Lithocholic Acid Alleviates Deoxynivalenol-Induced Inflammation and Oxidative Stress via PPARγ-Mediated Epigenetically Transcriptional Reprogramming in Porcine Intestinal Epithelial Cells.

Deoxynivalenol (DON) is a common mycotoxin that induces intestinal inflammation and oxidative damage in humans and animals. Given that lithocholic acid (LCA) has been suggested to inhibit intestinal inflammation, we aimed to investigate the protective effects of LCA on DON-exposed porcine intestinal epithelial IPI-2I cells and the underlying mechanisms. Indeed, LCA rescued DON-induced cell death in IPI-2I cells and reduced DON-stimulated inflammatory cytokine levels and oxidative stress. Importantly, the nuclear receptor PPARγ was identified as a key transcriptional factor involved in the DON-induced inflammation and oxidative stress processes in IPI-2I cells. The PPARγ function was found compromised, likely due to the hyperphosphorylation of the p38 and ERK signaling pathways. In contrast, the DON-induced inflammatory responses and oxidative stress were restrained by LCA via PPARγ-mediated reprogramming of the core inflammatory and antioxidant genes. Notably, the PPARγ-modulated transcriptional regulations could be attributed to the altered recruitments of coactivator SRC-1/3 and corepressor NCOR1/2, along with the modified histone marks H3K27ac and H3K18la. This study emphasizes the protective actions of LCA on DON-induced inflammatory damage and oxidative stress in intestinal epithelial cells via PPARγ-mediated epigenetically transcriptional reprogramming, including histone acetylation and lactylation.

Obeticholic acid protects against lithocholic acid-induced exogenous cell apoptosis during cholestatic liver injury.

AIMS: Lithocholic acid (LCA)-induced cholestasis was accompanied by the occurrence of apoptosis, which indicated that anti-apoptosis was a therapeutic strategy for primary biliary cholangitis (PBC). As an agonist of (Farnesoid X receptor) FXR, we supposed that the hepatoprotection of Obeticholic acid (OCA) against cholestatic liver injury is related to anti-apoptosis beside of the bile acids (BAs) regulation. Herein, we explored the non-metabolic regulating mechanism of OCA for resisting LCA-induced cholestatic liver injury via anti-apoptosis. MAIN METHODS: LCA-induced cholestatic liver injury mice were pretreated with OCA to evaluate its hepatoprotective effect and mechanism. Biochemical and pathological indicators were used to detect the protective effect of OCA on LCA-induced cholestatic liver injury. The bile acids (BAs) profile in serum was detected by LC-MS/MS. Hepatocyte BAs metabolism, apoptosis and inflammation related genes and proteins alteration were investigated by biochemical determination. KEY FINDINGS: OCA improved LCA-induced cholestasis and hepatic apoptosis in mice. The BA profile in serum was changed by OCA mainly manifested as a reduction of taurine-conjugated bile acids, which was due to the upregulation of FXR-related bile acid efflux transporters bile salt export pump (BSEP), multi-drug resistant associated protein 2 (MRP2), MRP3 and multi-drug resistance 3 (MDR3). Apoptosis related proteins cleaved caspase-3, cleaved caspase-8 and cleaved PARP were obviously reduced after OCA treatment. SIGNIFICANCE: OCA improved LCA-induced cholestatic liver injury via FXR-induced exogenous cell apoptosis, which will provide new evidence for the application of OCA to ameliorate PBC in clinical.

Synergistic anti-inflammatory effect of gut microbiota and lithocholic acid on liver fibrosis.

BACKGROUND: Bile acids can regulate liver disease progression by affecting the functions of gut microbiota and immune cells. As the most potent natural agonist of G-protein coupled bile acid receptor 5 (TGR5) (expressed in macrophages, HSCs, and monocytes), lithocholic acid (LCA) has multiple functions, such as inhibiting inflammation and regulating metabolism. Therefore, this study aims to investigate the effects of LCA on immune cells and HSCs in liver fibrosis. METHODS: A liver fibrosis mouse model was induced by carbon tetrachloride followed by gavage of LCA, and the effects of LCA were evaluated by serum biochemical analysis, liver histology, and western bolt. Plasma cytokine levels and the number of immune cells were determined by cytometric bead array and flow cytometry, respectively. RESULTS: LCA could inhibit the activation of HSCs by inducing apoptosis and reducing the activation of transforming growth factor-ß (TGF-ß) Smad-dependent and Smad-independent pathways. Meanwhile, LCA inhibited glycolysis and promoted oxidative phosphorylation, leading to the differentiation of macrophages to M2 type and inhibiting their differentiation to M1 type. Furthermore, LCA increased the recruitment of NK cells and reduced the activation of NKT cells. However, these effects of LCA were attenuated after antibiotics reduced the diversity and abundance of the gut microbiota. CONCLUSIONS: Gut microbiota and LCA exerted synergistic anti-inflammatory effects on liver fibrosis. The combined intervention of gut microbiota and LCA will be a new strategy for treating liver fibrosis.

Cholestatic models induced by lithocholic acid and α­naphthylisothiocyanate: Different etiological mechanisms for liver injury but shared JNK/STAT3 signaling.

α­naphthylisothiocyanate (ANIT) is used to induce intrahepatic cholestasis and it is frequently used for investigations into the disease mechanism. The lithocholic acid (LCA) cholestatic model has also been extensively used in various studies; however, to the best of our knowledge, a comparative study determining the hepatotoxic mechanisms induced by these two models has not been previously conducted. In the present study, ICR mice were treated with ANIT or LCA to induce cholestatic liver injury. Biochemical analysis was used to determine the serum. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bile acid (TBA) levels, and histopathological assessment was used to examine the liver tissue. Metabolomic analysis was used for the serum biomarker identification. Reverse transcription­quantitative PCR analysis and western blotting were used to analyze the inflammation biomarkers. The serum metabolome of the ANIT group clustered away from of the LCA group, which was demonstrated by the different modifications of the BA components. ALP level was found to be preferentially increased in the ANIT group from 24 to 48 h. Total BA levels was only increased in the ANIT group at 24 h. In contrast, AST and ALT activity levels were preferentially increased in the LCA group. The bile ducts in the hepatic tissues of the ANIT group were observed to be severely dilated, whereas the presence of edematous hepatocytes around the necrotic lesions and neutrophil infiltration were identified in the LCA group. The expression levels of cholesterol 7α­hydroxylase and sterol 12α­hydroxylase genes were significantly downregulated in the ANIT group compared with the LCA group, where a stronger adaptation of BA metabolism was supported by major differences in the concentration of the BA components. Despite the aforementioned etiological differences in the cholestasis induced by each treatment, the activation of the JNK/STAT3 signaling pathway was similar between the two cholestatic models. In conclusion, these data suggested that the liver injury induced by ANIT may be cholestatic, while the liver injury caused in the LCA model may be hepatocellular. Moreover, the downstream cholestatic liver injury in both models was indicated to be mediated by the JNK/STAT3 signaling pathway.

Lithocholic acid activates mTOR signaling inducing endoplasmic reticulum stress in placenta during intrahepatic cholestasis of pregnancy.

AIMS: Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disorder, which increases risks of adverse fetal outcomes. However, the pathophysiology is not fully understood. Here, we explored the roles of mTOR signaling and ER stress in placenta during ICP. MATERIALS AND METHODS: Placental tissues were collected from normal and ICP pregnancies. mTOR signaling and endoplasmic reticulum stress were detected by immunohistochemistry in the placenta. The human placenta trophoblast cell line HTR-8/SVneo was used in vitro experiment. KEY FINDINGS: ICP placenta displayed histological abnormalities with fewer trophoblasts. Moreover, the expression of Bip and the phosphorylation of pS6(S235/236) or pAkt(S473) were higher comparing with normal placenta. In in vitro studies, the bile acids specifically to lithocholic acid rather than taurocholic acid or ursodeoxycholic acid, drastically increased the phosphorylation of pS6K1(T389), pS6(S235/236), or pAkt(S473), whereas the mTOR inhibitor can prohibit the upregulation. Similarly, the expressions of IRE1α and BiP increased sharply under lithocholic acid (20 µM) administration, while the same inhibitor can also decrease the expression. Additionally, transmission electron microscopy showed enlarged endoplasmic reticulum lumen under the lithocholic acid treatment. Furthermore, the cell viability reduced sharply under treatment with different dose of lithocholic acid. The mTOR inhibitor can reverse the decrease of cell viability to some extent. SIGNIFICANCE: Bile acid can activate mTOR signaling which resulted in endoplasmic reticulum stress, leading to trophocyte viability decrease. mTOR pathway activation may be associated with the pathophysiology of ICP.

Glycyrrhetic Acid Derivative TY501 Protects Against Lithocholic Acid-Induced Cholestasis.

The aim of the study is to investigate the protective effects of TY501 against LCA-induced cholestasis in mice and to explore the potential mechanisms. It was demonstrated that TY501(5, 15 or 45 mg/kg, i.g.) can markedly reduced the level of ALT, AST and ALP which increased by LCA treatment. Meanwhile, TY501 also lowered total bile acids, total bilirubin and total cholesterol levels in serum. Furthermore, TY501 can protect HepG2 cell cultures from LCA-induced cytotoxicity. RT-PCR and Western Blot analysis showed that TY501 recovered the expression of BSEP, MRP2 and NTCP which were down-regulated by LCA. Moreover, mRNA and protein of FXR was also observed in TY501 treated mice significantly accumulation in nucleus. Taken together, It can be concluded that TY501 exerted beneficial effects on LCA-induced cholestasis, possibly via activation of FXR mediated upregulation of BSEP, MRP2 and NTCP.

Hepatoprotective Effects of Schisandra sphenanthera Extract against Lithocholic Acid-Induced Cholestasis in Male Mice Are Associated with Activation of the Pregnane X Receptor Pathway and Promotion of Liver Regeneration.

We previously reported that the ethanol extract of Schisandra sphenanthera [Wuzhi (WZ) tablet] significantly protects against acetaminophen-induced hepatoxicity. However, whether WZ exerts a protective effect against cholestasis remains unclear. In this study, the protective effect of WZ on lithocholic acid (LCA)-induced intrahepatic cholestasis in mice was characterized and the involved mechanisms were investigated. WZ pretreatment (350 mg/kg) with LCA significantly reversed liver necrosis and decreased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activity. More importantly, serum total bile acids and total bilirubin were also remarkably reduced. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis showed that hepatic expression of pregnane X receptor (PXR) target genes such as CYP3A11 and UDP-glucuronosyltransferase (UGT) 1A1 were significantly increased by WZ treatment. Luciferase assays performed in LS174T cells illustrated that WZ extract and its six bioactive lignans could all activate human PXR. In addition, WZ treatment significantly promoted liver regeneration via inhibition of p53/p21 to induce cell proliferation-associated proteins such as cyclin D1 and proliferating cell nuclear antigen. In conclusion, WZ has a protective effect against LCA-induced intrahepatic cholestasis, partially owing to activation of the PXR pathway and promotion of liver regeneration.

Hepatoprotective effect of vitamin C on lithocholic acid-induced cholestatic liver injury in Gulo(-/-) mice.

Prevention and restoration of hepatic fibrosis from chronic liver injury is essential for the treatment of patients with chronic liver diseases. Vitamin C is known to have hepatoprotective effects, but their underlying mechanisms are unclear, especially those associated with hepatic fibrosis. Here, we analyzed the impact of vitamin C on bile acid induced hepatocyte apoptosis in vitro and lithocholic acid (LCA)-induced liver injury in vitamin C-insufficient Gulo(-/-) mice, which cannot synthesize vitamin C similarly to humans. When Huh-BAT cells were treated with bile acid, apoptosis was induced by endoplasmic reticulum stress-related JNK activation but vitamin C attenuated bile acid-induced hepatocyte apoptosis in vitro. In our in vivo experiments, LCA feeding increased plasma marker of cholestasis and resulted in more extensive liver damage and hepatic fibrosis by more prominent apoptotic cell death and recruiting more intrahepatic inflammatory CD11b(+) cells in the liver of vitamin C-insufficient Gulo(-/-) mice compared to wild type mice which have minimal hepatic fibrosis. However, when vitamin C was supplemented to vitamin C-insufficient Gulo(-/-) mice, hepatic fibrosis was significantly attenuated in the liver of vitamin C-sufficient Gulo(-/-) mice like in wild type mice and this hepatoprotective effect of vitamin C was thought to be associated with both decreased hepatic apoptosis and necrosis. These results suggested that vitamin C had hepatoprotective effect against cholestatic liver injury.

The intake of a hazelnut skin extract improves the plasma lipid profile and reduces the lithocholic/deoxycholic bile acid faecal ratio, a risk factor for colon cancer, in hamsters fed a high-fat diet.

The effects on lipid and glucose metabolism of a hazelnut skin extract (FIBEROX™) administrated during 8 weeks (HFD-FBX8w group) or during the last 4 weeks of the study (HFD-FBX4w group) to Golden Syrian hamsters fed a high-fat diet (HFD) for 8 weeks were investigated. FIBEROX™ consumption reversed the increase in total and LDL plasma cholesterol induced by the HFD feeding in both HFD-FBX groups and decreased the circulating levels of free fatty acids and triglycerides in the HFD-FBX4w animals. The higher excretion of bile acids found in the faeces of both groups of hamsters fed the FIBEROX™ suggests that this mechanism is involved in the cholesterol-lowering effects of the extract. Furthermore, FIBEROX™ intake sharply decreased the lithocholic/deoxycholic bile acid faecal ratio, a risk factor for colon cancer, in both HFD-FBX groups. In conclusion, the consumption of FIBEROX™ improves different risk factors associated with cardiovascular disease and colon cancer.

Pregnane X receptor mediated-transcription regulation of CYP3A by glycyrrhizin: a possible mechanism for its hepatoprotective property against lithocholic acid-induced injury.

Licorice (LE) has been commonly used in traditional Chinese medicine (TCM) for over 4000 years to reconcile various drugs and for hepatic disorders. Glycyrrhizin is the main bioactive component isolated from LE herbs. In the present study we examined the effects of glycyrrhizin on pregnane X receptor (PXR)-mediated CYP3A expression and its hepatoprotective activity. Treatment of HepG2 cells with glycyrrhizin resulted in marked increase in both CYP3A4 mRNA and protein levels. The transcriptional activation of the CYP3A4 gene through glycyrrhizin is PXR-dependent, as shown in transient transfection experiments. Glycyrrhizin activates the DNA-binding capacity of the PXR for the CYP3A4 element responding to xenobiotic signals, as measured by the electrophoretic-mobility shift assay (EMSA). These results indicate that the induction of the hepatic CYP3A4 by glycyrrhizin is mediated through the activation of PXR. The next aim of the current study was to determine whether the activation of PXR and induction of CYP3A by glycyrrhizin prevents hepatotoxicity during cholestasis as a mechanism of hepatoprotection. Mice were pretreated with glycyrrhizin prior to induction of intrahepatic cholestasis using lithocholic acid (LCA). Pre-treatment with glycyrrhizin, as well as the PXR activator pregnenolone 16α-carbontrile (PCN), prevents the increase in plasma ALT and AST activity, multifocal necrosis and prevents an increase in a level of serum LCA level in mice, as compared with the results in the mice treated with LCA alone. Activation of the PXR by glycyrrhizin results in induction of CYP3A11 (CYP3A4 for human) expression and inhibition of CYP7A1 through an increase in small heterodimer partner (SHP) expression. Glycyrrhizin regulates the expression of the gene mentioned above to prevent toxic accumulation of bile acids in the liver and it also protects mouse livers from the harmful effects of LCA. In conclusion, PXR-mediated effects on CYP3A and CYP7A may contribute to the hepatoprotective property of glycyrrhizin against LCA-induced liver injury.

Activation of LXRs prevents bile acid toxicity and cholestasis in female mice.

UNLABELLED: Liver X receptors (LXRs) have been identified as sterol sensors that regulate cholesterol and lipid homeostasis and macrophage functions. In this study, we found that LXRs also affect sensitivity to bile acid toxicity and cholestasis. Activation of LXRalpha in transgenic mice confers a female-specific resistance to lithocholic acid (LCA)-induced hepatotoxicity and bile duct ligation (BDL)-induced cholestasis. This resistance was also seen in wild-type female mice treated with the synthetic LXR ligand TO1317. In contrast, LXR double knockout (DKO) mice deficient in both the alpha and beta isoforms exhibited heightened cholestatic sensitivity. LCA and BDL resistance in transgenic mice was associated with increased expression of bile acid-detoxifying sulfotransferase 2A (Sult2a) and selected bile acid transporters, whereas basal expression of these gene products was reduced in the LXR DKO mice. Promoter analysis showed that the mouse Sult2a9 gene is a transcriptional target of LXRs. Activation of LXRs a l so suppresses expression of oxysterol 7alpha-hydroxylase (Cyp7b1), which may lead to increased levels of LXR-activating oxysterols. CONCLUSION: We propose that LXRs have evolved to have the dual functions of maintaining cholesterol and bile acid homeostasis by increasing cholesterol catabolism and, at the same time, preventing toxicity from bile acid accumulation.

Comparative effects of secondary bile acids, deoxycholic and lithocholic acids, on aberrant crypt foci growth in the postinitiation phases of colon carcinogenesis.

The objective of this study was to investigate the effect of deoxycholic (DCA) and lithocholic (LCA) acids on the postinitiation phases of colon cancer. Male Sprague-Dawley rats (n = 170) were injected with azoxymethane (2 injections at 15 mg/kg body wt sc given 1 wk apart) and fed a control (CON) AIN-93 diet. Two weeks after the second azoxymethane injection, 10 animals were killed and aberrant crypt foci (ACF) were enumerated. The remaining animals were randomly assigned to four diet groups: 1) CON, 2) DCA, 3) LCA, and 4) high fat (HF, a positive control group). Bile acid diets consisted of 0.2% by weight DCA or LCA; HF diets consisted of 20% fat (5% soybean oil + 15% beef tallow by weight). Animals were killed at Weeks 3, 12, and 20 (from 1st carcinogen injection), and number and growth features of ACF and adenomatous lesions were enumerated in the colon. At Week 12, ACF number and small, medium, and large (1-3, 4-6, and > or = 7 crypts/focus, respectively) ACF were higher in the HF group than in the DCA, LCA, and CON groups (p < or = 0.05). By Week 20, ACF number and small, medium, and large ACF were similar in the LCA and HF groups, whereas the response was similar in the DCA and CON groups. Average crypt multiplicity was higher in the HF and LCA groups than in the DCA and CON groups (p < or = 0.05). Microadenoma (MA) incidence was higher in the HF group than in the CON and LCA groups (p < or = 0.05). Regional distribution patterns for ACF number were similar to MA and tumor distribution patterns within the CON, DCA, and HF groups. In the LCA group, ACF number and MA showed a proximal predominance in regional distribution, whereas tumors showed a distal predominance. HF diets provided the most stimulatory environment, immediately enhancing the number and growth of ACF and MA incidence. In conclusion, HF and LCA diets exerted distinct effects on postinitiation phases of colon cancer, whereas the DCA diet did not.

Tight junction permeability and liver plasma membrane fluidity in lithocholate-induced cholestasis.

The present study correlated the reversibility of bile flow (BF) impairment with biochemical and morphological changes in the liver after injection of a cholestatic dose (12 mumole/100 g body weight) of lithocholic acid (LCA). BF declined maximally at 60 min but recovered totally at 210 min after LCA treatment. During the cholestatic period, there was an increase in tight junction permeability as measured by the bile to plasma (B/P) ratio of inulin and using lanthanum as a tracer. Cholesterol content and the cholesterol/phospholipid ratio in liver plasma membranes (LPM) were augmented while the fluidity of bile canalicular membranes (BCM) was decreased at 30 and 60 min after LCA injection. These changes in cholesterol content and membrane fluidity seemed to be correlated with LCA incorporation in LPM; their reversal at 120 min preceded the recovery of BF (210 min). Some biochemical disorders were evident after LCA injection, but they did not correlate with the variation in BF. These data suggest that increased tight junction permeability and decreased BCM fluidity are important pathogenic steps in LCA-induced cholestasis.

The relation of periportal fibrosis to portal hypertension.

To clarify the relation of periportal fibrosis to portal hypertension, the present study was undertaken in rats given lithocholate. Portal vascular resistance measured by an isolated liver perfusion method increased with the length of lithocholate administration. In vivo portal vein pressure elevation was closely correlated to the increase in portal vascular resistance. The blood pressure difference between the portal vein and the terminal portal venule increased compared to control rats. The blood pressure differences between the terminal portal venule and the terminal hepatic venule, and between the terminal hepatic venule and the inferior vena cava, were almost the same as those in control rats. Lithocholate administration induced narrowing and meandering of the portal vein branches due to periportal fibrosis with proliferation of bile ductules. There was no perceptible change in the sinusoids and the hepatic vein branches. These data suggest that periportal fibrosis causes deformation of the peripheral branches of the portal vein and leads to presinusoidal portal hypertension.

Hepatic alterations during total parenteral nutrition in patients with inflammatory bowel disease: a possible consequence of lithocholate toxicity.

In order to understand the mechanism of hepatic abnormalities appearing during total parenteral nutrition, biliary bile acid composition and liver function tests were examined serially in 15 patients undergoing total parenteral nutrition for inflammatory bowel disease. In all 12 patients who underwent duodenal intubation before initiation of total parenteral nutrition, lithocholic acid accounted for less than 1% of total biliary bile acids. After 11-22 days of total parenteral nutrition, lithocholic acid accounted for 7%-15% of biliary bile acids in 5 patients and less than 1% of biliary bile acids in 10 patients. In the 5 patients with elevated levels of biliary lithocholic acid, serum alkaline phosphatase and aminotransferase activities rose progressively at serial determinations, the increase being significant after 2 wk of total parenteral nutrition. These results suggest that lithocholic acid may be involved in the hepatic lesions observed in patients undergoing total parenteral nutrition.