基于PI3K/Akt/FoxO1通路探讨脂肝清方对非酒精性脂肪性肝病小鼠胰岛素抵抗及糖异生的影响

Exploration on the effects of Zhiganqing Prescription on insulin resistance and gluconeogenesis in NAFLD mice based on the PI3K/Akt/FoxO1 signaling pathway

目的观察脂肝清方对高脂饲料诱导的非酒精性脂肪性肝病(NAFLD)C57BL/6J小鼠肝脏的保护作用及对PI3K/Akt/叉头盒转录因子1(FoxO1)信号通路、胰岛素抵抗(IR)、糖异生的影响。方法将48只8周龄雄性C57BL/6J小鼠按随机数字表法分为正常组(n=8)和造模组(n=40)。正常组普通饲料喂养,造模组饲喂高脂饲料8周建立NAFLD模型。将造模组小鼠按随机数字表法分为模型组、吡格列酮组及脂肝清方低、中、高剂量组,每组8只,相应药物干预8周。给药期间定期称重,分别于给药0、8周测定空腹血糖(FBG)水平,并进行口服葡萄糖耐量试验(OGTT)。实验结束后,检测血清GPT、GOT、TC、TG、LDL-C、HDL-C、胰岛素(FINS)、C肽(C-P)水平,并计算胰岛素抵抗指数(HOMA-IR);采用HE和过碘酸-雪夫(PAS)染色观察肝组织病理形态;免疫组化染色检测PI3K、p-Akt蛋白表达;Western blot检测PI3K、Akt、p-Akt、FoxO1、p-FoxO1、葡萄糖-6-磷酸酶(G6PC)、磷酸烯醇丙酮酸羧激酶(PCK1)蛋白表达。结果与模型组比较,给药第4、6、8周,各给药组小鼠体重下降(P<0.01或P<0.05);给药第8周各给药组小鼠FBG水平、OGTT曲线下面积降低(P<0.01或P<0.05),GPT、TC、TG、LDL-C水平降低(P<0.01),脂肝清方中、高剂量组GOT水平降低(P<0.01),脂肝清方中剂量组HDL-C水平降低(P<0.01或P<0.05),脂肝清方低、中剂量组HOMA-IR降低(P<0.05或P<0.01);各给药组FINS、C-P水平升高(P<0.05或P<0.01),肝组织PI3K蛋白表达及p-Akt/Akt、p-FoxO1/FoxO1升高(P<0.05或P<0.01),G6PC、PCK1蛋白表达降低(P<0.05或P<0.01)。结论脂肝清方可有效控制NAFLD小鼠体重、血糖、肝功能及血脂水平,改善IR及糖异生,其作用机制可能与激活PI3K/Akt/FoxO1信号通路有关。

Objective To observe the protective effects of Zhiganqing Prescription on the liver of C57BL/6J non-alcoholic fatty liver disease(NAFLD)mice induced by high fat diet and its effects on PI3K/Akt/FoxO1 signaling pathway,insulin resistance(IR)and gluconogenesis.Methods A total of 488-week-old male C57BL/6J mice were divided into control group(n=8)and modeling group(n=40)according to random number table method.The control group was fed with ordinary diet,and the model group was fed with high-fat diet.The NAFLD model was established after 8 weeks of feeding.The modeling group was divided into model group,Pioglitazone group,Zhiganqing Prescription low-,medium-,and high-dosage group(n=8 in each group)according to random number table method,and drug intervention lasted for 8 weeks.The body mass of mice was measured regularly during administration.Fasting blood glucose(FBG)levels were measured at 0 and 8 weeks of administration,and oral glucose tolerance tests(OGTT)were conducted.After the experiment,serum levels of GPT,GOT,TC,TG,LDL-C,HDL-C,FINS and C-P were detected and HOMA-IR was calculated.The pathological morphology of liver was observed by HE and PAS staining.The expression levels of PI3K and p-Akt were detected by IHC staining.The protein expression levels of PI3K,Akt,p-Akt,FoxO1,p-FoxO1,G6PC and PCK1 were detected by Western blot.Results Compared with model group,the body weight of mice in each administration group decreased at 4,6 and 8 weeks(P<0.05 or P<0.01).At the 8th week of administration,the levels of FBG and OGTT AUC in each administration group decreased(P<0.05 or P<0.01),the levels of GPT,TC,TG and LDL-C decreased(P<0.01),and the GOT levels in Zhiganqing Prescription medium-and high-dosage groups decreased(P<0.01).The HDL-C level in Zhiganqing Prescription medium-dosage group decreased(P<0.05 or P<0.01),and the HOMA-IR level in Zhiganqing Prescription low-and medium-dosage groups decreased(P<0.05 or P<0.01).The levels of FINS and C-P in each administration group increased(P<0.05 or P<0.01),and the expressions of PI3K protein and p-Akt/Akt,p-FoxO1/FoxO1 protein in liver tissues increased(P<0.05 or P<0.01).The protein expressions of G6PC and PCK1 decreased(P<0.05 or P<0.01).Conclusion Zhiganqing Prescription can effectively control the body mass,blood glucose,liver function and blood lipids of NAFLD mice,improve IR and gluconeogenesis,the mechanism of which may be related to the activation of PI3K/Akt/FoxO1 signaling pathway.