摘要
目的:从内皮细胞及细胞能量代谢角度,探讨益肾活血方对肾间质纤维化(RIF)的作用机制。方法:采用单侧输尿管梗阻(UUO)方法造模成功后将75只SPF级C57BL/6小鼠随机分为模型组、白藜芦醇组(50 mg·kg^(-1)·d^(-1))、益肾活血方低、中、高剂量组(7.1、14.2、28.4 g·kg^(-1)·d^(-1)),每组15只。另设15只作为假手术组。假手术组及模型组予等体积生理盐水灌胃。所有小鼠于造模术后第7、14、21天(d7、d14、d21)处死并取材,采用免疫组织化学S-P法检测血小板内皮细胞黏附分子31(CD31)蛋白表达,蛋白免疫印迹法(Western blot)检测肾组织α-平滑肌肌动蛋白(α-SMA)、Ⅳ型胶原蛋白(Col-Ⅳ)、促血管生成素-1(Ang-1)/酪氨酸激酶受体-2(Tie-2)、血管内皮生长因子(VEGF)、血管内皮钙黏蛋白(VE-cadherin)、闭合蛋白(Occludin)蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测肾组织Ang-1/Tie-2、VEGF、VE-cadherin、Occludin mRNA表达,酶联免疫吸附测定法(ELISA)检测小鼠活性氧(ROS)水平。结果:与假手术组比较,模型组肾组织中CD31表达明显减少且随造模时间延长而加重(P<0.05),α-SAM、Col-Ⅳ蛋白表达水平显著升高(P<0.01);但d14~d21间CD31表达趋于平稳;ROS水平显著升高(P<0.01);Ang-1/Tie-2、VEGF、VE-cadherin、Occludin蛋白表达及mRNA表达显著下调(P<0.01)。与模型组比较,白藜芦醇组、益肾活血方中、高剂量组治疗后CD31表达量明显增加(P<0.05),α-SAM、Col-Ⅳ显著下降(P<0.01),ROS含量显著减少(P<0.01),Ang-1/Tie-2、VEGF、VE-cadherin、Occludin的蛋白表达及mRNA表达量均显著上调(P<0.01)。与白藜芦醇组比较,益肾活血方低剂量组与益肾活血方中剂量组Ang-1/Tie-2、VEGF、VE-cadherin、Occludin蛋白表达具有显著差异(P<0.01),益肾活血方高剂量组CD31、Ang-1/Tie-2 mRNA表达量差异无统计学意义,益肾活血方中剂量组ROS水平差异无统计学意义。结论:益肾活血方抗RIF作用可能与促进血管内皮修复、调控线粒体ROS减轻氧化应激,保护肾脏内皮结构完整性,延缓细胞凋亡,维持细胞能量代谢有关。
Objective: To explore the mechanism of Yishen Huoxue prescription in renal interstitial fibrosis(RIF)from the perspective of endothelial cell and cell energy metabolism.Method: The model was successfully established by unilateral ureteral obstruction(UUO).Seventy-five SPF C57BL/6 mice were randomly divided into a model group,a resveratrol group(50 mg·kg^(-1)·d^(-1)),three Yishen Huoxue prescription low,medium,and high-dose groups(7.1,14.2,28.4 g·kg^(-1)·d^(-1)),with 15 mice in each group.In addition,another 15 mice were used to prepare sham operation model.Mice in the sham operation group and the model group were gavaged with equal volume of normal saline.All mice were sacrificed on 7,14,and 21 d after modeling.The protein expression of platelet endothelial cell adhesion molecule 31(CD31)was detected by immunohistochemical S-P method.The expression ofα-smooth muscle actin(α-SMA),collagenⅣ(Col-Ⅳ),angiopoietin 1(Ang-1)and tyrosine kinase receptors 2(Tie-2),vascular endothelial growth factor(VEGF),vascular endothelial cadherin(VE-cadherin),and occludin in renal tissues was detected by Western blotting.The mRNA expressions of Ang-1/Tie-2,VEGF,VE-cadherin,and occludin in renal tissues were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and the levels of reactive oxygen species(ROS)in mice were detected by enzyme linked immunosorbent assay(ELISA).Result: As compared with the sham operation group,the expression of CD31 in renal tissues of the model group was significantly decreased and worsened with the extension of modeling time(P<0.05),α-SAM and Col-Ⅳprotein expression levels were significantly increased(P<0.01),but the expression of CD31 was stable in 14-21 d.ROS levels were significantly increased(P<0.01),and the protein and mRNA expressions of Ang-1/Tie-2,VEGF,VE-cadherin,and occludin were significantly down-regulated(P<0.01).As compared with the model group,the expression of CD31 was increased(P<0.05),andα-SAM and Col-Ⅳin the resveratrol group and the medium and high-dose Yishen Huoxue prescription groups were significantly decreased(P<0.01).The ROS content was significantly decreased(P<0.01),and the protein and mRNA expressions of Ang-1/Tie-2,VEGF,VE-cadherin,and occludin were up-regulated(P<0.01),As compared with the resveratrol group,the protein expressions of Ang-1/Tie-2,VEGF,VE-cadherin,and occludin in the medium and low-dose Yishen Huoxue prescription groups were significantly different(P<0.01).There was no significant difference in the mRNA expressions of CD31 and Ang-1/Tie-2 in the high-dose Yishen Huoxue prescription group,and no significant difference in the ROS level in the medium-dose Yishen Huoxue prescription group.Conclusion: The anti-RIF effect of Yishen Huoxue prescription may be related to promoting vascular endothelial repair,regulating mitochondrial ROS to reduce oxidative stress,protecting the integrity of renal endothelial structure,delaying cell apoptosis,and maintaining cell energy metabolism.
作者
钟建
罗芳
方桂玉
卢登勇
石晓冬
吴慧敏
翁雪莹
陈宇珊
左金巾
ZHONG Jian;LUO Fang;FANG Guiyu;LU Dengyong;SHI Xiaodong;WU Huimin;WENG Xueying;CHEN Yushan;ZUO Jinjin(The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530023,China;Graduate School of Guangxi University of Chinese Medicine,Nanning 530001,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第15期70-77,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81760807,82060820)。
关键词
益肾活血方
单侧输尿管梗阻(UUO)小鼠
肾间质纤维化
内皮细胞
Yishen Huoxue prescription
unilateral ureteral obstruction(UUO)mice
renal interstitial fibrosis
endothelial cells
