基于Nrf2/FPN1信号通路探究活血地龟汤对糖尿病肾病小鼠肾脏足细胞铁积累和铁死亡的影响

Effects of Huoxue Digui Decoction on Iron Accumulation and Ferroptosis in Renal Podocytes of Diabetic Nephropathy Mice Based on Nrf2/FPN1 Signaling Pathway

目的观察活血地龟汤对糖尿病肾病(DN)小鼠肾脏足细胞铁积累及铁死亡的影响。方法将50只C57BL/6J小鼠随机分为正常组6只和造模组44只,采用链脲佐菌素注射建立DN小鼠模型,将成模小鼠随机分为模型组8只,萝卜硫素组及活血地龟汤低、中、高剂量组各6只。萝卜硫素组予萝卜硫素腹腔注射,活血地龟汤各剂量组分别予相应溶液灌胃,连续12周。检测小鼠空腹血糖、血尿素氮、血肌酐、24 h尿白蛋白含量,HE染色观察肾组织形态,PAS染色观察肾组织糖原沉积,Masson染色观察肾组织纤维化情况,透射电镜观察肾组织超微结构,免疫组化检测肾组织Podocin、Nephrin表达,Western blot检测肾组织谷胱甘肽过氧化物酶4(GPX4)、ACSL4、核因子E2相关因子2(Nrf2)、膜铁转运蛋白1(FPN1)蛋白表达,普鲁士蓝染色观察肾组织铁离子沉积情况。结果与正常组比较,模型组小鼠空腹血糖、血尿素氮、血肌酐、24 h尿白蛋白含量均明显升高(P<0.01),肾组织毛细血管团萎缩,糖原沉积增加,纤维化加重,肾组织Podocin、Nephrin表达下调,GPX4、Nrf2、FPN1蛋白表达明显降低(P<0.01),ACSL4蛋白表达明显升高(P<0.01),肾组织铁离子沉积增多;与模型组比较,活血地龟汤各剂量组及萝卜硫素组小鼠空腹血糖、血尿素氮、血肌酐、24 h尿白蛋白含量均不同程度降低(P<0.05,P<0.01),肾组织病理损伤不同程度减轻,糖原沉积减少,纤维化减轻,肾组织Podocin、Nephrin表达上调,GPX4、Nrf2、FPN1蛋白表达明显升高(P<0.01),ACSL4蛋白表达明显降低(P<0.05,P<0.01),肾组织铁离子沉积减少。结论活血地龟汤能减轻DN小鼠肾组织病理损伤,降低血糖水平,延缓DN进展,其机制可能与调节Nrf2、FPN1表达,抑制DN小鼠肾脏足细胞铁积累和铁死亡有关。

Objective To observe the effects of Huoxue Digui Decoction on iron accumulation and ferroptosis in renal podocytes of diabetic nephropathy(DN)mice.Methods Totally 50 C57BL/6J mice were randomly divided into normal group(6 mice)and modeling group(44 mice).The DN model was established by streptozotocin injection.Mice conforming to DN model were randomly divided into model group(8 mice),and sulforaphane group,Huoxue Digui Decoction low-,medium-and high-dosage groups(6 mice for each group).The sulforaphane group was injected with sulforaphane intraperitoneally, while Huoxue Digui Decoction low-, medium- and high-dosage groupswere orally administered with corresponding solution for 12 consecutive weeks. The fasting blood glucose, bloodurea nitrogen, serum creatinine, 24 h urinary albumin were detected, HE staining was used to observe the morphologyof renal tissue, PAS staining was used to observe glycogen deposition in renal tissue, Masson staining was used toobserve fibrosis in renal tissue;transmission electron microscopy was used to observe the ultrastructure of renaltissue, immunohistochemistry was used to detect the expressions of Podocin and Nephrin in renal tissue, Western blotwas used to detect the protein expressions of glutathione peroxidase 4 (GPX4), ACSL4, nuclear factor E2 relatedfactor 2 (Nrf2) and ferroportin 1 (FPN1) in renal tissue, Prussian blue staining was used to observe the deposition offerric ion in renal tissue. Results Compared with the normal group, the fasting blood glucose, blood urea nitrogen,serum creatinine and 24 h urinary albumin in model group significantly increased (P<0.01), with renal tissue capillaryatrophy, increased glycogen deposition, and worsening fibrosis, the expressions of Podocin and Nephrin in renaltissue were down-regulated, while the expressions of GPX4, Nrf2 and FPN1 protein were significantly decreased (P<0.01), the expression of ACSL4 protein significantly increased (P<0.01), and the deposition of ferric ion in renaltissue increased. Compared with the model group, the fasting blood glucose, blood urea nitrogen, serum creatinineand 24 h urinary albumin in sulforaphane group and Huoxue Digui Decoction groups were reduced to differentdegrees (P<0.05, P<0.01), renal tissue pathological damage was reduced to varying degrees, glycogen deposition wasreduced, and fibrosis was alleviated, the expressions of Podocin and Nephrin in renal tissue were up-regulated,while the expressions of GPX4, Nrf2 and FPN1 protein significantly increased (P<0.01), the expression of ACSL4protein significantly decreased (P<0.05, P<0.01), and the deposition of ferric ion in renal tissue was reduced.Conclusion Huoxue Digui Decoction can alleviate renal pathological injury, reduce blood glucose and delay theprogression of DN mice. The mechanism may be related to regulating the expressions of Nrf2 and FPN1, inhibitingiron accumulation and ferroptosis in renal podocytes of DN mice.