加味升降散通过介导RIP1/RIP3/MLKL通路抑制坏死性凋亡减轻糖尿病肾病大鼠肾脏纤维化

Modified Shengjiangsan Inhibits Necroptosis by Mediating RIP1/RIP3/MLKL Signaling Pathway and Reduces Renal Fibrosis in Rats with Diabetic Nephropathy

目的:探讨加味升降散基于刺激受体相互作用蛋白(RIP)1/RIP3/混合谱系激酶结构域样假激酶(MLKL)通路对糖尿病肾病大鼠坏死性凋亡及肾纤维化的干预机制。方法:75只SD大鼠分为正常组、模型组、加味升降散低、中、高剂量组(4.365、8.73、17.46 g·kg^(-1))和厄贝沙坦组(0.013 5 g·kg^(-1)),灌胃给药干预4周后,检测各组大鼠24 h尿蛋白定量(UTP),血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)水平,肾脏病理变化程度;实时荧光定量聚合酶链式反应(Real-time PCR)法和免疫组化分别检测大鼠肾组织中IL-1β、TNF-α、单核细胞趋化蛋白-1(MCP-1)、转化生长因子-β1(TGF-β1)、核转录因子-κB(NF-κB)mRNA和蛋白表达水平;蛋白免疫印迹法(Western blot)检测RIP1/RIP3/MLKL信号通路关键蛋白的表达水平。结果:与正常组比较,模型组大鼠肾脏间质纤维化明显,24 h UTP、IL-1β、TNF-α水平明显升高(P<0.05),肾组织中IL-1β、TNF-α、MCP-1、TGF-β1、NF-κB mRNA和蛋白表达水平明显升高(P<0.05),RIP1、RIP3、磷酸化(p)-MLKL、MLKL蛋白表达量明显增多(P<0.05);与模型组比较,加味升降散各剂量组及厄贝沙坦组大鼠肾脏间质纤维化水平均有不同程度改善,24 h UTP水平均有不同程度降低(P<0.05),血清中IL-1β、TNF-α含量明显降低(P<0.05),肾组织中IL-1β,TNF-α,MCP-1,TGF-β1,NF-κB mRNA和蛋白表达水平明显下调(P<0.05),RIP1、RIP3、p-MLKL、MLKL蛋白表达量明显下调(P<0.05)。结论:加味升降散可以改善糖尿病肾病大鼠肾损伤,其机制可能与下调RIP1/RIP3/MLKL信号通路,阻止肾组织坏死性凋亡,抑制肾纤维化有关。

Objective:To observe the mechanism of modified Shengjiangsan in necroptosis and renal fibrosis of rats with diabetic nephropathy based on receptor-interacting protein(RIP)1/RIP3/mixed lineage kinase domain-like protein(MLKL)signaling pathway.Method:Seventy-five SD rats were randomly divided into a model group,a normal group,three high,medium,and low-dose modified Shengjiangsan groups(4.365,8.73,17.46 g·kg^(-1)),and an irbesartan group(0.013 5 g·kg^(-1)).After 4 weeks of intragastric administration,the levels of 24 h urine protein(UTP),tumor necrosis factor-α(TNF-α),and interleukin-1β(IL-1β)of rats in each group were determined,as well as the changes in degree of renal pathology.Real-time quantitative polymerase chain reaction(Real-time PCR)and immunohistochemistry were used to detect the mRNA and protein expression levels of IL-1β,TNF-α,monocyte chemoattractant protein-1(MCP-1),transforming growth factor-β1(TGF-β1),and nuclear factor kappa B(NF-κB)in kidney tissues of rats.Western blot assay was used to detect the expression levels of key proteins in the RIP1/RIP3/MLKL signaling pathway.Result:As compared with the normal group,the renal interstitial fibrosis in the model group was obvious,and the 24 h UTP,IL-1β,TNF-α levels were significantly increased(P<0.05).In the model group,the mRNA and protein expression levels of IL-1β,TNF-α,MCP-1,TGF-β1,and NF-κB in the kidney tissues were significantly increased(P<0.05),and protein expression levels of RIP1,RIP3,p-MLKL,and MLKL were significantly increased(P<0.05).Compared with the model group,all modified Shengjiangsan groups and the irbesartan group improved the levels of renal interstitial fibrosis in rats to varying degrees.As compared with the model group,the 24 h UTP levels in all modified Shengjiangsan groups and the irbesartan group were decreased to varying degrees(P<0.05),the content of IL-1β and TNF-α in the serum were decreased(P<0.05),the mRNA and protein expression levels of IL-1β,TNF-α,MCP-1,TGF-β1,and NF-κB in renal tissues was downregulated(P<0.05),and the protein expression levels of RIP1,RIP3,p-MLKL,and MLKL were downregulated(P<0.05).Conclusion:Modified Shengjiangsan ameliorates renal injury of rats with diabetic nephropathy,and the mechanism may be related to the down-regulation of the RIP1/RIP3/MLKL signaling pathway,the prevention of renal tissue necroptosis,and the inhibition of renal fibrosis.