Physiologically based pharmacokinetic modeling for confirming the role of CYP3A1/2 and P-glycoprotein in detoxification mechanism between glycyrrhizic acid and aconitine in rats.

Fuzi, an effective common herb, is often combined with Gancao to treat disease in clinical practice with enhancing its efficacy and alleviating its toxicity. The major toxic and bioactive compounds in Fuzi and Gancao are aconitine (AC) and glycyrrhizic acid (GL), respectively. This study aims to elucidate detoxification mechanism between AC and GL from pharmacokinetic perspective using physiologically based pharmacokinetic (PBPK) model. In vitro experiments exhibited that AC was mainly metabolized by CYP3A1/2 in rat liver microsomes and transported by P-glycoprotein (P-gp) in Caco-2 cells. Kinetics assays showed that the Km and Vmax of AC towards CYP3A1/2 were 2.38 µM and 57.3 pmol/min/mg, respectively, whereas that of AC towards P-gp was 11.26 µM and 147.1 pmol/min/mg, respectively. GL markedly induced the mRNA expressions of CYP3A1/2 and MDR1a/b in rat primary hepatocytes. In vivo studies suggested that the intragastric and intravenous administration of GL significantly reduced systemic exposure of AC by 27% and 33%, respectively. Drug-drug interaction (DDI) model of PBPK predicted that co-administration of GL would decrease the exposure of AC by 39% and 45% in intragastric and intravenous dosing group, respectively. The consistency between predicted data and observed data confirmed that the upregulation of CYP3A1/2 and P-gp was the crucial detoxification mechanism between AC and GL. Thus, this study provides a demonstration for elucidating the compatibility mechanisms of herbal formula using PBPK modeling and gives support for the clinical co-medication of Fuzi and Gancao.

Gancao decoction attenuates hepatic necroptosis via activating caspase 8 in cholestatic liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Gancao Decoction (GCD) is widely used to treat cholestatic liver injury. However, it is unclear whether is related to prevent hepatocellular necroptosis. AIM OF THE STUDY: The purpose of this study is to clarify the therapeutic effects of GCD against hepatocellular necroptosis induced by cholestasis and its active components. MATERIALS AND METHODS: We induced cholestasis model in wild type mice by ligating the bile ducts or in Nlrp3-/- mice by intragastrical administering Alpha-naphthylisothiocyanate (ANIT). Serum biochemical indices, liver pathological changes and hepatic bile acids (BAs) were measured to evaluate GCD's hepatoprotective effects. Necroptosis was assessed by expression of hallmarkers in mice liver. Moreover, the potential anti-necroptotic effect of components from GCD were investigated and confirmed in ANIT-induced cholestasis mice and in primary hepatocytes from WT mouse stimulated with Tumor Necrosis Factor alpha (TNF-α) and cycloheximide (CHX). RESULTS: GCD dose-dependently alleviated hepatic necrosis, reduced serum aminotranferase activity in both BDL and ANIT-induced cholestasis models. More importantly, the expression of hallmarkers of necroptosis, including MLKL, RIPK1 and RIPK3 phosphorylation (p- MLKL, p-RIPK1, p-RIPK3) were reduced upon GCD treatment. Glycyrrhetinic acid (GA), the main bioactive metabolite of GCD, effectively protected against ANIT-induced cholestasis, with decreased expression of p-MLKL, p-RIPK1 and p-RIPK3. Meanwhile, the expression of Fas-associated death domain protein (FADD), long isoform of cellular FLICE-like inhibitory protein (cFLIPL) and cleaved caspase 8 were upregulated upon GA treatment. Moreover, GA significantly increased the expression of active caspase 8, and reduced that of p-MLKL in TNF-α/CHX induced hepatocytes necroptosis. CONCLUSIONS: GCD substantially inhibits necroptosis in cholestatic liver injury. GA is the main bioactive component responsible for the anti-necroptotic effects, which correlates with upregulation of c-FLIPL and active caspase 8.

Glycyrrhetinic acid attenuates endoplasmic reticulum stress-induced hepatocyte apoptosis via CHOP/DR5/Caspase 8 pathway in cholestasis.

Bile acid (BA)-induced apoptosis is a common pathologic feature of cholestatic liver injury. Glycyrrhetinic acid (GA) is the hepatoprotective constituent of licorice. In the present study, the anti-apoptotic potential of GA was investigated in wild type and macrophage-depleted C57BL/6 mice challenged with alpha-naphthyl isothiocyanate (ANIT), and hepatocytes stimulated with Taurocholic acid (TCA) or Tumor necrosis factor-alpha (TNF-α). Apoptosis was determined by TUNEL positive cells and expression of executioner caspases. Firstly, we found that GA markedly alleviated liver injury, accompanied with reduced positive TUNEL-staining cells, and expression of caspases 3, 8 and 9 in mice modeled with ANIT. Secondly, GA mitigated apoptosis in macrophage-depleted mice with exacerbated liver injury and augmented cell apoptosis. In vitro study, pre-treatment with GA reduced the expression of activated caspases 3 and 8 in hepatocytes stimulated with TCA, but not TNF-α. The ability of GA to ameliorate apoptosis was abolished in the presence of Tauroursodeoxycholic Acid (TUDCA), a chemical chaperon against Endoplasmic reticulum stress (ER stress). Furthermore, GA attenuated the over-expression of Glucose regulated protein 78 (GRP78), and blocked all three branches of Unfolded protein reaction (UPR) in cholestatic livers of mice induced by ANIT. GA also downregulated C/EBP homologous protein (CHOP) expression, accompanied with reduced expression of Death receptor 5 (DR5) and activation of caspase 8 in both ANIT-modeled mice and TCA-stimulated hepatocytes. The results indicate that GA inhibits ER stress-induced hepatocyte apoptosis in cholestasis, which correlates with blocking CHOP/DR5/Caspase 8 pathway.

Yinchen decoction protects against cholic acid diet-induced cholestatic liver injury in mice through liver and ileal FXR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Cholestasis is a pathophysiological syndrome characterized by the accumulation of bile acids (BAs) that leads to severe liver disease. Artemisia capillaris is documented in Chinese Pharmacopoeia as the authentic resources for Yinchen. Although Yinchen (Artemisia capillaris Thunb.) decoction (YCD) has been used in China for thousands of years to treat jaundice, the underlying mechanisms to ameliorate cholestatic liver injury have not been elucidated. AIM OF THE STUDY: To investigate the molecular mechanism of how YCD protects against 1% cholic acid (CA) diet-induced intrahepatic cholestasis through FXR signaling. MATERIALS AND METHODS: Wild-type and Fxr-deficient mice were fed a diet containing 1% CA to establish the intrahepatic cholestasis model. The mice received low-, medium-, or high-dose YCD for 10 days. Plasma biochemical markers were analyzed, liver injury was identified by histopathology, and hepatic and plasma BA content was analyzed. Western blot was used to determine the expression levels of transporters and enzymes involved in BA homeostasis in the liver and intestine. RESULTS: In wild-type mice, YCD significantly improved plasma transaminase levels, multifocal hepatocellular necrosis, and hepatic and plasma BA contents, upregulated the expression of hepatic FXR and downstream target enzymes and transporters. Meanwhile, YCD significantly induced the expressions of intestinal FXR and FGF15 and hepatic FGFR4. In contrast, the hepatic protective effect of YCD on cholestasis was abolished in Fxr-deficient mice. CONCLUSION: YCD protects against cholestatic liver injury induced by a CA diet by restoring the homeostasis of BAs via activation of the liver FXR/SHP and ileal FXR/FGF15 signaling pathways. Furthermore, chlorogenic acid and caffeic acid may be the pharmacological agents in YCD responsible for protecting against cholestatic liver injury.

The choleretic role of tauroursodeoxycholic acid exacerbates alpha-naphthylisothiocyanate induced cholestatic liver injury through the FXR/BSEP pathway.

The aim of this study was to determine the effect of tauroursodeoxycholic acid (TUDCA) on the alpha-naphthylisothiocyanate (ANIT)-induced model of cholestasis in mice. Wild-type and farnesoid X receptor (FXR)-deficient (Fxr-/- ) mice were used to generate cholestasis models by gavage with ANIT. Obeticholic acid (OCA) was used as a positive control. In wild-type mice, treatment with TUDCA for 7 days resulted in a dramatic increase in serum levels of alanine aminotransferase (ALT), with aggravation of bile infarcts and hepatocyte necrosis with ANIT-induction. TUDCA activated FXR to upregulate the expression of bile salt export pump (BSEP), increasing bile acids (BAs)-dependent bile flow, but aggravating cholestatic liver injury when bile ducts were obstructed resulting from ANIT. In contrast, TUDCA improved the liver pathology and decreased serum ALT and alkaline phosphatase (ALP) levels in ANIT-induced Fxr-/- mice. Furthermore, TUDCA inhibited the expression of cleaved caspase-3 and reduced the area of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in the model mice. TUDCA also upregulated anion exchanger 2 (AE2) protein expression, protecting cholangiocytes against excessive toxic BAs. Our results showed that TUDCA aggravated cholestatic liver injury via the FXR/BSEP pathway when bile ducts were obstructed, although TUDCA inhibited apoptotic activity and protected cholangiocytes against excessive toxic BAs.

Potential therapeutic action of tauroursodeoxycholic acid against cholestatic liver injury via hepatic Fxr/Nrf2 and CHOP-DR5-caspase-8 pathway.

Cholestasis is a pathophysiologic syndrome with limited therapeutic options. Tauroursodeoxycholic acid (TUDCA) has been employed to treat hepatobiliary disorders and is as effective as UDCA in alleviating cholestatic liver disease in clinical trials. Until now, TUDCA's mechanism of action toward cholestasis remains unclear. In the present study, cholestasis was induced with a cholic acid (CA)-supplemented diet or α-naphthyl isothiocyanate (ANIT) gavage in wild-type and Farnesoid X Receptor (FXR) deficient mice, using obeticholic acid (OCA) as control. The effects of TUDCA on liver histological changes, transaminase level, bile acid composition, hepatocyte death, expression of Fxr and nuclear factor erythroid 2-related factor 2 (Nrf2) and target genes, as well as apoptotic signaling pathways, were investigated. Treating CA-fed mice with TUDCA markedly alleviated liver injury, attenuated bile acids retention in liver and plasma, increased Fxr and Nrf2 nuclear levels and modulated the expression of targets regulating synthesis and transportation of bile acids, including BSEP, MRP2, NTCP and CYP7A1. TUDCA, but not OCA, activated Nrf2 signaling and exerted protective effects against cholestatic liver injury in Fxr-/- mice fed with CA. Furthermore, in both mice with CA- and ANIT-induced cholestasis, TUDCA decreased expression of GRP78 and CCAAT/enhancer-binding protein homologous protein (CHOP), reduced death receptor 5 (DR5) transcription, caspase-8 activation, and BID cleavage, and subsequently inhibited activation of executioner caspases and apoptosis in liver. We confirmed that TUDCA protected against cholestatic liver injury by alleviating BAs burden of dually activating hepatic Fxr and Nrf2. Moreover, inhibiting CHOP-DR5-caspase-8 pathway contributed to the anti-apoptotic effect of TUDCA in cholestasis.

15,16-dihydrotanshinone I in Danshen ethanol extract aggravated cholestasis by inhibiting Cyp3a11 mediated bile acids hydroxylation.

Our previous study found that high-dose Tanshinones Capsule (TC) aggravated cholestasis in mice. To explore its underlying mechanism, main tanshinones components (15,16-dihydrotanshinone I (DTI), cryptotanshinone (CTS) and tanshinone IIA (TSA)) form TC were studied separately. Bile acids (BAs) that were primarily metabolized by hydroxylation were identified, and then the inhibitory effect of each tanshinones on their hydroxylation were evaluated. The anti-cholestasis effect of each tanshinones were studied in mice, the hepatic concentrations of BAs and tanshinones were measured and analyzed as well. The effect of tanshinones on Cyp3a11 protein expression was investigated. DTI exhibited inhibitory effect on the hydroxylation of lithocholic acid (LCA), taurolithocholic acid (TLCA) and taurochenodeoxycholic acid (TCDCA), their IC50 values were 0.81, 0.36 and 1.29 µM, respectively. The hydroxylation of LCA, TLCA and TCDCA were mediated by Cyp3a11. Low-dose DTI, CTS and TSA ameliorated cholestatic liver injury in mice, while high-dose DTI didn't exhibit anti-cholestatic effect. The hepatic BAs profiles indicated that hydroxylation of BAs was inhibited in high-dose DTI group. DTI and TSA up-regulated the protein expression of Cyp3a11. As the hepatic concentration of DTI increased, the inhibitory effect at enzymatic activity level overwhelmed its up-regulation effect at protein level, thus resulted in worsening of cholestasis.

Hepatoprotective Effects of Glycyrrhetinic Acid on Lithocholic Acid-Induced Cholestatic Liver Injury Through Choleretic and Anti-Inflammatory Mechanisms.

Cholestasis is a clinical syndrome triggered by the accumulation and aggregation of bile acids by subsequent inflammatory responses. The present study investigated the protective effect of glycyrrhetinic acid (GA) on the cholestatic liver injury induced by lithocholic acid (LCA) from both anti-inflammatory and choleretic mechanistic standpoints. Male C57BL/6 mice were treated with LCA twice daily for 4 days to induce intrahepatic cholestasis. GA (50 mg/kg) and pregnenolone 16α-carbonitrile (PCN, 45 mg/kg) were intraperitoneally injected 3 days before and throughout the administration of LCA, respectively. Plasma biochemical indexes were determined by assay kits, and hepatic bile acids were quantified by LC-MS/MS. Hematoxylin and eosin staining of liver sections was performed for pathological examination. Protein expression of the TLRs/NF-κB pathway and the mRNA levels of inflammatory cytokines and chemokines were examined by Western blotting and PCR, respectively. Finally, the hepatic expression of pregnane X receptor (PXR) and farnesoid X receptor (FXR) and their target genes encoding metabolic enzymes and transporters was evaluated. GA significantly reversed liver necrosis and decreased plasma ALT and ALP activity. Plasma total bile acids, total bilirubin, and hepatic bile acids were also remarkably preserved. More importantly, the recruitment of inflammatory cells to hepatic sinusoids was alleviated. Additionally, the protein expression of TLR2, TLR4, and p-NF-κBp65 and the mRNA expression of CCL2, CXCL2, IL-1ß, IL-6, and TNF-α were significantly decreased. Moreover, GA significantly increased the expression of hepatic FXR and its target genes, including BSEP, MRP3, and MRP4. In conclusion, GA protects against LCA-induced cholestatic liver injury by inhibiting the TLR2/NF-κB pathway and upregulating hepatic FXR expression.

Clarify the potential cholestatic hepatotoxicity components from Chinese Herb Medicine and metabolism's role via hBSEP vesicles and S9/hBSEP vesicles.

In this study, the inhibitory effect of components from Chinese Herb Medicine (CHMs) with potential hepatotoxicity was assessed by human bile salt export pump (hBSEP) vesicles with and without S9 metabolism. Sixty-three compounds from 22 hepatoxicity CHMs were selected as the test articles. In hBSEP vesicles, eighteen of them were found to have moderate or strong inhibitory effect towards BSEP. Further studies were performed to determine the IC50 values of strong inhibitors. For the compounds belong to CHMs reported to cause cholestasis and strong inhibitors defined in hBSEP vesicles, their relative transport activities of Taurocholic acid (TCA) were evaluated in hBSEP vesicles as well as hBSEP vesicles with S9 system (S9/hBSEP vesicles). The differences of their relative transport activities of TCA between the above two system were compared to reveal the net effect of metabolism on BSEP's activity. It was found that the inhibitory effect of Saikogenin A (SGA), Saikogenin D (SGD), Diosbulbin B (DB) and rhein were significantly increased; while the inhibitory effect of isobavachalcone, saikosaponin d and saikosaponin b2 were significantly decreased after S9 metabolizing. Identification of metabolic pathways suggested that CYP3A4 was responsible for aggravating inhibitory effect of SGA and SGD against BSEP.

Effect of Different Ratios of Yinchen and Gancao Decoction on ANIT-Treated Cholestatic Liver Injury in Mice and Its Potential Underlying Mechanism.

Cholestasis is a pathological state that leads to serious liver disease; however, therapeutic options remain limited. Yinchen and Gancao are often used in combination at different ratios in traditional Chinese formulae for the treatment of jaundice and cholestasis. In the present study, we investigated the effect of decoctions containing different ratios of Yinchen and Gancao (YGD) on alpha-naphthyl isothiocyanate (ANIT)-treated intrahepatic cholestasis (IC) in mice, and further explored the underlying mechanism. Treatment with 0:4 and 1:4 YGD significantly reduced plasma total bile acid (TBA), total bilirubin (TBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities; decreased unconjugated and conjugated bile acid levels; and improved hepatocyte necrosis and inflammatory cells recruitment to hepatic sinusoids. Moreover, the expression levels of Toll-like receptor 4 (TLR4), interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor alpha (TNF-α), C-C ligand 2 (CCL2), and C-X-C ligand 2 (CXCL2) in the liver were significantly reduced. However, treatment with 4:1 and 4:0 YGD increased plasma TBA, TBIL, AST, ALT, and ALP activities and aggravated liver cell injury and inflammation. Moreover, the mRNA expression of the bile salt export pump (BSEP) in the liver was significantly increased in mice treated with 4:0 YGD. The present study demonstrates that YGD containing a high proportion of Gancao, which inhibits the TLR4/NF-κB pathway and reduces the inflammatory response, had protective effects against ANIT-treated IC in mice. However, YGD containing a high proportion of Yinchen aggravated the ANIT-treated IC in mice, which may be related to upregulation of BSEP and boosting bile acid regurgitation from damage cholangiocytes to liver in ANIT-treated IC mice.