二甲双胍、吡格列酮对非酒精性脂肪性肝病大鼠肝脏CYP2E1表达的影响

Metformin and pioglitazone reduce CYP2E1 expression in the liver of rats with nonalcoholic fatty liver disease

目的:观察二甲双胍、吡格列酮对非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)大鼠肝脏细胞色素P4502E1(CYP2E1)的影响.方法:SD大鼠随机分为正常对照组(普通饲料喂养)、非酒精性脂肪肝组(高脂饮食喂养)、二甲双胍干预组[高脂饮食喂养加二甲双胍500mg/(kg·d)灌胃]、吡格列酮干预组[高脂饮食喂养加吡格列酮15mg/(kg·d)].饲养12wk末处死大鼠,检测血清丙氨酸氨基转移酶(aspartate aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)、碱性磷酸酶(bioactive alkaline phosphatase,ALP)活性、总胆固醇(total cholesterol,TC)、甘油三酯(total glyceride,TG)、游离脂肪酸(floating free acid,FFA)含量;测量空腹血糖(fasting blood-sugar,FBG)及胰岛素(level and insulin,FINS),计算胰岛素抵抗指数(insulin resistance index,HOMA-IR);采用HE染色及Masson染色(显示胶原纤维),观察肝脏病理形态学的改变;采用RT-qPCR和免疫组织化学法分别检测肝脏中CYP2E1mRNA及蛋白的表达.结果:与非酒精性脂肪肝组相比,二甲双胍干预组的ALT(79.86U/L±10.40U/Lvs143.80U/L±26.34U/L)、AST(221.30U/L±34.21U/Lvs358.76±97.20U/L)、ALP(153.32U/L±24.20U/Lvs207.47U/L±35.39U/L)、TC(1.70mmol/L±0.34mmol/Lvs2.76mmol/L±0.28mmol/L)、TG(3.96mmol/L±0.23mmol/Lvs5.67mmol/L±0.54mmol/L)、FFA(493.19μmol/L±38.89μmol/Lvs654.40μmol/L±71.74μmol/L)以及FBG(5.47mmol/L±0.42mmol/Lvs8.05mmol/L±0.48mmol/L)、FINS(20.89mU/L±3.12mU/Lvs27.51mU/L±4.02mU/L)、HOMA-IR(5.08±1.20vs9.84±2.11)均明显降低(P<0.05),吡格列酮干预组的ALT(84.73U/L±9.18U/Lvs143.80U/L±26.34U/L)、AST(235.23U/L±33.62U/Lvs358.76U/L±97.20U/L)、ALP(160.02U/L±25.32U/Lvs207.47U/L±35.39U/L)、TC(1.68mmol/L±0.26mmol/Lvs2.76mmol/L±0.28mmol/L)、TG(3.60mmol/L±0.30mmol/Lvs5.67mmol/L±0.54mmol/L)、FFA(495.21μmol/L±43.67μmol/Lvs654.40μmol/L±71.74μmol/L)以及FBG(5.32mmol/L±0.37mmol/Lvs8.05mmol/L±0.48mmol/L)、FINS(21.01mU/L±2.86mU/Lvs27.51mU/L±4.02mU/L)、HOMA-IR(4.97±0.97vs9.84±2.11)均明显降低(P<0.05),肝脏脂肪变性及纤维化程度均明显改善,CYP2E1mRNA及蛋白的表达明显降低(P<0.05),且两干预组间差异无统计学意义(P>0.05).结论:二甲双胍、吡格列酮均通过降低CYP2E1的表达,预防NAFLD的发生.

AIM： To observe the preventive effects of metformin and pioglitazone on nonalcoholic fatty liver disease （NAFLD） and their influence on CYP2E1 expression in the liver. METHODS： SD rats were randomly divided into four groups： normal control group （fed a normal diet）, NAFLD group （fed a fat-rich diet）, metformin group [fed a fat-rich diet and intragastrically given metformin 500 mg/（kg·d）], pioglitazone group [fed a fat-rich diet and intragastrically given pioglitazone 15 mg/（kg·d）]. The rats were killed at the end of 12 wk. The contents of alanine transferase （ALT）, aspartate aminotransferase （AST）, bioactive alkaline phosphatase （ALP）, total cholesterol （TC）, total glyceride （TG）, and floating free acid （FFA） were determined. Fasting blood sugar （FBG） level and insulin （FINS） level were measured to calculate the insulin resistance index （HOMA-IR）. HE staining and Masson staining were carried out to observe the pathological changes in the liver. The expression levels of CYP2E1 mRNA and protein in the liver were determined by RT-PCR and immunohistochemistry, respectively. RESULTS： Compared to the NAFLD group, treatment with metformin significantly reduced the bioactivity of ALT （79.86 U/L ± 10.40 U/L vs 143.80 U/L ± 26.34 U/L）, AST （221.30 U/L ± 34.21 U/L vs 358.76 U/L ± 97.20 U/L）, and ALP （153.32 U/L ± 24.20 U/L vs 207.47 U/L ± 35.39 U/L）, the contents of TC （1.70 mmol/L ± 0.34 mmol/L vs 2.76 mmol/L ± 0.28 mmol/L）, TG （3.96 mmol/L ± 0.23 mmol/L vs 5.67 mmol/L ± 0.54 mmol/L）, and FFA （493.19 μmol/L ± 38.89 μmol/L vs 654.40 μmol/L ± 71.74 μmol/L）, FBG （5.47 mmol/L ± 0.42 mmol/L vs 8.05 mmol/L ± 0.48 mmol/L）, FINS （20.89 mU/L ± 3.12 mU/L vs 27.51 mU/L ± 4.02 mU/L）, and HOMA-IR （5.08 ± 1.20 vs 9.84 ± 2.11） （all P 〈 0.05）; treatment with pioglitazone significantly reduced the bioactivity of ALT （84.73 U/L ± 9.18 U/L vs 143.80 U/L ± 26.34 U/L）, AST （235.23 U/L ± 33.62 U/L vs 358.76 U/L ± 97.20 U/L）, and ALP （160.02 U/L ± 25.32 U/L vs 207.47 U/L ± 35.39 U/L）, the contents of TC （1.68 mmol/L ± 0.26 mmol/L vs 2.76 mmol/L ± 0.28 mmol/L）, TG （3.60 mmol/L ± 0.30 mmol/L vs 5.67 mmol/L ± 0.54 mmol/L）, and FFA （495.21 μmol/L ± 43.67 μmol/L vs 654.40 μmol/L ± 71.74 μmol/L）, FBG （5.32 mmol/L ± 0.37 mmol/L vs 8.05 mmol/L ± 0.48 mmol/L）, FINS （21.01 mU/L ± 2.86 mU/L vs 27.51 mU/L ± 4.02 mU/L）, and HOMA-IR （4.97 ± 0.97 vs 9.84 ± 2.11） （all P 〈 0.05）. Treatment with both metformin and pioglitazone ameliorated pathological changes in the liver and reduced the expression levels of CYP2E1 mRNA and protein in the liver （both P 〈 0.05）. However, there were no significant differences in the above parameters between the two treatment groups （all P 〉 0.05）. CONCLUSION： Both metformin and pioglitazone can prevent the occurrence of NAFLD by reducing the expression of CYP2E1.