Advances in the mechanism of bile acids in liver regeneration

Sheng-Lan Zeng, Na Wang, Rong-Zhen Zhang, Cong Wu, Ting-Shuai Wang, Ying-Yu Le, De-Wen Mao?

1. The First Clinical Medical College of Guangxi University of Traditional Chinese Medicine, Nanning 530022, China

2. The First Division of Liver Disease First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, Nanning 530022, China

ABSTRACT Hepatocytes can divide rapidly and proliferate in the absence of inflammation and fibroplasia in the damaged or partial hepatectomy (PHx) of the liver, which is essential for the recovery of related patients. Recent studies have found that bile acids (BA) play an important role in the process of liver regeneration. In the early stages of PHx, bile acid overload occurs, and liver injury is aggravated by loading. Later bile acids can induce protective and proliferative responses in the liver and promote liver regeneration. In this paper, we summarize the negative effects after bile acid overload and its positive role as a signaling molecule involved in related signaling pathways on liver regeneration, including protection of the liver and promotion of liver regeneration, and its double?edswordged "in Liver regeneration. This provides a theoretical basis for subsequent in?depth study of the mechanism and benefit avoidance in clinical treatment.

Keywords:Liver regeneration Bile acids FXR Cell proliferation TGR5

The liver strictly maintains its own size in order to complete various physiological functions. The organism can sense the loss of liver tissue, and can carry out regenerative response and return to its normal size. The process can be called "reversion to the liver mechanism". The recovery process after PHx surgery in patients with liver tumors in clinical practice is a typical case of liver regeneration. Obstruction of regeneration of the residual liver may lead to postoperative liver failure (PLF), one of the main causes of postoperative death. It is necessary to understand the mechanism related to liver regeneration after PHx.

Liver regeneration involves many genes and signaling pathways,and the main pathways involved in the process are: cytokines,growth factors, and metabolic signaling [1]. In addition to this, BA and its receptors constitute a complex signaling network in repair processes and liver regeneration, with multiple effects on liver regeneration. Studies have shown that BA may play a central role in liver regeneration . The effect of BA on liver regeneration is mediated by activation of stellate cells, interaction with cholesterol,and various BA receptors. BA receptors include ligand?activated nuclear hormone receptors, G?protein?coupled receptors, and other BA?sensing molecules, such as integrins and ion channels, which are mainly expressed in human and rodent liver [3]. BA although has many positive effects on liver regeneration, such as protection and promotion of liver regeneration. However, at the early stage after PHx, when BA is overloaded in the body, it will cause damage to the remaining liver, which in turn hinders the process of liver regeneration. So, it is essential to maintain the homeostasis of BA.

1. BA overload

BA is synthesized in the liver from cholesterol and is the main component of bile. BA and hepatic secretions can produce bile flow and promote the elimination of endogenous compounds and metabolites, such as bilirubin and hormones [4]. Prior to export from the liver, BA is conjugated with glycine and taurine to generate bile salts that are excreted into the gallbladder as a "transfer station". BA is absorbed from the intestine and reinfused into the liver via the portal vein, with a small amount excreted in the feces. BAs returned to the liver enter the next cycle [5]. Primary BAs, such as cholic and chenodeoxycholic acids, are produced in the liver and converted by gut microbes to secondary BAs, such as deoxycholic and lithocholic acids [6].

Following PHx, BA is refluxed from the gut via the portal circulation, exceeding the capacity of the remaining liver to process,resulting in acute BA overload in the liver and systemic circulation[7-9]. Merlen et al.10 suggested that BA overload is not only a consequence of the cholestatic disease itself but also a marker of significant liver injury of any type. After PHx, the liver parenchyma is reduced, and the phenomenon of BA overload has been observed in both mouse experiments and humans [9, 11, 12].

After BA overload, the liver injury induced by it includes direct destruction of cells by the toxicity of BA as well as indirect processes that induce inflammation associated with BA [13, 14].Direct disruption of BA?induced cell death includes plasma membrane damage, mitochondrial damage, production of reactive oxygen species (ROS), and induction of apoptosis. But this cell death mechanism shows differences between species and there are still many controversies. Several mechanisms of BA?induced hepatocyte injury have been summarized by Arab[15] et al.

Cai [16] and others have found that the accumulation of intracellular BA leads to mitochondrial damage and ER stress, and triggers an innate immune response by activating Toll?like receptor 9,which initiates an inflammatory response and triggers liver injury.Accumulated BA can induce ER stress, activate cell death pathways,and trigger apoptosis. Experiments using hydrophobic bile salts to treat hepatocytes in vitro have demonstrated this idea [17].

More exact mechanisms of BA?induced liver injury are not fully understood, and more related studies are needed. Although BA overload has a negative effect, it also has a positive effect on liver repair and has a "double?edged sword" role in the process of liver regeneration. That is, on the one hand, it has potential toxicity,and on the other hand, it can transmit signals of protection and proliferation. Positive effects are detailed below.

2. BA homeostasis and hepatoprotective mechanisms

BA overload?induced liver parenchymal injury, has a hindering effect on liver proliferation after PHx. In the repair process after injury, the remaining liver must maintain normal physiological functions to meet peripheral needs. Fine regulation of BA is therefore required to maintain BA homeostasis during repair.

Pean et al8 found in mouse experiments that BA overload in mouse liver cells occurred several hours after PHx, and BA levels peaked after approximately 24 hours. Within 24 hours to 48 hours after PHx,intrahepatic BA levels gradually returned to pre?PHx levels[18]. In the process of restoring homeostasis, BA and its receptors constitute a signaling network to cope with the massive and potential hazards that BA overload after PHx poses to the residual liver, with the effect of protecting the remaining liver.

2.1 Farnesoid X Receptor (FXR)

FXR is a BA?responsive ligand?activated transcription factor that is a member of the nuclear receptor superfamily. FXR plays a key role in controlling BA homeostasis and is a major regulator of BA homeostasis. Cariello et al. [19] found that in the liver and terminal ileum, FXR can trigger the expression of its target gene fibroblast growth factor 19 and inhibit the synthesis of BA through the expression of fibroblast growth factor 19 and its receptor fibroblast growth factor receptor 4 on hepatocytes. FXR can also induce the expression of small heterodimer partners, which interact with hepatic receptor homologs, resulting in reduced expression of cholesterol 7α hydroxylase, which catalyzes the breakdown of cholesterol into BA,which ultimately leads to reduced BA synthesis.

FXR is involved in immune regulation and barrier function in the intestine, thereby reducing the damage of endotoxin to the liver and protecting the liver. It specifically regulates the degree of inflammatory response and maintains the integrity and function of the intestine [16]. FXR plays an important role in mucosal immune responses and has an important impact on immune regulation [20].Vavassori et al [21] found that proinflammatory cytokine mRNA expression levels were significantly increased in FXR?deficient mice. Raybould et al. [22] showed that FXR activation by INT?747 prevented intestinal inflammation caused by dextran sulfate sodium and trinitrobenzene sulfonic acid, improved colitis symptoms,inhibited epithelial permeability and reduced goblet cell loss.Second, FXR regulates the involvement of intestinal bacteria in intestinal mucosal barrier function. BA has antibacterial activity by destroying the bacterial cell membrane, thereby inhibiting the growth of bacteria [23]. Inagaki et al[24] found that mice lacking FXR develop bacterial overgrowth, increased intestinal mucosal permeability,bacterial translocation, as well as intestinal inflammation. FXR plays a key role in limiting bacterial overgrowth and thus protecting the intestine from damage caused by bacteria.

2.2 Takeda G-protein coupled receptor 5 (TGR5)

TGR5 is a cell surface G protein?coupled receptor responsive to BA. TGR5 mRNA was highly expressed in the gallbladder, liver, and intestine. In the liver, TGR5 is present in sinusoidal endothelial cells,Kupffer cells, and cholangiocytes. TGR5 also has a role in regulating BA homeostasis, but the mechanism between it and BA is not clear.There are several hypotheses: (1) TGR5 can directly control BA synthesis in hepatocytes; (2) TGR5 can control BA conversion in the intestine through effects on gut bacteria function or composition;and (3) TGR5 may regulate BA transepithelial flux throughout the enterohepatic circulation in the ileum, biliary tract, and kidney,thereby regulating the composition of the BA pool [25].

One mechanism by which TGR5 protects the liver from BA overload is related to the receptor's properties. In previous studies,many scholars have found that BA?mediated proinflammatory effects have an important role in BA?induced liver injury [15, 26].Activation of BA TGR5 stimulates nitric oxide production by rat liver endothelial cells and decreases lipopolysaccharide?induced cytokine induction in rat Kupffer cells and mouse macrophages[27]. In terms of specific mechanisms, Wang [28] et al found that specific TGR5 stimulation in macrophages blunted IkB and p65 nuclear translocation in a cyclic adenosine monophosphate (cAMP)?dependent manner. Perino [29] et al suggested that TGR5 activation also reduces chemokine secretion and macrophage migration through AKT?mTOR complex 1 pathway and CEBP?β induction. Some scholars have suggested that BA becomes an endogenous modulator of NLRP3 inflammasome activity through a complex mechanism involving TGR5 [30,31]. One study found that chenodeoxycholic acid transactivates NLRP3 via TGR5?dependent EGFR during cholestasis[30]. In contrast, Guo et al.31 found that lithocholic acid inhibits NLRP3 by interacting with the phosphorylation and ubiquitination of NLRP3 protein through a TGR5?cAMP?PKA dependent pathway.Therefore, more studies are needed on TGR5 interaction with inflammasomes to more clearly define its protective role in BA overload.

2.3 Other Routes

Cholesterol has an important role in maintaining BA homeostasis.The cholesterol metabolites 25?hydroxycholesterol?3?sulfate and 25?glycol disulfate were shown to be effective modulators of lipid metabolism, inflammatory response, apoptosis, and cell survival.Ning et al. [32] showed protective effects in the damaged liver,lung, and kidney in a mouse model of lipopolysaccharide?induced acute liver failure using administration of 25?hydroxycholesterol?3?sulfate and 25?glycol disulfate in a mouse experiment. Yula et al.[33] tested the effect of cholesterol on mice with high BA levels in an experiment and found that mice fed cholesterol attenuated symptoms such as liver fibrosis caused by high BA levels and improved hepatocyte survival, thus confirming that cholesterol intake can protect the liver from BA toxicity.

HCO3? secreted by hepatocytes also has a role in maintaining BA homeostasis. Bicarbonate regulates the PH value of bile to protect cells, so the liver is a way for the liver to protect itself by adjusting the secretion of bicarbonate in the bile. Van [34] et al found that cholangiocytes and hepatocytes form a protective apical basic barrier by secreting bicarbonate into the bile duct lumen, which allows bile salts to remain polar, deprotonated, and impermeable to avoid excessive invasion of non?polar toxic BA. Huang [35] et al found experimentally that changing the cholic acid pool size can significantly improve the survival rate of mice after PHx, and the study of cholic acid pool may also help to understand the protective mechanism after PHx.

Ibrahim et al [36] found that the expression and activity of CYP7A1 mRNA were reduced through the pathway of signal transducer and activator of transcription 3 (STAT3) activation and hepatocyte nuclear factor 4a (HNF4α) reduction, and CYP7A1 was the ratelimiting enzyme in the BA synthesis pathway. Overexpression of short liver regeneration enhancer can reduce BA accumulation as well as BA?induced apoptosis, thereby alleviating the adverse effects of BA elevation.

After BA overload, various mechanisms to maintain BA homeostasis and protect residual hepatocytes are triggered, but some effects are not yet fully understood, and the relevant protective mechanisms obtained in some animal experiments need to be more observed and studied in clinical practice.

3. BA promotes proliferation

3.1 BA and Stellate Cells

Stellate cells are retinoid storage cells that are present in multiple organs [37]. Hepatic stellate cells can promote the formation of fibrous scars in chronic liver disease [38]. It has been found that hepatic stellate cells are mesenchymal stem cells resident in the liver, which can differentiate not only into adipocytes and osteocytes, but also into hepatic epithelial cells, such as hepatocytes and cholangiocytes[39], and into mesenchymal stem cells in bone marrow or adipose tissue, and stellate cells promote liver regeneration through the above differentiation mechanisms [40]. During hepatocyte differentiation,stellate cells and other mesenchymal stem cells transiently develop into hepatic progenitor cells with epithelial characteristics [38]. In stem cell?based models of liver injury, transplanted stellate cells from the liver and pancreas are able to promote liver regeneration,and transplanted stellate cells can also be found in the bone marrow[38, 40]. Baroni et al [41] found that BA at concentrations greater than 25 micromol·L?1induced a 2.5? to 3?fold increase in stellate cell proliferation by activating epidermal growth factor receptors.

BA can activate signaling pathways in hepatic stellate cells,and the detailed mechanism of this process is not clear. Elevated BA concentrations induce hepatocyte apoptosis and necrosis in cholestatic liver disease, but elevated BA concentrations can induce epidermal growth factor receptor activation and stimulate hepatic stellate cell proliferation, which is inhibitory to BA?mediated apoptosis.

3.2 Nuclear Receptors

In the study of liver regeneration after PHx, FXR has been relatively intensively studied. FXR can regulate BA metabolism,lipid metabolism, as well as glucose metabolism, which is essential during liver regeneration [42]. FXR is expressed at high levels in the liver and intestine, and in the case of liver regeneration, this constitutes a component of enterohepatic signaling communication[43]. BA can trigger mitotic and metabolic signals through hepatocyte FXR to promote liver regeneration. Under the combined action of the above mechanisms, liver cells can proliferate.Activated hepatocellular FXR signals proliferation through FOXM1B[44], a transcription factor that regulates DNA replication and mitosis by stimulating protein expression of cyclin?dependent kinase 2 (CDK2) and CDK1 [45]. CDK2 is essential for G1/S transition (46), while CDK1 progresses the cell cycle from S phase to mitotic M phase (47). FOXM1B of hepatocytes affects cell proliferation at the cell cycle level, and BA induces this process through hepatocyte FXR.

In addition to promoting mitosis, liver regeneration in PHx may be associated with metabolic signaling induced by FXR in hepatocytes.Xie et al. [48] found that hepatocyte FXR can activate pyruvate dehydrogenase kinase 4 (PDK4) in mitochondria. PDK4 has been implicated in the metabolism of glucose and certain amino acids to biomass, which is essential in cell proliferation. However, there is no direct evidence that PDK4 enzyme activity increases after PHx, and PDK4 is regulated by a variety of signals, and the specific mechanism of the FXR?PDK4 axis in liver regeneration remains to be further studied.

FXR is also expressed in enterocytes, and BA stimulates the expression of fibroblast growth factor 15/19, which is released into the portal blood and regulates BA synthesis by activating FGFR4 on hepatocytes, fibroblast growth factor 15/19, and fine?tunes liver regeneration as part of the "reversion mechanism" (2).

4. Clinical Significance

With the increasing understanding of the mechanisms by which BA mediates liver protection and prompts liver regeneration,relevant pathways in the mechanism can be used to regulate liver regeneration. FXR agonists have the therapeutic potential to accelerate liver regeneration after PHx, and targeting FXR or TGR5 receptors by synthetic agonists may promote regeneration and protect the liver [49]. Meng et al. [50] observed a dose?dependent stimulation of liver regeneration after administration of a plant triterpenoid with FXR?activating properties in mice. In an experiment in aged mice,the synthetic FXR agonist Px20350 overcame the regenerative defect in aged mice [51]. In another study, the use of fibroblast growth factor 15/19 analogues enhanced liver regeneration after portal vein embolization and prevented postoperative failure [52]. Understanding the relevant mechanisms is significant for clinical treatment, and current studies focus on the clinical treatment of liver regeneration with FXR agonists and fibroblast growth factor 15/18, and the therapeutic modalities to promote liver regeneration through other mechanisms need to be more studied.

5. Summary and Prospect

It is particularly important to protect the liver and stimulate liver regeneration after hepatectomy and liver injury. This article describes the "two sides" of BA in the process of liver regeneration,including the damage of BA overload to the liver and some specific mechanisms of protection and proliferation in the liver. A detailed understanding of the mechanism can help to reduce factors that inhibit regeneration or directly stimulate liver regeneration in rational targeted therapy. However, many studies have not yet been deepened, including BA?induced apoptosis, the specific mechanism of the FXR?PDK4 axis, and the pathway of action of TGR5. In addition, although animal models of liver regeneration are important,the clinical relevance of some animal models remains to be further improved to recognize the differences between model systems and clinical practice. The interaction between BA and liver regeneration needs to be further studied later.

Author’s Contributions

Zeng Shenglan: put forward research ideas, write papers and revise them;

Wu Cong, Wang Tongshuai, Le Qianyu: Collecting literatures and sorting out documents;

Wang Na, Zhang Rongzhen, Mao Dewen: Propose revision comments and provide guidance.