保肾通络方对KK-Ay小鼠及高糖培养下足细胞凋亡及p53通路的作用研究  

Research of Baoshentongluo Formula on Podocyte Apoptosis and p53 Pathway in KK-Ay Mice and Podocytes Cultured with High Glucose

作　　者：郭弋凡 张宁[3] 刘羽飞 庞彦余 赵婧文 崔方强 刘梦超 王梦迪 赵文景[2] GUO Yifan;ZHANG Ning;LIU Yufei(School of Clinical Medicine,Beijing University of Chinese Medicine,Beijing,100029;Department of Nephrology,Beijing Hospital of Traditional Chinese Medicine,Capital Medical University,Beijing,100010)

机构地区：[1]北京中医药大学临床医学院,北京100029 [2]首都医科大学附属北京中医医院肾病科,北京100010 [3]中国中医科学院望京医院肾病内分泌科,北京100102

出　　处：《中国中西医结合肾病杂志》2024年第3期200-205,I0002,I0003,共8页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：北京市自然科学基金资助项目(No.7222271);首都医科大学附属北京中医医院职工助推项目(No.ZT202116)。

摘　　要：目的:探讨保肾通络方对KK-Ay小鼠及高糖培养下足细胞凋亡及p53通路的影响。方法:将KK-Ay小鼠高脂饲料喂养建立糖尿病模型,造模成功后随机分为模型组(10只)与保肾通络方组(10只),另设10只C57BL/6J小鼠作为对照组;根据培养条件不同将体外小鼠肾小球足细胞(MPC5)分为正常对照组、高糖组、保肾通络方含药血清组。测定小鼠24 h尿蛋白定量(24 h-UTP)水平并进行肾组织病理学检查,CCK-8法检测足细胞活力,Western blot检测足细胞凋亡相关蛋白Bcl-2、Bax、cleaved-caspase3及p53、p-p53的表达,TUNEL染色观察各组足细胞凋亡情况。结果:与对照组相比,模型组小鼠24 h-UTP显著升高(P<0.01),肾小球系膜细胞增生,细胞外基质增多,肾小球凋亡细胞比例及cleaved-caspase3蛋白表达增加(P<0.05),肾组织抗凋亡蛋白Bcl-2表达降低,促凋亡蛋白Bax表达增加,p53磷酸化水平增加(P<0.01);保肾通络方干预使KK-Ay小鼠24 h-UTP显著降低(P<0.01),肾脏病理改善,肾小球凋亡细胞比例及cleaved-caspase3蛋白表达减少(P<0.05),Bcl-2、Bax及p-p53表达恢复(P<0.05)。体外研究显示,高糖组足细胞活力明显降低(P<0.01),足细胞凋亡比例及cleaved-caspase3蛋白表达增加(P<0.05);Bcl-2表达减少,Bax和p-p53表达增加(P<0.05);而保肾通络方含药血清能够恢复足细胞活力(P<0.01),抑制细胞凋亡发生并降低高糖诱导的足细胞p-p53的表达(P<0.05)。结论:保肾通络方能够减轻KK-Ay小鼠及高糖诱导的足细胞凋亡,恢复促凋亡与抗凋亡失衡,抑制p53通路的激活,这可能是其发挥足细胞保护作用的潜在机制。Objective:To investigate the effect of Baoshentongluo formula on podocyte apoptosis and p53 pathway in KK-Ay mice and podocytes cultured with high glucose.Methods:KK-Ay mice were fed with high-fat diet to establish a diabetic model.After successful modeling,the mice were randomly divided into model group(10 mice)and kidney-protecting formula group(10 mice),and 10 C57BL/6J mice were set up as the control group;in vitro mouse glomerular foot cells(MPC5)were divided into normal control group,high glucose group and kidney-protecting formula containing drug serum group according to different culture conditions.The 24 h urine protein quantification(24 h-UTP)levels of mice were measured and histopathological examination of kidney was performed.The viability of podocytes was measured by CCK-8 method,the expression of apoptosis-related proteins Bcl-2,Bax,cleaved-caspase3 and p53,p-p53 were detected by Western blot,and the apoptosis of podocytes in each group was observed by TUNEL staining.Results:Compared with the control group,the model group showed significantly increased 24 h-UTP(P<0.01),proliferated glomerular mesangial cells,increased the extracellular matrix,elevated the proportion of apoptotic cells and the expression of cleaved-caspase3 protein(P<0.05),reduced the expression of anti-apoptotic protein Bcl-2,increased the expression of pro-apoptotic protein Bax and the phosphorylation level of p53(P<0.01).Baoshentongluo formula intervention significantly reduced the 24 h-UTP level in KK-Ay mice(P<0.01),improved renal pathological state,restored the proportion of glomerular apoptotic cells and the expression of cleaved-caspase3(P<0.05),decreased the expression of Bcl-2,recovered the expression of Bax and p-p53(P<0.05).In vitro study showed that high glucose group were significantly decreased the activity of podocytes(P<0.01),increased the proportion of podocyte apoptosis and the expression of cleaved-caspase3(P<0.05),decreased the expression of Bcl-2,elevated the expression of Bax and p-p53(P<0.05);Baoshentongluo for

关 键 词：保肾通络方 糖尿病肾脏疾病 足细胞 凋亡 p53 

分 类 号：R285.5[医药卫生—中药学]