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

作　　者：李媛 舒瑞辰 陈红光[2] 尹毅青 Li Yuan;Shu Ruichen;Chen Hongguang;Yin Yiqing(Department of Anesthesiology,Tianjin Medical University Cancer Institute and Hospital,National Clinical Research Center for Cancer,Tianjin′s Clinical Research Center for Cancer,Key Laboratory of Cancer Prevention and Therapy,Tianjin 300060,China;Department of Anesthesiology,Tianjin Medical University General Hospital,Tianjin Research Institute of Anesthesiology,Tianjin 300052,China)

机构地区：[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

出　　处：《中华麻醉学杂志》2024年第3期334-338,共5页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(81801889,82101351);天津市医学重点学科(专科)建设项目(TJYXZDXK-009A)。

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1μg/ml;E组于加入LPS前2 h加入C3酶,终浓度3μg/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。Objective To evaluate the role of Ras homolog gene family member A(RhoA)in hydrogen-induced alleviation of lipopolysaccharide(LPS)-caused damage to pulmonary microvascular endothelial 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 divided into 6 groups(n=36 each)using a random number table method:control group(group A),hydrogen-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 1μg/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 incubation 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 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 C(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 D(P<0.05).Compared with group C,the expression of VE-cadherin and occludin was significantly up-r

关 键 词：GTP结合蛋白质类 氢 脂多糖类 肺 微血管 内皮细胞 

分 类 号：R614[医药卫生—麻醉学]