补肾活血方抑制PERK/ATF4/CHOP通路改善db/db小鼠糖尿病肾病的机制研究  

Mechanism of Inhibiting PERK/ATF4/CHOP Pathway to Improve Diabetic Nephropathy in db/db Mice by Bushen Huoxue Formula

作　　者：韦茂英 曲莲莲 高云逸 赵琦瑶 方泽阳 潘欣渝 魏军平[1] WEI Mao-ying;QU Lian-lian;GAO Yun-yi;ZHAO Qi-yao;FANG Ze-yang;PAN Xin-yu;WEI Jun-ping(Guang′anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100053;Dongzhimen Hospital of Beijing University of Chinese Medicine,Beijing 100700;Traditional Chinese Medicine Hospital of Penglai City,Penglai Shandong 265600)

机构地区：[1]中国中医科学院广安门医院,北京100053 [2]北京中医药大学东直门医院,北京100700 [3]蓬莱市中医医院,山东蓬莱265600

出　　处：《世界中西医结合杂志》2025年第3期425-432,共8页World Journal of Integrated Traditional and Western Medicine

基　　金：北京市自然科学基金面上项目(7202172);中央高水平中医医院临床科研业务费资助(HLCMHPP2023098);国家资助博士后研究人员计划项目(GZC20230324);中国博士后科学基金面上项目(2024M750263);中国中医科学院科技创新工程项目(CI2021A01617);中国中医科学院中医药循证能力建设项目(60104)。

摘　　要：目的探讨补肾活血方对db/db小鼠肾损伤的影响和作用机制。方法将48只雄性db/db小鼠按随机数字表法分为模型组、补肾活血方低剂量组(0.78 g/kg)、补肾活血方高剂量组(3.12 g/kg)和缬沙坦组(10.40 mg/kg),每组各12只。另设12只同周龄同性别db/m小鼠作为对照组。各给药组给予相应药物,对照组和模型组给予同等体积0.5%羧甲基纤维素钠溶液,灌胃1次/d,连续给药12周。给药期间每4周检测1次空腹血糖(Fasting blood glucose,FBG),实验结束后检测各组尿素氮(Blood urea nitrogen,BUN)、血肌酐(Serum creatinine,Scr)和尿微量白蛋白排泄率(Urinary albumin excretion rate,UAER)。透射电镜观察肾小管上皮细胞超微结构;免疫组化法检测肾脏中GRP78阳性细胞表达;RT-PCR法检测肾组织GRP78 mRNA表达;Western blot法检测肾组织磷酸化蛋白激酶样内质网激酶(phospho-protein kinase r-like endoplasmic reticulum kinase,p-PERK)/蛋白激酶样内质网激酶(Protein kinase r-like endoplasmic reticulum kinase,PERK)、磷酸化真核翻译起始因子2α(phosphorylated eukaryotic translation initiation factor 2 alpha,p-eIF2α)/真核起始因子2α(eukaryotic initiation factor 2 alpha,eIF2α)、活化转录因子4(Activating transcription factor 4,ATF4)、C/EBP同源蛋白(C/EBP-homologous protein,CHOP)、B淋巴细胞瘤2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、切割型半胱天冬酶-3(Cleaved-caspase-3)和切割型半胱天冬酶-9(Cleave-caspase-9)蛋白表达。结果与对照组比较,模型组FBG、BUN、Scr、UAER水平明显升高(P<0.01);肾小管区域GRP78阳性表达增加;肾小管上皮细胞出现核膜皱缩,染色质凝聚,细胞核变形等改变;肾组织p-PERK/PERK、p-eIF2α/eIF2α、ATF4、CHOP、Bax、Cleaved-caspase-3、Cleave-caspase-9蛋白和GRP78 mRNA表达明显增加,Bcl-2表达明显减少(P<0.01)。与模型组比较,补肾活血方低剂量组、补肾活血方高剂量组和缬沙坦组FBG无明显变Objective To investigate the effects and action mechanism of Bushen Huoxue formula on renal injury in db/db mice.Methods 48 male db/db mice were divided into a control group,a model group,a low-dose Bushen Huoxue formula group(0.78 g/kg),a high-dose Bushen Huoxue formula group(3.12 g/kg),and a valsartan group(10.40 mg/kg)using random number table,with 12 mice in each group.In addition,12 db/m mice of the same age and sex were set up as the antrol group.Each administration group was given the corresponding drug.The control and model groups were given equal volumes of 0.5%sodium carboxymethylcellulose solution by gavage once a day for 12 weeks.During administration,fasting blood glucose(FBG)was tested every 4 weeks.At the end of the experiment,blood urea nitrogen(BUN),serum creatinine(Scr),and urinary microalbumin excretion rate(UAER)were measured in each group.The ultrastructure of renal tubular epithelial cells was detected by transmission electron microscopy.The expression of glucose regulated protein 78(GRP78)-positive cells in the kidney was detected by immunohistochemistry.The expression of GRP78 mRNA in renal tissues was detected by RT-PCR.Western blot assay was employed to detect the expression of phosphorylated-protein kinase r-like endoplasmic reticulum kinase(p-PERK)/protein kinase r-like endoplasmic reticulum kinase(PERK),phosphorylated eukaryotic translation initiation factor 2 alpha(p-eIF2α)/eukaryotic initiation factor 2 alpha(eIF2α),activating transcription factor 4(ATF4),C/EBP-homologous protein(CHOP),B-cell lymphoma-2(Bcl-2),Bcl2 associated X protein(Bax),Cleaved-caspase-3,and Cleaved-caspase-9 proteins in renal tissues.Results Compared with the control group,the model group demonstrated significantly higher levels of FBG,BUN,Scr,and UAER(P<0.01).Besides,GRP78-positive expression was increased in the renal tubular region and renal tubular epithelial cells exhibited changes including wrinkled nuclear membranes,condensed chromatin,and deformed nuclei.In renal tissues,the expression of the followin

关 键 词：糖尿病肾病 补肾活血方 内质网应激 细胞凋亡 肾脏保护 

分 类 号：R285.5[医药卫生—中药学]