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.

Development and validation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of eight Xiakucao Oral liquid-related compounds in rat plasma and its application in pharmacokinetic study.

Xiakucao Oral Liquid (XKCOL) has been widely used for treating mammary gland hyperplasia and goiter in China. However, its pharmacokinetic data have been missing to date. To conduct its pharmacokinetic study, we established an LC-tandem mass spectrometry method for the simultaneous determination of eight XKCOL-related compounds in rat plasma. Liquid-liquid extraction was used for the sampling process. Chromatographic separation was performed on a Phenomenon Luna C18 column with a mobile phase of methanol and 2 mM ammonium acetate, using gradient elution at a flow rate of 0.8 mL/min. Detection was performed in the multiple reaction monitoring mode using negative electrospray ionization (ESI-) with optimized MS parameters. Endogenous substances and carryover did not interfere in the detection of analytes. The calibration curves showed a good linear relationship within the linear ranges. The intra- and inter-batch accuracy and precision were 94.8%-110.0% and ≤11.2%, respectively. There was no significant matrix effect and the recovery was reproducible. The dilution of samples did not affect the accuracy and precision. The solution and plasma samples were stable under the various test conditions. The major components of XKCOL absorbed into the blood were salvianic acid A and rosmarinic acid. They demonstrated linear kinetics over the dose range used in this study.

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.

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.

Salvianolic acid B attenuates tubulointerstitial fibrosis by inhibiting EZH2 to regulate the PTEN/Akt pathway.

CONTEXT: Salvianolic acid B (SAB) can alleviate renal fibrosis and improve the renal function. OBJECTIVE: To investigate the effect of SAB on renal tubulointerstitial fibrosis and explore its underlying mechanisms. MATERIALS AND METHODS: Male C57 mice were subjected to unilateral ureteric obstruction (UUO) and aristolochic acid nephropathy (AAN) for renal fibrosis indication. Vehicle or SAB (10 mg/kg/d, i.p.) were given consecutively for 2 weeks in UUO mice while 4 weeks in AAN mice. The serum creatinine (Scr) and blood urine nitrogen (BUN) were measured. Masson's trichrome staining and the fibrotic markers (FN and α-SMA) were used to evaluate renal fibrosis. NRK-49F cells exposed to 2.5 ng/mL TGF-ß were treated with SAB in the presence or absence of 20 µM 3-DZNep, an inhibitor of EZH2. The protein expression of EZH2, H3k27me3 and PTEN/Akt signaling pathway in renal tissue and NRK-49F cells were measured by Western blots. RESULTS: SAB significantly improved the levels of Scr by 24.3% and BUN by 35.7% in AAN mice. SAB reduced renal interstitial collagen deposition by 34.7% in UUO mice and 72.8% in AAN mice. Both in vivo and in vitro studies demonstrated that SAB suppressed the expression of FN and α-SMA, increased PTEN and decreased the phosphorylation of Akt, which were correlated with the down-regulation of EZH2 and H3k27me3. The inhibition of EZH2 attenuated the anti-fibrotic effects of SAB in NRK-49Fs. CONCLUSION: SAB might have therapeutic potential on renal fibrosis of CKD through inhibiting EZH2, which encourages further clinical trials.

Influence of Zuojin Pill on the Metabolism of Venlafaxine in Vitro and in Rats and Associated Herb-Drug Interaction.

Venlafaxine (VEN), a first-line antidepressant, and Zuojin Pill (ZJP), a common Chinese herbal medicine consisting of Rhizoma Coptidis and Fructus Evodiae, have a high likelihood of combination usage in patients with depression with gastrointestinal complications. ZJP exhibits inhibitory effects on recombinant human cytochrome P450 isoenzymes (rhP450s), especially on CYP2D6, whereas VEN undergoes extensive metabolism by CYP2D6. From this perspective, we investigated the influence of ZJP on the metabolism of VEN in vitro and in rats for the first time. In this study, ZJP significantly inhibited the metabolism of VEN in both rat liver microsomes (RLM) and human liver microsomes (HLM); meanwhile, it inhibited the O-demethylation catalytic activity of RLM, HLM, rhCYP2D6*1/*1, and rhCYP2D6*10/*10, primarily through CYP2D6, with IC50 values of 129.9, 30.5, 15.4, and 2.3 µg/ml, respectively. Furthermore, the inhibitory effects of ZJP on hepatic metabolism and pharmacokinetics of VEN could also be observed in the pharmacokinetic study of rats. The area under drug concentration-time curve0-24 hour of VEN and its major metabolite O-desmethylvenlafaxine (ODV) increased by 39.6% and 22.8%, respectively. The hepatic exposure of ODV decreased by 57.2% 2 hours after administration (P = 0.014). In conclusion, ZJP displayed inhibitory effects on hepatic metabolism and pharmacokinetics of VEN in vitro and in rats mainly through inhibition of CYP2D6 activity. The human pharmacokinetic interaction between ZJP and VEN and its associated clinical significance needed to be seriously considered. SIGNIFICANCE STATEMENT: Zuojin Pill, a commonly used Chinese herbal medicine, demonstrates significant inhibitory effects on hepatic metabolism and pharmacokinetics of venlafaxine in vitro and in rats mainly through suppression of CYP2D6 activity. The human pharmacokinetic interaction between Zuojin Pill and venlafaxine and its associated clinical significance needs to be seriously considered.

Metabolic characteristics of Tanshinone I in human liver microsomes and S9 subcellular fractions.

Tanshinone I (TSI) is a lipophilic diterpene in Salvia miltiorrhiza with versatile pharmacological activities. However, metabolic pathway of TSI in human is unknown. In this study, we determined major metabolites of TSI using a preparation of human liver microsomes (HLMs) by HPLC-UV and Q-Trap mass spectrometer. A total of 6 metabolites were detected, which indicated the presence of hydroxylation, reduction as well as glucuronidation. Selective chemical inhibition and purified cytochrome P450 (CYP450) isoform screening experiments revealed that CYP2A6 was primarily responsible for TSI Phase I metabolism. Part of generated hydroxylated TSI was glucuronidated via several glucuronosyltransferase (UGT) isoforms including UGT1A1, UGT1A3, UGT1A7, UGT1A9, as well as extrahepatic expressed isoforms UGT1A8 and UGT1A10. TSI could be reduced to a relatively unstable hydroquinone intermediate by NAD(P)H: quinone oxidoreductase 1 (NQO1), and then immediately conjugated with glucuronic acid by a panel of UGTs, especially UGT1A9, UGT1A1 and UGT1A8. Additionally, NQO1 could also reduce hydroxylated TSI to a hydroquinone intermediate, which was immediately glucuronidated by UGT1A1. The study demonstrated that hydroxylation, reduction as well as glucuronidation were the major pathways for TSI biotransformation, and six metabolites generated by CYPs, NQO1 and UGTs were found in HLMs and S9 subcellular fractions.

Different effects of ursodeoxycholic acid on intrahepatic cholestasis in acute and recovery stages induced by alpha-naphthylisothiocyanate in mice.

The aim of this study was to determine the effect of ursodeoxycholic acid (UDCA) on the alpha-naphthylisothiocyanate (ANIT)-induced acute and recovery stage of cholestasis model mice. In the acute stage of model mice, pretreatment with UDCA (25, 50, and 100 mg·kg-1, ig) for 12 days prior to ANIT administration (50 mg·kg-1, ig) resulted in the dramatic increase in serum biochemistry, with aggrevation of bile infarcts and hepatocyte necrosis. The elevation of beta-muricholic acid (ß-MCA), cholic acid (CA), and taurocholic acid (TCA) in serum and liver, and reduction of these bile acids (BAs) in bile was observed. In contrast, in the recovery stage of model mice, treatment with UDCA (25, 50, and 100 mg·kg-1, ig) for 7 days after ANIT administration (50 mg·kg-1, ig) resulted in the significant decrease in levels of serum alanine aminotransferase (ALT) and total bile acid (TBA). Liver injury was attenuated, and the levels of TBA, CA, TCA, and ß-MCA in the liver were significantly decreased. Additionally, UDCA can upregulate expression of BSEP, but it cannot upregulate expression of AE2. UDCA, which induced BSEP to increase bile acid-dependent bile flow, aggravated cholestasis and liver injury when the bile duct was obstructed in the acute stage of injury in model mice. In contrast, UDCA alleviated cholestasis and liver injury induced by ANIT when the obstruction was improved in the recovery stage.

Charactering the metabolism of cryptotanshinone by human P450 enzymes and uridine diphosphate glucuronosyltransferases in vitro.

Cryptotanshinone (CT) is the main active component in the root of Salvia miltiorrhiza Bunge (SMB) that displays antibacterial, anti-inflammatory and anticancer activities. In this study, we characterized phase I and phase II metabolism of CT in human liver microsomes in vitro and identified the metabolic enzymes (CYPs and UGTs) involved. The metabolites of CT generated by CYPs were detected using LC-MS/MS and the CYP subtypes involved in the metabolic reactions were identified using chemical inhibitors of CYP enzymes and recombinant human CYP enzymes (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Glucuronidation of CT was also examined, and the UGT subtypes involved in the metabolic reactions were identified using recombinant human UGT enzymes (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). After adding NADPH to the human liver microsomes incubation system, CT was transformed into 6 main dehydrogenation and hydroxylation metabolites. CYP2A6, CYP3A4 and CYP2C19 were the major contributors to the transformation of its hydroxylation metabolites. CYP2C19, CYP1A2 and CYP3A4 were the major contributors to the transformation of its hydrogenation metabolites in human liver microsomes. This study showed that the metabolites at m/z of 473 were mediated by UGT1A9 and that the metabolites at m/z of 489 were mediated by UGT2B7 and UGT2B4. CT was extensively metabolized by UGTs following metabolism by CYPs in the liver.

Effects of the Chinese herbal formula "Zuojin Pill" on the pharmacokinetics of dextromethorphan in healthy Chinese volunteers with CYP2D6*10 genotype.

OBJECTIVE: Zuojin Pill has been shown to inhibit the cytochrome P450 (CYP) 2D6 isoenzyme in vitro. In Chinese individuals, CYP 2D6*10 is the most common allele with reduced enzyme activity. In this study, we investigated the pharmacokinetic interaction between Zuojin Pill and the sensitive CYP2D6 probe dextromethorphan in healthy Chinese volunteers with CYP2D6*10 genotype. METHODS: A pharmacokinetics interaction study was carried out in three groups with CYP2D6*1/*1 (n = 6), CYP2D6*1/*10 (n = 6), and CYP2D6*10/*10 (n = 6) genotypes. Each participant received a single oral dose of dextromethorphan (15 mg) followed by Zuojin Pill (3 g twice daily) for 7 days, and received 3 g Zuojin Pill with 15 mg dextromethorphan in the last day. Blood samples (0-24 h) and urine samples (0-12 h) were collected at baseline and after the administration of Zuojin Pill, and the samples' concentration of dextromethorphan and its main metabolite dextrorphan was determined. RESULTS: Compared to baseline values, co-administration of Zuojin Pill (3 g twice daily) for 7 days increased the AUC0-24 of dextromethorphan [mean (90 % CI)] by 3.00-fold (2.49∼3.61) and 1.71-fold (1.42∼2.06), and decreased oral clearance(CL/F) by 0.27-fold (0.2-0.40) and 0.57-fold (0.48-0.67) in the participants with CYP2D6*1/*1 and CYP2D6*1/*10 genotypes, respectively. In contrast, no significant change was observed in these pharmacokinetic parameters of the participants with CYP2D6*10/*10 genotype. CONCLUSION: These data demonstrated that administration of Zuojin Pill inhibited moderately CYP2D6-mediated metabolism of dextromethorphan in healthy volunteers. The inhibitory influence of CYP2D6 was greater in CYP2D6*1/*1 and CYP2D6*1/*10 groups than CYP2D6 *10/*10 group.

IL-1 receptor-associated kinase family proteins: An overview of their role in liver disease.

The interleukin-1 receptor-associated kinase (IRAK) family is a group of serine-threonine kinases that regulates various cellular processes via toll-like receptor (TLR)/interleukin-1 receptor (IL1R)-mediated signaling. The IRAK family comprises four members, including IRAK1, IRAK2, IRAK3, and IRAK4, which play an important role in the expression of various inflammatory genes, thereby contributing to the inflammatory response. IRAKs are key proteins in chronic and acute liver diseases, and recent evidence has implicated IRAK family proteins (IRAK1, IRAK3, and IRAK4) in the progression of liver-related disorders, including alcoholic liver disease, non-alcoholic steatohepatitis, hepatitis virus infection, acute liver failure, liver ischemia-reperfusion injury, and hepatocellular carcinoma. In this article, we provide a comprehensive review of the role of IRAK family proteins and their associated inflammatory signaling pathways in the pathogenesis of liver diseases. The purpose of this study is to explore whether IRAK family proteins can serve as the main target for the treatment of liver related diseases.

Simultaneous determination of 11 oral targeted antineoplastic drugs and 2 active metabolites by LC-MS/MS in human plasma and its application to therapeutic drug monitoring in cancer patients.

Interindividual exposure differences have been identified in oral targeted antineoplastic drugs (OADs) owing to the pharmacogenetic background of the patients and their susceptibility to multiple factors, resulting in insufficient efficacy or adverse effects. Therapeutic drug monitoring (TDM) can prevent sub-optimal concentrations of OADs and improve their clinical treatment. This study aimed to develop and validate an LC-MS/MS method for the simultaneous quantification of 11 OADs (gefitinib, imatinib, lenvatinib, regorafenib, everolimus, osimertinib, sunitinib, tamoxifen, lapatinib, fruquintinib and sorafenib) and 2 active metabolites (N-desethyl sunitinib and Z-endoxifen) in human plasma. Protein precipitation was used to extract OADs from the plasma samples. Chromatographic separation was performed using an Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) column with a gradient elution of the mobile phase composed of 2 mM ammonium acetate with 0.1 % formic acid in water (solvent A) and methanol (solvent B) at a flow rate of 0.8 mL/min. Mass analysis was performed using positive ion mode electrospray ionization in multiple-reaction monitoring mode. The developed method was validated following FDA bioanalytical guidelines. The calibration curves were linear over the range of 2-400 ng/mL for gefitinib, imatinib, lenvatinib, regorafenib, and everolimus; 1-200 ng/mL for osimertinib, sunitinib, N-desethyl sunitinib, tamoxifen, and Z-endoxifen; and 5-1000 ng/mL for lapatinib, fruquintinib, and sorafenib, with all coefficients of correlation above 0.99. The intra- and inter-day imprecision was below 12.81 %. This method was successfully applied to the routine TDM of gefitinib, lenvatinib, regorafenib, osimertinib, fruquintinib, and sorafenib to optimize the dosage regimens.

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.

Pharmacokinetic study of multiple active constituents after oral gavage of Guizhi decoction in rats using a LC-MS/MS method.

Guizhi decoction (GZD) is a classic traditional Chinese medicine formula, clinically used for the treatment of influenza, common cold, and other pyretic conditions. A sensitive, specific, and validated liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed to investigate the pharmacokinetic properties of cinnamic acid, hippuric acid, paeoniflorin, and glycyrrhetic acid in rat. After single dose oral administration of 7.9 g extract/kg body weight GZD in rats, plasma concentrations of cinnamic acid, hippuric acid, paeoniflorin, and glycyrrhetic acid were measured by LC-MS/MS. Pharmacokinetic parameters were calculated from the plasma concentration-time data. The values of AUC0-t, half-life (t 1/2), and C max were 7.2 ± 2.3 µg h/mL, 1.2 ± 0.3 h, and 9.2 ± 5.2 µg/mL for cinnamic acid, 53 ± 31 µg h/mL, 2.8 ± 2.0 h, and 17 ± 3 µg/mL for hippuric acid, 1.1 ± 0.5 µg h/mL, 1.9 ± 1.1 h, and 0.6 ± 0.3 µg/mL for paeoniflorin, and 11 ± 6 µg h/mL, 6.6 ± 2.5 h, and 0.9 ± 0.6 µg/mL for glycyrrhetic acid, respectively. The results would offer useful information for effective components of GZD in vivo.

Screening of major hepatotoxic components of Tripterygium wilfordii based on hepatotoxic injury patterns.

BACKGROUND: Tripterygium wilfordii Hook. F. (TwHF), a traditional Chinese medicine, is widely used in the treatment of rheumatoid arthritis. Due to multiorgan toxicity, particularly hepatotoxicity, the application of TwHF is restricted. To clarify the hepatotoxic substances, zebrafish, hepatocytes and macrophages were used for screening based on hepatotoxic injury patterns. This study provides a basis for further elucidation of the hepatotoxic mechanism of TwHF. METHODS: First, 12 compounds were selected according to the chemical categories of TwHF. The fluorescence area and fluorescence intensity of zebrafish livers were observed and calculated. The viability of two hepatocyte lines was detected by CCK8 assay. TNF-α and IL-1ß mRNA expression in bone marrow-derived macrophages was used to evaluate macrophage activation, a factor of potential indirect hepatotoxicity. Finally, the hepatotoxic characteristics of 4 representative components were verified in mice in vivo. RESULTS: Parthenolide, triptolide, triptonide, triptobenzene H, celastrol, demethylzeylasteral, wilforlide A, triptotriterpenic acid A and regelidine significantly reduced the fluorescence area and fluorescence intensity of zebrafish livers. The viability of L-02 or AML-12 cells was significantly inhibited by parthenolide, triptolide, triptonide, celastrol, demethylzeylasteral, and triptotriterpenic acid A. Parthenolide, triptolide, triptonide, celastrol, demethylzeylasteral and triptobenzene H significantly increased TNF-α and IL-1ß mRNA levels in macrophages, while triptophenolide, hypodiolide and wilforine significantly reduced TNF-α and IL-1ß mRNA levels. Triptotriterpenic acid A, celastrol and triptobenzene H at a dose of 10 mg/kg significantly increased the levels of mouse serum alanine aminotransferase and aspartate aminotransferase and aggravated liver inflammation. CONCLUSIONS: Parthenolide, triptolide, triptonide, celastrol, demethylzeylasteral, triptotriterpenic acid A and triptobenzene H might be the main hepatotoxic components of TwFH. Among them, only triptotriterpenic acid A presents direct hepatotoxicity. Triptobenzene H exerts indirect liver damage by activating macrophages. Parthenolide, triptolide, triptonide, celastrol, and demethylzeylasteral can directly and indirectly cause liver injury.

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.

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.

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.

Increased systemic exposure to rhizoma coptidis alkaloids in lipopolysaccharide-pretreated rats attributable to enhanced intestinal absorption.

Rhizoma coptidis is a rhizome commonly used in traditional Chinese medicine. After oral administration of rhizoma coptidis extract, the plasma concentrations of its effective alkaloid constituents are so low that their systemic therapeutic actions cannot be explained. This study aimed to investigate the influence of lipopolysaccharide (LPS) on the pharmacokinetics of the rhizoma coptidis alkaloids. Pharmacokinetic experiments were performed with rats; both in vitro absorption and efflux experiments were carried out with everted rat gut sacs, whereas in vitro metabolism experiments were conducted with rat liver microsomes and intestinal S9 fractions. Mucosal changes were evaluated with light microscopy and transmission electron microscopy. The results showed that, in rat plasma, LPS pretreatment increased systemic alkaloid exposure. LPS pretreatment increased the in vitro absorption of the alkaloids and decreased their efflux. The efflux of vinblastine and rhodamine 123, P-glycoprotein substrates, also was decreased. The absorption of fluorescein isothiocyanate-labeled dextran (average molecular mass, 4 kDa), a gut paracellular permeability probe, was not influenced. Obvious damage was observed in the mucosa, but the tight junctions between epithelial cells remained intact. Intestinal, rather than hepatic, alkaloid metabolism was decreased. These findings indicated that LPS pretreatment increased systemic exposure to the alkaloids through enhancement of their absorption, which was related to decreased intestinal efflux and metabolism. The results add to the understanding of why rhizoma coptidis is active despite the low plasma concentrations of the rhizoma coptidis alkaloids measured in normal subjects and experimental animals.

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.