Supplementary Material for: Curcumin Affects Leptin-Induced Expression of Methionine Adenosyltransferase 2A in Hepatic Stellate Cells by Inhibition of JNK Signaling

Introduction: Obese patients are often accompanied by hyperleptinemia and prone to develop liver fibrosis. Accumulating data including those obtained from human studies suggested the promotion role of leptin in liver fibrosis. The remodeling of the DNA methylation is an epigenetic mechanism for regulating gene expression and is essential for hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis. Leptin increases the expression of methionine adenosyltransferase 2A (MAT2A) which is associated with DNA methylation and HSC activation. Curcumin, an active polyphenol of the golden spice turmeric, inhibits leptin-induced HSC activation and liver fibrogenesis. Thus, the present research aimed to investigate the influence of curcumin on the roles of leptin in MAT2A expression in HSCs. Methods: The in vivo experiments were conducted by using leptin-deficient obese mice. The gene expressions were examined by Western blot, real-time PCR, promoter activity assay, and immunostaining analysis. Results: Curcumin reduced leptin-induced MAT2A expression. JNK signaling contributed to leptin-induced increase in MAT2A level, which could be interrupted by curcumin treatment. Curcumin inhibited leptin-induced MAT2A promoter activity by influencing MAT2A promoter fragments between −2,847 bp and − 2,752 bp and between −2,752 bp and +49 bp. The effect of curcumin on leptin-induced MAT2A expression paralleled the reductions in leptin-induced activated HSCs and liver fibrosis. Conclusion: These results might have implications for curcumin inhibition of the liver fibrogenesis in obese patients with hyperleptinemia.

引言：肥胖患者常伴随高瘦素血症（hyperleptinemia），且易进展为肝纤维化。现有包括人体研究在内的大量研究数据表明，瘦素（leptin）在肝纤维化进程中发挥促进作用。DNA甲基化重塑是调控基因表达的表观遗传机制，对肝星状细胞（hepatic stellate cell, HSC）的激活至关重要，而该过程正是肝纤维化发生的关键步骤。瘦素可上调甲硫氨酸腺苷转移酶2A（methionine adenosyltransferase 2A, MAT2A）的表达，该酶与DNA甲基化及肝星状细胞激活密切相关。姜黄素（curcumin）作为黄金香料姜黄的活性多酚成分，可抑制瘦素诱导的肝星状细胞激活与肝纤维化发生。因此本研究旨在探讨姜黄素对瘦素调控肝星状细胞中MAT2A表达的影响。
方法：本研究通过瘦素缺陷型肥胖小鼠开展体内实验。采用蛋白质印迹法（Western blot）、实时荧光定量PCR（real-time PCR）、启动子活性分析及免疫染色实验检测基因表达情况。
结果：姜黄素可降低瘦素诱导的MAT2A表达。JNK信号通路参与瘦素介导的MAT2A水平升高，该作用可被姜黄素处理阻断。姜黄素通过作用于MAT2A启动子的-2847 bp至-2752 bp区域以及-2752 bp至+49 bp区域，抑制瘦素诱导的MAT2A启动子活性。姜黄素对瘦素诱导的MAT2A表达的抑制作用，与瘦素诱导的活化肝星状细胞减少及肝纤维化程度减轻的结果相一致。
结论：本研究结果可为高瘦素血症肥胖患者的姜黄素抗肝纤维化治疗提供潜在的理论参考。