BBR, or 2,3-methylenedioxy-9,10-dimethoxyprotoberberine chloride, is a quaternary ammonium salt with a molar mass of 336.36122 g/mol. (Caliceti et al., 2016). It is one of a group of benzylisoquinoline alkaloids found in plants of the genus Berberis, such as B. vulgaris (barberry), Phellodendron amurense (Amur cork tree), and Coptis chinensis (Chinese goldthread), and the latter two species are used in Chinese herbal medicines. (Yin et al., 2008). BBR has a plethora of biological activities, such as antibacterial, antiinflammatory, (Kumar et al., 2015) anticancer, (Pandey et al., 2008) antidiabetic, (Zhang Y. et al., 2008) and hypolipidemic (Kong et al., 2004a) activities. However, BBR self-aggregates, does not effectively permeate into tissues, is subject to efflux and hepatobiliary re-excretion, (Feng et al., 2015) and undergoes first-pass processing in the small intestine. Thus, BBR is poorly absorbed in the body, and has an absolute bioavailability of 0.36%. (Liu et al., 2010a). BBR is metabolized in the liver by oxidative demethylation, which is performed by the cytochrome P450 enzyme system (mainly by CYP2D6, CYP1A2 and CYP3A4), to yield four major phase I metabolites (demethyleneberberine, berberrubine, jatrorrhizine, and thalifendine) (Liu et al., 2016); these are subsequently glucuronidated via UDP-glucuronosyltransferase (UGT) to their corresponding phase II metabolites. (Guo et al., 2016; Liu et al., 2016). These BBR metabolites act on the same targets as BBR (e.g., AMPK and the low-density lipoprotein receptor (LDLR)) but with a lower potency. (Li et al., 2011a). Ultimately, BBR and its derivatives are excreted primarily by hepatobiliary and renal pathways. Thus, there is a need for effective strategies to improve the oral bioavailability of BBR to enable its effective use in clinical settings.

The fundamental feature of liver fibrosis is the abnormal activation of HSCs, and BBR has been shown to be a potential treatment for thioacetamide (TAA)-, CCl₄-, ethanol- and high cholesterol-induced liver fibrosis models; in these contexts, it likely acts by suppressing HSC activation and downregulating alpha-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) levels. (Sun et al., 2009; Domitrovic et al., 2013; Eissa et al., 2018; Bansod et al., 2021). Previous studies have indicated that the direct beneficial effects of BBR involving modulation of the expression of multiple genes involved in HSC activation, cholangiocyte proliferation and liver fibrosis are linked to the downregulation of two important ribonucleotide molecules that promote liver fibrosis progression: microRNA34a and long noncoding RNA H19. (Wang et al., 2021). Another commonly reported mechanism is the induction of HSC cycle arrest in G1 phase, which inhibits HSC activation and prevents liver fibrosis. (Zhou et al., 2021). In addition, BBR has been revealed to have direct antifibrotic activity in bile duct ligation-induced liver fibrosis, due to its suppression of HSCs activation, and (partly) due to its inhibition of the AMPK signalling pathway. (Wang et al., 2016). However, other studies have found that BBR exerts hepatoprotective effects and prevents liver fibrosis by activating the AMPK signalling pathway. (Li et al., 2014; Wang et al., 2016; Bansod et al., 2021). BBR was also shown to activate the AMP-activated protein kinase (AMPK) pathway and inhibit macrophage polarisation and TGF-β1/Smad3 signalling, thereby alleviating tissue fibrosis. (Xu X. et al., 2021). ER stress may be another target of BBR treatment, and it has indeed been confirmed that a reduction in ER stress was the most logical explanation for the fact that BBR hinders the progression of hepatic steatosis to fibrosis. (Zhang et al., 2016). Moreover, BBR was shown to directly relieve liver injury-induced hepatic metabolic disorders by decreasing ER stress in hepatocytes (Yang et al., 2021), and the inhibition of Akt/FoxO1 signalling-mediated reduction of oxidative ER stress has been associated with BBR treatment of liver fibrosis. (Bansod et al., 2021). In other work, Zhang et al. reported that BBR prevents hepatic fibrosis by regulating the antioxidant system and lipid peroxidation in multiple hepatotoxic factor-induced fibrosis models, which was reflected by improved liver function, an increased antioxidant index and a decrease in fibrosis markers. (Zhang BJ. et al., 2008; Bansod et al., 2021). BBR-mediated normalization of liver function, suppression of inflammation, amelioration of ECM deposition and prevention of fibrosis correlate with NF-κB- and PPARγ-regulation. (Cao H. et al., 2018).

Many anticancer agents, such as methotrexate, (Sadeghian et al., 2018) doxorubicin (Zhao et al., 2012) and cyclophosphamide, (Germoush and Mahmoud 2014) are hepatotoxic (and thus cause hepatitis, steatohepatitis, liver cell necrosis, liver fibrosis or cirrhosis), and it is imperative to identify ways to limit this hepatotoxicity. It is therefore encouraging that anticancer drug-induced liver histopathological changes, including fibrosis, are significantly decreased by BBR treatment in animal studies. (Zhao et al., 2012; Germoush and Mahmoud, 2014).

Orally administered BBR displayed therapeutic effects in cirrhotic patients in a 1982 Japanese clinical study, with these effects being due to BBR inhibiting intestinal bacterial tyrosine decarboxylase. (Watanabe et al., 1982). Moreover, some randomized, placebo-controlled trials have found that BBR has positive effects in hyperlipidemic patients with virus hepatitis-related cirrhosis. (Riccioni et al., 2018). However, there are few clinical reports proving that BBR can alleviate cirrhosis, and properly designed clinical trials must be performed to determine this. To this end, our group is currently performing a randomized controlled trial to assess whether BBR can trigger the regression of hepatitis B-related fibrosis (ChiCTR1900023426), and our preliminary results are encouraging.

As the absolute bioavailability of BBR is extremely low (<1%) and more than half of the pro-BBR is not absorbed by the intestine, BBR is converted by intestinal flora into absorbable metabolites such as dihydroberberine (dhBBR), oxyberberine (OBB), canadine and other compounds. (ENRIZ et al., 2006; Liu et al., 2010a; Chen W. et al., 2011; Feng et al., 2015). Two of these metabolic products of BBR, dhBBR and OBB, exhibit superior anti-inflammatory and anti-oxidant functions compared to pro-BBR as they modulate intestinal flora and inhibit TLR4-MyD88-NF-κB and MAPK signalling, resulting in the reduction of levels of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-17, interferon (IFN)-γ, IL-1β and IL-6 and immunoglobulin IgA. (Li et al., 2019; Tan et al., 2019; Li et al., 2020). Previous studies have revealed that dhBBR reduces inflammation via an NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome-related mechanism, which likely reduces the release of caspase-1,apoptosis-associated speck-like protein (ASC) and IL-1β to inhibit pyroptotic cell death, which is a form of programmed cell death that occurs in liver fibrosis. (Xu et al., 2021a; de Carvalho Ribeiro and Szabo, 2021). dhBBR may even have better anti-sclerotic effects than BBR. (Chen et al., 2014).

It has been reported that OBB treatment enhances superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and decreases reactive oxygen species (ROS), malondialdehyde (MDA) and myeloperoxidase (MPO) concentrations to reduce oxidative stress. Liver function recovery mediated by OBB might therefore hinder the progression of liver diseases and promote liver regeneration. (Dou et al., 2021). OBB has also been shown to ameliorate the pathological deterioration of adipocytes and hepatocytes via the AMPK pathway and stimulate energy expenditure to control lipid homeostasis at smaller dosages than BBR. Moreover, OBB was demonstrated to inhibit macrophage migration and trigger a phenotypic conversion of M1 macrophages to M2 macrophages to relieve the inflammatory burden of the liver. (Li et al., 2021). Surprisingly, the BBR derivatives dhBBR, canadine, stylopine and coptisine were reported to inhibit TGF-β1-induced collagen secretion in vitro fibrotic conditions and possess anti-inflammatory, anti-fibrotic, wound-healing promoting and cytoprotective activities. (Pietra et al., 2015).

In summary, the metabolites of BBR appear to have similar effects to those of BBR prodrug in terms of anti-inflammatory, anti-oxidant and anti-fibrosis activities. Moreover, the former appears safer and more efficacious than BBR itself. Thus, BBR and its derivatives must be examined in future research on liver fibrosis.

Elevated concentrations of triglycerides are a key contributor to lipid profile disorders and metabolic syndrome, and the ability of BBR to decrease hepatic and blood concentrations of triglycerides has been convincingly proven in both animal experiments and human studies.(Hu et al., 2012; Li et al., 2018). As such, BBR is used in many countries as a lipid-lowering drug for hyperlipidemia treatment. (Affuso et al., 2010). Animal experiments demonstrated that the pre-administration of BBR can reduce triglyceride accumulation in the liver caused by tunicamycin administration, thus treating liver injury. In particular, compared with the control group, the BBR group downregulated lipid metabolism-related gene expression of stearoyl-Coenzyme A desaturase 1 (SCD1). (Yang et al., 2021). The triglyceride-reducing efficacy of BBR is attributable to its decreasing triglyceride biosynthesis and increasing triglyceride oxidation. AMPK is essential for the control of lipid metabolism in terms of lipogenesis or lipid degradation, due to its affecting transcription factors and metabolic enzymes. (Fryer and Carling 2005). BBR also decreases the deposition of lipids in the liver by regulating AMPK, which balances fatty acid biosynthesis and oxidation. (Boudaba et al., 2018). Zhu et al. discovered that BBR can activate the AMPK-SREBP-1c pathway, which results in downregulation of the expression of hepatic stearoyl CoA desaturase 1 and other triglyceride (TG)-biosynthesis related genes, and in the attenuation of hepatic TG deposition, which alleviates NAFLD. (Zhu et al., 2019). Animal studies show that BBR can improve high fat diet-induced increases in serum triglyceride concentrations, thereby ameliorating hepatic steatosis and fibrosis via the SIRT3/AMPK/ACC pathway. (Yu-pei et al., 2019). Moreover, high-fat diet-induced hepatic steatosis is significantly inhibited by BBR treatment as reflected by the upregulated expression of proteins implicated in fatty acid oxidation, including ACC and carnitine palmitoyltransferase IA. (Zhang et al., 2019b). BBR can also regulate the LDLR pathway, by which BBR downregulates fatty-acid biosynthesis genes and upregulates fatty acid oxidation genes in adipocytes. (Lee et al., 2006; Lee et al., 2007). Xu et al. also found that BBR could improve systematic lipid homeostasis by promoting fatty acid β-oxidation; specifically, it causing deacetylation of long-chain acyl-CoA dehydrogenase via SIRT3 activation. (Xu et al., 2019). A meta-analysis of clinical trials was consistent with the evidence reviewed above, as it found that BBR lowered blood TG concentrations in a dose-dependent manner. (Dong et al., 2013). Therefore, the regulation of triacylglycerol metabolism may be a critical part of the mechanism of action of BBR (Figure 2).

The ability of BBR to decrease cholesterol concentrations was first described in human, animal and cell test in 2004, (Kong et al., 2004a) and BBR was subsequently found to have the same effect in diabetes mellitus (Zhang Y. et al., 2008) and cirrhosis (Zhao et al., 2008a) patients. It was also found that the phase I BBR metabolite berberrubine may decrease cholesterol concentrations by targeting LDLR expression. (Cao S. et al., 2018). The utility of BBR as a cholesterol-lowering drug has been consistently validated in clinical research, and it is widely used as a drug in Asian populations. Clinical trials and diverse disease models have been designed to confirm the beneficial effects of BBR on the regulation of cholesterol homeostasis. Abnormal cholesterol homeostasis was reversed to varying degrees after BBR intervention, and this was extensively probed in human studies and in hyperlipidemic and diabetic animal models. (Wang and Zidichouski 2018). Numerous randomized controlled trials have demonstrated that BBR supplementation improves blood profiles of total cholesterol, LDL-C, and high-density lipoprotein C (HDL-C), but some studies have not observed in HDL-C. (Kong et al., 2004a; Derosa et al., 2013; Wei et al., 2016). Moreover, the ability of statins to reduce cholesterol concentrations is significantly enhanced by BBR. (Moss and Ramji 2016).

The mechanisms by which BBR regulates cholesterol concentrations are related to anti-inflammatory processes or the post-transcriptional upregulation of LDLR expression.(Kong et al., 2004a; Pirillo and Catapano 2015). Proprotein convertase subtilisin/kexin type 9, which controls LDLR degradation, is inhibited by BBR, and is thus linked to the ability of BBR to decrease cholesterol concentrations. (Barbagallo et al., 2015). In addition, the adenosine triphosphate-binding cassette transporter A1 gene, which is involved in cholesterol efflux, is upregulated by BBR administration. (Lee et al., 2010). Similarly, BBR accelerates cholesterol excretion by inhibiting adipocyte enhancer-binding protein 1 (Huang et al., 2012) or augmenting cholesterol-binding receptor, which account for its hepatoprotective properties. (Zarei et al., 2015) (Figure 2).

Arachidonic acid is an essential unsaturated fatty acid that is stored under physiological conditions in cell membranes in the form of phospholipids. It is released from the phospholipid pool under various stimuli with the aid of phospholipase A2 (PLA2), and is subsequently converted into a wide variety of biologically active metabolites that induce the inflammatory cascade, such as 15-deoxy-Δ12,14-prostaglandin J2 (15 days-PGJ2), thromboxane B2 and prostaglandin E2 (PGE2). (Funk 2001). Cycloxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP450) are key enzymes in the metabolism of arachidonic acid. (Funk 2001; Calder 2009). Some metabolic enzymes involved in the arachidonic acid pathway can be inhibited by BBR, and this has been illustrated in various pathological processes. Specifically, BBR can affect the activity of metabolic enzymes such as PLA2(Huang et al., 2002; Li et al., 2015; Yarla et al., 2016a; Zhao et al., 2017), arachidonate 5-lipoxygenase (5-LOX) (Li et al., 2012) and COX-2 (Guo et al., 2008; Feng et al., 2012; Li et al., 2012; Wang and Zhang 2018), and in turn affects the production of downstream metabolites such as PGE2 and 5-hydroxyeicosatetraenoic acid to regulate disease course. (Huang et al., 2002; Zeng et al., 2011). A MetaboAnalyst system analysis showed that the arachidonic acid pathway affects biological activity of BBR in cancer interventions. (Li et al., 2017). For example, the anti-hepatocellular carcinoma effects of BBR involve inhibition of cytosolic PLA2 and COX-2. (Li et al., 2015). Extensive studies by various research groups have proven that BBR inhibits COX-2 expression and thereby decreases the production of PGE2. (Kuo et al., 2005; Singh et al., 2011). BBR is also a major element of a traditional Chinese medicine recipe, and in this form has been found to inhibit the expression of COX-2 and 5-LOX, and decrease the production of the inflammatory metabolites PGE2 and leukotriene B4, thereby ameliorating the effects of the inflammatory cascade. (Li et al., 2012).

Furthermore, studies of metabolic enzymes (particularly COX-2) have suggested that BBR benefits liver fibrosis in an arachidonic acid pathway-dependent manner. Domitrović et al. discovered that BBR provides protection against CCl₄-induced liver injury in a concentration-dependent manner, which is partly attributable to a reduced COX-2 related-inflammatory response. (Domitrović et al., 2011). Similarly, the suppression of COX-2-driven inflammatory responses is also involved in the protective effects of BBR against drug-induced hepatotoxicity. (Germoush and Mahmoud 2014). Liver fibrosis is preceded by inflammation and can ultimately lead to liver cancer, and both of the latter have been widely reported to benefit from BBR treatment, due to its inhibition of the arachidonic acid pathway, (Domitrović et al., 2011; Germoush and Mahmoud 2014; Li et al., 2015; Yarla et al., 2016a; Zhang F. et al., 2019) which contains calcium-independent PLA2 and COX-2. These findings regarding the pathological development of chronic liver diseases and the biological function of BBR suggest that BBR and its derivatives could be used to treat liver fibrosis via arachidonic acid related mechanisms (Figure 2).

It is well accepted that PPARγ is a key molecule involved in the pathogenesis of liver fibrosis. (Zardi et al., 2013). PPARγ is encoded by the gene PPARG, and agonists of PPARG (e.g., IFC305 and pioglitazone) obstruct liver fibrosis by inhibiting HSC activation and regulating the expression of adipogenic- and fibrogenic-related genes. (Yuan et al., 2004; Perez-Carreon et al., 2010). PPARγ is also a crucial transcriptional regulator of genes involved in lipid metabolism, liver fibrosis, fat metabolism and adipocyte differentiation for adipose tissue development and functional maintenance. (Hazra et al., 2004; Ueki et al., 2004). Interestingly, BBR regulates lipid metabolism by precisely controlling the transcription and translation of nuclear reporters. (Zhou et al., 2008). PPARs have one of two different types of effects on fatty acid metabolic process, depending on their subtype. On the one hand, BBR inhibits TG production by acting with PPARα to enhance the expression of the gene coding for the fatty-acid oxidation enzyme carnitine palmitoyltransferase IA. (Zhou et al., 2008; Yu et al., 2016). On the other hand, PPARγ supports de novo fatty acid synthesis and fatty acid uptake. (Zhou et al., 2008). Zhou et al. showed that reduced PPARγ expression may be associated with the downregulation of adipogenic genes in the presence of BBR. (Zhou et al., 2008). High-throughput screening assays have also suggested that natural extract of BBR contains potential agonists of all PPAR subtypes (Xia et al., 2013; Tu et al., 2016; Yu et al., 2016) and that these can regulate the progression of liver diseases by acting as ligands. Interestingly, arachidonic acid metabolic products have also been reported to be PPARγ ligands and transcriptional activators (Xu et al., 1999; Choi and Bothwell 2012) that inhibit the activation of inflammatory signals, thereby modulating hepatic fibrosis via PPARγ regulation (Figure 2). The anti-fibrosis effect of PPARγ agonists (such as 15 days-PGJ2) has been observed in scarring models and has manifested as TGF-β-induced decreases in the extracellular matrix of HSCs. These findings imply that BBR acts on liver fibrosis via arachidonic acid pathway-mediated PPARγ activation. This is supported by research showing that arachidonic acid derived 15dPGJ2 attenuates fibrotic diseases by activating PPARγ, and that this effect is potentiated by co-administration of 15dPGJ2 and BBR. (Guan et al., 2018). Thus, it appears that PPARγ is a key target of BBR.

In conclusion, studies have confirmed the relationship between BBR, lipid metabolism pathways and subsequent signalling cascades, especially the arachidonic acid pathway. BBR may therefore relieve fibrosis by regulating PPARγ and restoring lipid homeostasis via modulation of arachidonic acid metabolism. More comprehensive studies on the effects of BBR on PPARγ, enzymes and downstream metabolites in the arachidonic acid pathway are needed to further elucidate appropriate clinical applications.

Although the oral bioavailability of BBR is limited, intestinal flora promotes the absorption and enhance the efficacy of BBR. The BBR metabolites generated by intestinal flora are considered to be crucial to the biological activity of BBR; in particular, dihydroberberine (dhBBR), which has less biological activity than BBR but approximately five times the intestinal absorption rate of BBR. (Feng et al., 2015). Thus, the conversion of BBR to dhBBR, which is catalysed by nitroreductase, is the rate-limiting step that controls the amount of BBR entering the blood. Nitroreductase is present in many intestinal bacteria, such as Staphylococcus aureus, Enterococcus faecium, Lactobacillus casei and L. acidophilus. (Feng et al., 2015). The role of intestinal nitroreductase in potentiating BBR bioavailability is supported by the fact that BBR has greater efficacy in individuals with higher nitroreductase activity. (Wang et al., 2017b). Moreover, BBR increases the populations of probiotics containing nitroreductase, such as Clostridia. (Lemmon et al., 1997; Cui et al., 2018). After entering intestinal tissues, dhBBR is immediately reoxidised to BBR. (Feng et al., 2015). These findings indicate that intestinal flora derived nitroreductase may be a biomarker of the therapeutic efficacy of BBR (Figure 3).

BBR also restores bile acid homeostasis by targeting multiple pathways that markedly inhibit inflammation, thereby alleviating non-alcoholic steatohepatitis and liver fibrosis. (Wang et al., 2021). Bile acids serve as key regulators of lipid and glucose homeostasis, energy consumption and inflammation. (Yuan and Bambha 2015). Additionally, bile acids play critical roles in the homeostasis of intestinal flora, which may in turn regulate the size and composition of the bile acid pool that maintains normal bile acid excretion and hepatoenteral circulation. (Hofmann 1999; Ridlon et al., 2006; Rajilic-Stojanovic 2013). However, abnormal biliary secretion results in the destruction of microfloral structure, which adversely effects the abundance of bacteria responsible for bile acid catabolism, resulting in the improper excretion and reabsorption of conjugated bile acid. (Hedenborg et al., 1991). Nuclear receptor FXR and cell-surface receptor Takeda G protein-coupled receptor 5 (TGR5) can alter bile acid-mediated metabolism by binding to bile acids. (Pathak et al., 2018). Thus, BBR continues to be pursued as a potential agonist of FXR (Sun et al., 2017) and TGR5 (Yang et al., 2016) binding of bile acids, as this may offer ways to increase the abundance of bacteria that promote the decomposition of conjugated bile acids and regulate bile acid signalling. Furthermore, BBR significantly increases the abundance of intestinal Firmicutes, especially Clostridium scindens, which primarily maintain metabolism and the hepatoenteral circulation of bile acids. (Gu et al., 2015). Studies have revealed that the lipid-modification function of BBR is possibly achieved via the modulation of bile acid metabolism, (Sun et al., 2017; Meng et al., 2018) given that BBR regulates intestinal flora. (Gu et al., 2015). Thus, crosstalk between bile acid metabolism and intestinal flora might affect the absorption efficiency of BBR, which could be exploited in treatments for cirrhosis (Figure 3).