Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation.

OBJECTIVE: This study explores the mechanism of action of Danhongqing formula (DHQ), a compound-based Chinese medicine formula, in the treatment of cholestatic liver fibrosis. METHODS: In vivo experiments were conducted using 8-week-old multidrug resistance protein 2 knockout (Mdr2-/-) mice as an animal model of cholestatic liver fibrosis. DHQ was administered orally for 8 weeks, and its impact on cholestatic liver fibrosis was evaluated by assessing liver function, liver histopathology, and the expression of liver fibrosis-related proteins. Real-time polymerase chain reaction, Western blot, immunohistochemistry and other methods were used to observe the effects of DHQ on long non-coding RNA H19 (H19) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in the liver tissue of Mdr2-/- mice. In addition, cholangiocytes and hepatic stellate cells (HSCs) were cultured in vitro to measure the effects of bile acids on cholangiocyte injury and H19 expression. Cholangiocytes overexpressing H19 were constructed, and a conditioned medium containing H19 was collected to measure its effects on STAT3 protein expression and cell activation. The intervention effect of DHQ on these processes was also investigated. HSCs overexpressing H19 were constructed to measure the impact of H19 on cell activation and assess the intervention effect of DHQ. RESULTS: DHQ alleviated liver injury, ductular reaction, and fibrosis in Mdr2-/- mice, and inhibited H19 expression, STAT3 expression and STAT3 phosphorylation. This formula also reduced hydrophobic bile acid-induced cholangiocyte injury and the upregulation of H19, inhibited the activation of HSCs induced by cholangiocyte-derived conditioned medium, and decreased the expression of activation markers in HSCs. The overexpression of H19 in a human HSC line confirmed that H19 promoted STAT3 phosphorylation and HSC activation, and DHQ was able to successfully inhibit these effects. CONCLUSION: DHQ effectively alleviated spontaneous cholestatic liver fibrosis in Mdr2-/- mice by inhibiting H19 upregulation in cholangiocytes and preventing the inhibition of STAT3 phosphorylation in HSC, thereby suppressing cell activation. Please cite this article as: Li M, Zhou Y, Zhu H, Xu LM, Ping J. Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation. J Integr Med. 2024; 22(2): 188-198.

Yin-chen Wu-ling powder alleviate cholestatic liver disease: Network pharmacological analysis and experimental validation.

BACKGROUND: Yin-chen Wu-ling Powder (YWP) has potential therapeutic effects on cholestatic liver disease (CLD), however, its active compounds and conceivable mechanism are as yet indistinct. METHODS: The network pharmacology and gene function annotation examined the multiple active ingredients, potential targets, and possible mechanisms of YWP in CLD treatment. Then the molecular docking reassured the reliability of the core compounds including the key genes and farnesoid X receptor (FXR). Finally, The Mdr2-/- mice were used to test the effect and mechanism of YWP against CLD. RESULTS: The network analysis identified nine main active ingredients, including quercetin, capillarisin, eupalitin, isorhamnetin, skrofulein, genkwanin, cerevisterol, gederagenin, and sitosterol. The PPI network predicted the ten hub genes involved were AKT1, MAPK1, MAPK14, IL6, RXRA, ESR1, IL10, NCOA1, CAV1, and EGFR. The KEGG and GO analysis showed that YWP might contribute to CLD treatment through the PI3K/Akt and MAKP signalings to manage pathological reactions, for instance, inflammatory responses. The molecular docking displayed a functional similarity among the core compounds with ursodeoxycholic acid (UDCA) and Obeticholic acid (OCA) on the effects on AKT1, MAPK1, MAPK14, RXRA, and ESR, and the affinity to FXR. In addition, the YWP could significantly attenuate hepatic injury and improve inflammatory response in Mdr2-/- mice. The mechanism exploration showed that YWP mainly decreased inflammatory response by inhibiting AKT/P38MAPK signaling. CONCLUSION: This study firstly revealed the multiple active ingredients, potential targets, and possible mechanism of YWP to treat CLD based on network pharmacology Analysis and molecular docking. YWP could alleviate cholestasis in Mdr2-/- mice by impairing inflammation via inhibiting AKT/P38MAPK Signaling.

Compound Heterozygous Myosin 5B (Myo5b) Mutation with Early Onset Progressive Cholestasis and No Intestinal Failure.

Background: Exome sequencing studies have recently identified novel genes implicated in normal or low GGT pediatric cholestasis including myosin 5B (MYO5B). Case report: We identified novel compound heterozygote mutations in exon 14 and exon 19 of the MYO5B gene in an 18-month-old Indian child with history of fluctuating jaundice and severe pruritus. His liver biopsy showed portal and perivenular fibrosis with focal bridging septa and mild activity. He is currently on UDCA, cholestyramine and vitamin supplements. There is no history of diarrhea. His asymptomatic mother showed heterozygous mutation in exon 19 of the MYO5B gene and his asymptomatic father showed heterozygous mutation in exon 14 of the MYO5B gene. Conclusion: Our report confirms that patients with compound heterozygote mutations in MYO5B develop progressive cholestasis with no intestinal disease.

miRNA and miRNA target genes in intervention effect of Zhuyu pill on cholestatic rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhuyu pill (ZYP), an effective prescription of traditional Chinese medicine, is composed of Coptis chinensis Franch. and Tetradium ruticarpum (A. Jussieu) T. G. Hartley and has shown potential anticholestatic effects. However, its mechanism of action in treating cholestasis remains unclear. Since post-transcriptional control of mRNA by micro-RNAs (miRNAs) represents an important mechanism of gene regulation, it is promising to explore this in relation to ZYP and cholestasis. AIM OF THE STUDY: To confirm the anticholestatic effect of ZYP and to explore its potential biological mechanism. MATERIALS AND METHODS: In this study, a cholestasis rat model was induced by α-naphthyl-isothiocyanate (ANIT, 50 mg/kg) and treated with ZYP (low dose: 0.6 g/kg, high dose: 1.2 g/kg). Serum biochemistry indices and liver histopathology were used to evaluate the model and efficacy, and miRNA sequencing was used to measure differences in miRNA expression in the liver between the control, model, low-dose ZYP, and high-dose ZYP groups. To verify the accuracy of sequencing results and explore the potential anti-cholestasis mechanism of ZYP, RT-PCR was used to identify differentially expressed miRNAs and their target genes. RESULTS: Both high- and low-dose ZYP exhibited significant anticholestatic effects, with the high-dose showing better effects than low-dose ZYP. Additionally, four differentially expressed miRNAs, rno-miR-147, rno-miR-20b-5p, rno-miR-29b-3p, and rno-miR-3586-3p, were found to be upregulated in cholestasis and downregulated after ZYP intervention. Eight target genes of the above miRNAs, including ABCG8, CLOCK, PLEC, SLC4A2, NEB, ADAMTS12, TTN and FAM174B were inhibited in cholestatic rats, exhibiting up-regulated expression tendencies after ZYP intervention, and the expression tendencies were significant negatively correlated with serum biochemical indices. CONCLUSIONS: ZYP can significantly reduce liver biochemical indices and improve liver tissue damage in cholestasis rats through the regulation of miRNA expression in the liver, producing a positive regulatory effect on bile excretion-related genes.

Network pharmacology assessment of Qingkailing injection treatment of cholestatic hepatitis.

OBJECTIVE: To investigate the targets and mechanisms of action of Qingkailing injection (，QKL) in the treatment of cholestatic hepatitis. METHODS: A network pharmacology method was implemented using drug and disease databases to target QKL and cholestasis hepatitis, respectively. The functional protein association network STRING database was used to construct a protein-protein interaction network using R language and the Bioconductor toolkit. The org.Hs.eg.db and clusterProfiler packages were used for gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, which explored biological functions and pathways of potential targets. Targets were then visualized using Cytoscape 3.6.0 software. RESULTS: We screened 121 compounds in QKL and identified 112 targets for the treatment of cholestatic hepatitis. QKL played a role in the treatment of cholestatic hepatitis through 305 biology process terms, 15 cellular component and 29 molecular function terms. The mechanism of QKL action was mainly related to tumor necrosis factor, mitogen-activated protein kinase, and PI3K-Akt signaling pathways. CONCLUSION: The treatment of cholestatic hepatitis by QKL involved multiple targets, biological functions, and signaling pathways that are closely associated with the disease.

[Study on protective mechanism of Tibetan medicine Ershiwuwei Songshi Pills on cholestatic liver injury in rats based on FXR signaling pathway].

This paper aimed to investigate the protective effect and mechanism of the Tibetan medicine Ershiwuwei Songshi Pills on α-naphthalene isothiocyanate(ANIT)-induced cholestatic liver injury in rats based on the farnesol X receptor(FXR) signaling pathway. SD rats were randomly divided into blank group, model group, ursodeoxycholic acid(UDCA) group, Tibetan medicine Ershiwuwei Songshi Pills low, medium and high dose groups(0.09, 0.18, 0.36 g·kg~(-1)). A prophylactic dosing regimen was used in the experiment. From the 1 st to 4 th days, the UDCA group and the Tibetan medicine Ershiwuwei Songshi Pills suspension groups received prophylactic gavage administration; on the 5 th day, the blank control group was given an equal volume of olive oil blank reagent, and the remaining groups were given ANIT modeling reagent. Administration was continued on day 5 to 6 in each administration group. Forty-eight hours after modeling on the 7 th day, blood was collected from the femoral artery of rats. Serum alkaline phosphatase(ALP), alanine aminotransferase(ALT), aspartate aminotransferase(AST), direct bilirubin(DBIL), total bilirubin(TBIL), and total bile acid(TBA) levels were detected, and liver histopathological changes were observed. The relative expression changes of FXR, SHP, CYP7 A1, MRP2, MRP3, NTCP, BSEP mRNA in liver tissues were detected by Real-time fluorescence quantitative method, and the expression changes of FXR, SHP, UGT2 B4 protein in liver tissues were detected by Western blot. The results showed that the Tibetan medicine Ershiwuwei Songshi Pills significantly reduced the levels of ALT, ALP, DBIL, TBIL and TBA in the serum of the ANIT mo-del rats(P<0.01, P<0.05), significantly up-regulated the mRNA expressions of SHP and NTCP(P<0.01, P<0.05), significantly down-regulated the mRNA expression of CYP7 A1 and MRP3(P<0.01, P<0.05); and significantly up-regulated the protein expressions of FXR and SHP(P<0.01, P<0.05). The Tibetan medicine Ershiwuwei Songshi Pills have an obvious protective effect on ANIT-induced cholestatic liver injury in rats, and its mechanism may be related to the bile acid metabolism mediated by the FXR signaling pathway.

Hepatic stem cell Numb gene is a potential target of Huang Qi Decoction against cholestatic liver fibrosis.

Numb is a negative regulator of Notch signal pathway. Previous study has demonstrated that Notch signal pathway activation is required for hepatic progenitor cell (HPC) differentiating into cholangiocytes in cholestatic liver fibrosis (CLF), and Huang Qi Decoction (HQD) could prevent CLF through inhibition of the Notch signal pathway. However, the role of Numb in HQD against CLF is yet unclear. Thus, CLF rats transplanted into rat bone marrow-derived mesenchymal stem cells with knocked down Numb gene (BMSCNumb-KD) were treated with HQD. Simultaneously, Numb gene knockdown was also performed in WB-F344 cell line and then treated with refined HQD in vitro. In vivo study revealed that liver fibrosis was inhibited by HQD plus BMSCNumb-KD treatment, while Hyp content in liver tissue, the gene and protein expression of α-SMA, gene expression of Col I, TNF-α, and TGF-ß1 were increased compared to that in HQD group. Furthermore, Notch signal pathway was inhibited by HQD plus BMSCNumb-KD, while the protein expression of Numb was decreased and RBP-Jκ and Hes1 was increased compared to that in HQD group. In vitro, HQD reduced the differentiation of WB-F344 cells into cholangiocyte phenotype, while this effect was attenuated after Numb-knockdown. This study highlights that the absence of hepatic stem cell Numb gene decreases effect of HQD against CLF, which give rise the conclusion that Numb might be a potential target for HQD against CLF.

Effect of cholestasis and NeuroAid treatment on the expression of Bax, Bcl-2, Pgc-1α and Tfam genes involved in apoptosis and mitochondrial biogenesis in the striatum of male rats.

Cholestasis means impaired bile synthesis or secretion. In fact, it is a bile flow reduction following Bile Duct Ligation (BDL). Cholestasis has a main role in necrosis and apoptosis. Apoptosis is a form of programmed cell death that has intrinsic and extrinsic pathways. The intrinsic pathway is mediated by Bcl-2 (B cell lymphoma-2) proteins which integrate death and survival signals. Bcl-2 has anti-apoptotic and Bax has pro-apoptotic effects. Also, striatum is one of the brain regions that has high expressions of Bcl-2 proteins. Moreover, Tfam and Pgc-1α are involved in mitochondrial biogenesis. On the other hand, NeuroAid, is a drug that has neuroprotective and anti-apoptosis effects. In this study, using quantitative PCR, we measured the expression of all these genes in the striatum of male rats following BDL and NeuroAid administration. Results showed, BDL increased the expression of Bax and Tfam and decreased the expression of Bcl-2. NeuroAid restored the effect of BDL on the expression of Bax, while did not alter the effect of BDL on Bcl-2. In addition, it increased the expression of Tfam that was previously elevated by BDL and raised the expression of Tfam in normal rats. Both BDL and NeuroAid, had no effect on Pgc-1α. In conclusion, cholestasis increased the expression of Bax and decreased the expression of Bcl-2, and this effect may have related to enhanced susceptibility of mitochondrial pathways following oxidative stress. Tfam expression was increased following cholestasis and this effect may have related to cellular compensatory mechanisms against high accumulation of free radicals or mitochondrial biogenesis failure. Furthermore, NeuroAid may play a role against apoptosis and can be used to increase mitochondrial biogenesis.

Development of an in vitro cholestatic drug-induced liver injury evaluation system using HepG2-hNTCP-C4 cells in sandwich configuration.

Toxicological approaches in screening drugs that cause drug-induced liver injury (DILI) are urgently needed to reduce the risk of developing DILI and avoid immense costs resulting from late-stage drug withdrawal from clinical trials. Cholestatic DILI is characterized by bile acid (BA) accumulation in hepatocytes, typically caused by drug-induced inhibition of important bile transporters, such as bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4). Therefore, NTCP expression is essential for construction of an in vitro hepatocellular toxicity evaluation system. Here, we investigated whether sandwich-cultured HepG2-hNTCP-C4 (SCHepG2-hNTCP-C4) cells were applicable for evaluation of cholestatic DILI. In SCHepG2-hNTCP-C4 cells, NTCP and MRP2/4 expression levels were comparable to those in human primary hepatocytes; however, BSEP expression was low. In addition, the substrates tauro-nor-THCA-24 DBD and CDF confirmed the functionality of NTCP and MRP2, respectively. When 22 known hepatotoxins were exposed to BAs to evaluate cholestatic DILI, cytotoxicity in SCHepG2-hNTCP-C4 cells was more frequent than that in SCHepG2 cells. Thus, SCHepG2-hNTCP-C4 cells may be useful preclinical screening tools to predict the risk of cholestatic DILI induced by drug candidates. However, further studies are needed to determine why the cholestatic cytotoxicity of some compounds would be still insufficient in SCHepG2-hNTCP-C4 cells.

Severe and protracted cholestasis in 44 young men taking bodybuilding supplements: assessment of genetic, clinical and chemical risk factors.

BACKGROUND: Bodybuilding supplements can cause a profound cholestatic syndrome. AIM: To describe the drug-Induced liver injury network's experience with liver injury due to bodybuilding supplements. METHODS: Liver injury pattern, severity and outcomes, potential genetic associations, and exposure to anabolic steroids by product analysis were analysed in prospectively enrolled subjects with bodybuilding supplement-induced liver injury with causality scores of probable or higher. RESULTS: Forty-four males (mean age 33 years) developed liver injury with a median latency of 73 days. Forty-one per cent presented with hepatocellular pattern of liver injury as defined by the R > 5 ([Fold elevation of ALT] ÷ [Fold elevation of Alk Phos] (mean, range = 6.4, 0.5-31.4, n = 42) despite all presenting with clinical features of cholestatic liver injury (100% with jaundice and 84% with pruritus). Liver biopsy (59% of subjects) demonstrated a mild hepatitis and profound cholestasis in most without bile duct injury, loss or fibrosis. Seventy-one per cent were hospitalised, and none died or required liver transplantation. In some, chemical analysis revealed anabolic steroid controlled substances not listed on the label. No enrichment of genetic variants associated with cholestatic syndromes was found, although mutations in ABCB11 (present in up to 20%) were significantly different than in ethnically matched controls. CONCLUSIONS: Patients with bodybuilding supplements liver injury uniformly presented with cholestatic injury, which slowly resolved. The ingested products often contained anabolic steroids not identified on the label, and no enrichment in genetic variants was found, indicating a need for additional studies.

Beyond an Obvious Cause of Cholestasis in a Toddler: Compound Heterozygosity for ABCB11 Mutations.

A 27-month-old girl presented with a short history of jaundice initially attributed to drug-induced liver injury. During the preceding 20 days, she had received a 10-day course of cefprozil and 2 doses of a homeopathic preparation of cantharidin for cystitis. Severe conjugated hyperbilirubinemia was present with normal γ-glutamyl transpeptidase activity. Liver biopsy revealed marked canalicular and hepatocellular cholestasis, with moderate hepatocellular disarray, as well as evidence of chronicity, including moderate portal-tract and perisinusoidal fibrosis. Immunohistochemical studies revealed that bile salt export pump expression was preserved, whereas canalicular γ-glutamyl transpeptidase expression was largely absent. An inherited cholestatic disorder was suspected. The entire coding region of ABCB11, encoding bile salt export pump, was analyzed. The patient was found to be a compound heterozygote for the missense mutation c.3148C>T (p.Arg1050Cys) associated with benign recurrent intrahepatic cholestasis type 2 in the homozygous state and for the nonsense mutation c.3904G>T (p.Glu1302Ter) associated with progressive familial intrahepatic cholestasis type 2. Despite initial improvement with ursodeoxycholic acid, over the course of 5 years the patient developed cirrhosis that required liver transplant. Our report emphasizes the need for molecular studies even in patients with putatively "explained" cholestasis to reveal the entire spectrum of inherited cholestatic disorders.

Therapeutic Effect of Glycyrrhizin Arginine Salt on Rat Cholestatic Cirrhosis and its Mechanism.

To investigate the therapeutic effect of glycyrrhizin arginine salt on rat cholestatic cirrhosis, we subjected male Sprague Dawley rats to common bile duct ligation for 14 days and treated them with distilled water (model group), arginine, or a low or high dose of glycyrrhizin arginine salt by gavage. A sham-operated group was used as a control group. Treatment with glycyrrhizin arginine salt substantially improved animal growth rates, reduced the ratio of liver weight to body weight and decreased total bilirubin, aspartate aminotransferase, 8-isoprostane and malondialdehyde compared with the values measured in the model group. The progress of liver fibrosis, as detected by hematoxylin and eosin and Masson's trichrome staining, was slower in the glycyrrhizin arginine salt groups than in the model group or the arginine group. Reductions of bile salt pool size, hepatic hydroxyproline content and fibrosis score were also seen in the glycyrrhizin arginine salt groups compared with the model group. Furthermore, glycyrrhizin arginine salt significantly reduced the expression of transforming growth factor [Formula: see text]1 (TGF-[Formula: see text]1), [Formula: see text]-smooth muscle actin, tumor necrosis factor-[Formula: see text] and matrix metalloproteinases 2 and 9. Glycyrrhizin arginine salt also inhibited the expression of [Formula: see text]-SMA and matrix metalloproteinases 2 and 9 in response to TGF-[Formula: see text]1 in LX-2 cells and primary rat hepatic stellate cells and mitigated the cytotoxicity induced by rat bile in HepG2 cells and primary rat hepatocytes.

Transient receptor potential canonical 5 channels plays an essential role in hepatic dyslipidemia associated with cholestasis.

Transient receptor potential canonical 5 (TRPC5), a calcium-permeable, non-selective cation channel is expressed in the periphery, but there is limited knowledge of its regulatory roles in vivo. Endogenous modulators of TRPC5 include a range of phospholipids that have an established role in liver disease, including lysophosphatidylcholine (LPC). Cholestasis is characterized by impairment of excretion of bile acids, leading to elevation of hepatic bile acids. We investigated the contribution of TRPC5 in a murine model of cholestasis. Wild-type (WT) and TRPC5 knock-out (KO) mice were fed a diet supplemented with 0.5% cholic acid (CA) for 21 days. CA-diet supplementation resulted in enlargement of the liver in WT mice, which was ameliorated in TRPC5 KO mice. Hepatic bile acid and lipid content was elevated in WT mice, with a reduction observed in TRPC5 KO mice. Consistently, liver enzymes were significantly increased in cholestatic WT mice and significantly blunted in TRPC5 KO mice. Localized dyslipidaemia, secondary to cholestasis, was investigated utilizing a selected lipid analysis. This revealed significant perturbations in the lipid profile following CA-diet feeding, with increased cholesterol, triglycerides and phospholipids, in WT, but not TRPC5 KO mice. Our results suggest that activation of TRPC5 contributes to the development of cholestasis and associated dyslipidemia. Modulation of TRPC5 activity may present as a novel therapeutic target for liver disease.

PXR- and CAR-mediated herbal effect on human diseases.

The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are two members of the nuclear receptor superfamily that regulate a broad range of genes involved in drug metabolism and transport. A variety of naturally occurring compounds present in herbal medicines were identified as ligands of PXR and CAR. Recently, accumulative evidences have revealed the PXR- and CAR-mediated herbal effect against multiple human diseases, including inflammatory bowel disease (IBD), cholestatic liver disease, and jaundice. The current review summarized the recent progress in identifying the expanding libraries of herbal medicine as ligands for PXR and CAR. Moreover, the potential for herbal medicines as promising therapeutic agents which were mainly regulated through PXR/CAR signaling pathways was also discussed. The discovery of herbal medicines as modulators of PXR and CAR, and their PXR- and CAR-mediated effect on human diseases will provide a basis for rational drug design, and eventually be explored as a novel therapeutic approach against human diseases. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

[Screening of effective ingredients of Yinzhihuang injection against cholestasis and their mechanism].

Chinese herbal medicinal formulation Yinzhihuang injection is widely used in clinic for jaundice and chronic liver diseases in eastern Asian countries. However, the pharmacologically active components and the underlying mechanism are unclear. In this study, 30 male ICR mice were randomly assigned into 6 groups： normal control, model control, chlorogenic acid group, geniposide group, baicalin group and wogonoside group. The liver function, liver pathological changes and bile acid metabolism-related gene expression in mice were assayed. The serum levels of ALT, AST, ALP, TBA in chlorogenic acid group (15.89±2.53), (18.32±2.56), (26.38±9.87) U•L⁻¹, (40.63±7.67) µmol•L⁻¹, respectively and geniposide group (20.54±2.36), (24.28±5.19), (35.09±5.03) U•L⁻¹, (42.86±7.11) µmol•L⁻¹, respectively were lower than those in the model control group (59.52±10.94), (128.37±17.97),(169.52±9.62) U•L⁻¹, (132.50±33.00) µmol•L⁻¹, respectively. Hematoxylin-eosin staining showed necrosis, infiltration of inflammation cell in chlorogenic acid group and geniposide group were milder than those in the model control group. Q-PCR analysis revealed the expression of bile acid metabolism-related genes was normalized after treatment with chlorogenic acid or geniposide. Chlorogenic acid and geniposide improved the liver injury and cholestasis effectively, and reversed the mRNA expression of bile acid metabolism-related genes induced by ANIT. So, chlorogenic acid and geniposide were protective for cholestasis, suggesting their pharmacodynamic effect in Yinzhihuang injection.

Promising toxicological biomarkers for the diagnosis of liver injury types: Bile acid metabolic profiles and oxidative stress marker as screening tools in drug development.

Promising biomarkers were identified in adult male Crl:CD (SD) rats for the screening of new chemical entities for their potential to cause liver injury. We examined the serum biochemistry, liver histopathology, and bile acid profiles by LC-MS/MS, and the mRNA expression of transporters and CYPs by an RT-PCR after the following treatments to male Crl:CD (SD) rats: (a) bile duct ligation (BDL); (b) a single oral dose of 150 mg/kg α-naphthylisothiocyanate (ANIT); and (c) repeated oral doses of a novel pyrrolidinecarboxylic acid derivative (abbreviated as PCA) at 30, 300, and 1000 mg/kg. The serum total bile acid levels and bilirubin concentrations were found to be elevated in all of the groups. However, the bile acid component profiles of the PCA group differed significantly from BDL and ANIT models: deoxycholic acid, lithocholic acid, and sulfated bile acids were upregulated in a dose-dependent manner only in the PCA group. In addition, the PCA group demonstrated high levels of hepatic heme oxygenase-1 expression, whereas the profiles of the mRNA levels of the hepatic transporters and CYPs of all groups were found to be similar. The histopathological findings, for both the BDL and ANIT groups, were of bile duct hyperplasia, hepatocyte degeneration and necrosis. In contrast, only bile duct hyperplasia and hepatocyte degeneration were observed in the PCA group, even at a lethal dose. These results indicated that PCA induced a cholestatic condition and the increase of oxidative stress markers implies that this will also lead hepatocellular injury. In conclusion, the serum bile acid components and sulfated bile acid levels, and the expression of oxidative stress markers could provide information that aids in the diagnosis of liver injury type and helps to elucidate the mechanisms of hepatotoxicity. These findings can be extrapolated into our clinical investigation. The analysis of these crucial biomarkers is likely to be a useful screening tool in the lead optimization phase of drug discovery.

Xiayuxue decoction reduces renal injury by promoting macrophage apoptosis in hepatic cirrhotic rats.

Renal pathological changes in cirrhotic rat have not been extensively reported. The aim of this study was to investigate whether Xiayuxue decoction (XYXD) could attenuate renal injury induced by bile duct ligation (BDL), with special focus on the mechanisms promoting renal macrophage apoptosis. The rats were treated with BDL for 5 weeks and administered 0.36 g/kg XYXD intragastrically from day 1 of initiating BDL. Renal tissue was monitored by hematoxylin-eosin and Sirius red staining. Macrophage infiltration and proinflammatory cytokines such as tumor necrosis factor and chemokine ligand 2 were detected by quantitative polymerase chain reaction. Macrophage apoptosis was detected by double immunofluorescence staining. Blood urea nitrogen, creatinine, and glomerulus diameter increased significantly after a 5-week BDL treatment in XYXD (BDL-XYXD) rats. CD68 and pro-inflammatory cytokine mRNA increased in the kidneys of control (BDL-water) rats. Fluorescence microscopy analysis showed that XYXD promoted apoptosis in renal CD68+ macrophages. Collogen1 (Col 1), pro-fibrogenic cytokines, and α-smooth muscle actin in kidneys of BDL-water rats increased significantly compared to the sham group. XYXD inhibited Col 1 and pro-fibrotic factors in BDL-XYXD rats. Our results demonstrated that XYXD significantly reduced renal injury by, at least in part, promoting macrophage apoptosis in rats with damaged renal histopathology due to BDL-induced cirrhosis.

Bile acids: chemistry, physiology, and pathophysiology.

The family of bile acids includes a group of molecular species of acidic steroids with very peculiar physical-chemical and biological characteristics. They are synthesized by the liver from cholesterol through several complementary pathways that are controlled by mechanisms involving fine-tuning by the levels of certain bile acid species. Although their best-known role is their participation in the digestion and absorption of fat, they also play an important role in several other physiological processes. Thus, genetic abnormalities accounting for alterations in their synthesis, biotransformation and/or transport may result in severe alterations, even leading to lethal situations for which the sole therapeutic option may be liver transplantation. Moreover, the increased levels of bile acids reached during cholestatic liver diseases are known to induce oxidative stress and apoptosis, resulting in damage to the liver parenchyma and, eventually, extrahepatic tissues. When this occurs during pregnancy, the outcome of gestation may be challenged. In contrast, the physical-chemical and biological properties of these compounds have been used as the bases for the development of drugs and as pharmaceutical tools for the delivery of active agents.

Sodium retention and ascites formation in a cholestatic mice model: role of aldosterone and mineralocorticoid receptor?

UNLABELLED: Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal-intact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium- and potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium- and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum- and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium- and potassium-dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. CONCLUSION: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium- and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium- and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids.