芍药苷能通过调节Src/血管内皮-钙黏蛋白通路改善心脏微血管内皮细胞通透性  

Paeoniflorin improves the permeability of cardiac microvascular endothelial cells by regulating Src/vascular endothelial-cadherin pathway

作　　者：洪秀芳[1,2] 李莉 郭冬阳[1] 杨舟鑫 严静[1] Hong Xiufang;Li Li;Guo Dongyang;Yang Zhouxin;Yan Jing(Department of Critical Care Medicine,Zhejiang Hospital,Hangzhou 310013,Zhejiang,China;The Second Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China)

机构地区：[1]浙江医院重症医学科,杭州310013 [2]浙江中医药大学第二临床医学院,杭州310053

出　　处：《中华危重病急救医学》2020年第1期83-87,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81772051);浙江省中医药科学研究基金项目(2019ZB004)。

摘　　要：目的探讨芍药苷对脓毒症心脏微血管内皮细胞(CMECs)通透性的影响及机制。方法体外分离并原代培养大鼠CMECs细胞,待细胞进入对数生长期用于实验。采用四甲基偶氮唑盐比色法(MTT)筛选出芍药苷的安全有效浓度10、20、40μmol/L。将细胞分为空白对照组、脂多糖(LPS)组及低、中、高浓度芍药苷预处理组。空白对照组用完全培养基培养;LPS组在完全培养基中加入1 mg/L的LPS刺激细胞;芍药苷预处理各组分别于LPS刺激前4 h加入10、20、40μmol/L芍药苷进行预处理。各组细胞于LPS刺激后继续培养24 h,采用辣根过氧化物酶(HRP)法检测大鼠CMECs细胞通透性;采用酶联免疫吸附试验(ELISA)检测细胞上清液中CXC趋化因子配体(CXCL1、CXCL2)水平;采用实时荧光定量反转录-聚合酶链反应(RT-qPCR)检测细胞中CXCL1、CXCL2的mRNA表达;采用蛋白质免疫印迹试验(Western Blot)检测细胞中磷酸化Src(p-Src)、血管内皮-钙黏蛋白(VE-cadherin)、磷酸化丝裂素活化蛋白激酶(p-MAPK)的蛋白表达。结果与空白对照组相比,LPS组大鼠CMECs细胞通透性明显增加;经不同浓度芍药苷预处理后细胞通透性均有一定程度改善,以40μmol/L芍药苷组改善最明显,与LPS组比较差异有统计学意义(A值:1.61±0.07比2.13±0.06,P<0.01)。ELISA结果显示,空白对照组大鼠CMECs细胞上清液中有适量CXCL1、CXCL2分泌;但在LPS诱导下,细胞上清液中CXCL1、CXCL2分泌量明显增加;给予不同浓度芍药苷预处理后细胞上清液中CXCL1、CXCL2的分泌量均明显减少,其中40μmol/L芍药苷对CXCL1的抑制作用最佳,20μmol/L芍药苷对CXCL2的抑制作用最佳,与LPS组比较差异均有统计学意义〔CXCL1(ng/L):337.51±68.04比829.86±65.06,CXCL2(ng/L):4.48±0.11比9.41±0.70,均P<0.01〕。RT-qPCR结果显示,大鼠CMECs细胞中CXCL1、CXCL2的mRNA表达与ELISA结果一致,表现为LPS能够诱导大鼠CMECs细胞中CXCL1、CXCL2的mRNA表达增加;而不同浓度Objective To investigate the effect and mechanism of paeoniflorin on the permeability of cardiac microvascular endothelial cells(CMECs)in sepsis.Methods Primary rat CMECs were isolated and cultured in vitro,and the cells in the logarithmic growth phase were used for experiments.Tetramethylazozolium colorimetry(MTT)was used to screen the safe and effective concentrations of paeoniflorin at 10,20,and 40μmol/L.The cells were divided into blank control group,lipopolysaccharide(LPS)group and low,medium and high concentration paeoniflorin pretreatment group.The cells in the blank control group were cultured in complete medium;the cells in the LPS group were challenged with LPS(1 mg/L)in complete medium;and the cells in the paeoniflorin pretreatment groups were pretreated with 10,20,and 40μmol/L paeoniflorin at 4 hours before LPS stimulation.The cells in each group were further cultured for 24 hours after LPS stimulation.The horseradish peroxidase(HRP)method was used to detect the permeability of rat CMECs.The enzyme-linked immunosorbent assay(ELISA)was used to detect the CXC chemokine ligand(CXCL1,CXCL2)levels in the cell supernatant.The real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-qPCR)was used to detect the mRNA expressions of CXCL1 and CXCL2 in the cells.Western Blot was used to detect phosphorylated Src(p-Src),vascular endothelial-cadherin(VE-cadherin)and phosphorylated mitogen activated protein kinase(p-MAPK).Results Compared with the blank control group,the permeability of rat CMECs in the LPS group was significantly increased.The cell permeability was improved to some extent after paeoniflorin pretreatment at different concentrations,and the improvement was most obvious in the 40μmol/L paeoniflorin group,with statistically significant difference as compared with the LPS group(A value:1.61±0.07 vs.2.13±0.06,P<0.01).ELISA results showed that there were moderate amounts of CXCL1 and CXCL2 in the cell supernatant of rat CMECs in the blank control group.However,the secre

关 键 词：芍药苷 脓毒症 心肌损伤 心脏微血管内皮细胞 血管内皮-钙黏蛋白 

分 类 号：R459.7[医药卫生—急诊医学]