中药药理与临床

目的:探讨加减补阳还五汤是否通过激活自噬促进核因子E2相关因子2(Nrf2)核转移改善糖尿病肾病(DKD)小鼠足细胞损伤。方法:选取11～12周龄的db/db小鼠24只，经适应性喂养1 w并检测尿蛋白阳性后，随机分为模型对照组、加减补阳还五汤16 g/kg组、雷帕霉素-海藻糖2 mg/kg组，设db/m组小鼠作为正常对照组，每组8只，连续给药8 w。检测小鼠血糖(FBG)、总胆固醇(TC)、甘油三脂(TG)、超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-PX)、尿素氮(BUN)、肌酐(Scr)、肾损伤分子-1(KIM-1)、尿微量白蛋白(mALB)含量或活力；HE染色观察肾脏的病理形态学；qRT-PCR法检测肾组织Wilms肿瘤基因(Wt1)、nephrin、podocin mRNA表达；Western blot法检测小鼠肾组织P62/SQSTM1抗体、Beclin1抗体、微管相关蛋白1轻链3-II抗体(LC3-II)、溶酶体相关膜蛋白1抗体(LAMP1)、Nrf2、Kelch样ECH关联蛋白1(Keap1)、醌氧化还原酶1(NQO-1)、血红素加氧酶-1(HO-1)的蛋白表达；免疫荧光观察Nrf2核转移变化。结果:与正常对照组比较，模型对照组小鼠肾组织出现病理损伤，血清FBG、TC、TG、MDA、BUN、Scr、KIM-1及mALB含量显著升高，SOD、GSH-PX活力显著降低(P<0.01),肾组织P62表达升高，LC3-II、beclin1、LAMP1表达下调(P<0.01),Wt1、Nephrin、Podocin mRNA表达下调(P<0.01),肾组织Keap1蛋白表达上调，NQO-1、HO-1、总Nrf2、核Nrf2蛋白表达下调(P<0.01);与模型对照组比较，经加减补阳还五汤干预后，DKD小鼠肾脏病理损伤显著改善(P<0.01),血清TC、TG、MDA、BUN、Scr、KIM-1及mALB含量显著降低，SOD、GSH-PX活力升高(P<0.01),肾组织P62蛋白表达下调，LC3-II、Beclin1、LAMP1蛋白表达上调(P<0.01),Wt1、Nephrin、Podocin mRNA表达上调(P<0.01),肾组织Keap1蛋白表达下调，NQO-1、HO-1、总Nrf2、核Nrf2蛋白表达上调(P<0.01)。结论:加减补阳还五汤可能通过激活DKD小鼠肾脏细胞自噬，疏通自噬通路，促进Nrf2核转移，抑制氧化应激，保护足细胞，降低尿蛋白排泄，改善DKD肾脏损伤。

Objective:To investigate whether modified Buyang Huanwu(补阳还五) Decoction can ameliorate podocyte injury in the mouse model of diabetic kidney disease(DKD) by activating autophagy to promote the nuclear translocation of nuclear factor-erythroid 2-related factor 2(Nrf2). Methods:A total of 24 db/db mice of 11-12 weeks old were selected. After one week of adaptive feeding and positive urinary protein monitoring, mice were randomized into model, modified Buyang Huanwu Decoction(16 g/kg),and rapamycin-trehalose groups, while 8 db/m mice were included in the control group. Each group contained 8 mice. After 8 weeks of administration, the levels of fasting blood glucose(FBG),total cholesterol(TC),triglyceride(TG), superoxide dismutase(SOD),malondialdehyde(MDA),glutathione peroxidase(GSH-PX),blood urea nitrogen(BUN),serum creatinine(Scr),kidney injury molecule-1(KIM-1),and microalbuminuria(mALB) were measured. The kidney tissue was stained with hematoxylin-eosin to reveal the pathological changes. The mRNA levels of wilms tumor 1 protein(Wt1),nephrin,and podocin were determined by real-time PCR. Western blotting was employed to assess the protein levels of P62/SQSTM1,Beclin1,microtubule-associated protein 1 light chain 3B(LC3-II),lysosomal associated membrane protein 1(LAMP1),Nrf2,Kelch-like ECH-associated protein 1(Keap1),quinone oxidoreductase 1(NQO1),and hemeoxygenase 1(HO-1) in the kidney tissue of mice. Immunofluorescence was utilized to identify the nuclear translocation of Nrf2. Results:Compared with the control group, the model group showed pathological injury in kidneys, elevated serum levels of FBG,TC,TG,MDA,BUN,Scr, KIM-1,and mALB,lowered levels of SOD and GSH-PX(P<0.01). Moreover, the model group presented increased expression of P62,decreased expression of LC3-II,Beclin1,and LAMP1(P<0.01),down-regulated mRNA levels of Wt1,nephrin,and podocin(P<0.01),and activated Nrf2-Keap1 pathway in the renal tissue(P<0.01). Compared with the model group, modified Buyang Huanwu Decoction alleviated the pathological injury in the renal tissue(P<0.01),lowered the serum levels of TC,TG,MDA,BUN,Scr, KIM-1,and mALB(P<0.01), elevated the levels of SOD and GSH-PX(P<0.01). Additionally, the treatment decreased the expression of P62,increased the expression of LC3-II,Beclin1,and LAMP1(P<0.01),restored the mRNA levels of Wt1,nephrin,and podocin,and activated the Nrf2-Keap1 pathway in the renal tissue(P<0.01). Conclusion:Modified Buyang Huanwu Decoction can activate renal cell autophagy, dredge the autophagy pathway, promote the nuclear translocation of Nrf2,inhibit oxidative stress, protect podocytes, and reduce urinary protein excretion, thereby ameliorating the renal injury in DKD.