To explore the mechanism of melatonin (Mel) alleviating cholestatic liver injury in a mouse cholestasis model induced by cholic acid (CA) feeding.

A total of 15 8-week-old male C57BL/6J mice were randomly divided into control group, 1%CA group, and 1%CA+Mel group, with 5 animals in each group. The control group was fed with normal chow diet, and the other 2 groups were fed with a diet containing 1%CA for 14 d to construct a model of cholestasis, and intraperitoneal injection with 100 mg/kg Mel was given to the mice from the 1%CA+Mel group. Immunohistochemical assay of α-SMA was applied for the liver tissues in the 1%CA group and the 1%CA+Mel group. The mRNA expression levels of fibrosis-related indicators in mouse liver tissue were examined by RT-qPCR. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and automated biochemical analyzer were used to detect the contents of bile acids in the liver tissues and the serum of mouse, respectively. Then real-time qPCR and Western blotting were applied to detect the expression of bile acid synthesis and liver detoxification enzymes related indicators at mRNA and protein levels, respectively, to further investigate the mechanism of bile acid metabolism.

Compared with the 1%CA group, the mRNA levels of liver fibrosis indicators (such as Tgfβ1, ColⅠa1, ColⅡa1 and α-SMA) were significantly reduced (P<0.05), and activation of stellate cells was obviously weakened displayed by immunohistochemical staining in the 1%CA+Mel group. The 1%CA+Mel group had notably decreased contents of bile acids in the serum and liver tissues, especially taurocholic acid and reduced mRNA levels of cholesterol 7α-hydroxylase (Cyp7a1) and Cyp8b1, while enhanced mRNA levels of hepatic detoxification enzymes Cyp2b10 and udp-glucuronosyltransferase (Ugt1a1) as well as protein levels of Cyp2b10 and sulfotransferase family 2A member 1 (Sult2a1/2) when compared with the 1%CA group (P<0.05).

Mel exerts its therapeutic effect on cholestasis by decreasing bile acid synthesis and increasing hepatic detoxification enzymes.

To screen and verify the Hub genes involved in conjugated bile acid-induced hepatocyte metabolism through transcriptome sequencing analysis.

Primary mouse hepatocytes were treated with taurocholic acid（TCA）, and the gene expression of TCA-induced hepatocytes was detected by RNA-Seq analysis. Differential expression genes（DEGs）were identified using a fold change ≥1.5 and P value<0.05 as the screening criteria. The protein-protein interaction（PPI）network of DEGs was constructed using the STRING database, and Hub genes were screened using Cytoscape software based on the PPI network. Real-time qPCR was used to validate the identified Hub gene in vitro and in vivo. Western blotting was employed to detect the expression of proteins related to PSAT1/AKT/GSK3β signaling pathway. The glycogen level in the hepatocytes and liver tissues were detected using glycogen detection kit.

RNA-Seq analysis identified 293 DEGs in primary mouse hepatocytes upon TCA treatment, including 122 up-regulated genes and 171 down-regulated genes. Gene Ontology（GO）analysis, Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis, and Gene Set Enrichment Analysis（GSEA）revealed that these DEGs were mainly enriched in metabolism, inflammatory and tumorigenic pathways. Cytohubba plugin identified 16 Hub genes, including Ccnd1, Ppara, Irs1, Ccl2, Ddit3, Serpine1, Nr0b2, Atf3, Phgdh, Trib3, Lepr, G6pc, Psat1, Cxcl2, Asns, and Fgf21. RT-qPCR results demonstrated that various conjugated bile acids, glycocholic acid（GCA）, glycochenodeoxycholic acid（GCDCA）, and TCA can induce a significant increase in Psat1 mRNA level in primary mouse hepatocytes（P<0.05）. In vivo experiments also showed that the mRNA level of Psat1 was increased significantly in the liver tissues of mouse cholestatic model induced by bile duct ligation（BDL）when compared to the control group（P<0.05）. Western blotting indicated that TCA treatment induced the expression of Psat1 and activation of AKT/GSK3β signaling pathway in primary mouse hepatocytes. TCA treatment also resulted in an increase in glycogen synthesis in primary mouse hepatocytes, which was consistent with an increase in glycogen synthesis in the liver tissue and a decrease in blood glucose level in the mouse model established with BDL（P<0.05）.

Psat1 may be a gene involved in alteration of glucose metabolism induced by bile acid, and its high expression may be associated with these changes in glucose metabolism in hepatocytes.

To explore the effect and molecular mechanism of artesunate（ART）on cholestatic liver fibrosis induced by 3, 5-diethoxycarbonyl-1, 4-dihydro-2, 4, 6-collidine（DDC）.

8-week-old male C57BL/6J mice（n=24）were randomly divided into 3 groups: the control group（n=6）, the 0.1% DDC group（n=9）and the 0.1% DDC+ART group（n=9）. Mice have been fed with diet containing 0.1% DDC for 2 weeks to establish cholestatic mouse model, and ART was given intramuscular injection（30.0 mg/kg）once a day in the 0.1% DDC+ART group. The indicators of liver injury in serum of mice were detected. HE staining and Sirius red staining were performed, and histopathological scores were obtained according to Scheuer scoring system. RT-qPCR was used to detect the levels of α-SMA, Col1a1, Col1a2 and Tgfβ1 in mouse liver. Hydroxyproline（HYP）content was detected with HYP detection kit. LX2 cells were treated with TGF-β combined with ART, and the proteins related to PI3K/Akt/mTOR pathway were detected by Western blot assay. The expression of α-SMA, hepatic stellate cell（HSC）activation marker, was detected after ART and pathway inhibitors were used to treated with LX2 cells.

ART significantly ameliorated DDC-induced cholestatic liver injury and liver fibrosis（P<0.05）. ART markedly decreased the levels of ALT, AST, ALP and TBA in serum of mice（P<0.05）. ART inhibited the mRNA expression of α-SMA, Col1a1, Col1a2 and Tgfβ1, and reduced the content of HYP in liver tissues of mice with cholestasis（P<0.05）. In addition, ART obviously inhibited TGF-β-induced activation of PI3K/Akt/mTOR pathway in LX2 cells（P<0.05）. PI3K/Akt/mTOR signaling pathway inhibitors enhanced the inhibitory effect of HSC activation induced by TGF-β（P<0.05）.

ART observably improves DDC-induced cholestatic liver fibrosis, and it can inhibit HSC activation by regulating PI3K/Akt/mTOR signaling pathway, thus exerting anti-fibrosis effect.

To investigate the underlying molecular mechanism by which melatonin suppresses bile acids (BAs)-initiated expression of chemokine C-X-C motif chemokine ligand 2 (Cxcl2).

Mice were fed a 1% cholic acid (CA)-supplemented diet for 2 weeks to establish a model of cholestatic disease, and were given an intraperitoneal injection of melatonin (100 mg/kg) once a day for 2 weeks. Then, the liver tissues were collected for HE staining, and the liver histology was scored using the Scheuer scoring system. Effects of melatonin on the expression of Cxcl2, Cxcr2, myeloperoxidase (Mpo) and c-Jun in the liver tissues at mRNA and protein levels were detected by real-time qPCR and Western blotting. The expression of c-Jun in the liver tissues was measured by immunohistochemical assay. Primary mouse hepatocytes were isolated and cultured, and then treated with taurine-conjugated bile acid, taurocholic acid (TCA) and melatonin in order to explore the mechanism of melatonin reducing BAs-initiated Cxcl2 expression. Furthermore, these primarily cultured cells were treated with anisomycin (JNK agonist) and melatonin to further detect the expression of Cxcl2.

Melatonin treatment significantly ameliorated cholestasis-induced inflammatory liver injury (P<0.05), and reduced the elevated expression of Cxcl2, Cxcr2, Mpo (a biomarker of neutrophils) and the transcription factor c-Jun in the livers of cholestatic disease mice (P<0.05). In addition, melatonin suppressed TCA-stimulated Cxcl2 in primary mouse hepatocytes (P<0.05). Anisomycin reversed melatonin-inhibited Cxcl2 (P<0.05).

Melatonin treatment can decrease BAs-initiated expression of Cxcl2 via inhibition of JNK/c-Jun signaling pathway in mouse hepatocytes.