大黄糖络丸通过AMPK/mTOR/ULK1通路调控糖尿病肾病小鼠足细胞自噬的作用机制研究

Mechanism of Dahuangtang pellets in regulating podocyte autophagy of diabetic nephropathy mice through AMPK/mTOR/ULK1 signaling pathway

目的:探究大黄糖络丸(DHT)基于腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白/unc-51样激酶1(AMPK/mTOR/ULK1)信号通路对糖尿病肾病(diabetic nephropathy,DN)小鼠的干预作用。方法:40只造模成功的C57BL/KSJ-db/db(以下简称db/db)小鼠随机分为模型组,达格列净组(1.5 mg·kg^(-1)·d^(-1)),DHT高、中、低剂量组(3.6、1.8、0.9 g·kg^(-1)·d^(-1)),每组8只;另取10只C57BL/KSJ-db/dm(以下简称db/m)小鼠为正常组,正常组和模型组给予生理盐水,治疗组小鼠分别给予相应药物,连续给药10周,1次/d。于给药0、4、8、10周固定时间,禁食不禁水12 h,取尾静脉血检测空腹血糖(FBG);于给药0、5、10周末收集尿液检测尿中白蛋白、肌酐含量,计算尿白蛋白肌酐比值(ACR);给药10周后,检测各组小鼠24 h尿总蛋白,血肌酐(Scr),尿素氮(BUN)含量;蛋白免疫印迹法检测肾脏组织p-AMPK、p-mTOR及p-ULK1蛋白的表达水平,以及自噬关键分子酵母Atg6同系物1(Beclin-1)、微管相关蛋白1轻链3(LC3)、P62蛋白的表达水平;免疫组化法检测肾脏组织足细胞裂孔膜蛋白(Nephrin、Podocin)的表达水平;采用光学显微镜和透射电镜观察肾脏组织病理形态学变化。结果:与模型组比较,达格列净组和DHT组小鼠FBG、ACR、24 h尿总蛋白均降低,Scr、BUN无统计学差异;肾组织中p-AMPK、p-ULK1表达水平升高,p-mTOR表达水平降低及LC3Ⅱ/LC3Ⅰ、Beclin-1表达水平升高,P62表达水平降低(P<0.01,P<0.05);肾小球的足细胞裂孔膜蛋白Nephrin、Podocin表达水平升高(P<0.01,P<0.05);肾脏病理损害减轻;透射电镜显示自噬小体、自噬溶酶体数量增加。结论:DHT可能通过调控AMPK/mTOR/ULK1信号通路,增强足细胞自噬,保护肾小球,延缓DN发展进程。

AIM:To explore the intervention effect of Dahuangtang pellets(DHT)on diabetic nephropathy(DN)based on the AMP-activated protein kinase/mammalian target of rapamycin/unc-51-like kinase 1(AMPK/mTOR/ULK1)signaling pathway.METHODS:Eight mice were randomly assigned to the model group,the dapagliflozin group,and the DHT(high,medium,and low dosage)group out of a total of 40 C57BL/KSJ-db/db(hereafter referred to as db/db)mice;another 10 C57BL/KSJ-db/dm mice were used as the normal group,saline was provided to the normal and model groups,and the mice in the treatment group received the appropriate medications.The medications were given for 10 consecutive weeks,once per day,to the mice in the treatment group.At weeks 0,4,8,and 10 of administration,fasting blood glucose(FBG)was assessed by drawing blood at a predetermined time from the tail vein;Urine samples were taken at 0,5,and 10 weeks after treatment to evaluate the levels of albumin and creatinine,and the urinary albumin-creatinine ratio(ACR)was computed.After 10 weeks,mice in each group were assayed for 24 h total urine protein,serum creatinine(Scr),urea nitrogen(BUN)levels;Western blotting analysis was conducted to detect the expression of p-AMPK,p-mTOR,and p-ULK1,as well as the expression of autophagy related proteins homolog of yeast Atg6(Beclin-1),autophagy-related proteins microtubule-associated protein 1 light chain 3(LC3),P62 in renal tissue;Immunohistochemistry was used to measure the expression of podocyte lacunar membrane proteins(Nephrin,Podocin)in renal tissues;The pathological morphology of renal tissue was observed by light microscopy and transmission electron microscopy.RESULTS:Compared with the model group,FBG,ACR,and 24 h total urine protein were reduced in the dapagliflozin group and DHT groups of mice,and there was no statistically significant difference in Scr and BUN;In renal tissues,there is increased expression of p-AMPK and p-ULK1,decreased expression of pmTOR,increased expression of LC3II/LC3I and Beclin-1,and decreased expression of P62(P<0.01,P<0.05);differentially upregulated in glomeruli are the podocyte lacunar membrane proteins Nephrin and Podocin(P<0.01,P<0.05);renal pathologic damage was reduced to varying degrees;transmission electron microscopy showed an increase in the number of autophagic vesicles and autophagic lysosomes.CONCLUSION:DHT can delay the development of DN by regulating the AMPK/mTOR/ULK1 signaling pathway,enhancing podocyte autophagy,and protecting glomeruli.