高血糖诱导肝星状细胞5-羟色胺降解在2型糖尿病致肝脏炎症和纤维化时的作用

目的: 探讨5-羟色胺(5-hydroxytryptamine，5-HT)在2型糖尿病(type 2 diabetes mellitus，T2DM)引起肝脏炎症及纤维化时的作用。方法: 雄性C57BL/6J小鼠，通过高脂饲料喂养结合腹腔注射链脲佐菌素，建立T2DM模型；将已形成高血糖的小鼠继续用高脂饲料喂养9周或同时用5-HT_2A受体(5-HT 2A receptor，5-HT_2AR)拮抗剂盐酸沙格雷酯(sarpogrelate hydrochloride，SH)及5-HT合成抑制剂卡比多巴(carbidopa，CDP)分别或联合给药进行治疗。细胞实验用人肝星状细胞(hepatic stellate cells，HSCs)株LX-2，高浓度葡萄糖刺激或同时用SH、CDP或单胺氧化酶A(monoamine oxidase A，MAO-A)抑制剂氯吉兰(clorgyline，CGL)处理细胞，观察高糖诱导LX-2细胞肌成纤维细胞化时5-HT的作用。用苏木精-伊红(hematoxylin & eosin，HE)及马松(Masson)染色法检测肝组织切片病理变化，免疫组织化学及Western blot分析蛋白表达，ELISA或酶试剂法检测生化指标，荧光探针法检测细胞内活性氧(reactive oxygen species，ROS)含量。结果: T2DM小鼠肝脏的5-HT_2AR、5-HT合成酶和MAO-A表达上调，且5-HT含量升高。SH和CDP治疗在降低肝脏5-HT含量及下调MAO-A表达的同时，可有效地改善肝脏病变：不仅改善肝功能及肝脂肪变性，还明显抑制肝脏ROS(H₂O₂)含量升高，改善氧化应激，并抑制转化生长因子β1(transforming growth factor β1，TGF-β1)的产生，以及炎症和纤维化的发生，且SH和CDP的作用呈协同效应。LX-2细胞研究表明，高糖可上调5-HT_2AR、5-HT合成酶和MAO-A表达，升高细胞内5-HT含量，使细胞的ROS产生增多及肌成纤维细胞化，从而增加TGF-β1合成及炎症和纤维化因子的产生。通过SH拮抗5-HT_2AR，高糖作用被明显抑制；通过CGL抑制线粒体5-HT降解，高糖作用被强烈抑制。SH还可抑制高糖诱导的5-HT合成酶及MAO-A表达上调。结论: 高糖诱导HSCs肌成纤维细胞化和TGF-β1产生，从而导致T2DM小鼠肝脏炎症及纤维化损伤，其病理机制可能是诱导了5-HT_2AR表达上调，5-HT合成及降解增加，使线粒体的ROS产生增多，其中，5-HT_2AR的作用是参与了对5-HT合成酶和MAO-A表达的调控。

Role of hyperglycemia-induced 5-hydroxytryptamine degradation of hepatic stellate cells in hepatic inflammation and fibrosis induced by type 2 diabetes mellitus

Objective: To explore the role of 5-hydroxytryptamine (5-HT) in type 2 diabetes mellitus (T2DM)-related hepatic inflammation and fibrosis. Methods: Male C57BL/6J mice were used to establish T2DM model by high-fat diet feeding combined with intraperitoneal injection of streptozotocin. Then, the mice with hyperglycemia were still fed with high-fat diet for nine weeks, and treated with or without 5-HT_2A receptor (5-HT_2AR) antagonist sarpogrelate hydrochloride (SH) and 5-HT synthesis inhibitor carbidopa (CDP) (alone or in combination). To observe the role of 5-HT in the myofibroblastization of hepa-tic stellate cells (HSCs), human HSCs LX-2 were exposed to high glucose, and were treated with or without SH, CDP or monoamine oxidase A (MAO-A) inhibitor clorgiline (CGL). Hematoxylin & eosin and Masson staining were used to detect the pathological lesions of liver tissue section, immunohistochemistry and Western blot were used to analyze protein expression, biochemical indicators were measured by ELISA or enzyme kits, and levels of intracellular reactive oxygen species (ROS) were detected by fluorescent probe. Results: There were up-regulated expressions of 5-HT_2AR, 5-HT synthases and MAO-A, and elevated levels of 5-HT in the liver of the T2DM mice. In addition to reduction of the hepatic 5-HT levels and MAO-A expression, treatment with SH and CDP could effectively ameliorate liver lesions in the T2DM mice, both of which could ameliorate hepatic injury and steatosis, significantly inhibit the increase of hepatic ROS (H₂O₂) levels to alleviate oxidative stress, and markedly suppress the production of transforming growth factor β1 (TGF-β1) and the development of inflammation and fibrosis in liver. More importantly, there was a synergistic effect between SH and CDP. Studies on LX-2 cells showed that high glucose could induce up-regulation of 5-HT_2AR, 5-HT synthases and MAO-A expression, increase intracellular 5-HT level, increase the production of ROS, and lead to myofibroblastization of LX-2, resulting in the increase of TGF-β1 synthesis and production of inflammatory and fibrosis factors. The effects of high glucose could be significantly inhibited by 5-HT_2AR antagonist SH or be markedly abolished by mitochondrial 5-HT degradation inhibitor CGL. In addition, SH significantly suppressed the up-regulation of 5-HT synthases and MAO-A induced by high glucose in LX-2. Conclusion: Hyperglycemia-induced myofibroblastization and TGF-β1 production of HSCs, which leads to hepatic inflammation and fibrosis in T2DM mice, is probably due to the up-regulation of 5-HT_2AR expression and increase of 5-HT synthesis and degradation, resulting in the increase of ROS production in mitochondria. Among them, 5-HT_2AR is involved in the regulation of 5-HT synthases and MAO-A expression.