Intestinal flora: A new target for traditional Chinese medicine to improve lipid metabolism disorders

Lipid metabolism disorders (LMD) can cause a series of metabolic diseases, including hyperlipidemia, obesity, non-alcoholic fatty liver disease (NAFLD) and atherosclerosis (AS). Its development is caused by more pathogenic factors, among which intestinal flora dysbiosis is considered to be an important pathogenic mechanism of LMD. In recent years, the research on intestinal flora has made great progress, opening up new perspectives on the occurrence and therapeutic effects of diseases. With its complex composition and wide range of targets, traditional Chinese medicine (TCM) is widely used to prevent and treat LMD. This review takes intestinal flora as a target, elaborates on the scientific connotation of TCM in the treatment of LMD, updates the therapeutic thinking of LMD, and provides a reference for clinical diagnosis and treatment.


Introduction
With the development of society and the improvement of living conditions, the incidence of lipid metabolism disorders (LMDs) is increasing year by year and showing a trend of rejuvenation, which seriously threatens human health. Abnormal lipid metabolism is a pathological process of elevated blood lipid levels and ectopic lipid deposition caused by genetic or acquired factors, and is an important risk factor for many metabolic diseases. Lipid metabolism is central to this process, and when lipid biosynthesis and degradation are abnormal, or when lipoprotein synthesis, metabolism, and transport are impaired, this can lead to disorders of lipid metabolism, contributing to hyperlipoproteinemia (Karr, 2017), obesity (Aron-Wisnewsky et al., 2021), non-alcoholic liver disease (NAFLD) (Katsiki et al., 2016), and atherosclerosis (Zhang et al., 2022b). Furthermore, dyslipidemia also has an effect on other target organs such as the brain and kidneys (Ross, 1999). Therefore, it is crucial to investigate the biological mechanisms underlying the development of LMD and to seek effective therapeutic targets for effective regulation of lipid metabolism to prevent and treat LMD.
The intestinal flora is an essential component of the intestinal micro-ecosystem and controls numerous metabolic processes with the host immune system, such as energy balance, glucose and lipid metabolism (Sonnenburg and Backhed, 2016). In recent years, numerous studies have further elucidated the connection between intestinal flora and its metabolites and LMD (Woting and Blaut, 2016;Jonsson and Backhed, 2017;Duttaroy, OPEN ACCESS EDITED BY Juei-Tang Cheng, Chang Jung Christian University, Taiwan 2021), finding that dysregulation of intestinal flora and its metabolites contribute to the development of LMD (Ridaura et al., 2013;Matey-Hernandez et al., 2018;Jia et al., 2021). LMD can also be improved by altering the intestinal flora by fecal transplantation or probiotic administration (Falcinelli et al., 2018;Allegretti et al., 2020;Witjes et al., 2020). In addition, LMD can affect intestinal flora homeostasis and have a negative impact on the organism's health. Numerous studies have shown that the intestinal flora structure of animals with high-fat diet (HFD) -induced LMD is altered, as evidenced by a decrease in intestinal flora diversity, a reduction in Bacteroidetes, and an increase in Firmicutes, Proteobacteria, and Verrucomicrobia (Tomas et al., 2016), or a significant decrease in the abundance of beneficial bacteria like Bifidobacterium and Lactobacillus (Wang B. et al., 2020). Meanwhile, some key intestinal metabolites such as lipopolysaccharides (LPS) (Cani et al., 2007b), bile acids (BAs) (Gillard et al., 2022), short-chain fatty acids (SCFAs) (Wang G. et al., 2021), branched-chain amino acids (BCAAs) (Nie et al., 2018) and trimethylamine-N-oxide (TMAO) (Janeiro et al., 2018) were discovered to play a role in the host's lipid metabolism function. Therefore, intestinal flora dysbiosis may take a significant part in the progression of LMD, and reshaping the structure of intestinal flora can contribute to correcting LMD.
Studies have found that Lipid-lowering drugs can treat LMD by altering the intestinal flora. Simvastatin can alter the abundance and diversity of intestinal flora from phylum to genus level, modulate the downstream metabolic pathways of intestinal flora and ultimately exert a hypolipidemic effect (Zhang S. et al., 2020). In another research investigating the influence of intestinal flora on the atorvastatin's hypolipidemic effect, it was found that atorvastatin significantly reduced serum levels of total cholesterol (TC) and low-density lipoprotein (LDL) in mice with intact intestinal flora but did not have the same effects in mice with depleted intestinal flora. Besides, we observed changes in the abundance of several sphingolipids after treatment with atorvastatin in mice with intact intestinal flora, which was not present in mice with depleted intestinal flora, suggesting that the lipid-lowering efficacy of atorvastatin also depends on the composition of intestinal flora before treatment (Zimmermann et al., 2020). However, in addition to the financial burden, longterm use of statins may trigger side effects such as myopathy (muscle pain or muscle weakness), hyperglycemia and liver enzyme abnormalities (Bellosta and Corsini, 2018), limiting the treatment options for patients. Therefore, it is crucial to provide patients with therapies that are effective and have few side effects.
TCM has been practiced for several thousand years in China, and now its use has expanded globally. TCM has a significant role in improving human subhealth, controlling metabolism and preventing major diseases. For a long time, TCM has been playing a significant role in improving metabolic disorders due to its multi-component and multi-target properties (Zhang et al., 2014;. Research has revealed that TCM (natural medicine extracts and Chinese herbal formulas) can significantly improve the components and metabolite function of intestinal flora, as well as help to maintain of intestinal flora homeostasis, thereby regulating lipid metabolism. For example, baicalin improved HFD-induced abnormalities in glucose and lipid metabolism via enhancing the number of bacteria that produce SCFA (Ju et al., 2019). Poria cocos (Schw.) Wolf [Polyporaceae; Poria] water insoluble polysaccharide (WIP) improved markedly glucose and lipid metabolism in ob/ob mice, and the underlying mechanism may be associated with increased numbers of butyrate-producing bacteria Lachnospiracea and Clostridium, elevated butyrate levels in the intestine, improved intestinal barrier function, and activated the intestinal peroxisome proliferator-activated receptor γ (PPAR-γ) pathway . Si Miao Formula, a classic TCM formula, played an anti-NAFLD role by altering the composition of intestinal flora, especially by upping the proportion of Akkermansia muciniphila and down-regulating the production of pro-inflammatory proteins . Resveratrol (RSV) inhibited TMAO production by remodeling intestinal flora, depressed the enterohepatic farnesoid X receptor/fibroblast growth factor 15 (FXR/FGF15) pathway, enhanced BA hydrolase activity, and promoted the synthesis of hepatic BAs, resulting in its anti-AS effects (Chen et al., 2016). On the other hand, intestinal flora may convert effective ingredients of TCM in a variety of ways to generate secondary metabolites, thus exerting the therapeutic effects of TCM in regulating lipid metabolism-related pathways and gene expression. It was found that treatment with Dingxin Recipes (DXR) IV resulted in increased levels of metabolites related to fatty acid metabolism, such as acetate and butyrate, downregulation of the Liver X Receptor α/sterol regulatory element-binding protein 1 (LXR-α/ SREBP1) axis, decreased blood lipid levels, as well as suppression of excessive cholesterol deposition in the aorta in HFD-fed mice . Notably, lack of Blautia greatly reduced the cholesterollowering efficacy of berberine (BBR), revealing that intestinal flora plays a crucial part in BBR's hypolipidemic impact .
In this review, we aimed to elucidate the role of intestinal flora and its metabolites in the occurrence and prevention of LMDs, and to systematically evaluate the multiple mechanisms involved in the regulation of intestinal flora in the improvement of LMDs in TCM, with the aim of providing new approaches for the prevention and treatment of LMDs in TCM and evidence for the development of novel anti-LMD drugs.

Relationship between intestinal flora and LMD
A growing majority of research have revealed an interaction between lipid metabolism and intestinal flora (Matey-Hernandez et al., 2018;Aron-Wisnewsky et al., 2021). Dysbiosis of the intestinal flora can disrupt lipid metabolism, and dyslipidemia can in turn cause imbalances in intestinal flora, but the exact mechanisms between the two remain to be investigated in depth. In recent years, researchers have also identified intestinal flora metabolites that may also be involved in regulating lipid metabolism, including LPS, SCAFAs, BAs, TMAO (Liang et al., 2020;Zhou X. et al., 2021;Jia et al., 2021). These metabolites derived from the intestinal flora can act not only on the host local tissues, but also affect the host endocrine and organ functions the in various ways (Figure 1).

The role of intestinal flora structure in the development of LMD
Intestinal flora is an important influence on energy absorption and metabolism, and a decrease in intestinal flora diversity is associated with an increased relative risk of LMD (Rastelli et al., 2018). Studies have shown that the α diversity of intestinal flora in high-fat rats is significantly reduced, as demonstrated through a drop in the intestinal flora abundance and a decrease in the structural stability of intestinal flora (Zhao J. et al., 2022). This phenomenon is consistent with the findings on intestinal flora of patients with LMD (Mu et al., 2020), but the specific composition of intestinal flora in healthy and obese people is still debated (Kasai et al., 2015). The human intestinal flora is mainly composed of Firmicutes and Bacteroidetes, which account for more than 98% of intestinal flora abundance and are involved in lipid and BA metabolism to maintain energy balance in the host. Among them, Firmicutes can induce hepatic steatosis via regulating fatty acid inflow and lipogenesis (Chen Y. H. et al., 2019), while Bacteroidetes can suppress obesity by promoting the catabolism of BCAAs in brown adipose tissue (BAT) (Yoshida et al., 2021). In addition, Bacteroidetes also produce bile salt hydrolase (BSH), which hydrolyzes conjugated BAs and regulates BAs-mediated lipid metabolism (Tian et al., 2019). Therefore, Firmicutes are hypothesized to be positively correlated with LMD, and Bacteroidetes are negatively correlated with LMD. A lower Firmicutes/Bacteroidetes (F/B) ratio represents a healthier gut microbial environment, and the risk of obesity and LMD may be increased by an elevated F/B ratio (Magne et al., 2020). Firmicutes were increased in the intestinal tract of obese individuals compared to healthy individuals, and F/B ratio decreased with an increase in Bacteroidetes and a decrease in Firmicutes following weight loss treatment (Damms-Machado et al., 2015). Akkermansia is a mucusdegrading bacteria that reverses HFD-induced increase in adiposity, metabolic endotoxemia, and adipose tissue inflammation (Everard et al., 2013). Bifidobacterium also promotes changes in lipid metabolism and glucose homeostasis (Salazar et al., 2019). Long-term HFD-induced changes in the structure of intestinal flora in animal models, mainly in the form of a significant reduction in the content of beneficial bacteria like Akkermansia, Bifidobacterium, Lactobacillus, Blautia, and Faecalibaculum, which have the ability to alleviate LMD (Shin et al., 2014;Wang B. et al., 2020). In another study, a substantial growth in the abundance of Bacteroides and Parabacteroides was found in hyperlipidemic rats (Zhao J. et al., 2022). Bacteroides and Parabacteroides contain a large number of conditionally pathogenic bacteria that can increase the risk of infection. They can also produce acetic acid, which promotes appetite and increases food intake by activating the parasympathetic nervous system and promoting the release of insulin and ghrelin, leading to elevated TG levels, liver and muscle fat accumulation and the development of obesity-related metabolic diseases (Perry et al., 2016). LMD influenced by changes in the intestinal flora have been found extensively in animals and are also largely reflected in humans (Maruvada et al., 2017). Therefore, the management of LMD focused on the modulation of intestinal flora structure is crucial.

The role of LPS in the development of LMD
into other tissues, organs or body circulation via the cellular bypass pathway, resulting in metabolic endotoxemia, inflammation and metabolic disorders (Zheng et al., 2022). LPS is the central aspect of Gram-negative (G-) bacteria's outermost layer of cell wall, and is an important inflammatory stimulus that can cause severe damage to the intestinal barrier and contribute to a chronic inflammatory response in the body, leading to the development and progression of metabolic diseases (Zhang-Sun et al., 2015). It has been found that a HFD disrupts the structure of intestinal flora and increases the abundance of LPS-producing bacteria, such as Desulfovibrio, which further enhances intestinal permeability and contributes to large amounts of LPS to enter the circulation from the intestine, triggering metabolic endotoxemia (Cani et al., 2007a), which is linked to the development of LMDs including dyslipidemia, obesity, NAFLD and cardiovascular disease (Manco et al., 2010). LPS can exacerbate lipid metabolism by binding to Toll-like receptor 4 (TLR4), a receptor expressed on macrophages, hepatocytes and adipocytes, and inducing the secretion of pro-inflammatory factors like interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1). Secretion, exacerbating lipid metabolic disorders (Robbins et al., 2014). Furthermore, LPS increases intestinal permeability through chronic inflammation mediated by MAPK or NF-κB pathways , triggering circulating metabolic endotoxemia and leading to lipid dysfunction (Sultana et al., 2016). Myeloid differentiation factor 88 (MyD88), a central adapter molecule for most TLRs, regulates fat storage and inflammatory responses (Everard et al., 2014), influences the activity of transcription factors related to lipid metabolism and BA profiles, and is involved in regulating the body's inflammatory response and lipid metabolism (Duparc et al., 2017). Additionally, LPS forms a triple complex with CD14 and LPS-binding protein (LBP), which activates the MyD88/NF-B pathway and increases the production of numerous inflammatory proteins. Moreover, cluster of differentiation 36 (CD36) is an important lipid absorption regulator in the intestine, and its expression is mediated by derivatives of Desulfovibrio and Clostridium. Loss of CD36 may lead to excessive lipid accumulation and activation of inflammatory factors, triggering the risk of LMD (Plociennikowska et al., 2015;Petersen et al., 2019). Thus, chronic low-level inflammation in the organism may be a key link in the pathogenesis of LMD, and disorders of intestinal flora may also contribute to chronic inflammation.

The role of SCFAs in the development of LMD
SCFAs are metabolites derived from the fermentation of undigested carbohydrates or proteins by intestinal flora and include butyrate and acetate, propionate. Among them, butyrate is primarily performed by Firmicutes, while acetate and propionate are mostly generated by bacteria belonging to Bacteroidetes (Walker et al., 2005), and they can play a crucial part in controlling host appetite, lipid metabolism, inflammatory response, and maintenance of energy balance through various pathways (Sanchez et al., 2020). SCFAs, represented by butyric acid and propionic acid, not only stimulate the release of leptin from adipose tissue, suppressing hunger and reducing host feeding activity (Naraoka et al., 2018), but also inhibit hepatic FAS activity and reduce serum lipid (TG and TC) levels (Larkin et al., 2009). As key regulators of host lipid metabolism, about 95% of SCFAs are absorbed in the intestine or utilized by intestinal flora, acting as important regulators of host lipid metabolism. These molecules not only providing the host energy, but also acting as signal transduction molecules to initiate G protein-coupled receptors 43 and 41 (GPR43 and GPR41) in intestinal epithelial cells. Activated GPR43 and GPR41 promote the secretion of peptide tyrosine-tyrosine (PYY) and glucagon-like peptide-1 (GLP-1) from enteroendocrine L cells, which reduce appetite, increase energy release and inhibit adipocyte synthesis through the "brain-gut axis" (Eslick et al., 2022). SCFAs also promote beige lipogenesis, leading to enhanced fat oxidation and energy expenditure, a process associated with the activation of GPR43 or GPR41 (Lu et al., 2016). And butyric acid also improves serum lipid metabolism by increasing fatty acid oxidation in brown adipose tissue (BAT) through thermogenesis, significantly reducing serum TG levels (Li Z. et al., 2018). In addition, SCFAs may also be involved in lipid metabolism by regulating related pathways and gene expression. For example, butyric acid downregulates the expression of proliferator-activated receptor γ (PPARγ), upregulates the expression of uncoupling protein 2 (UCP2), promotes mitochondrial proton efflux, which in turn activates the AMPK pathway, decreases lipid synthesis, and increases lipid oxidation. Acetic acid decreases SREBP-1 expression and ATP citrate lyase (ATP-CL) mRNA levels, decreases acetyl coenzyme A supply, and contributes to a decrease in cholesterol and fatty acid synthesis; it also enhances acyl-CoA oxidase (AOX) gene expression, promotes fatty acid β-oxidation, and increases energy expenditure (Fushimi et al., 2006). While another study speculated that acetate regulation of lipid metabolism may be associated with an increase in the AMPK/PGC-1α/PPARα axis (Araujo et al., 2020). Additionally, SCFAs can regulate the intestinal barrier and inflammatory levels and are indirectly implicated in lipid metabolism regulation. According to studies, SCFAs can elevate intestinal trans-epithelial resistance and reduce LPS production, thereby upregulating tight junction protein expression and mucin secretion, reducing intestinal mucosal permeability and alleviating intestinal barrier disorders . Meanwhile, SCFAs inhibit the expression of TNF-α/NF-κB inflammation-related genes, reduce the secretion of pro-inflammatory molecules like IL-6 and IL-12, as well as increase the secretion of anti-inflammatory molecules like IL-10 and IL-4, alleviating chronic inflammatory response and avoiding the formation of hyperlipidemia (Chang et al., 2014;Yuan et al., 2019).

The role of BAs in the development of LMD
The synthesis of BAs is the main pathway of cholesterol degradation and metabolism in humans, which is regulated by intestinal flora. The majority of primary BAs are converted from cholesterol by the enzymatic action of three cholesterol hydroxylases, CYP7A1, CYP8B1 and CYP27A1 in the liver (classical pathway), and a small proportion is processed by Frontiers in Pharmacology frontiersin.org 04 CYP27A1 and CYP7B1 in extrahepatic sites (alternative pathway). Primary BAs, which including cholic acid (CA) and chenodeoxycholic acid (CDCA), can bind to glycine or taurine to form conjugated BAs like glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), taurocholic acid (TCA) and taurochenodeoxycholic acid (TCDCA). The bile salt export pump (BSEP) or ATP-binding cassette subfamily G member 5 (ABCG5)/ABCG8 subsequently actively transfers these conjugated BAs into the bile, where they contribute to lipid emulsification when the food enters the intestine (Jia et al., 2018). The apical sodium-dependent bile acid transporter (ASBT) reabsorbs around 95% of BAs from the intestine; The apical sodium-dependent bile acid transporter (ASBT) reabsorbs around 95% of BAs from the intestine; these are subsequently released into the portal circulation by the organic solute transporter α/β (OST α/β), which finally transports them back to the liver (Vourakis et al., 2021). Intestinal flora can reduce cholesterol levels by producing BSH and CYP7A1, which decouple conjugated BAs into unconjugated BAs including deoxycholic acid (DCA) and lithocholic acid (LCA), preventing their reabsorption through ASBT and promoting BAs excretion through the feces (Wahlström et al., 2016). It is known that BSH-producing bacteria include Bacteroides, Bifidobacterium, Clostridium, Eubacterium and Lactobacillus, while Clostridium and Eubacterium also possess 7α-dehydroxylation activity (Vourakis et al., 2021). BAs can also act as signaling molecules, such as conjugated BA T-βMCA, unconjugated BAs CDCA and LCA, which regulate lipid metabolism by interacting to the BA receptors like FXR and Takeda G protein-coupled receptor 5 (TGR5) to regulate BA selfsynthesis, transport, as well as lipid digestion and absorption (Schoeler and Caesar, 2019). In summary, BAs play an important part in lipid metabolism homeostasis. Increasing intestinal BA production can block the reverse cycle from BA to cholesterol; it can also accelerate the transformation of cholesterol to BA, lowering serum TC levels. It is worth noting that the synthesis and excretion of BAs are related to the regulation of intestinal flora.

The role of TMAO in the development of LMD
Trimethylamine oxide (TMAO) is an intestinal floradependent metabolite formed by the oxidation of trimethylamine (TMA) in the liver by flavin-containing monooxygenase (FMO), especially FMO3 (Chen Y. et al., 2019). Studies have shown that serum TMAO levels are determined by genetics, diet and intestinal flora, and may be a biomarker of AS (Randrianarisoa et al., 2016;Canyelles et al., 2018). Another observational study found that TMAO levels were positively and significantly related with body mass index (BMI), fatty liver index and visceral obesity index, and could be used as a predictor of NAFLD and metabolic syndrome (Barrea et al., 2018). Additionally, 8 consecutive weeks of supplementation with a high TMAO diet may lead to elevated the levels of plasma TC, TG and LDL-C and induce hyperlipidemia (Koeth et al., 2013). Knockdown of FMO3 may downregulate circulating TMAO levels and acts as a preventive measure against hyperlipidemia (Miao et al., 2015). As a result, intestinal flora metabolite TMAO is essential for lipid metabolism. The underlying mechanism of TMAO-induced disruption of lipid metabolism is unknown; however, it may be associated with host cholesterol and BA metabolism. It has been investigated that TMAO reduces the expression of CYP7A1 and CYP27A1, two key enzymes necessary for the synthesis of BAs, and various BA transport proteins, decreases BA biosynthesis, inhibits the reverse cholesterol transport (RCT) pathway, and affects BA metabolism and cholesterol homeostasis in the hepaticintestinal tract (Koeth et al., 2019). In turn, BAs can also lead to an increase in serum TMAO by mediating the upregulation of FMO3 expression by FXR (Bennett et al., 2013). Moreover, TMAO is able to induce CD36 and scavenger receptor A1 (SR-A1), which are involved in stimulating macrophages to bind ox-LDL, promoting macrophage foaminess and causing intracellular cholesterol accumulation (Wang et al., 2011). This process may also be mediated through MAPK and NF-κB signaling pathways that promote vascular inflammation, one of the earliest cellular signals in the atherosclerotic process (Ma et al., 2017;Geng et al., 2018).

2.6
The role of intestinal flora and its metabolites in the clinical monitoring and treatment of LMD Obesity, NAFLD and AS are frequently associated with LMD. Therefore, for the efficient implementation of atherosclerotic cardiovascular disease (ASCVD) preventive and treatment methods, early diagnosis of dyslipidemia and monitoring changes in its levels are key foundations.
Routine lipid testing to identify the "hidden" high-risk groups will not only facilitate better treatment decisions, but more importantly, it will provide early intervention for these high-risk individuals or patients with LMD, so that they can have more cardiovascular benefit. The basic items of clinical lipid monitoring are TC, TG, LDL-C and HDL-C. It is noteworthy that LDL-C is an important causative factor of ASCVD and has an important significance in lipid monitoring. More and more clinical laboratories are using ApoAⅠ, ApoB, and Lp(a) as routine lipid testing items. In addition, the clinical testing items of oxLDL, FFA and non-HDL-C are also receiving increasing attention. In recent years, assays for intestinal flora and its metabolites have also been increasingly used to assist in monitoring lipid levels. Usually healthy people have low F/B ratio, while patients with LMD tend to have high F/B ratio (Human Microbiome Project, 2012). The main core of LMD such as hyperlipidemia is chronic low-level inflammation in the body, which is also caused by intestinal flora, often manifested as elevated serum LPS levels (Dai and Fu, 2019). SCFA is inversely correlated with lipid levels and can reduce serum TG and TC levels by inhibiting FAS activity (Larkin et al., 2009). BA is produced by cholesterol conversion, and its increased synthesis and impaired transport frequently lead to the development of LMD (Haeusler et al., 2016). Serum TMAO levels predict the risk of cardiovascular disease and are closely related to the regulation of Frontiers in Pharmacology frontiersin.org cholesterol . Therefore, the detection of intestinal flora and its metabolites can also be used as a reference item for clinical lipid monitoring, but it still needs to be combined with the items of lipid testing for comprehensive assessment, so it is not yet popular in the clinic. Currently, the guidelines recommend three main classes of lipidlowering drugs (Du, 2022): 1) Statins, which reduce cholesterol synthesis in hepatocytes mainly through competitive inhibition of endogenous cholesterol synthesis rate-limiting enzymes, leading to a significant decrease in blood cholesterol levels, and are the cornerstone of primary prevention of ASCVD. 2) Ezetimibe, which reduces cholesterol by inhibiting the absorption of cholesterol in the intestinal tract. 3) Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, which lower cholesterol levels by inhibiting the binding of PCSK9 to LDL receptors and preventing LDL receptor degradation, are the most potent cholesterol-lowering drugs available and have a good safety profile, offering another option for high-risk patients who are statin intolerant (Koren et al., 2019). The intestinal flora and its metabolites serve as a key link in lipid metabolism, and the therapeutic effect of lipid-lowering drugs may be related to the regulation of intestinal flora. Statins were found to modulate the abundance of SCFA, SBA, TMAO and LPS-producing intestinal flora, which in turn affected AMPK-SREBP, FXR, PXR, FFAR2 and TLR4-Myd88 signaling pathways, acting to regulate cholesterol metabolism and LDL-C levels . In addition, different statins, such as atorvastatin, simvastatin and resevastatin, have different effects on different intestinal flora. Desulfurization was negatively correlated with TG and HDL-C levels, and desulfurization was significantly reduced after ezetimibe intervention (Jin et al., 2022). Desulfovibrio was considerably decreased following ezetimibe treatment and had a negative correlation with TG and HDL-C levels (Jin et al., 2022). PCSK9 inhibitors can promote the clearance of LDL-C and LPS from the blood, regulate intestinal microecology, alleviate intestinal inflammation and endotoxemia, and reduce the risk of cardiovascular disease (Morelli et al., 2019). Furthermore, probiotic supplementation alone was able to significantly reduce plaque area in the full-length aorta and aortic sinus, lower plasma TMAO and cecum TMA levels, improve lipid disorders, reduce serum oxLDL and inflammatory factor levels in mice, increase cecum acetate and butyric acid levels, and reduce inflammatory responses in the aorta and liver of mice by inhibiting the levels of key proteins in the TLR4/MyD88/NF-kB/NLRP3 pathway (Wang, 2022). In contrast, mice on diets supplemented with choline or TMAO showed increased cholesterol levels in peritoneal macrophages, elevated TMAO plasma levels, and enhanced aortic atherosclerotic plaques (Wang et al., 2011). Thus, intestinal flora and its metabolites also play an important role in the treatment of LMD, but further studies are needed to validate this in order to maximize the clinical benefits of drugs.
3 Mechanisms of regulating intestinal flora in TCM for LMD TCM is widely utilized to treat metabolic diseases in China. The effective components of TCM can directly interact with the intestinal flora once they enter the intestinal system by oral administration and produce therapeutic effects. Studies have shown that TCM (natural medicine extracts, Chinese herbal formulas and proprietary Chinese medicines) can effectively modify the host intestinal flora structure and its metabolite levels, repair the intestinal mucosal barrier, reduce inflammatory infiltration, as well as regulate lipid metabolism-related pathways and genes expression to correct LMD (Dai and Fu, 2019). Therefore, we systematically summarized the possible mechanisms involved in TCM to modulate intestinal flora and its metabolites in order to prevent and treat LMD (obesity, hyperlipidemia, NAFLD, and AS) ( Figure 2).
Faecalibaculum, a SCFAs-producing bacteria, while Burdock inulin (Wang, 2020) and Yinchenhao Decoction (Li Z. H. et al., 2019) can upregulate the relative abundance of Faecalibaculum to promote the production of SCFAs and alleviate inflammation to improve lipid metabolism. In contrast, the opposite result was observed after Caffeic acid (Mu et al., 2021) treatment, which showed a decrease in the abundance of Faecalibaculum. This suggests that it may be the role of multiple intestinal flora in regulating lipid metabolism and inflammation levels, and that Faecalibaculum may not be a core flora.

TCM modulates the level of SCFAs to improve LMD
Although the strong connection between intestinal flora, SCFAs and LMDs are still unclear, an increasing number of studies have shown that LMDs are directly correlated with both the number of SCFAproducing bacteria and the level of SCFAs. To play its part in treating LMDs, TCM can control the prevalence of bacteria that produce SCFAs and encourage the generation of SCFAs. LC , DLT , NXT (Lu et al., 2022), IPTS (Ba et al., 2022), LJP (Zhang et al., 2021d), LBPs (Gao et al., 2021), Zanthoxylum bungeanum Maxim. [Rutaceae; Zanthoxyli pericarpium] (ZB) (You, 2016), Ginkgo biloba extract (GbE) , Inulin (Bao, 2021;, NFP , TP , Bilberry anthocyanins (Nakano et al., 2020) and COSM (Feng et al., 2022) significantly improved serum lipid levels (TC, TG) as well as reducing lipid accumulation, which may be associated with modifying intestinal flora and increasing SCFAs levels. In-depth research revealed that Compound Danshen Dripping Pills  and JZG  intervention elevated the abundance of Lachnospiraceae; Qingxin Jieyu Granule (QXJY) (Wang, 2019) and water insoluble polysaccharide WIP  intervention increased Clostridium abundance; Macroalgae Laminaria japonica (MLJ) (Zhang Q. et al., 2020), Quercetin (Porras et al., 2017), Myricetin (Sun et al., 2021), MC (Bai, 2019) and GLP (Sang et al., 2021) administration improved the abundance of Allobaculum; Erchen decoction (ECD)  and EGCG (Zuo, 2020) administration enhanced the abundance of Roseburia; HGQZ (Tang, 2019) and DG (Li Y. et al., 2018) treatment increased the abundance of Ruminococcaceae; SLBZ (Hong, 2021) and DZSM (Guo et al., 2022) administration enriched the abundance of Bifidobacterium, Lactobacillus; Guanxinning Tablet (GXNT) (Yang Q. et al., 2022), Black tea polyphenols (BTP) (Henning et al., 2018) and RLPs (Zhang X. J., 2020) treatment all increased the relative abundance of Prevotella; G. pentaphyllum saponins (GPS) (Zhong F. W. et al., 2022) administration enriched the abundance of Bacteroides. The above mentioned Ruminococcaceae, Lachnospiraceae, Clostridium, Allobaculum, Bifidobacterium, Lactobacillus, Prevotella, Roseburia, Bacteroides are all SCFAs-producing bacteria. In addition, TCM can promote the synthesis of key synthetic enzymes of SCFAs to elevate the level of SCFAs, or mediate SCFAs to regulate energy metabolism and inflammation-related pathways to improve LMD. GXNT treatment was also found to promote the production of butyrate kinase, propionate kinase, the key synthetic enzymes of SCFAs, thereby increasing the levels of butyric acid and propionic acid (Yang Q. et al., 2022). BTP treatment increased the level of SCFAs and activated the AMPK signaling pathway to increase energy expenditure, thus exerting a fat-lowering and weight-reducing effect (Henning et al., 2018). WIP treatment enhanced intestinal butyric acid levels, improved intestinal mucosal integrity, and regulated PPAR-γ pathway in the intestine . Dingxin Recipe IV (DXR) intervention promoted acetate, butyrate, propionate production, inhibited LXR-α/SREBP1 pathway and improved lipid metabolism . The mechanism through which GLP intervention ameliorated LMD may be indirectly engaged in regulating lipid metabolism by promoting the production of acetate and butyrate, activating the GPR43 receptor and modulating the intestinal barrier and inflammatory response (Sang et al., 2021).

TCM regulates BAs metabolism to improve LMD
Studies have shown that GXNT (Yang Q. et al., 2022) and Quercetin (Nie et al., 2019) can reduce serum or intestinal BAs levels. HYQT (Sui et al., 2021b), HZRG (Li C. et al., 2022), Proanthocyanidin (Fu et al., 2013), Theabrownins , Radix scutellariae water extract (Zhao et al., 2021), ZB (You, 2016), GbE , QGE  and Pae (He, 2021) promoted the conversion of cholesterol to BA and significantly increased the excretion of BA in feces. In-depth studies have revealed that the hypolipidemic effects of TCM are closely related to the modulation of BA synthesis and transport pathways. Among them, HYQT can increase BA synthesis-related genes expression, upregulate hepatic CYP7A1 expression, promote BA biosynthesis, and reduce serum lipid levels (Sui et al., 2021b). XXD may be achieved hypolipidemic effects by upregulating CYP8B1 expression (Lei, 2020), and Baicalein exhibited the same effects (Li P. et al., 2022). HZRG aimed to reduce serum TC and TG levels mainly by decreasing the expression of BA transporter-related genes ASBT and OSTβ mRNA and reducing the reabsorption of harmful BA such as LCA, DCA and HCA (Li C. et al., 2022). QGE promoted BA transport in the liver by increasing BSEP expression and hepatic TGR5 receptor expression to attenuate the inflammatory response and regulate lipid metabolism in the liver . RC alkaloids reduced lipid and TBA levels involving multiple BA receptor pathways, and were associated with reduced ASBT expression and elevated CYP7A1 and TGR5 expression (He et al., 2016). NXT decreased BSH enzyme activity, modulated the Frontiers in Pharmacology frontiersin.org Xiaoyao San (XYS) Granules NAFLD rats induced by HFD (8 weeks BA profile in the intestine, and reduced lipid deposition, but the exact mechanism has not been elucidated (Lu et al., 2022). It was found that treatment with THF (Yang L. et al., 2022), WESB (Zhao et al., 2021), HQF (Sui et al., 2021b), TB , Pae (He, 2021), Naringin (Wang F. et al., 2020) and Penthorum chinense Pursh. (PCP) extract (Li X. et al., 2022) decreased the abundance of intestinal flora that produces BSH and BSH enzyme activity, leading to a weakened hydrolysis of BAs, increased the level of FXR antagonist T-βMCA, decreased FXR agonist CDCA and LCA levels, inhibited FXR/ FGF15 axis in the intestine, activated CYP7A1 and CYP7B1 expression in the liver, and promoted hepatic BA synthesis, thereby reducing serum and hepatic TC levels. In addition, RSV enhanced BSH enzyme activity via boosting the number of BSH-producing bacteria, including Bifidobacterium and Lactobacillus, to promote BA catabolism and fecal excretion in the intestine, downregulated OSTα, OSTβ expression to reduce BA reabsorption transport, and reduced CDCA content to suppress enterohepatic FXR/FGF15 axis in order to promote hepatic BA synthesis (Chen et al., 2016).

TCM adjusts TMAO production to improve LMD
It was found that serum lipid and TMAO levels were significantly decreased in HFD-induced AS model animals after treatment with Ginkgolide B (GB) (Lv et al., 2021), Eucommia ulmoides extract (Sun, 2020), HYQT (Sui et al., 2021a) and AOB (Zhu, 2021). The possible mechanism is to reduce TMA production, decrease hepatic FMO3 expression and inhibit the oxidation of TMA to TMAO by adjusting intestinal flora, which is a process consistent with the study of Xiangsha Liujunzi Decoction (XSLJZ) for the treatment of hyperlipidemia model rats . BBA reduces TMAO biosynthesis by decreasing the abundance of TMAO-related enzymes, such as carnitine oxygenase (CntA), choline-trimethylamine lyase (CutC), FMO and betaine reductase, effectively reducing serum TMAO levels Ma et al., 2022). The content of TMAO in the blood was shown to be associated to the severity of AS. Treatment with Tongmai Zhuyu decoction (TMZY)  or Tanyutongzhi Formula (TYTZ) (Wang, 2016) led to the reduction of TMAO levels as well as the decrease of atherosclerotic plaque area in AS model animals, and its anti-AS effect may be connected with TMAO reduction and the reduction of endothelial damage and inflammation levels. Another study showed that GXNT treatment was effective in reducing the production of TMAO, a TMA metabolite, lowering ox-LDL levels and inhibiting macrophage foaminess, while reducing NF-κB expression in AS plaques and alleviating the inflammatory response, thus preventing and preventing the formation of AS, which was correlated with reduced Escherichia abundance (Yang Q. et al., 2022). Furthermore, Jianpi Huazhuo Tiaozhi Granule (JPHZTZ) (Huang Q., 2021) and RSV (Chen et al., 2016) also exerted significant efficacy in anti-AS by a mechanism that may be achieved by inhibiting the enterohepatic FXR/FGF15 pathway, upregulating hepatic CYP7A1 expression, promoting BA biosynthesis, reducing TMAO production, and maintaining cholesterol metabolic homeostasis.

Discussion
Lipid metabolism, which is an essential component of an organism's fundamental metabolism, and LMD is linked inextricably to numerous illnesses and their consequences. With the rapid development of sequencing technology, significant advancements in our study of intestinal flora have been achieved, and it is also clear that lipid metabolism is closely related to intestinal flora. When LMD occur, the structure and composition of intestinal flora are abnormal, while dysbiosis of intestinal flora can further aggravate LMD. Therefore, the relationship between intestinal flora metabolites and LMD (hyperlipidemia, obesity, NAFLD and AS) can provide new perspectives for understanding LMD, and promote the development of research and treatment methods of metabolism disorders.
In this review, we systematically analyzed the previous related literature and found that intestinal flora and its metabolites may be major targets of TCM for treating LMD (Tables 1, Supplementary  Table S1). The composition of Chinese herbal formulas and proprietary Chinese medicines is shown in the Supplementary  Table S2. TCM can reshape the composition and structure of intestinal flora by enhancing the abundance of beneficial intestinal bacteria, for example, increasing the abundance of Akkermansia to repair intestinal barrier function (Park et al., 2019); upregulating Prevotella, a bacteria that produces SCFAs, to improve lipid metabolism of the host and alleviate inflammatory responses ; promoting the growth of Lactobacillus and Bifidobacterium, and enhancing BSH enzyme activity to promote BA catabolism and fecal excretion in the intestine to accelerate cholesterol excretion (Yang L. et al., 2022). At the same time, TCM can also reduce the abundance of harmful bacteria by inhibiting the growth of pathogenic bacteria such as Escherichiacoli and Enterococcus, down-regulating the number of Desulfovibrio, bacteria that produces LPS, alleviating low chronic inflammatory response and metabolic endotoxemia in the intestine, and maintaining intestinal microecological health Kou et al., 2022). Notably, TCM also reduces the risk of abnormal lipid metabolism diseases by lowering the F/B ratio, reducing fat accumulation in the body and accelerating fat metabolism. For example, Luteolin modulated intestinal flora (decreased F/B ratio, elevated Bifidobacterium, Lactobacillus) against liver fatty lesions in rats (Liu, 2022), while BBR successful in boosting Akkermansia abundance, it also enhanced intestinal tight junction protein expression and colonic mucus layer thickness, reduced HFD-induced metabolic endotoxemia and decreased expression of pro-inflammatory factors and chemokines (Zhu et al., 2018). After SLBZ supplementation treatment, the abundance of Desulfovibrio was reduced in NAFLD rats, and LPS production as well as the secretion of inflammatory factors were inhibited, resulting in improvement of LMD (Hong, 2021). NXT reduced BSH enzyme activity in hyperlipidemic rats and modulated the BA profile in the intestine to reduce lipid levels in serum and liver (Lu et al., 2022). RLPs increased the relative abundance of Prevotella, promoted the production of SCFAs such as Propionic acid and butyric acid, and reduced lipid levels in NAFLD mice (Zhang X. J., 2020). In addition, TCM can also modulate intestinal flora metabolites to play a role in correcting LMD, including LPS, SCFAs, BAs and TMAO. Among them, LPS and SCFAs-mediated inflammatory pathways are key aspects of herbal agents for lipid lowering. BAs have a vital part in maintaining lipid metabolism homeostasis, involving lipid synthesis, transport, and excretion. TMAO is positively correlated with visceral obesity index and is not only involved in the biosynthesis of BAs, but also serves as a biomarker for AS (Barrea et al., 2018). Given its multi-component and multi-target of action characteristics, TCM often also affects MAPK or TLR4/NF-κB pathway-mediated chronic inflammatory responses and FXR/FGF15 pathway-mediated metabolism of BAs to regulate lipid metabolism. For instance, Quercetin reduced LPS translocation, promoted SCFA production, increased intestinal Occludin, Claudin-1 expression, repaired intestinal barrier function, and inhibited TLR4 pathway to improve inflammation levels by regulating NAFLD intestinal flora (Porras et al., 2017). Myricetin significantly reduced LPS-induced metabolic endotoxemia and systemic inflammation, corrected dyslipidemia and restored liver function in NAFLD rats by regulating the TLR4/ NF-κB signaling pathway (Sun et al., 2021). After treatment with BTP, it can effectively increase the level of SCFAs in obese mice, and at the same time activate AMPK signaling pathway to increase energy consumption for weight loss (Henning et al., 2018). QGE promoted BA transport in the liver by increasing BSEP expression, in addition to upregulating hepatic TGR5 receptor expression, attenuating hepatic inflammatory response and regulating lipid metabolism in NAFLD mice . RSV regulated lipid levels mainly by regulating BA metabolism, which not only promoted the catabolism of BA in the intestine and fecal excretion, but also downregulated OSTα, OSTβ expression and reduced BA reabsorption IPTS (30/60/ 120 mg/kg/d) by gavage (n = 10/10/10) The same volume of 0.5% CMC-Na by gavage (n = 10) Simvastatin (5 mg/kg/ d) by gavage (n = 10)

weeks Colorimetric method
Bai (2019) Tea seed saponins Hyperlipidemia rats induced by HFD (4 weeks) Tea seed saponins (50/100/150 mg/kg/ d) by gavage (n = 10/ 10/10) The same volume of 0.5% CMC-Na by gavage (n = 10) Simvastatin (10 mg/kg/ d) by gavage (n = 10) 4 weeks - Lin et al. (2020) Frontiers in Pharmacology frontiersin.org 27 transport (Chen et al., 2016). HYQT may reduce TMA production, decrease hepatic FMO3 expression and inhibit TMAO synthesis by altering the structure of the intestinal flora; meanwhile, it can inhibit the intestinal and hepatic FXR/FGF15 axis, promote the synthesis and excretion of BAs and maintain the balance of cholesterol metabolism, and finally achieve the purpose of preventing and treating AS (Sui et al., 2021a;Sui et al., 2021b). It can be seen that TCM and its effective ingredients can improve the metabolic indexes of blood lipids directly or indirectly by acting on intestinal flora and its metabolites, thus playing a comprehensive role in preventing and treating LMD.
The study of the interaction between TCM and intestinal microorganisms has an important prospect, and the in-depth study of the interaction between TCM and intestinal flora is conducive to the elucidation of the potential mechanisms of TCM in preventing and treating LMD, as well as to the enrichment of TCM theory. In recent years, a few reviews have been reported on the regulation of intestinal flora by TCM to improve metabolic diseases, but there are shortcomings such as few included studies and few diseases involved . Based on previous studies, this review enriches the number of included literature and adds the amount of LMD, including hyperlipidemia, obesity, NAFLD and AS. Also, we illustrate potential mechanisms of TCM for regulating intestinal flora to improve LMD by category, which helps to minimize the issue of biased reporting. In this review, we have conducted a full quality assessment of the included original literature, which includes the dosage, periodicity and quality control of the use of TCM prescriptions (Chinese herbal formulas and proprietary Chinese medicines) and natural medicine extracts in animal experiments and clinical trials (Tables 2-4). However, there are some limitations in the included studies. 1) The impact of TCM on LMD based on intestinal flora has gained attention in the scientific community, and the number of related clinical studies is growing. However, there are issues such as small sample sizes, poor quality of single studies, as well as few reports of large-scale clinical trials.
2) There were differences in the specific content of the control group between the included original literature, and although the experimental groups were Chinese herbal formulas or natural medicine extracts that could regulate intestinal flora, the specific groups were different, which was also a source of heterogeneity and may have affected the results of the review assessment. 3) The majority of the original literature that was considered did not mention any allocation concealment, blinding, or other concerns, which made the results less reliable. 4) Despite the extensive search, the currently included literature is mainly in English and Chinese, which may have regional bias and language bias. 5) The current research on the treatment of diseases related to LMD by TCM is mainly focused on obesity, hyperlipidemia and NAFLD, while there are fewer studies on diseases like AS, which may be related to the bioavailability of TCM and to the stage and severity of the disease. 6) The number of research on various intestinal metabolites is significantly skewed. Most studies of TCM against LMD have taken the chronic low levels of inflammation as their entry point, focusing mainly on the regulation of LPS levels and the levels of SCFAs in stool, while fewer studies have been conducted on BAs and TMAO, which are lipid metabolism-related risk factors. 7) Studies on intestinal flora are mostly correlational, while relatively few studies on causality are available. 8) Most of the research is focused on natural medicine extracts, but there is still considerable opportunity for research on proprietary Chinese medicines and Chinese herbal formulas. Therefore, Future research should focus on how the effective ingredients of TCM are bio-transformed by intestinal flora, and whether these biotransformation metabolites have synergistic or antagonistic effects on the treatment of LMD in TCM, which will help discover new favorable metabolites of intestinal flora. Furthermore, it is necessary to conduct more high-quality studies to verify the safety of TCM in modulating intestinal flora to improve LMD, so as to promote clinical application and provide new ways and references for the intervention targets of TCM in preventing and treating LMD.
In summary, intestinal microecology plays a significant part in the development of LMD, and the regulation of intestinal flora and its metabolites is a potential new therapeutic target for LMD. TCM has obtained some achievements in improving lipid metabolism disorder diseases, probably by regulating intestinal flora and its metabolites, but in order to clarify the precise mechanism of action, more extensive research will still be required in the future.

Author contributions
ML, YW, and KW designed the research protocol; WS and YH collected and organized the literature; ML drafted the original manuscript; YW and KW revised the manuscript.

Funding
This study was supported by the National Natural Science Foundation of China (Nos 81973684 and 81873334).

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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