基于AMPK/mTOR/ULK1通路探讨解毒通络保肾方改善糖尿病肾脏疾病的作用机制

Jiedu Tongluo Baoshen Formula exerts beneficial effects on diabetic kidney disease by regulating the AMPK/mTOR/ULK1 pathway

目的通过动物实验及细胞实验观察解毒通络保肾方对糖尿病肾脏疾病(DKD)大鼠肾脏病理形态及大鼠肾小球系膜细胞(HBZY-1)增殖的影响,探讨其改善DKD的作用机制。方法(1)从85只健康雄性SD大鼠中任取10只纳入正常组,其余大鼠采用高脂饲料喂养与链脲佐菌素注射诱导为DKD模型。造模成功的大鼠按体质量随机分为模型组,厄贝沙坦组(16 mg/kg),解毒通络保肾方低、中、高剂量组(0.5、1.0、2.0 g/kg)。正常组及模型组给予等量生理盐水灌胃。每日1次,连续灌胃8周。采用光学显微镜和透射电镜观察肾脏组织病理形态,蛋白质印迹法检测肾脏组织自噬关键分子酵母Atg6同系物(Beclin-1)、自噬蛋白微管相关蛋白1轻链3(LC3Ⅱ/LC3Ⅰ)、泛素结合蛋白P62(P62)、自噬相关蛋白5(ATG5)的表达。(2)培养HBZY-1细胞,分为空白对照组(5.5 mmol/L葡萄糖),模型组(30 mmol/L葡萄糖),解毒通络保肾方低、中、高剂量组(50、100、200 mg/L),厄贝沙坦组(5μmol/L)。采用CCK8法检测细胞增殖率,划痕实验检测细胞迁移能力,蛋白质印迹法检测LC3Ⅱ/LC3Ⅰ、P62、ATG5与腺苷酸活化蛋白激酶(AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)、unc-51样激酶1(ULK1)及磷酸化蛋白(p-ULK1、p-AMPK及p-mTOR)的表达。结果(1)解毒通络保肾方可改善DKD大鼠肾脏基底膜、系膜、肾小管上皮细胞病理形态与超微结构,增加自噬小体、自噬溶酶体数量。与正常组比较,模型组Beclin-1、LC3Ⅱ/LC3Ⅰ、ATG5蛋白表达降低,P62蛋白表达增加(P<0.01);解毒通络保肾方干预后,肾组织中Beclin-1、LC3Ⅱ/LC3Ⅰ、ATG5蛋白表达增加,P62蛋白表达降低(P<0.05,P<0.01)。(2)解毒通络保肾方可抑制高糖诱导的HBZY-1细胞增殖,增加LC3Ⅱ/LC3Ⅰ、ATG5、p-ULK1、p-AMPK蛋白表达,降低P62、p-mTOR蛋白表达(P<0.05,P<0.01)。结论解毒通络保肾方可改善DKD大鼠肾脏组织病理与超微结构,抑制高糖诱导的HBZY-1细胞增殖,其机制可能是通过调控AMPK/mTOR/ULK1通路,改善细胞自噬实现的。

Objective We aimed to observe the effects and underlying mechanism of Jiedu(removing toxicity)Tongluo(dredging collateral)Baoshen(protecting kidney)Formula on renal pathology in diabetic kidney disease(DKD)rats and on HBZY-1 cell proliferation.Methods(ⅰ)From 85 healthy Sprague-Dawley male rats,10 rats were randomly selected as the normal group.Other rats were fed with a high-fat diet and injected with streptozotocin(STZ)to induce DKD.DKD rats were randomly divided into the model group,the irbesartan group(16 mg/kg),and the Jiedu Tongluo Baoshen Formula groups(low-,medium-,and high-dose groups at 0.5,1.0,and 2.0 g/kg,respectively).The normal group and the model group were given the same volume of normal saline.All rats were continuously dosed for 8 weeks.The pathological morphology of renal tissue was observed by light microscopy and transmission electron microscopy.Western blotting analysis was conducted to detect the expression of autophagy-related proteins homolog of yeast Atg6(Beclin-1),autophagy-related proteins microtubule-associated protein 1 light chain 3(LC3Ⅱ/LC3Ⅰ),ubiquitin-binding protein P62(P62),and autophagy related 5 homolog(ATG5)in renal tissue.(ⅱ)HBZY-1 cells were cultured.The cells were divided into the control group(low glucose,5.5 mmol/L),the model group(high glucose,30 mmol/L),the Jiedu Tongluo Baoshen Formula groups(low-,medium-,and high-dose groups,at 50,100,and 200 mg/L,respectively),and the irbesartan group(5μmol/L).The cell proliferation rate was detected by the CCK8 assay,and the cell migration ability was detected by the scratch assay.The expression levels of autophagy-related proteins(LC3Ⅱ/LC3Ⅰ,P62,and ATG5)were determined by Western blotting analysis.At the same time,the expression levels of AMP-activated protein kinase(AMPK),mammalian target of rapamycin(mTOR),unc-51 like kinase 1(ULK1),and their phosphorylated proteins(p-AMPK,p-mTOR,and p-ULK1)were detected.Results(ⅰ)Compared with the model group,Jiedu Tongluo Baoshen Formula improved the pathology and ultrastructure of renal basement membrane,mesangial,and tubular epithelial cells in rats with DKD and increased the number of autophagosomes and autolysosomes.Compared with the normal group,the protein expression levels of Beclin-1,LC3Ⅱ/LC3Ⅰ,and ATG5 in the model group decreased and the protein expression of P62 increased(P<0.01);after intervention with Jiedu Tongluo Baoshen Formula,the protein expression levels of Beclin-1,LC3Ⅱ/LC3Ⅰ,and ATG5 in renal tissue increased and the protein expression levels of P62 decreased(P<0.05,P<0.01).(ⅱ)In HBZY-1 cells induced by high glucose,Jiedu Tongluo Baoshen Formula inhibited cell proliferation,increased the protein expression levels of LC3Ⅱ/LC3Ⅰ,ATG5,p-ULK1,and p-AMPK and decreased the protein levels of P62 and p-mTOR compared with the model group(P<0.05,P<0.01).Conclusion The mechanism of Jiedu Tongluo Baoshen Formula in improving renal pathology and inhibiting the proliferation of HBZY-1 cells induced by high glucose may be related to the regulation of AMPK/mTOR/ULK1 pathway and the improvement of autophagy.