目的 探讨双氢青蒿素(dihydroartemisinin,DHA)治疗2型糖尿病(type 2 diabetes mellitus,T2DM)小鼠肝脏糖代谢紊乱、炎症反应的作用及作用机制。方法 利用高糖高脂饲料喂养联合ip链脲佐菌素(40 mg/kg)诱导建立T2DM小鼠模型,设置对照组、模型组、二甲双胍(200 mg/kg)组和DHA低、中、高剂量(30、60、120 mg/kg)组,每组6只。给予药物干预4周后,检测小鼠体质量、空腹血糖(fasting blood glucose,FBG)、胰岛素抵抗指数(homeostatic model assessment for insulin resistance,HOMA-IR)、血脂四项、肝功能指标;采用ELISA法检测肝组织白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;采用苏木素-伊红(hematoxylin-eosin,HE)、PAS、油红O染色观察胰岛、肝脏组织病理变化;采用免疫组化法检测肝组织F4/80阳性表达;采用Western blotting检测肝组织糖代谢和炎症通路相关蛋白表达。采用高浓度胰岛素诱导建立肝细胞胰岛素抵抗(insulin resistance-human hepatocellular carcinoma,IR-HepG2)模型,给予DHA和己糖激酶(hexokinase,HK)抑制剂3-溴丙酮酸(3-bromopyruvic acid,3-BrPA)干预后,检测细胞存活率和葡萄糖消耗量。结果 与模型组比较,DHA显著降低小鼠FBG(P＜0.05、0.01),改善糖耐量损伤(P＜0.05、0.01),减少HOMA-IR(P＜0.01、0.001),升高高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)水平(P＜0.05、0.01),降低三酰甘油(triglyceride,TG)水平(P＜0.001),降低丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)活性(P＜0.05),减少肝脏炎症因子水平(P＜0.05、0.01、0.001)。病理染色结果显示,DHA能够增加小鼠胰岛面积(P＜0.001),改善β细胞损伤和肝细胞萎缩,减少肝脏脂质堆积,增加肝糖原含量。免疫组化结果显示,DHA能够减少肝脏F4/80阳性表达。Western blotting结果显示,DHA显著下调肝脏叉头框蛋白1(forkhead box O1,FOXO1)、葡萄糖-6-磷酸酶(glucose-6-phosphatase,G6PC)、髓样分化因子88(myeloid differentiation factor 88,MyD88)、核因子-κB(nuclear factor-κB,NF-κB)、F4/80蛋白表达(P＜0.05、0.01、0.001),上调HK2蛋白表达(P＜0.01)。细胞实验结果显示,1 μmol/L胰岛素处理HepG2细胞36 h,可成功构建IR-HepG2模型;给予DHA干预后,葡萄糖消耗量显著增加(P＜0.05、0.01);给予DHA和3-BrPA同时干预后,3-BrPA可部分抑制DHA对葡萄糖消耗量上调的作用。结论 DHA通过调节FOXO1/HK2/G6PC和MyD88/NF-κB通路降低FBG,改善胰岛素抵抗和糖耐量,保护胰岛和肝细胞,进而改善T2DM小鼠肝脏糖代谢紊乱和炎症反应。

Objective To investigate the effect and mechanism of dihydroartemisinin (DHA) on hepatic glucose metabolism disorder and inflammatory reaction in mice with type 2 diabetes (T2DM). Methods A T2DM mouse model was established by feeding a high sugar and high-fat diet combined with ip streptozotocin (40 mg/kg). Control group, model group, metformin (200 mg/kg) group, DHA low-, medium-, and high-dose (30, 60, 120 mg/kg) groups were set up, with six mice in each group. After four weeks of drug intervention, body weight, fasting blood glucose (FBG), homeostatic model assessment for insulin resistance (HOMA-IR), blood lipids, and liver function indicators of mice were measured; ELISA was used to detect the levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in liver tissue; Hematoxylin-eosin (HE), PAS and Oil Red O staining were used to observe pathological changes in pancreatic islets and liver tissues; Immunohistochemistry was used to detect F4/80 positive expression in liver tissue; Western blotting was used to detect the expressions of glucose metabolism and inflammatory pathway related proteins in liver tissue. An insulin resistance human hepatocellular carcinoma (IR-HepG2) model induced by high concentration insulin was established, after intervention with DHA and hexokinase (HK) inhibitor 3-bromopyruvic acid (3-BrPA), cell survival rate and glucose consumption were measured. Results Compared with model group, DHA significantly reduced FBG (P < 0.05, 0.01), improved glucose tolerance injury (P < 0.05, 0.01), reduced HOMA-IR (P < 0.01, 0.001), increased high-density lipoprotein cholesterol (HDL-C) level (P < 0.05, 0.01), decreased triglyceride (TG) level (P < 0.001), decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities (P < 0.05), and reduced inflammatory factor levels in liver of mice (P < 0.05, 0.01, 0.001). Pathological staining results showed that DHA could increase the pancreatic islet area in mice (P < 0.001), improve beta cell damage and liver cell atrophy, reduce lipid accumulation in liver, and increase liver glycogen content. Immunohistochemistry results showed that DHA could reduce the positive expression of F4/80 in liver. Western blotting results showed that DHA significantly down-regulated the expressions of forkhead box O1 (FOXO1), glucose-6-phosphate (G6PC), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB) and F4/80 protein in liver (P < 0.05, 0.01, 0.001), and up-regulated the expression of HK2 protein (P < 0.01). The cell experiment results showed that treating HepG2 cells with 1 μmol/L insulin for 36 h could successfully construct IR-HepG2 model; After DHA intervention, glucose consumption was significantly increased (P < 0.05, 0.01); After simultaneous intervention with DHA and 3-BrPA, 3-BrPA partially inhibited the up-regulation effect of DHA on glucose consumption. Conclusion DHA reduces FBG, improves insulin resistance and glucose tolerance, protects pancreatic islets and liver cells by regulating FOXO1/HK2/G6PC and MyD88/NF-κB pathways, thereby improving hepatic glucose metabolism disorders and inflammatory responses in T2DM mice.

R285.5

中国中医科学院科技创新团队项目（CI2021B015）；国家自然科学基金面上项目（82274181）；博士科研启动资金项目（2024BSJJ04）