褪黑素通过JNK/c-Jun信号通路抑制胆汁酸诱导Cxcl2表达的机制研究

Melatonin inhibits bile acids-initiated Cxcl2 expression through JNK/c-Jun signalling pathway

目的 研究褪黑素抑制胆汁酸诱导趋化因子Cxcl2表达的分子调控机制。方法 喂养小鼠含有1%胆酸(cholic acid, CA)的饲料2周,构建胆汁淤积小鼠模型,给予褪黑素腹腔注射(100 mg/kg, 1次/d)。收集肝组织后HE染色,根据Scheuer系统进行病理组织学评分。采用Real-time qPCR及Western blot方法检测褪黑素对小鼠肝脏内Cxcl2、Cxcr2、Mpo、c-Jun表达的影响。使用免疫组化方法检测小鼠肝脏内c-Jun的表达。分离并培养小鼠原代肝细胞,使用结合型胆汁酸TCA联合褪黑素处理小鼠原代肝细胞,在体外水平进一步探索褪黑素抑制胆汁酸诱导的Cxcl2表达的机制。使用JNK激动剂Anisomycin和褪黑素共同处理小鼠原代肝细胞,检测趋化因子Cxcl2表达。结果 褪黑素给药明显减轻胆汁淤积诱导的炎症性肝损伤(P<0.05)。褪黑素明显抑制胆汁淤积小鼠肝脏内趋化因子Cxcl2及其受体Cxcr2表达,并抑制肝脏内中性粒细胞标志物Mpo和转录因子c-Jun的表达(P<0.05)。褪黑素显著减少TCA诱导的小鼠原代肝细胞内趋化因子Cxcl2的表达(P<0.05)。JNK激动剂Anisomycin逆转褪黑素对Cxcl2的抑制作用(P<0.05)。结论 褪黑素通过抑制JNK/c-Jun信号通路来降低胆汁酸诱导的趋化因子Cxcl2表达。

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