芪蛭通络方通过调控PI3K/Akt/TSC2/mTOR途径对糖尿病肾病大鼠自噬及肾功能的影响

Effect of Qilee Tongluo Formula on Autophagy and Renal Function in Rats with Diabetic Nephropathy by Regulating PI3K/Akt/TSC2/mTOR Pathway

目的:观察芪蛭通络方对糖尿病肾病大鼠的自噬及肾功能的干预作用及对磷酯酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/结节性硬化症复合物2(TSC2)/哺乳动物雷帕霉素(mTOR)信号的调控作用,研究芪蛭通络方对糖尿病肾病的保护机制。方法:85只大鼠随机选取15只为正常组,其余大鼠采用高糖高脂饲料喂养联合小剂量链脲佐菌素(STZ)腹腔注射诱导糖尿病肾病大鼠模型。将造模成功的大鼠随机分为模型组、芪蛭通络方低、中、高剂量组(1.215、2.43、4.86g/kg)及厄贝沙坦组(0.0135g/kg),每组各10只。各组分别灌胃相应药物或生理盐水,1次/d,连续给药8w后,检测大鼠血、尿、肾组织病理及相关蛋白的表达。结果:与正常组比较,模型组大鼠24h尿蛋白定量(UTP)、血清总胆固醇(TC)及甘油三酯(TG)水平均升高(P<0.05);与模型组比较,芪蛭通络方可以降低糖尿病肾病大鼠的24h尿蛋白定量(UTP)、尿素氮(BUN)、血肌酐(Scr)、空腹血糖(FBG)、总胆固醇(TC)及甘油三酯(TG)等指标(P<0.05,P<0.01),减轻肾脏病理改变,降低p-PI3K、p-Akt、p-TSC2、p-mTOR蛋白表达,且芪蛭通络低、中、高剂量组与厄贝沙坦组间比较差异无统计学意义(P>0.05)。此外,芪蛭通络方能抑制DN大鼠肾组织p-PI3K、p-Akt、p-TSC2、p-mTOR的表达,进而提高自噬标志蛋白LC3B的表达水平和LC3B-Ⅱ/LC3B-Ⅰ比值。结论:芪蛭通络方能抑制糖尿病肾病大鼠24h UTP的升高并改善其肾脏病理变化,发挥以上肾脏保护作用的机制可能与其抑制PI3K/Akt//TSC2/mTOR信号通路,进而促进肾脏自噬有关。

Objective:To observe the interventional effects of Astragalus Tongluo Formula on autophagy and renal function and the regulation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/tuberous sclerosis complex 2(TSC2)/mammalian rapamycin signalling in diabetic nephropathy rats,and to study the protective mechanism of Astragalus Tongluo Forournal of Sichuan of Traditional Chinese Medicine mula against diabetic nephropathy.METHODS:85rats were randomly selected 15as normal group,and the rest were fed with high sugar and high fat chow combined with small-dose streptozotocin(STZ)intraperitoneally to induce diabetic nephropathy rat model.The rats were randomly divided into the model group,the low,medium and high dose groups(1.215,2.43and 4.86g/kg)of Astragalus Hirudinea Tongluo Formula and the irbesartan group(0.0135g/kg),with 10rats in each group.Each group was gavaged with the corresponding drugs or saline once/d for 8w.The histopathology of blood,urine and kidney and the expression of related proteins were detected.Results:Compared with the normal group,the quantitative 24h urine protein(UTP),serum total cholesterol(TC)and triglyceride(TG)levels were increased in the model group(P<0.05);compared with the model group,Astragali Hirudinea and Tongluo Formula could reduce 24h urine protein(UTP),the blood creatinine(Scr),urea nitrogen(BUN),fasting blood glucose(FBG),total cholesterol(TC)and triglyceride(TG)in rats with diabetic nephropathy.and 24h urine protein quantification(UTP)(P<0.05,P<0.01),attenuate renal pathological changes,reduce p-PI3k,p-Akt,p-TSC2,p-mTOR protein expression,and the difference between Astragalus Tongluo low,medium,and high dosage groups and Irbesartan group was not statistically significant(P>0.05).In addition,Astragalus Tongluo Formula inhibited the expression of p-PI3K,p-Akt,p-TSC2,and p-mTOR in renal tissues of DN rats,which in turn increased the expression level of LC3B,an autophagy marker protein,and the ratio of LC3B-II/LC3B-Ⅰ.CONCLUSION:Astragalus Tongluo Formula can inhibit the elevation of 24h UTP and improve the renal pathological changes in rats with diabetic nephropathy,and the mechanism of the above nephroprotective effects may be related to its inhibition of the PI3K/Akt//TSC2/mTOR signalling pathway,which then promotes renal autophagy.