目的 探討雙氫青蒿素(dihydroartemisinin,DHA)治療2型糖尿病(type 2 diabetes mellitus,T2DM)小鼠肝臟糖代謝紊亂、炎癥反應(yīng)的作用及作用機(jī)制。方法 利用高糖高脂飼料喂養(yǎng)聯(lián)合ip鏈脲佐菌素(40 mg/kg)誘導(dǎo)建立T2DM小鼠模型,設(shè)置對(duì)照組、模型組、二甲雙胍(200 mg/kg)組和DHA低、中、高劑量(30、60、120 mg/kg)組,每組6只。給予藥物干預(yù)4周后,檢測(cè)小鼠體質(zhì)量、空腹血糖(fasting blood glucose,FBG)、胰島素抵抗指數(shù)(homeostatic model assessment for insulin resistance,HOMA-IR)、血脂四項(xiàng)、肝功能指標(biāo);采用ELISA法檢測(cè)肝組織白細(xì)胞介素-1β(interleukin-1β,IL-1β)、IL-6、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)水平;采用蘇木素-伊紅(hematoxylin-eosin,HE)、PAS、油紅O染色觀察胰島、肝臟組織病理變化;采用免疫組化法檢測(cè)肝組織F4/80陽(yáng)性表達(dá);采用Western blotting檢測(cè)肝組織糖代謝和炎癥通路相關(guān)蛋白表達(dá)。采用高濃度胰島素誘導(dǎo)建立肝細(xì)胞胰島素抵抗(insulin resistance-human hepatocellular carcinoma,IR-HepG2)模型,給予DHA和己糖激酶(hexokinase,HK)抑制劑3-溴丙酮酸(3-bromopyruvic acid,3-BrPA)干預(yù)后,檢測(cè)細(xì)胞存活率和葡萄糖消耗量。結(jié)果 與模型組比較,DHA顯著降低小鼠FBG(P＜0.05、0.01),改善糖耐量損傷(P＜0.05、0.01),減少HOMA-IR(P＜0.01、0.001),升高高密度脂蛋白膽固醇(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),降低丙氨酸氨基轉(zhuǎn)移酶(alanine aminotransferase,ALT)、天冬氨酸氨基轉(zhuǎn)移酶(aspartate aminotransferase,AST)活性(P＜0.05),減少肝臟炎癥因子水平(P＜0.05、0.01、0.001)。病理染色結(jié)果顯示,DHA能夠增加小鼠胰島面積(P＜0.001),改善β細(xì)胞損傷和肝細(xì)胞萎縮,減少肝臟脂質(zhì)堆積,增加肝糖原含量。免疫組化結(jié)果顯示,DHA能夠減少肝臟F4/80陽(yáng)性表達(dá)。Western blotting結(jié)果顯示,DHA顯著下調(diào)肝臟叉頭框蛋白1(forkhead box O1,FOXO1)、葡萄糖-6-磷酸酶(glucose-6-phosphatase,G6PC)、髓樣分化因子88(myeloid differentiation factor 88,MyD88)、核因子-κB(nuclear factor-κB,NF-κB)、F4/80蛋白表達(dá)(P＜0.05、0.01、0.001),上調(diào)HK2蛋白表達(dá)(P＜0.01)。細(xì)胞實(shí)驗(yàn)結(jié)果顯示,1 μmol/L胰島素處理HepG2細(xì)胞36 h,可成功構(gòu)建IR-HepG2模型;給予DHA干預(yù)后,葡萄糖消耗量顯著增加(P＜0.05、0.01);給予DHA和3-BrPA同時(shí)干預(yù)后,3-BrPA可部分抑制DHA對(duì)葡萄糖消耗量上調(diào)的作用。結(jié)論 DHA通過(guò)調(diào)節(jié)FOXO1/HK2/G6PC和MyD88/NF-κB通路降低FBG,改善胰島素抵抗和糖耐量,保護(hù)胰島和肝細(xì)胞,進(jìn)而改善T2DM小鼠肝臟糖代謝紊亂和炎癥反應(yīng)。

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

中國(guó)中醫(yī)科學(xué)院科技創(chuàng)新團(tuán)隊(duì)項(xiàng)目（CI2021B015）；國(guó)家自然科學(xué)基金面上項(xiàng)目（82274181）；博士科研啟動(dòng)資金項(xiàng)目（2024BSJJ04）