氧化苦参碱改善非酒精性脂肪肝病的作用及转录组学研究

Ameliorating non-alcoholic fatty liver disease of oxymatrine and transcriptomics study

中文关键词:  非酒精性脂肪肝  氧化苦参碱  转录组学  信号通路

英文关键词:non-alcoholic fatty liver disease  oxymatrine  transcriptomics  signaling pathway

中文摘要:

      目的 研究氧化苦参碱(oxymatrine, Oxy)的抗非酒精性脂肪性肝病(non-alcoholic fatty liver disease, NAFLD)作用及机制。方法 将大鼠随机分为正常组、模型组、白藜芦醇组(50 mg/kg)和Oxy低、高剂量组(50 mg/kg和100 mg/kg); 采用高脂饮食(high-fat diet, HFD)饲喂16周诱导NAFLD大鼠模型, 从第8周开始按分组对应灌胃给药; 测定各组大鼠体重、肝脏重量和肝脏指数; 采用试剂盒检测大鼠血清天门冬氨酸氨基转移酶(aspartate transaminase, AST)、丙氨酸氨基转移酶(alanine transaminase, ALT)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol, LDL-c)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol, HDL-c)、总胆固醇(total cholesterol, TC)和甘油三酯(triglyceride, TG)水平; 采用苏木素-伊红(hematoxylin-eosin, HE)、油红O和Masson染色检查大鼠肝脏病理变化; 采用高通量测序技术分析大鼠肝脏基因表达。结果 与正常组比较, 模型组大鼠体重、肝脏重量及肝脏指数显著增加(P<0.01); 血清中AST、ALT、LDL-c、TC和TG水平显著升高, HDL-c水平显著降低(P<0.01); 肝脏肝小叶结构出现损伤, 呈现弥漫性小泡、大泡脂肪变性及气球样变; 肝脏中脂滴和蓝色胶原纤维显著增多(P<0.01)。与模型组比较, Oxy高剂量组大鼠体重、肝脏重量及肝脏指数显著降低(P<0.01); 血清中肝损伤及脂代谢相关指标水平显著恢复(P<0.01); 肝脏结构趋于正常, 肝脏仅有轻微的小泡脂肪变性; 肝脏中脂滴和蓝色胶原纤维显著减少(P<0.01)。转录组结果显示, 正常组、模型组和Oxy组样本各自聚集在一起; 正常组与模型组间有499个差异基因, 模型组与Oxy组间有434个差异基因, 共有差异基因218个, 集中于环磷酸鸟苷(cyclic guanosine monophosphate, cGMP)/蛋白激酶G (protein kinase G, PKG)、磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase, PI3K)/蛋白激酶B (protein kinase B, Akt)、环磷酸腺苷(cyclic adenosine monophosphate, cAMP)、松弛素、胰岛素抵抗、Rap1和Hippo信号通路。结论 Oxy具有明确的抗NAFLD作用, 具体表现为对HFD诱导的NAFLD大鼠的肥胖的缓解以及肝功能、脂代谢紊乱、肝脂肪变性和肝纤维化的改善, 机制可能与调控cGMP/PKG、PI3K/Akt、cAMP、松弛素、胰岛素抵抗、Rap1和Hippo信号通路有关。

英文摘要:

      Objective To study the anti-non-alcoholic fatty liver disease (NAFLD) effect and mechanism of oxymatrine (Oxy). Methods Rats were randomly divided into normal, model, resveratrol (50 mg/kg) and low- and high-dose Oxy groups (50 mg/kg and 100 mg/kg); high-fat diet (HFD) was used to induce NAFLD model for 16 weeks, and the drug was administered by gavage from week 8 onwards. The body weight, liver weight, and liver index of rats in each group were measured; the kits were used to detect the levels of aspartate transaminase (AST), alanine transaminase (ALT), low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-c), total cholesterol (TC) and triglyceride (TG) in serum of rats; hematoxylin-eosin (HE), oil red O and Masson staining were used to detect the pathological changes of rat liver; the gene expression of rat liver was analyzed by high-throughput sequencing technology. Results Compared with the normal group, the body weight, liver weight and liver index of the model group rats were significantly increased (P<0.01); AST, ALT, LDL-c, TC and TG levels in serum were significantly increased (P<0.01) and HDL-c levels were significantly decreased (P<0.01); hepatic lobular structures of the liver were damaged, showing diffuse microvesicular and macrovesicular steatosis and balloon-like changes; lipid droplets and blue collagen fibers in the liver became significantly more abundant (P<0.01). Compared with the model group, the body weight, liver weight and liver index were significantly lower in the high-dose Oxy group rats (P<0.01); the levels of liver injury and lipid metabolism-related indexes in the serum were significantly restored (P<0.01); the liver structure tended to be normal, with only slight microvesicular steatosis in the liver; lipid droplets and blue collagen fibers in the liver were significantly reduced (P<0.01). The transcriptome results showed that the normal, model and Oxy group samples were each clustered together; there were 499 differential genes between the normal and model groups and 434 differential genes between the model and Oxy groups, of which there were 218 shared differential genes, concentrated in cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), cyclic adenosine monophosphate (cAMP), relaxin, insulin resistance, Rap1 and Hippo signaling pathways. Conclusion Oxy has a clear anti-NAFLD effect, as evidenced by the alleviation of obesity and improvement of liver function, lipid metabolism disorders, hepatic steatosis and liver fibrosis in rats with HFD-induced NAFLD. The mechanism may be related to the regulation of cGMP/PKG, PI3K/Akt, cAMP, relaxin, insulin resistance, Rap1 and Hippo signaling pathways.