RhoA在氢减轻脂多糖致小鼠肺微血管内皮细胞屏障功能损伤中的作用
Role of RhoA in hydrogen-induced alleviation of lipopolysaccharide-caused damage to pulmonary microvascular endothelial cell barrier function in mice
李媛 1舒瑞辰 1陈红光 2尹毅青1
作者信息
- 1. 天津医科大学肿瘤医院麻醉科 国家恶性肿瘤临床医学研究中心 天津市恶性肿瘤临床医学研究中心 天津市肿瘤防治重点实验室,天津 300060
- 2. 天津医科大学总医院麻醉科 天津市麻醉学研究所,天津 300052
- 折叠
摘要
目的 评价Ras同源家族成员A(RhoA)在氢减轻脂多糖(LPS)致小鼠肺微血管内皮细胞屏障功能损伤中的作用.方法 小鼠肺微血管内皮细胞培养于含10％胎牛血清和1％青链双抗的DMEM/F12培养基中,传代至第4～6代的细胞用于实验,采用随机数字表法分为6组(n=36):对照组(A组)、富氢液组(B组)、LPS组(C组)、LPS+富氢液组(D组)、LPS+RhoA抑制剂C3酶组(E组)和LPS+富氢液+RhoA激活剂U-46619组(F组).A组、C组和E组采用正常培养基培养,B组、D组和F组采用含饱和氢气培养基培养,C组、D组、E组和F组加入LPS,终浓度1 µg/ml;E组于加入LPS前2 h加入C3酶,终浓度3 μg/ml;F组于加入LPS前3 h加入U-46619,终浓度10 mg/ml.分别于LPS孵育后6、12和24 h时采用Western blot法检测血管内皮钙黏蛋白(VE-cadherin)和紧密连接蛋白(occludin)的表达;于LPS孵育后24 h时采用LDH法测定细胞LDH释放率,MTT法测定细胞活力,GST pull-down法测定RhoA活性.结果 与A组比较,C组孵育6、12和24 h时occludin和VE-cadherin 表达下调,孵育24 h时细胞活力降低,LDH释放率和RhoA活性升高(P＜0.05);与C组比较,D组孵育6、12和24 h时occludin和VE-cadherin表达上调,孵育24 h时细胞活力升高,LDH释放率和RhoA活性降低(P＜0.05);与C组比较,E组孵育6、12和24 h时occludin和VE-cadherin表达上调,孵育24 h时细胞活力升高,LDH释放率和RhoA活性降低(P＜0.05);与D组比较,F组孵育6、12和24 h时occludin和VE-cadherin表达下调,孵育24 h时细胞活力降低,LDH释放率和RhoA活性升高(P＜0.05).结论 RhoA参与了氢减轻LPS致小鼠肺微血管内皮细胞屏障功能损伤的过程.
Abstract
Objective To evaluate the role of Ras homolog gene family member A(RhoA)in hy-drogen-induced alleviation of lipopolysaccharide(LPS)-caused damage to pulmonary microvascular endothe-lial cell(PMVEC)barrier function in mice.Methods PMVECs were cultured in DMEM/F12 medium containing 10％fetal bovine serum and 1％penicillin/streptomycin until 4-6 passage.These cells were di-vided into 6 groups(n=36 each)using a random number table method:control group(group A),hydro-gen-rich medium group(group B),LPS group(group C),LPS+ hydrogen-rich medium group(group D),LPS+RhoA inhibitor C3 enzyme group(group E)and LPS+hydrogen-rich medium+RhoA agonist U-46619 group(group F).Cells were cultured within normal medium in group A,group C and group E and within hydrogen-rich medium in group B,group D and group F.LPS at a final concentration of 1 μg/ml was simultaneously added in group C,group D,group E and group F.C3 enzyme at a final concentration of 3μg/ml was added at 2 h before addition of LPS in group E.U-46619 at a final concentration of 10 mg/ml was added at 3 h before addition of LPS in group F.The expression of vascular endothelial(VE)-cadherin and occludin was determined by Western blot at 6,12 and 24 h after incubation with LPS.At 24 h after incuba-tion with LPS,the release rate of LDH was measured by LDH method,cell viability was measured by MTT method,and the activity of RhoA was determined by GST pull-down method.Results Compared with group A,the expression of VE-cadherin and occludin was significantly down-regulated at 6,12 and 24 h of incu-bation,the cell viability was decreased at 24 h of incubation,and the release rate of LDH and activity of RhoA were increased in group C(P＜0.05).Compared with group C,the expression of VE-cadherin and oc-cludin was significantly up-regulated at 6,12 and 24 h of incubation,the cell viability was increased at 24 h of incubation,and the release rate of LDH and activity of RhoA were decreased in group D(P＜0.05).Compared with group C,the expression of VE-cadherin and occludin was significantly up-regulated at 6,12 and 24 h of incubation,the cell viability was increased at 24 h of incubation,and the release rate of LDH and activity of RhoA were decreased in group E(P＜0.05).Compared with group D,the expression of VE-cadherin and occludin was significantly down-regulated at 6,12 and 24 h of incubation,the cell viability was decreased at 24 h of incubation,and the release rate of LDH and activity of RhoA were increased in group F(P＜0.05).Conclusions RhoA is involved in hydrogen-induced alleviation of LPS-caused damage to PMVEC barrier function in mice.
关键词
GTP结合蛋白质类/氢/脂多糖类/肺/微血管/内皮细胞Key words
GTP-binding proteins/Hydrogen/Lipopolysaccharides/Lung/Microvessels/Endothelial cells引用本文复制引用
基金项目
国家自然科学基金(81801889)
国家自然科学基金(82101351)
天津市医学重点学科(专科)建设项目(TJYXZDXK-009A)
出版年
2024
NETL
NSTL
维普
万方数据