol (42) selectively activated LXRβ, further suppressing SREBP-1 to regulate metabolic syndrome (Hoang et al., 2015). Formononetin (43), an isoflavone constituent from Brazilian red propolis, enhanced LXR transcription and promoted ABCG1 activity in THP-1 macrophages, indicating agonistic function of both LXRα and LXRβ (Daugsch et al., 2008;Iio et al., 2012b). Nelumbo nucifera Gaertn., usually regarded as tea in Japan, has been used in traditional medicine; the leaves of Nelumbo nucifera Gaertn. contains quercetin glycosides, especially quercetin-3-O-glucuronide (44). It was proven to increase ABCA1 expression via activating LXRα, providing a potential direction on arteriosclerosis prevention (Ohara et al., 2013). Rhus verniciflua Stokes is used in TCM for the therapy of gastritis, stomach cancer, and atherosclerosis. One of the effective compounds, butein (45), increased CYP7A1 luciferase activity in AML 12 cells via activating LXRα dependently and upregulating the transcriptional expression level of LXRα, which was borne out using the knockdown of LXRα (Jeong et al., 2015). Scutellarein (46), widely found in Erigeron breviscapus (vant.), is a traditional agent for the treatment of inflammation and obesity. Cholesterol output was reduced by 46 by activating the PPARγ-LXRα-ABCA1 pathway, suggesting a potential function in the cholesterol metabolism. Beyond that, treatment with 46 attenuated high-fat diet (HFD)-induced obesity and the associated diseases as it reduced the body weight, inflammatory state, and visceral index as well as improved Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 772435 hyperlipidemia and hepatic dysfunction in C57 mice fed on the HFD-developed obesity (Lin et al., 2019). Puerarin (47), obtained from Radix Pueraria or Pueraria lobate, attenuated acute lung injury via activating LXRα and suppressed LPS-induced inflammatory response Wang et al., 2018d). Isoliquiritigenin (48), isolated from Glycyrrhizae species, could promote the activation of LXRα and repressed LXRα-dependent hepatic steatosis, further protecting hepatocytes against oxidative injury inflicted caused by fat accumulation (Kim YM. et al., 2010). The n-butanol fraction isolated from Zanthoxylum bungeanum Maxim., widespread in China as an edible aroma and a traditional medicine, mainly including rutin (49) and hyperin (50), was demonstrated to regulate the lipid metabolism in apolipoprotein E knockout mice via promoting the expression of genes involved in RCT, such as CYP27A1, LXRα, and ABCG1 (Wu et al., 2015). SPF1 (51) and SPF2 (52), two flavonoids derived from the root of Sophora tonkinensis Gagnep. induced the ABCA1 protein in RAW264.7 cells. A more detailed investigation revealed their neuroprotective effect by activating RXR/LXRs heterodimers as a novel approach to the treatment or prevention of Alzheimer's disease (Inoue et al., 2014;Wang et al., 2019). A recent study using APOE −/− mice model, kuwanon G (53) isolated from the root bark of Morus alba L., significantly decreased intracellular lipid accumulation as well as inflammatory cytokines through activating the LXRα-ABCA1/ ABCG1 pathway, which upregulated cholesterol efflux-related proteins, ABCA1 and ABCG1, in an LXRα-dependent way .

Alkaloids
Tetramethylpyrazine (54), an alkaloid diffusely widespread in foods like fermented Japanese food natto and Chinese black vinegar, also present in medicinal plant like Ligusticum chuanxiong, could directly regulate the expression of PPAR and LXRα gene by elevating the PPARγ-LXRα-ABCA1 pathway, indicating its potency of improving lipid profiles. In a small cohort of patients with pulmonary arterial hypertension or chronic thromboembolic pulmonary, the therapeutic effects of 54 were accompanied by inhibition of intracellular calcium homeostasis in rat distal pulmonary arterial smooth muscle Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 772435 cells as a novel and inexpensive medication for the treatment of pulmonary hypertension (Chen J. et al., 2017;Chen et al., 2020). Leonurine (55) present in Herba leonuri is traditionally used for therapy of gynecological disorders, dysmenorrhea, and menstrual disorders. In a study using Oil Red O staining and liquid scintillation counting assay, 55 mediated cholesterol efflux by promoting ABCA1/G1 induced by PPAR and LXRα, which inhibited lipid accumulation in THP-1 macrophage-derived foam cells via the PPAR and LXRα pathway . A fungus, Penicillium paxilli metabolite paxilline (56), was reported as an LXRα/β dual agonist. It activated LXRα and LXRβ with equivalent potency in HEK293 cells, subsequently leading to Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 772435 the expression of ABCA1 and SREBP (Bramlett et al., 2003). Oxepinamides D-G (57-60), four novel oxepin-containing pyrimidines derived from Aspergillus puniceus F02Z-1744, were revealed as LXRα selective agonists for their agonistic function of LXRα in chimeric receptor reporter gene assay with the EC 50 values of 10.6, 12.8, 13.6, and 12.1 μM, respectively (Lu et al., 2011). Oxepinamides H-K (61-64) and four 4-quinazolinone alkaloids, puniceloids A-D (65-68) were obtained from the deep-sea fungus A. puniceus SCSIO z021. Compounds 61-68 remarkably activated LXRα with EC 50 values of 1.7-50 μM, with 67 and 68 exhibiting the most potency . Scequinadoline D (69), an alkaloid derived from the marine fungus Scedosporium apiospermum F41-1, stimulated the mRNA expression of a series target genes, such as LXRα and PPARγ. It was found to facilitate TG accumulation with EC 50 values of 0.27 ± 0.03 μM as a potent insulin sensitizer targeting adipocytes and a promising potential for the treatment of T2D (Li CJ. et al., 2020).
There have been several marine-derived steroids reported as ligands of LXRs. Fucosterol (76), a steroid widespread in marine algae, could promote the transactivation of both LXRα (+155% at 200 μM) and LXRβ (+83% at 200 μM) serving as an LXRα/β dual agonist, further indicating nutritional implications in hypercholesterolemia and atherosclerosis (Hoang et al., 2012a). 24S-Saringosterol (77) (maybe same as 75) from an edible seaweed Sargassum fusiforme, promoted the transactivation of LXRα/β, and stimulated LXRβ by 14.4-fold, higher than LXRα by 3.8-fold, acting as a selective LXRβ agonist, the discovery of which also further confirmed that phytosterols in Sargassum fusiforme contributed to the well-known antiarteriosclerosis. Beyond that, 77 was in the observed effects on cognition and Aβ plaque load as an attractive option for the treatment of neurodegenerative disorders such as AD (Chen P. et al., 2014;Bogie et al., 2019;Hannan et al., 2020). Present in marine fish and plant roots is 4-Cholesten-3-one (78), but it is also found in a red marine alga Laurencia papillosa. Compound 78 at 12.5 μM resulted in a remarkable enhancement of the mRNA expression of LXRβ as well as its associated target genes compared with untreated cells in monocytic cell line THP-1. To confirm this effect, LXRs inverse agonist and LXRβ knockdown were employed, which resulted in the neutralization. Further study showed that 78 decreased the viability of two breast cancer cell lines, namely MCF-7 and MDA-MB-231, exerting promising antibreast cancer activity by altering LXRs-dependent lipid metabolism in breast cancer cells without increasing lipogenesis (Elia et al., 2019). 9α,5α,6β, and gorgost-5-ene-3β,9α,11α-triol (80) isolated from the Plexaura species possessed a better affinity with LXRα than LXRβ with EC 50 values of 0.45 and 0.05 μM .

Phenol derivatives
Ethyl 2,4,6-trihydroxybenzoate (81, ETB) was derived from Celtis biondii Pamp., which is traditionally used in the therapy of cardiovascular disease. In a reporter gene assay by timeresolved fluorescence resonance energy transfer as well as SPR analysis, ETB (81) suppressed cellular cholesterol accumulation via activating LXRα/β-responsive transcriptional genes as a LXRα/β dual agonist (Hoang et al., 2012b). Paeonol (82), a phenol present in Paeonia suffruticosa, is significantly beneficial for various inflammatory diseases, especially atherosclerosis. Treatment with 82 markedly attenuated cholesterol accumulation and suppressed the formation of foam cells in macrophages and APOE −/− mice as it activated LXRα to promote LXRα-ABCA1-dependent cholesterol efflux, providing a novel explanation for the antiatherogenic action of 82 (Zhao et al., 2013). Resveratrol (83), widespread in red wine, berries, and peanuts, elevated LXRα together with the mRNA levels of associated target genes such as ABCA1 and ABCG1. A more detailed investigation revealed that ABCA1-mediated cholesterol efflux and intracellular cholesterol could be mediated by 83 via the PPARγ/LXRα pathway. Pathologic analysis of treatment with 83 (30 mg/kg/day) in HBVX protein transgenic mice showed a therapeutic effect on HBVX proteininduced fatty liver and the early stages of liver damage, considering it as a potential preventive agent for HBVassociated hepatocellular carcinoma (Sevov et al., 2006;Voloshyna et al., 2013). Danshensu (84), rosmarinic acid (85), salvianolic acid A (86), and salvianolic acid B (87), bioactive compounds present in Salvia miltiorrhiza Bunge used for the therapy of angina pectoris, myocardial infarction, and stroke, functioned as LXRα/FXR dual agonists via the transactivation assays and improved the lipid profiles in hyperlipidemic rats (Ji and Gong, 2008;Li et al., 2015). Methyl gallate (88) and ethyl gallate (89) were isolated from Talisia nervosa Radlk, widespread in the tropical moist and wet forest. The evaluation of LXRα transcriptional activity showed that 88 and 89 were 3.16-and 2.62-fold activation of LXRα at a concentration of 100 µg/ml, indicating their potential utility against metabolic syndrome (Vásqueza et al., 2019).
The described biological activities of sesamol (95) found in sesame oil of Sesamum indicum L. mainly include antiinflammatory and antioxidative effects, protecting against hypertension, atherosclerosis, and aging. In a study using pGL3-TK-PPRE-X3-luciferase reporter assay, 95 prominently enhanced LXRα transcriptional activity for 2.6-fold at 100 μM (Periasamy et al., 2013;Majdalawieh and Ro, 2014). Magnolol (96), a natural lignin isolated from Magnolia officinalis Rehder & E.H.Wilson, has proved to activate LXRs and PPARγ target genes and inhibit the NF-κB (nuclear factor kappa-B) and mRNA expression of inflammatory cytokines under Aβ incubation in vitro studies to attenuate Aβ-induced AD (Xie et al., 2020). Honokiol (97), another lignin from M. officinalis Rehder & E.H.Wilson, activated LXRs transcriptional activity and increased its downstream gene ABCA1 expression. Interestingly, it also functions as a dual activator of LXRs/RXR heterodimer (Jung et al., 2010;Xie et al., 2020). Herniarin (98) is a 7methoxycoumarin derivative found in the aerial part of Artemisia dracunculus L., which is traditionally used to alleviate the symptomatic pain of spasmodic colitis. A detailed research indicated that 98 could upregulate LXRα/β to inhibit the development of breast cancer in Sprague-Dawley rats (Talbi et al., 2016;Pattanayak and Bose, 2019).
Emodin (99) is widespread in the TCM such as Rheum officinale Baill., R. palmatum L., and Polygonam cuspidatum, with its biological activities that range from cardioprotective to antioxidant, anticancer, antibacterial, antifibrotic, and antiinflammatory effects. Emodin (99) promoted cholesterol efflux from differentiated THP1 macrophages through activating the PPARγ/LXRs/ABCA1/ABCG1 signaling pathway. It prevented atherosclerosis in ApoE −/− mouse model, and in vitro evidence supported this effect commendably, highlighting the therapeutic potential in atherosclerosis (Luo et al., 2021). Six new octulosonic acid derivatives were obtained from the flower heads of Chamaemelum nobile L., while 100 and 101 among them exhibited an increase in LXRα activity at 30 μM (Zhao et al., 2014).
Our previous study revealed eight new ene-yne hydroquinones derived from marine fungus Pestalotiopsis neglecta SCSIO41403, one of them, pestalotioquinoside C (102), functioning as a potential LXRα agonist via upregulating the mRNA level of the target gene ABCA1. Using SPR assay, 102 interacted with LXRα dosedependently with the K D of 50.0 μM (Wang F. et al., 2020). Further study discovered pestalotiochromones A (103) as a potential LXRs agonist, remarkably upregulated LXRβ and downstream gene ABCA1. Moreover, 103 combined well with LXRα in a dose-dependent manner, the kinetic curves of which resembled those of the potent LXRs agonist GW3965 for binding with LXRα, with K D of 6.2 μM .

Others
Betaine (104), a natural trimethyl glycine in common foods, including wheat products, spinach, pretzels, and shrimp, has been used for the therapy of NAFLD via upregulating hepatic expression of LXRα and PPARα, along with attenuating the changes in their associated target genes in fructose-induced rat models. 101 also ameliorated hepatic lipid accumulation, gluconeogenesis, and inflammation through a battery of determinations, further confirming potential mechanisms involved in the treatment of NAFLD (Ge et al., 2016;Chen Q. et al., 2017). Allicin (105) is an essential ingredient of garlic, responsible for its favor, and its pharmacological activities range from anti-inflammatory to antioxidative stress and antihypertensive activities. 105 has also been confirmed to attenuate inflammation via increasing the expression of LXRα in a dose-dependent manner . Taurine (106), known as 2-aminoethanesulfonic acid is synthesized in the liver to a small extent, which is also isolated from seafood. Macrophage cells incubated with 106 inhibited cholesterol accumulation and regulated genes expression involved in RCT as an LXRα agonist (Hoang et al., 2012c). 5-Hydroxy-3-methoxy-5-methyl-4-bu-tylfuran-2(5H)-one (107), obtained from marine-derived fungus Setosphaeria sp. SCSIO41009, was reported as an LXRα agonist. It decreased ox-low-density lipoprotein-induced lipid accumulation and possessed a greater TG-lowering effect than the positive control T0901317 through targeting LXRα. Taken together, 107 displayed a weak cytotoxicity but had a powerful TClowering effect most likely through targeting PPARα, and it exhibited a potential application for the treatment of dyslipidemia (Li et al., 2020b). Setosphapyrone C (108) and D (109) derived from the same fungus, enhanced LXRα/ABC pathways to achieve a lipid-lowering effect, possessing potential application for the treatment of hyperlipidemia .
In addition, total jiaogulan saponins isolated from Gynostemma pentaphyllum were proven to upregulate LXRα and a series of target genes to increase BA and cholesterol excretions (Liu et al., 2016).

Natural antagonists targeting liver X receptors
Polyunsaturated fatty acids (PUFAs) are the dominating source of endogenous LXR antagonists, especially for arachidonic acid identified as a dual antagonist as interacting directly with LXRα and LXRβ, which also could be isolated from traditional medicine Acanthopanax koreanum Nakai. (Yoshikawa et al., 2002;Kuang et al., 2012). Prostaglandin F2α, one of the essential metabolites of compound arachidonic acid, has also been verified as a dual antagonist. Notably, prostaglandin F2α could function as an LXRs/RXR heterodimer antagonist (Zhuang et al., 2013). In addition, endogenous antagonist ursodeoxycholic acid, a key secondary BA, has been substantiated to inhibit LXRα-induced lipogenic gene expression as a negative modulator of LXRα signaling .

Terpenes
Iridoid (110) functions as a predominant compound of Valeriana jatamansi Jones. It has been shown that treatment with 110 resulted in the decrease of lipid biochemical indexes in hyperlipidemic rats by decreasing the expressions of SREBP-1c and LXRα (Zhu et al., 2016). Lucidone (111), a natural occurring terpene derivative present in a folk medicine Lindera erythrocarpa Makino, has been found to inhibit adipogenesis in 3T3-L1 cells by decreasing adipogenic genes transcription levels, including LXRα, indicating that it is a nutraceutical to guard against obesity and subsequent metabolic disorders (Hsieh and Wang 2013;Wong et al., 2014). Two sesquiterpenoids named paraconiothins C (112) and I (113), isolated from the endophytic fungus Paraconiothyrium brasiliense ECN258, were proven to inhibit LXRα at 50 μM .
Ursolic acid (114) is a common pentacyclic triterpenoid in many plants, such as Cornus officinalis Siebold & Zucc., and the described pharmacological activities range from anticancer to antioxidant, antiangiogenic, anti-inflammatory, immunoregulatory, hypolipidemic, and hepatoprotective effects. It was demonstrated to be an LXRα antagonist and displays efficacy in treating NAFLD as it significantly decreased TC accumulation and induced steatosis at 20 μM through modulation of LXRα, transcription factor SREBP-1c, and a battery of downstream target genes . Potentilla chinensis Ser. found in oriental countries, has been traditionally used in the therapy of immune disorders and liver diseases. Asiatic acid (115), a pivotal constituent isolated from P. chinensis Ser., significantly regulated the key factors associated with lipid metabolism including SREBP-1c and LXRα to restrain the production of hepatocyte lipogenesis. A further study elucidated that 115 effectively alleviated hepatic steatosis and hepatocyte damage, attributed to its ability to alleviate oxidative stress, recruit the antioxidative defense system, inhibit the NF-κB pathway, alleviate hepatocyte apoptosis, and lipid metabolism disorder and, thereby, for the treatment of NAFLD (Wang et al., 2018a). A clerodane diterpenoid borapetoside E (116), derived from Tinospora crispa (L.) Hook. f. & Thomson, which is used in traditional medicine for the therapy of diabetes and other diseases, suppressed the mRNA expressions of SREBP and LXRα in the liver, serving a potential therapy for diet-induced T2D. It improved hyperglycemia and oral glucose tolerance, promoted insulin signaling, improved insulin resistance, and improved lipid levels, etc., in high-fat diet-induced obese mice, these beneficial effects in vivo further demonstrated the promising therapy (Xu W. et al., 2017). A TCM Panax ginseng C. A. Meyer has been regarded as a panacea for centuries in the treatment of various diseases such as metabolic disorders. (117) is present in the root of this plant encompassing antiinflammatory and antioxidative stress bioactive activities. A synthetic agonist T0901317-dependent LXRα target genes and T0901317-induced TG accumulation were inhibited by 117 in primary hepatocytes, further suggesting it as a potential antilipogenic agent to treat NAFLD (Oh et al., 2015a).

Flavonoids
Luteolin (118), occurring in a broad range of vegetables, fruits, and grains like carrots, peppers, celery, parsley, and spinach, is a common dietary flavonoid exerting numerous biological activities including antioxidant, anticancer, antimicrobial, antiallergic, and antiinflammatory effects. It abrogated agonistinduced LXRα/β transcriptional activity and suppressed the expression of related target genes serving as an LXRα/β antagonist (Francisco et al., 2016). Treatment with 118 inhibited LXR activation in HepG2 cells and eliminated lipid accumulation induced by LXR-SREBP-1c activation, thereby decreasing TG accumulation and primary hepatocytes. Overall, lipid accumulation induced by LXRs-SREBP-1c activation was abolished both in vivo and in vitro after treatment with 118, indicating the potential as a therapeutic agent for treating NAFLD (Yin et al., 2017). Besides, 118 in combination with cisplatin could potentially be used as a new regimen for the treatment of ovarian cancer (Wang et al., 2018c). It exhibited that 118 could upregulate LXRα and downstream target gene expression to control cholesterol metabolism . Morin (119) present in many plants like mulberry, jackfruit, green tea, also in TCM like Tartary buckwheat, could reduce LXRα/β agonism induced by a synthetic agonist GW3965 (Gu et al., 2017a). Licochalcone A (120), present in the licorice root of Glycyrrhiza plant, such as Glycyrrhiza glabra L. and G. inflata Batalin, has been known as "Guolao" in China for thousands of years. The described bioactive properties range from antiparasitic to anticancer, antifungal, antiinflammatory, and osteogenic activities. As an LXRα antagonist, 120 restrained the transcription of lipogenic LXRα and resulted in the diminishment of accumulating TG in primary hepatocytes treated with a synthetic agonist T0901317 (Oh et al., 2015b). Isorhamnetin (121), an active ingredient of Hippophae rhamnoides L., downregulated the mRNA levels of LXRα and PPARγ (Lee et al., 2009). It also promoted the protein expression of PPARγ, LXRα, and CYP7A1 in qPCR analysis, indicating it as a potential agent in lipid homeostasis (Xiao et al., 2021). In HFD mice model, administration of alpinetin (122), derived from Alpinia katsumadai Hayata, efficiently suppressed LXRα and a series of receptors associated with lipid metabolism. Oral glucose and insulin tolerance tests, immunohistochemical and immunofluorescent analyses manifested that 122 attenuated insulin resistance and alleviated liver injury in HFD-induced mice as an ideal natural product in NAFLD (Zhou et al., 2018).
Sophoricoside (123), an LXRβ antagonist, present in the dried fruit of Styphnolobium japonicum (L.) Schott, has been reported to possess an antioxidant property. It could decrease the transcriptional activity of LXRβ concentration dependently (Zhang et al., 2020d). Xanthohumol (124), a critical flavonoid present in the female inflorescences of Humulus lupulus L., remarkably attenuated the mRNA expression of a direct target for LXRα transcriptional activation, inducible degrader of the low-density lipoprotein receptor (LDLR) in hepatic cells, which further inferenced a potential role of counteracting LXRs activation (Chen SF. et al., 2017). A flavonolignan silymarin (125), obtained from Silybum marianum (L.) Gaertn., has beneficial effects on liver diseases. It has been shown to reduce de novo hepatic lipogenesis, recover in insulin sensitivity, and protect against exacerbated myocardial ischemia reperfusion injury. Administration of 125 at 100 and 300 mg/kg depressed the upregulation of LXRβ and related genes expression in the liver of high fructose diet mice (Prakash et al., 2014;Chen et al., 2017b). Citrus fruits contain ample bioactive compounds including flavonoids, carotenoids, and coumarins. Citrange peel extract and citrange flesh and seed extract were proved to decrease LXRα/β and PPARγ level, emerging as a potential candidate for the treatment of obesity and related metabolic disorders (Lu et al., 2013).

Phenol derivatives
Curcumin (133), an active phenol derivative obtained from Curcuma longa L., could suppress the hepatic overexpression of LXRα, PPARγ, and fatty acid synthase. An immunoblot analysis also verified that 133 decreased the protein expression of LXRα and SREBP1c in the liver. Moreover, histological and serum biochemical analyses indicated that 133 apparently attenuated the hepatic lipid accumulation and decreased TG, TC, and nonesterified fatty acid levels in NAFLD mice model on account of the role for the prevention and treatment of NAFLD (Maithilikarpagaselvi et al., 2016;Chen et al., 2017b;Auger et al., 2018).
Cyclic bibenzyl derivative riccardin F (138), present in liverworts, such as Blasia pusilla L., suppressed LXREdependent luciferase transcription in HepG2 cells functioning as an LXRα antagonist as well, and its homologous compound 90 has been described above as an LXRβ antagonist (Tamehiro et al., 2005;Asakawa, 2008). An LXRα antagonist biacetophenone cynandione A (139) found in the dried roots of Cynanchum wilfordi is both an endemic tonic and a traditional herbal medicine to promote liver and renal function. In HepG2 cells, treatment with 139 inhibited the mRNA levels of SREBP-1c as well as the expression of each enzyme upregulation promoted by two extrinsic LXRα agonists, GW3954 and T0901317 . Guttiferone I (140) is a naturally occurring polyisoprenylated benzophenone found in Garcinia humilis (Vahl) C.D.Adams. It has been found to bind with LXRα as an LXRα antagonist earlier on .
Rhein (141) is a key phytochemical of Rheum palmatum L., which is used as a TCM for thousands of years to treat obesity, constipation, ulcers, and gastrointestinal hemorrhage. It resulted into LXRs antagonism, dose-dependently interacted with LXRα and LXRβ with K D values of 46.7 and 31.97 μM in SPR assay, respectively. Moreover, 141 suppressed the transcriptional activity of SREBP-1c and further dose-dependently reduced the transaction of LXRs induced by GW3965. A further detailed study showed that 141 suppressed the expression levels of LXRs target genes. According to a series pharmacological experiment in an HFD-induced obese mouse model such as intraperitoneal glucose tolerance test and serum, fecal, and liver lipid content analysis, 141 showed several beneficial treatment effects on NAFLD in mice model, including decreased body and fat weight, lowered serum and hepatic lipid levels, improved insulin resistance, and reversed hepatic steatosis (Sheng et al., 2011a;Sheng et al., 2011b;Xue et al., 2020).

Others
Our recent study revealed that piericidin A (142, PA) and its glycoside glucopiericidin A (143, GPA), isolated from a marinederived Streptomyces strain, could decrease the protein level of LXRα rather than mRNA expression. SPR assay was further employed to explore, along with several other piericidin analogs. As expected, PA presented a similar LXRα protein affinity (K D 3.24 × 10 −5 M) compared with GW3965 (K D 6.97 × 10 −5 M). Another piericidin aglycone, 10-ketone PA (144), could also serve as an LXRα ligand with a K D value of 7.22 × 10 −5 (M). However, the affinity for GPA-LXRα binding was much weaker in nonkinetic simulation binding mode, with the K D value of 3.492 (M). Other piericidin glycosides, such as 13-OH GPA and 6″-Gal GPA, also exhibited no binding activity. The molecular docking analysis also showed the significant differences in the binding effects of glycosides and glycosides with LXRα protein. Intriguingly, PA (142) and GPA (143) have been considered as antirenal cell carcinoma (RCC) drug candidates, whereas on account of LXRα, PA/GPA caused specific hepatotoxicity in RCC xenograft mice-induced ALT to 2.6-and 2.3-fold, as well as the AST to 1.6-and 2.2-fold higher than that of the control group, respectively. Further study demonstrated that PA/GPA aggravated hepatotoxicity in high cholesterol diet-fed LXRα-activated mice while exhibiting no toxicity in chow diet-fed mice, indicating that the accumulation as well as the delay metabolism process of cholesterol resulted in hepatotoxicity and cholestasis. Pharmacokinetic study discovered that GPA existed as a prodrug in the liver and exerted toxic effect due to transforming into PA, different from PA directly combined with LXRα as an inhibitor (Zhou et al., 2019;Liang et al., 2020).
It exhibited that schisandra polysaccharides, isolated from Schisandra chinensis (Turcz.) Baill , phenol, flavonoid, and sugar collected in the fruit of Abelmoschus esculentus , and total glycosides, isolated from Ligustrum lucidum L. Moench (Zhao and Liu, 2020), have the potency to downregulate LXRα and a set of associated genes, indicating both preventive and therapeutic role in regard to metabolic disorders.

NATURAL PRODUCTS TARGETING FARNESOID X RECEPTOR
In this section, we gather natural products regulating FXR expression in different models and categories. These are illustrated in Supplementary Tables S3 and S4.

Terpenes and steroids
Forman et al. identified and described an orphan nuclear receptor, farnesoid X-activated receptor, which is activated by a naturally occurring isoprenoid farnesol (148) and related derivatives. Farnesol (148), present in many essential oils, activated FXR-RXR at the concentrations ranging between 5 and 50 μM as an FXR agonist, further inhibiting hepatic TG synthesis via regulating the mRNA expressions of related molecules involved in lipid metabolism (Forman et al., 1995). Further study indicated that 148 activated both PPARα and FXR at similar concentrations (30 or 50 μM) in CV-1 cells .
Lepidozenolide (149), a sesquiterpenoid isolated from the liverwort Lepidozia fauriana, could significantly activate FXR in a dose-dependent manner (Lin, 2015). Cafestol (150), a wellknown ingredient of coffee, has been identified as a dual FXR/ PXR agonist, as assessed by a double FXR/PXR KO mice model. It increased the expression of FXR-target genes in pathways of BA biosynthesis and intestinal FGF15 via the contribution of FXR/ PXR activation (Ricketts et al., 2007). A naturally occurring terpenoid found in Ferula ovina Boiss. is tschimgine (151), capable of activating FXR and bind ideally with the LBD of FXR in molecular docking. Interestingly, 151 inhibited the induction of inflammation genes in an FXR-dependent manner (Zheng W. et al., 2017). Dihydroartemisinin (152), the derivative of artemisinin, is present in Artemisia carvifolia Buch.-Ham. ex Roxb. and emerged as a potential candidate for the treatment of liver cancer and fibrogenesis. Compound 152 activated FXR to inhibit contractility of cultured hepatic stellate cells as a potential natural product to attenuate portal hypertension in fibrotic rodents Xu Y. et al., 2017;Ma et al., 2020). Scrophularia dentatais, a folk herbal medicine found in Tibet, is emerging as a potential candidate for the treatment of smallpox, measles, high-heat plague, and poisoning. Zhang et al. isolated four compounds from the whole plant of Scrophularia dentatais Royle ex Benth., all of them being C 19 -diterpenoids. One is scrodentoids F (153) that dosedependently activated FXR in dual-luciferase reporter assay system in 293T cells . Picroside II (154), the major terpene in Picrorhiza scrophulariiflora Pennell, activated FXR via transient transfection in a dual-luciferase reporter assay. It decreased CYP7A1 and increased bile salt export pump and UDP-glucuronosyltransferase 1a1 mRNA expression, the latter two of which were inhibited upon knockdown of FXR. Meanwhile, 154 dose-dependently reversed α-naphthylisothiocyanate (ANIT)-induced alteration in serum markers and thereby exerted protective effect on ANIT-induced cholestasis, possibly through FXR activation Li et al., 2020e). Geniposide (155), a pivotal constituent in Gardenia jasminoides Ellis, activated FXR, PXR, and SHP. Blood biochemical determination discovered that 155 at 25, 50, or 100 mg/kg increased bile flow rate and reversed the ANIT-induced increases in serum markers for hepatotoxicity and cholestasis in a dose-dependent manner . Genet et al. screened betulinic acid (156) from the Betula species, oleanolic acid (157) from Olea europaea L., and ursolic acid (114) from Cornus officinalis Siebold & Zucc., and tested them regarding their ability to activate FXR. In the results, 156, 157, and 114 activated FXR along with potential impact in diabetes (Genet et al., 2010), and 157 also generated frail FXR agonism, along with moderate PXR agonism, which protected against LCA-induced hepatotoxicity and cholestasis (Chen Z. et al., 2014). Hedragonic acid (158), from the stem and root of Celastrus orbicalatus Thunb., remarkably activated FXR and possessed therapeutic effects on liver injury and inflammation in acetyl-para-aminophenol-induced mice model as an FXR agonist (Lu et al., 2018). Ergosterol peroxide (159) and four triterpenoids, ganoderiol F (160), ganodermanontriol (161), lucidumol A (162), and ganoderic acid TR (163), are bioactive lanostane metabolites of Ganoderma lucidum Karst., which has been used in Asian medicines since 2,000 years against a variety of diseases including tumor, inflammation, obesity, and diabetes. 159-163 were verified as FXR agonists as inducing FXR with the low micromolar range in HEK293 cells (Grienke et al., 2011). Ginsenoside Rg1 (29) from Panax ginseng C.A.Mey. is both FXR and LXRs agonist, the function of 29 on LXRs has been described above. Herein, 29 also was reported to activate FXR to promote bile secretion and Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 772435 normalize enzyme activity in the serum in intrahepatic cholestasis mice model (Xiao et al., 2020). Astragalus membranaceus (Fisch.) Bunge, widespread in Europe and Asia, is beneficial to the treatment of diabetes, hyperlipidemia, atherosclerosis, and cancers, with its key constituents like astragaloside IV and cycloastragenol (164). Compound 164 stimulated FXR transcription activities and regulated the expression of FXR target gene in HepG2 cells as a potential candidate for NAFLD. Meanwhile, it improved metabolic profiles, ameliorated hepatic steatosis, altered BA composition, and activated FXR signaling and feedback loops in diet-induced obesity mice, further confirming the promise in ameliorating NAFLD. Besides, 164 also alleviated hepatic steatosis in methionine and choline-deficient L-amino acid diet-induced NASH mice (Gu et al., 2017b). Alisol M 23acetate (165) and alisol A 23-acetate (166) present in Alisma orientalis (Sam.) Juzep. have been described as FXR agonists by transactivating FXR to modulate promoter action (Lin 2012). Another FXR agonist present in A. orientale (Sam.) Juzep and Rhizoma alismatis is alisol B 23-acetate (167), protecting against NASH and CCl 4 -induced hepatotoxicity via FXR activation (Meng et al., 2015;Meng et al., 2017;Huo et al., 2018). In addition, 167, aisol F (168), aisol A (169), and 25-anhydro alisol A (170) found in A. orientale (Sam.) Juzep. activated FXR in a dose-dependent manner (Huo et al., 2018).
Four tirucallanes xylocarpols E (173), agallochols A (174), B (175), and D (176) isolated from mangrove plants, activated FXR at the concentration of 10.0 µM in a luciferase reporter assay in HepG2 cells . Dioscoreae rhizome, also known as "Shaoyao," acts as either a food or a traditional medicine because of its protectivity of the kidneys and liver. Dioscin (177), a steroid saponin from D. rhizome, attenuated oxidative stress and inhibited inflammation to improve thioacetamide-induced acute liver injury in Sprague-Dawley rats as upregulating the expression levels of FXR and AMPKα via FXR/AMPK signal pathway, providing a new insight in the treatment of acute liver injury (Zheng L. et al., 2018).

Flavonoids
Calycosin (178), present in the dry roots of Radix Astragali, has been demonstrated to improve TG metabolism, mitigate obesity, and hyperlipidemia. It played an emerging role in protecting against liver injury induced by CCl 4 on account of activating FXR and its target genes as an FXR agonist (Chen et al., 2005b). A more detailed study showed that 178 attenuated TG accumulation and hepatic fibrosis to protect against NASH through FXR activation . Cryptocarya chinensis (Hance) Hemsl. is an endemic famous for its antioxidant, immunological, and antitumor effects in southern China, Japan, and Taiwan. Four naturally occurring tetrahydroflavanone cryptochinones A-D (179-182), obtained from this plant, transactivated FXR dose dependently in a transient transfection reporter assay, semblable to CDCA (Lin et al., 2014). Xanthohumol (124) is not only a counteractor of LXRs activation, but also an FXR modulator. It activated FXR dose-dependently in the transient transfection assay. Furthermore, 124-fed mice possessed lowered levels of SREBP-1c, gluconeogenetic genes, as well as targets involved in fatty acid synthesis in KKA-(y) mice, indicating a promising role in attenuating diabetes (Nozawa, 2005).
A flavanone glycoside abundantly found in lemons and oranges is hesperidin (183), treatment with which prevented cholestatic liver injury and reduced BA toxicity in HepaRG cells via activating FXR. Compound 183 dose-dependently protected against 75 mg/kg dose of ANIT-induced cholestasis and liver injury as reversing increases in the liver index, biliary index, serum AST, ALT, alkaline phosphatase, and total bilirubin, functioning as an effective agent for the prevention and therapy of cholestatic liver disease . Desmodium styracifolium has extensive biological activities, ranging from cholesterol level-lowering to cancer prevention and inflammatory inhibition. Schaftoside (184), a flavanone glycoside found in D. styracifolium, ameliorated oxidative injury and inflammation as an FXR agonist. Particularly, 184 attenuated acetaminophen-induced hepatotoxicity via restraining NF-κB signaling and fine-tuning the generation of pro-and antiinflammatory eicosanoids, providing a promising agent to alleviate liver injury induced by acetaminophen overdose (Liu et al., 2020a). In another study, HFDinduced lipid accumulation in the liver was decreased after treatment with 184, as indicated by reduced AST and cholesterol in serum, along with intracellular TG levels in the serum and liver tissue, subsequently resulting in attenuation of liver histopathological injury. As assessed by RT-PCR and Western blotting, 184 ameliorated HFD-induced NAFLD probably via FXR-SREBP1 signaling (Liu et al., 2020b). A crucial tea catechin, epigallocatechin-3-gallate (185, EGCG), is well known as a potential tonic for cardiovascular diseases and a variety of cancers. EGCG activated FXR with the EC 50 value of 2.99 μM, nevertheless, which seemed to be specific because of the nonactivation of other nuclear receptors. It was regarded as a selective SBARM (a selective bile acid receptor modulator) (Li et al., 2012). EGCG possessed the strongest anti-HBV activity toward HBsAg and HBeAg among the crude green tea catechin tests. Meanwhile, the interaction between 185 and FXR has been confirmed to decrease the transcriptional activation of HBV, serving as an FXR antagonist and anti-HBV agent (Xu W. et al., 2016).
Total flavonoids of Astmgali Radix (TFA) are the essential ingredients of A. Radix, which contributes to the pharmacological efficacy including antiinjury, antimutation, and antitumor effects of this herb. TFA reversed the decrease in mRNA and protein levels of FXR induced by dimethylnitrosamine in rats and mitigated liver function in rats with liver cirrhosis . Procyanidins is abundant in grapes, apples, red grape juice, and red wine, whose folkloric applications embody in preventing and ameliorating atherosclerosis and other cardiovascular disease. A SBARM grape seed procyanidin extract promoted FXR transcriptional activity induced by BA and FXR dependently resulted in a reduction in TG in vivo (Del Bas et al., 2009).
Papaverine (194) is a bioactive constituent present in Papaver somniferum L. and contributes to the pharmacological efficacy of this herb, appearing to be an FXR agonist in a full length FXR transactivation assay (Steri et al., 2012). Another famous natural product berberine (195), beneficial for diarrhea, isolated from the roots of Coptis chinensi, regulated BA metabolism and, thus, exerted a lipid-lowering effect, functioning as an intestinerestricted FXR agonist. It prevented diet-induced obesity and TG accumulation in the liver with a significant decrease in serum and hepatic TG by 19% and 47%, respectively. A further mechanism study discovered that the antiobesity and lipidlowering effects of 195 are primarily due to the activation of intestinal FXR and thereby result into a reduction in the CD36 expression Tian et al., 2019).
Three naturally occurring phenylpropanoids, nelumol A (196) and nelumal A (197), from Ligularia nelumbifolia Hand. Mazz. (Bruyere et al., 2011), and auraptene (198), from Citrus aurantium, exhibited significant agonistic activity of FXR as FXR agonists (Epifano et al., 2012). The activation of FXR via 198 also resulted in hepatoprotective effect against cholestatic liver injury and increasing effect of BA efflux from the liver into the intestine . Moreover, it showed that 198 significantly attenuated apparent collagen deposition between liver lobules in thioacetamide-induced liver fibrosis mice. Treatment with 198 at 30 mg kg −1 significantly lowered the grade of fibrosis compared with the thioacetamide group. Further gene expression involved in hepatic stellate cell activation and fibrosis also confirmed its efficacious potency. Overall, 198 seemed to be beneficial to hepatic fibrosis as reducing toxic BAs and inhibiting hepatic stellate cell activation and inflammation due to FXR activation (Gao et al., 2018). Another phenylpropanoid present in the rhizomes of the Podophyllum species, podophyllotoxin (199), could transactivate FXR at a concentration of 10 μM in HeLa cells (Hiebl et al., 2018).
"Se-Ji-Mei-Duo," recognized as having auspicious and beautiful flowers, is a traditional medicine, Herpetospermum pedunculosum (Ser.) C.B. Clarke, for which various biological properties including anti-HBV, anti-inflammatory, and antioxidant effects have been described. Five lignans, herpetotriol (200), spathulatd (201), lariciresinol (202), dehydrodiconiferyl alcohol (203), and herpetrione (204), have emerged with significant FXR agonistic activity in vitro and, thus, were identified as the main bioactive constituents in the seeds of H. pedunculosum (Ser.) C.B. Clarke (Wei et al., 2020). Furthermore, dehydrodiconiferyl alcohol (205) is also an FXR agonist found in the seeds of H. dunculosum (Ser.) C.B. Clarke. To confirm its agonistic activity, knockdown of the expression of FXR in L-02 cells was performed. The mRNA and protein levels of FXR as well as a range of keys downstream has been remarkably upregulated after treatment with 205, which were decreased in Si-FXR cells .
Soybean coumestrol (206) with 50 μM stimulated FXR transcriptional activity to 2.0-fold over the control group in HEK293T cells. ORO staining was employed to investigate the effect of 206 on differentiation of 3T3-L1 preadipocytes into mature adipocytes. It was observed that 206 dose-dependently suppressed MDI (mixture of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin)-induced lipid accumulation in a manner, 206 at 20 or 40 µM decreased MDI-induced lipid accumulation by 38.8% and 77.4%, respectively. Together with RT-PCR analysis, 206 could be considered as an agent to prevent adipogenesis (Takahashi et al., 2008;Jang et al., 2016). Yangonin (207), a kavalactone found in Kava Piper methysticum, has been demonstrated to bind with the LBD of FXR and activate FXR to alleviate estrogen-caused cholestasis. Moreover, 207 caused an increase in BA efflux and detoxification as well as a decrease in BA uptake and synthesis via activating FXR, thereby exerting potential hepatoprotective effect. Moreover, in estrogen-induced cholestasis rat model, 17α-ethinylestradiolinduced growth retardation was reduced in a dose-dependent manner by 207, and relative liver weight was significantly decreased after treatment with 207, thereby alleviating estrogeninduced cholestasis .

Others
Two secondary metabolites collected in Monascus-fermented rice, monascin (208) and ankaflavin (209), have been confirmed to upregulate FXR and PPARα expressions and, thus, result in fatty acid oxidation as food supplements to prevent against diabetes and obesity (Hsu et al., 2014).
Oleanolic acid (157) not only activates LXRs as a promising treatment for NAFLD but also functions as an antagonist of FXR. It could bind with the LBD of FXR and inhibit the activity of FXR-LBD induced upon an endogenous ligand CDCA (Liu and Wong 2010). It also ameliorated obstructive cholestasis in mice after treatment with bile duct ligation due to FXR antagonism. Further histological and biochemical assessment showed that pretreatment with 20 mg/kg of 157 significantly ameliorated pathological alteration, such as extrahepatic cholestatic liver injury, bile duct hyperplasia, and portal infiltrates along with extensive foci of liver necrosis in bile duct-ligation mice (Chen et al., 2015a). Schisandra glaucescens Diels is both a traditional medicine and a food locally, as a potential candidate for the therapy of spontaneous sweat, night sweat, chronic diarrhea, along with neurasthenia in Hubei and Sichuan provinces of China. Cycloartane triterpenoids 6β-hydroxynigranoic acid (213) and schiglausin N (214), from the stems of S. glaucescens Diels, exhibited significant inhibitory effect on FXR as antagonists. Compound 213 restrained FXR with an IC 50 of 1.50 μM, and simultaneously 214 emerged with the inhibitory rate of FXR of more than 20% at 25 μM (Zou et al., 2012a). Other six triterpenoids from S. glaucescens Diels, schiglausins D-G (215-218), kadsuric acid (219), and 3β-hydroxy-lanost-9(11),24-dien-26-oic acid (220), revealed antagonistic activity against FXR together with the inhibitory rate higher than 20% at 25 µM (Zou et al., 2012b). An FXR antagonist, platycodin D (22), from Platycodon grandiflorum (Jacq.) A.DC., is capable of modulating hepatic lipogenesis in high glucose-exposed HepG2 cells and in HFD-fed rats (Hwang et al., 2013). Treatment with 22 exhibited an antagonistic ratio at 40%-50%, compared with the control value of CDCA in a coactivator recruitment assay for FXR as a novel cholesterol-lowering and anti-atherogenic candidate (Zhao et al., 2006).
Marine organisms, such as sponges and tunicates, have received remarkable interests as a valuable source of novel bioactive natural products encompassing diverse chemical structures and pharmacological activities. Suvanine (221), a sesterterpene present in sponge Coscinoder mamathewsi, has been confirmed to be an FXR antagonist, effectively antagonizing FXR transactivation (Di Leva et al., 2013). In addition, found in the genus of Spongia also antagonized FXR transactivation. They decreased the affinity of FXR LBD for a coactivator peptide facilitated by CDCA in SPR assay, especially for 224-227, disrupted very strong interaction between FXR and a coactivator peptide (Nam et al., 2006;Nam et al., 2007). An isoprenoid tuberatolide A (228), and five meroterpenoids tuberatolide B (229), 2′-epi-tuberatolide B (230), yezoquinolide (231), R-sargachromenol (232), and S-sargachromenol (233), were isolated from Korean marine tunicate Botryllus tuberatus, and all of them exhibited significant antagonism on FXR with IC 50 values as low as 1.5 µM. Compounds 228-233 released the coactivator peptide from the CDCA-bound hFXR LBD in cell-free SPR experiments, indicating the characterization of their potent FXR antagonists (Choi et al., 2011).

Steroids
Guggulsterone, existing in two isomeric forms, namely, E-guggulsterone (234) and Z-guggulsterone (235), is the active agent isolated from the resin of Commiphora mukul (Arn.) Bhandari, responsible for antihyperlipidemic effect. E/ Z-guggulsterones 234 and 235 have been identified as FXR antagonists directly, to decrease hepatic cholesterol levels in rodent models. Moreover, Z-guggulsterone (235) exhibited remarkable FXR antagonism with an IC 50 of 1-5 μM in HepG2 cells, increased the cholesterol CYP7A1, and further decreased the circulating cholesterol level (Urizar et al., 2002;Urizar and Moore, 2003;Bhutani et al., 2007;Yu et al., 2009;Singh and Sashidhara, 2017). A very well established FXR antagonist from a natural source is stigmasterol acetate (226), a principal phytosterol extracted from soybeans. Compound 236 inhibited the expression of FXR target genes induced by CDCA in a Gal4-responsive luciferase reporter gene assay in HepG2 cells. Intriguingly, except for FXR, 236 also antagonized the LBD of PXR (Carter et al., 2007). Ergostane-3,4,21,26-tetrol 3,21-bis (hydrogensulfate) (237) is a sulfated polyhydroxy sterol present in the marine invertebrate Ophiolepis superba. It resulted in a potent antagonism of FXR transactivation concentration dependently, and treatment with 237 at 100 μM completely reversed the effect exerted by CDCA on FXR expression. Taken together, 237 might be a potential candidate in regulating BA metabolism (Sepe et al., 2011;Fiorucci et al., 2012b). Some of polyhydroxylated steroids isolated from marine sponge Theonella swinhoei have been identified as FXR antagonists.

Others
Three PUFAs FXR antagonists, arachidonic acid (146), docosahexaenoic acid (254) from Crocodylus siamensis, and linolenic acid (255) from Perilla frutesccns, exhibited antagonistic activity of CDCA-activated FXR with IC 50 ranging from 0.9 to 4.7 μM (Zhao et al., 2004). Naringin (256), one of the most abundant and common flavonoids in citrus fruits and grapefruits, has been reported to relieve atherosclerosis. In ApoE −/− mice fed with an HFD, 256 ameliorated atherosclerosis, lowered the serum and liver cholesterol levels by 24.04% and 28.37%, increased lipid and BA excretion in feces, and regulated the gut microbiota associated with cholesterol metabolism. Moreover, 256 upregulated CYP7A1 via suppressing FXR/FGF15 pathway, and facilitated BA synthesis, providing insight into the atherosclerosis-amelioration mechanisms (Wang J. et al., 2020). In addition, 256 also could exert hepato-and nephroprotective effect in rats via the role of FXR and renal injury molecule-1 (Adil et al., 2016).
Maninsigins A (257), isolated from Magnolia officinalis as a TCM for the therapy of asthma, dyspepsia, and asthmatic cough, suppressed FXR activation induced by CDCA with an IC 50 value of 55.6 μM as an FXR antagonist (Shang et al., 2013). An isoquinoline alkaloid palmatine (258, PAL) from Rhizoma coptidis contributes to the pharmacological efficacy of the herb such as obesity, diabetes mellitus, hyperlipidemia, and hyperglycemia-cardiovascular diseases. PAL-treated rats were characterized by decreased TC, TG, low-density lipoprotein cholesterol levels, serum total bile acid (TBA) levels, and increased fecal TBA and TC levels. It showed that 258 reduced HFD-induced lipid accumulation in the liver. So, it indicated the curative effect of 258 for hyperlipidemia (Ning et al., 2020).
Halicylindramides F (259), A (260), and C (261) are three naturally occurring depsipeptides from a Petrosia species marine sponge collected in Korean waters. In a cotransfection assay in CV-1 cells, 259-261 possessed potent inhibitory effects of CDCAinduced hFXR transactivation with IC 50 values of 6.0, 0.5, and 5.0 μM, respectively. Nevertheless, the function of hFXR antagonists they exerted did not bind directly to hFXR. However, SPR assay showed that 259-261 did not appear to have any significant inhibition of the recruitment of cofactor coactivator peptide to hFXR at concentrations higher than their IC 50 values determined in a cell-based cotransfection assay. Thereby, the antagonism between FXR and 259-261 might be achieved by an indirect manner, not by a direct binding to LBD of FXR (Hahn et al., 2016).

MOLECULAR DOCKING ANALYSIS OF LIVER X RECEPTORS AND FARNESOID X RECEPTOR
In silico molecular docking is an important virtual screening method in the drug discovery process, which mainly focuses on structures of targets and ligands. It predicts the binding geometries as well as the binding energy of the ligand-target complex. To better understand the above-reported active natural products targeting LXR/FXR, most of them were performed together with molecular docking analyses, using the same method and parameters, although similar work has been done on some compounds in the literatures. As results, some natural compounds, like 7, 47, 54, 58, 60, 76, 77, 92, 93, 94, 136, 145, 156, 170, 189, 223, 226, 230, 232, 236, 238, 240, 244, 249, 250, 257, and 258, exhibited optimal property for targeting LXRs or FXR in molecular docking evaluation, suggesting that these compounds are even more promising.

Molecular docking analysis of liver X receptors
Based on the abovementioned LXR ligands, we have then analyzed, by means of molecular docking calculations, the interactions between LXRs ligands and LXRs proteins to understand and shed light on the binding mechanism of LXRs ligands. The crystal structures of the LXRα LBD in a complex with T0901317 (PDB ID: 1UHL) (Svensson et al., 2014) and GW3965 (PDB ID: 3IPQ) (Fradera et al., 2010), along with the LXRβ LBD in a complex with 2 (PDB ID: 1P8D) (Williams et al., 2003), were selected and subjected to an in silico molecular docking analysis, using the induced-fit module in the Schrödinger software suite. Molecular docking results (S values listed in Supplementary Tables S1 and S2) indicated that majority of the compounds fitted comfortably into the binding pocket, and the most potential 2D and 3D binding models of each protein are displayed in Supplementary Figures S1-S3, respectively.
As the docking results shown in Supplementary Figure 1A, the hydroxy in the saccharide moiety of 27 formed hydrogen bonds with the active-site residue SER 228 of 1UHL, the S value of which was −8.779, followed by 130 (−8.770), and 47 (−8.732), comparative with the ligand T0901317 (−8.931). In line with the abovementioned SPR assay of 33, 81, 102, 103, and 141, they could directly combine with LXRα protein 1UHL with S values from −5.763 to −9.187 ( Supplementary Figures S1B-F). In the 2D binding models, the hydroxy or carbonyl groups in the hexatomic ring formed hydrogen bonds with the active site residue THR258, HID421, LYS434, THR302, TRP443, SRE264, or ARG305 of LXRα.
The docking model between LXRα 3IPQ and 58 along with 60 ( Supplementary Figures S2A,B) exhibited that 58 and 60 formed hydrogen bonds with the active-site residue PHE 257, and the hexatomic ring plays a key role to form a π-π stacking interaction with PHE 315 with the docking score of −10.776 and −10.567 individually. Compounds 33,81,102,103,141,142, and 144 also appeared to interact with LXRα protein 3IPQ perfectly with the S values from −5.763 to −9.187 (Supplementary Figures S2C-I), consistent with the corresponding SPR assay. 2D binding models showed that the phenolic hydroxy groups in the hexatomic ring formed hydrogen bonds with the active site residue ARG305, GLU267, MET298, THR302, PHE257, or SER264 of LXRα. Additionally, the aromatic hexatomic ring of 33, 103, and 141 formed a π-π stacking interaction with PHE 315 or PHE 326 of LXRα.
As depicted in Supplementary Figures S3A-D, 94, 76, 27hydroxycholesterol, and its own ligand 24(S),25-epoxycholesterol all fitted well into LXRβ 1P8D and formed a hydrogen bond with ASN 239. Moreover, 27-hydroxycholesterol contained an additional hydrogen bond with HID 435. In fact, with respect to compounds 76, 77, 92-94, and 125, the S values of which are from −10.056 to −11.457, were better than or comparative to 24(S),25-epoxycholesterol (−9.941). Overall, ASN 239, GLU 281, and HID 435 might be regarded as key residues for the interactions and activity of 1P8D. In accordance with the SPR assay of 33, 81, and 141, they could directly combine with LXRβ protein 1P8D with the S values from −5.578 to −8.715 (Supplementary Figures S3E-G). The phenolic hydroxy groups in the hexatomic ring formed hydrogen bonds with the active site residues ASN239, GLU281, THR316, or MET312 of LXRβ, and there exist the formation of π-π stacking and π cation with PHE239 or PHE 243.

Molecular docking analysis of farnesoid X receptor
To gain an insight into the molecular interactions between FXR ligands and FXR proteins as well as generate a structure and activity relationship at the atomic level, molecular docking calculations were carried out using the crystal structures of the FXR LBD in a complex with obeticholic acid (PDB ID: 1OSV and 1OT7) (Mi et al., 2003) and DB08220 (PDB ID: 3BEJ) (Soisson et al., 2008) to explore the molecular interactions (S values listed in Supplementary Tables S3, S4). The most potential 2D and 3D binding models of each protein are shown in Supplementary  Figures S4-S6, respectively.
As illustrated in Supplementary Figures S4A, B, 239 and 250 occupied the LBD similar to obeticholic acid (the S value −11.769) with similar interaction (Supplementary Figure S4B3), which bound to 1OSV with the S values of −10.917 and −10.415, respectively. The hydroxy groups in the steroidal ring moiety of these molecules formed hydrogen bonds with the active-site residue LEU 284 and SER 329. Overall, the docking models 235 also showed the most potency with a docking score over −10. Compounds 222-233 were a series of steroids isolated from the genus of Spongia. SPR assay indicated that they all decreased the affinity of FXR LBD for a coactivator peptide facilitated by CDCA. In line with SPR, docking indicated that they fitted comfortably into the binding pocket for the agonist obeticholic acid with the similar binding positions, with the S values from −8.941 to −9.179, except for 225 with the S value of −5.849 ( Supplementary Figures S4C-N). The ester and phenolic hydroxyl groups formed hydrogen bonds with the active-site residue ARG 328 or TYR358 of FXR. Furthermore, both 226 and 227 formed π-π stacking interaction with TRP451 and TYR328.
Compound 168 interacted with FXR 1OT7 with the highest S value of −10.884, comparative to the ligand obeticholic acid (docking score −11.569) and CDCA (docking score −10.610). The hydroxy groups formed hydrogen bonds with the active-site residue TYR 358, HID 444, LEU 284, and ARG 328 (Supplementary Figure 5A). Meanwhile, 163, 218, and 168 also ideally fitted into the LBD of 1OT7 along with a docking score over −10. 2D binding models of 222-233 with 1OT7 supporting the corresponding SPR results excellently with the S values from −7.906 to −9.352, except for 225 with the S value of −5.899 (Supplementary Figures S5B-M). The ester and phenolic hydroxyl groups of 226, 232, and 236 formed hydrogen bonds with the active-site residue ARG 328 or TYR358 of FXR.
Compound 233 docked into FXR 3BEJ with the highest score of −11.025, though only inferior to DB08220 (the S value of −12.625), both formed hydrogen bonds with the active-site residue THR 288 and ARG 331. Differently, there is a salt bridge between DB08220 and ARG 331 of 3BEJ. The docking models of 233,204,244,252,228,230,253, and 255 predicted the significant role of the active-site residue THR 288 and ARG 331. Interestingly, direct binding of 233, along with 222-232 to the LBD of FXR, was monitored by SPR assay. In molecular docking, they also fitted comfortably into the binding pocket for the agonist obeticholic acid with the similar binding positions as well, with the S values from −8.894 to −10.113, except for 228 with the S value of −5.861 (Supplementary Figures S6A-K). In the 2D binding models, the ester and hydroxyl groups formed hydrogen bonds with the active-site residue THR288, ARG331 or HID447 of FXR.

CONCLUSION AND PERSPECTIVE
Concurrent with our increasing knowledge of the roles of LXRs/ FXR in cholesterol, lipid, bile acid, and glucose metabolism, selective and potent LXRs/FXR ligands have gained significant ground for shedding light on the functions of LXRs and FXR in metabolic syndrome-related diseases as promising chemical biology tools (Jian et al., 2014;Pu et al., 2019).
In the recent two decades (2000-2020), 261 natural products were discovered from natural resources, such as LXRs/FXR modulators, 109 agonists and 38 antagonists targeting LXRs, while 72 agonists and 55 antagonists are targeting FXR. As shown in Figure 4 and Figure 5, LXR ligands exhibited higher chemical diversity than FXR ligands, terpenes, flavonoids, phenol derivatives, and steroids constituted 29%, 19%, 21%, and 10% of the reported LXR agonists, constituted 21%, 21%, 24%, and 18% of the reported LXR antagonists, respectively, while for FXR ligands, terpenoids accounted for a large proportion, close to half, for 49% and 47% of the reported FXR agonists and antagonists, respectively. Sterols, primary type of FXR antagonists, the reason of which is a series of endogenous BAs that possess distinguishing potency of targeting BA-receptor FXR. It is a remarkable fact that several of the mentioned natural products are food constituents, such as compounds 41, 42, 54, 95, 96, 104, 106, 125, 127, 208, 210-212, and 214-220, thus, potential candidates for dietary interventions for the prevention and treatment of corresponding diseases. Terrestrial plants are a promising source for the discovery of new drug leads, especially for TCMs having a history of more than 2,000 years. In recent years, marine organisms and microorganisms have gradually become a vital source of LXRs/FXR modulators with the development of marine natural product research. For instance, 38% of FXR antagonists originated from marine organisms. Especially for 222-227 and 228-233, a series of steroids isolated from the genus of Spongia have been verified as FXR antagonists via various representative assays, including SPR, indicating the significance of discovering marine natural steroids associated with FXR in the genus of Spongia. Overall, it is believed and prospective that LXR/FXR modulators from marine sources, especially marine microbial sources, will take a greater proportion in the future.
LXRs and FXR are highly expressed in the livers of patients with NAFLD, increasing with the severity of NASH (Zheng Z. et al., 2017, Mi et al., 2019. Some natural compounds especially for 2, 3, 37, 42, 83, 104, 106, 114, 115, 117, 118, 122, 132-134, 141, 157, 164, 167, 178, and 193, proved effective in ameliorating NAFLD and NASH by targeting LXR/FXR or through the LXR/FXR pathway, presenting a promising therapeutic prospect. Risk of cardiovascular diseases has been reported to be associated with cellular cholesterol levels, that is, with excess cellular cholesterol often increasing the risk of cardiovascular diseases . Our review discovered that 3, 22, 39, 40, 44, 54, 76, 77, 82, 83, 99, 107-109, 152, 210, 211, 256, and 258 played a vital role in hypercholesterolemia, hypertension, or atherosclerosis, indicating the prospect of further exploration in cardiovascular diseases focusing on LXR/FXR ligands. Additionally,25,38,47,48,83,152,154,155,157,177,[183][184][185] have represented tremendous potential for the treatment of various liver diseases including HBV, HCV, hepatic fibrosis, acute lung injury, cholestatic liver injury, liver cancer, and cholestasis. Previous reports have elicited that the ligands targeting the LXR/FXR might be one of the most effective ways to intervene with diabetes and obesity . It is suggested that 46,114,116,124,156,157,195,208, and 209 could be developed as prospective candidates to reduce the risk of diabetes and obesity. Intriguingly, 51, 52, 77, and 96 have proved to be beneficial for the treatment of AD; meanwhile, 22, 78, 98, and 99 have contributed to alleviate the development of mastitis and breast cancer. Overall, the abovementioned provides multiple alternative approaches to LXR/FXR-related drug development. At this point it needs, nevertheless, to be mentioned that most lead compounds failed in preclinical and clinical developments mostly due to toxicological and/or pharmacokinetic issues. For instance, the clinical application of LXRα agonist triptolide (19) is greatly constrained by hepatotoxicity, suggesting the risk of LXRs as toxicity targets. Our recent study revealed that, different from PA (142) directly combined with LXRα as an inhibitor, another anti-RCC drug candidate GPA (143) exists as a prodrug in the liver and exerts a toxic effect due to transformation into PA. Longer survival time of GPAtreated mice indicates that further exploration in anti-RCC drug research should focus on reducing glycosides transformed into PA and concentrating in kidney tumor rather than the liver for lowering hepatotoxicity risk.
Thereby, the therapeutic effects of these on diseases associated with LXRs and FXR expression remain to be further investigated in preclinical or clinical studies. Even if some compounds possessed grand effects on LXRs or FXR at a high concentration without toxicity in cell models, there exists a difference in vivo. Besides, overactivation or repression of LXRs and FXR is likely to cause other diseases, the discovery of the right balance between activating and inhibiting LXRs and/or FXR effects will be crucial for the development of targeting agents.

AUTHOR CONTRIBUTIONS
JS searched the literature and wrote the draft of the manuscript. TG and XP improved the manuscript. YL and LT revised the manuscript and discussed the conclusion. XZ established the concept and approved the final manuscript.