基于mTOR/p70S6K/4EBP1通路探讨白花丹素对糖尿病肾病大鼠足细胞凋亡的影响

Effects of Plumbagin on Apoptosis of Podocytes in Rats with Diabetic Nephropathy Based on mTOR/p70S6K/4EBP1 Pathway

目的:探究白花丹素对糖尿病肾病(DN)大鼠足细胞凋亡及哺乳动物雷帕霉素靶蛋白(mTOR)/p70核糖体蛋白S6激酶(p70S6K)/真核细胞翻译起始因子4E结合蛋白1(4EBP1)通路的影响。方法:SD大鼠分为正常对照组、模型对照组、雷帕霉素2 mg/kg组、白花丹素50、100 mg/kg组、白花丹素100 mg/kg+自噬抑制剂2 mg/kg组,每组12只。除正常对照组外,其余各组采用高脂高糖饲料喂养联合链脲佐菌素腹腔注射的方法建立DN大鼠模型,造模成功后,各组给予相应药物处理,1次/d,连续给药8 w。每日观察大鼠的一般状态,并比较给药前后大鼠的体质量变化;检测大鼠24 h尿微量白蛋白(U-mAlb)含量,并检测空腹血糖(FBG)、血肌酐(Scr)和血尿素氮(BUN)含量;过碘酸雪夫染色观察肾组织病理学变化;免疫组织化学法检测足细胞标志蛋白足细胞裂孔膜蛋白(Nephrin)、肾母细胞瘤基因1(WT-1)的表达;透射电镜观察肾小球足细胞超微结构变化;蛋白印迹法检测肾脏组织凋亡、自噬和mTOR/p70S6K/4EBP1通路相关蛋白的表达。结果:与正常对照组比较,模型对照组大鼠的一般状态较差,出现肾小球基底膜增厚、系膜基质增多等肾脏病理损伤以及肾小球足细胞足突倒伏、足突相互融合、部分足突消失,24 h U-mAlb、FBG、Scr、BUN含量、肾组织半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、Bcl-2相关X蛋白(BAX)蛋白和mTOR(p-mTOR)/mTOR、p70S6K(p-P70S6K)/P70S6K、4EBP1(p-4EBP1)/4EBP1蛋白比值明显升高(P<0.05),肾组织Nephrin、WT-1、B淋巴细胞瘤-2基因(BCL-2)、自噬相关基因12(Atg12)蛋白和微管相关蛋白1轻链3(LC3)-II/LC3-Ⅰ蛋白比值明显降低(P<0.05);与模型对照组比较,白花丹素50、100 mg/kg组大鼠的一般状态、肾脏病理和肾小球足细胞损伤明显改善,24 h U-mAlb、FBG、Scr、BUN含量、肾组织Caspase-3、BAX蛋白、p-mTOR/mTOR、p-P70S6K/p70S6K、p-4EBP1/4EBP1蛋白比值明显降低(P<0.05),肾组织Nephrin、WT-1、BCL-2、Atg12蛋白、LC3-II/LC3-Ⅰ蛋白比值明显升高(P<0.05);自噬抑制剂3-甲基腺嘌呤能减弱白花丹素100 mg/kg对DN大鼠的改善作用(P<0.05)。结论:白花丹素可能通过抑制mTOR/P70S6K/4EBP1信号通路,激活自噬,抑制足细胞凋亡,进而减轻DN大鼠肾脏损伤。

Objective:To explore the effects of plumbagin on podocyte apoptosis and mammalian target of rapamycin(mTOR)/p70 ribosomal protein S6 kinase(p70S6K)/eukaryotic translation initiation factor 4E binding protein 1(4EBP1)pathway in rats with diabetic nephropathy(DN).Methods:SD rats were divided into normal control group,model group,rapamycin(2 mg/kg)group,plumbagin(50 or 100 mg/kg)groups,plumbagin(100 mg/kg)+autophagy inhibitor(2 mg/kg)group,with 12 rats in each group.Except for normal control group,the rats in other groups were fed with high fat and high sugar diet combined with intraperitoneal injection of streptozotocin to establish the DN model.After successful modeling,the rats in each group received corresponding drug treatment once a day for 8 weeks.The general state of rats was observed daily,and the changes of body weight were compared before and after the drug administration.The levels of 24 h urine microalbumin(U-mAlb),fasting blood glucose(FBG),blood creatinine(Scr)and blood urea nitrogen(BUN)were detected.The pathological changes of kidney tissue were determined with Periodic Acid-Schiff staining.The expressions of podocyte slit diaphragm protein Nephrin and Wilm's tumor gene 1(WT-1)were detected with immunohistochemical method,and the ultrastructural changes of glomerular podocytes were observed under transmission electron microscope.The expressions of apoptosis-,autophagy-,and mTOR/p70S6K/4EBP1 pathway-related proteins in kidney tissues were detected by Western blot.Results:Compared with normal control group,the general state of rats in the model group was poor,with renal pathological injuries such as thickening of glomerular basement membrane and increased mesangial matrix,lodging of glomerular podocytes foot process,fusion of foot process and disappearance of part of foot process.The contents of 24 h U-mAlb,FBG,Scr and BUN,the protein expression levels of Caspase-3,Bcl-2-related X protein(Bax)and the ratios of phosphorylated mTOR(p-mTOR)/mTOR,phosphorylated p70S6K(p-p70S6K)/p70S6K,phosphorylated 4EBP1(p-4EBP1)/4EBP1 in kidney tissue were significantly increased(P<0.05).The protein expression levels of Nephrin,WT-1,B cell lymphoma-2(Bcl-2),autophagy related gene 12(Atg12)and the ratio of microtubule associated protein 1 light chain 3(LC3)-II/LC3-I in kidney tissue were significantly decreased(P<0.05).Compared with model group,the general state,renal pathology and glomerular podocyte injury of rats in plumbagin 50 and 100 mg/kg groups were significantly improved.The contents of 24 h U-mAlb,FBG,Scr and BUN,the protein expressions of Caspase-3 and Bax,and the ratios of p-mTOR/mTOR,pp70S6K/p70S6K and p-4EBP1/4EBP1 in kidney tissue were significantly decreased(P<0.05).The protein expressions of Nephrin,WT-1,Bcl-2,Atg12,and ratio of LC3-II/LC3-Ⅰin kidney tissue were significantly increased(P<0.05).The autophagy inhibitor 3-methyladenine could attenuate the effect of plumbagin(100 mg/kg)on DN rats(P<0.05).Conclusion:Plumbagin may reduce kidney injury in DN rats by inhibiting the mTOR/p70S6K/4EBP1 signaling pathway,activating autophagy and inhibiting podocyte apoptosis.