加味升降散对糖尿病肾病大鼠肾脏内质网应激和Sirt1/PERK通路的影响  

Effect of Modified Shengjiangsan on Renal Endoplasmic Reticulum Stress and Sirt1/PERK Pathway in Rat Model of Diabetic Nephropathy

作　　者：任美芳 吴振华 高飞[3] 袁国栋 张倩 郭晓玲 杨凤文 REN Meifang;WU Zhenhua;GAO Fei;YUAN Guodong;ZHANG Qian;GUO Xiaoling;YANG Fengwen(The First Affiliated Hospital of Hebei University of Chinese Medicine,Shijiazhuang 050011,China;Hebei University of Chinese Medicine,Shijiazhuang 050200,China;Dongzhimen Hospital of Beijing University of Chinese Medicine,Beijing 100700,China)

机构地区：[1]河北中医药大学第一附属医院,石家庄050011 [2]河北中医药大学,石家庄050200 [3]北京中医药大学东直门医院,北京100700

出　　处：《中国实验方剂学杂志》2024年第14期55-62,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：河北省中医药管理局科研计划课题(2022333);第七批全国老中医药专家学术经验继承工作(国中医药办人教函〔2021〕272号)。

摘　　要：目的:探讨加味升降散减轻糖尿病肾病(DN)大鼠内质网应激,降低尿蛋白的分子机制。方法:将75只SD大鼠随机分为正常组、模型组、加味升降散低、中、高剂量组(4.37、8.73、17.46 g·kg^(-1))和厄贝沙坦组(0.014 g·kg^(-1)),每组10只。分别给予相应剂量药物或者蒸馏水灌胃,每日1次,连续给药8周。末次给药后检测大鼠血糖(GLU)、24 h尿蛋白(UTP)含量,肾脏组织中超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)水平;苏木素-伊红(HE)染色、过碘酸-雪夫(PAS)染色和透射电镜观察大鼠肾脏病理学改变;免疫组化法(IHC)检测大鼠肾脏中肾病蛋白(Nephrin)、足细胞裂隙膜蛋白(Podocin)、葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)和激活转录因子4(ATF4)蛋白表达水平;蛋白免疫印迹法(Western blot)检测大鼠肾脏中沉默调节蛋白1(Sirt1)、磷酸化(p)-蛋白激酶RNA样内质网激酶(PERK)、p-真核翻译起始因子(eIF2α)蛋白表达水平。结果:与正常组比较,模型组大鼠肾脏病理损伤较重,GLU、UTP水平明显升高(P<0.05),大鼠肾脏中GRP78、CHOP、ATF4、p-PERK、p-eIF2α蛋白表达水平升高、Sirt1蛋白表达下降(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GLU、24 h UTP水平有不同程度的降低(P<0.05),肾脏病理损伤明显减轻,GRP78、CHOP、ATF4、p-PERK、p-eIF2α蛋白表达水平均下降(P<0.05),Sirt1蛋白表达上升(P<0.05)。结论:加味升降散上调DN大鼠肾脏组织中Sirt1表达,抑制PERK/eIF2α通路蛋白的磷酸化,减轻肾组织ER应激反应和氧化应激,是其减轻DN大鼠肾脏病理损伤和尿蛋白的作用机制。Objective:To explore the molecular mechanism of modified Shengjiangsan in alleviating endoplasmic reticulum(ER)stress and reducing urinary protein in the rat model of diabetic nephropathy(DN)Method:Seventy-five SD rats were randomized into normal,model,low-,medium-,and high-dose(4.37,8.73,17.46 g·kg^(-1),respectively)modified Shengjiangsan,and irbesartan(0.014 g·kg^(-1))groups,with 10 rats in each group.Rats were administrated with corresponding doses of medications or distilled water by gavage,once a day,for 8 consecutive weeks.After the last administration,the levels of glucose(GLU)in the blood,24-hour urinary protein(24 h-UTP),and superoxide dismutase(SOD),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)in the renal tissue were measured.Hematoxylin-eosin staining,periodic acid-Schiff staining,and transmission electron microscopy were employed to observe the pathological changes in rat kidneys.Immunohistochemistry was employed to measure the expression levels of nephrin,podocin,glucoseregulated protein 78(GRP78),C/EBP homologous protein(CHOP),and activating transcription factor 4(ATF4)in the kidneys of rats.Western blot was employed to measure the protein levels of silent information regulator 1(Sirt1),phosphorylated(p)-protein kinase RNA-like endoplasmic reticulum kinase(PERK),and p-eukaryotic translation initiation factor 2 alpha(eIF2α)in rat kidneys.Result:Compared with the normal group,the modeling caused pathological damage to the kidneys,elevated the levels of GLU and 24 h-UTP(P<0.05),up-regulated the protein levels of GRP78,CHOP,ATF4,p-PERK,and p-eIF2α(P<0.05),and down-regulated the protein level of Sirt1(P<0.05)in rat kidneys.Compared with the model group,modified Shengjiangsan and irbesartan lowered the GLU and 24 h-UTP levels(P<0.05),alleviated the pathological damage in the renal tissue,down-regulated the protein levels of GRP78,CHOP,ATF4,p-PERK,and p-eIF2α(P<0.05),and up-regulated the protein level of Sirt1(P<0.05).Conclusion:Modified Shengjiangsan up-regulates Sirt1 expression and inhib

关 键 词：加味升降散 糖尿病肾病 足细胞损伤 氧化应激 内质网应激 沉默调节蛋白1(Sirt1)/蛋白激酶RNA样内质网激酶(PERK)通路 

分 类 号：R2-0[医药卫生—中医学] R33R289R587.1R692