G蛋白通路抑制因子2在机械通气肺损伤中的调控作用及机制

Regulatory role of G protein pathway suppressor 2 in lung injury induced by mechanical ventilation and related mechanism

目的探讨G蛋白通路抑制因子2(G protein pathway suppressor 2,GPS2)在细胞机械牵张及小鼠大潮气量机械通气相关性肺损伤(ventilation‑induced lung injury,VILI)中的作用机制。方法取人肺微血管内皮细胞(human pulmonary microvascular endothelial cells,HPMVEC)按照随机数字表法分成4组(每组3孔):未进行机械牵张组(Nc组)、低表达GPS2组(GPS2 siRNA组)、机械牵张4 h组(CS 4 h组)和低表达GPS2并机械牵张4 h组(CS 4 h+GPS2 siRNA组)。GPS2 siRNA组与CS 4 h+GPS2 siRNA组HPMVEC转染GPS2干扰小RNA(small interfering RNA,siRNA),CS 4 h组和CS 4 h+GPS2 siRNA组进行幅度为20%、频率为0.5 Hz的循环拉伸的方法建立机械牵张模型。Western blot法检测连环蛋白(p120)、紧密连接蛋白(Occludin)和GPS2水平,免疫荧光法检测p120和Occludin的定位及表达,流式细胞术检测活性氧(reactive oxygen species,ROS)和线粒体膜电位(mitochondrial membrane potential,MMP)水平。健康雄性C56BL/6小鼠40只,采用随机数字表法分为4组(每组10只):对照组(C组)、GPS2敲低组(si‑GPS2组)、机械通气4 h组(MV 4 h组)、GPS2敲低+机械通气4 h组(MV 4 h+si‑GPS2组)。si‑GPS2组和MV 4 h+si‑GPS2组小鼠通过尾静脉注射GPS2 siRNA转染复合物,1周2次;MV 4 h组和MV 4 h+si‑GPS2组小鼠进行气管插管机械通气4 h。采用H‑E染色观察小鼠肺组织病理学改变,检测肺组织中丙二醛(malondialdehyde,MDA)和超氧化物歧化酶(superoxide dismutase,SOD)水平。结果细胞实验中:与Nc组比较,GPS2 siRNA组、CS 4 h组和CS 4 h+GPS2 siR‑NA组GPS2、p120、Occludin水平下降(P<0.05),ROS水平增加(P<0.05),MMP水平降低(P<0.05),p120、Occludin表达荧光强度减弱、分布减少、细胞间隙增大;与CS 4 h组比较,CS 4 h+GPS2 siRNA组GPS2、p120和Occludin水平进一步下降(P<0.05),ROS水平增加(P<0.05),MMP水平降低(P<0.05),p120、Occludin表达荧光强度进一步减弱、分布进一步减少、细胞间隙进一步增大。动物实验中:与C组比较,si‑GPS2组、MV 4 h组、MV 4 h+si‑GPS2组小鼠肺组织MDA水平升高(P<0.05),SOD水平降低(P<0.05),肺组织出现明显损伤改变;与MV 4 h组比较,MV 4 h+si‑GPS2组小鼠肺组织MDA水平升高(P<0.05),SOD水平降低(P<0.05),肺组织损伤改变加重。结论GPS2可通过调控线粒体功能参与细胞机械牵张及小鼠大潮气量VILI。

Objective To investigate the effect of G protein pathway suppressor 2(GPS2)in mechanical cell stretch and ventilation‑induced lung injury(VILI)in mice.Methods According to the random number table method,human pulmonary micro‑vascular endothelial cells(HPMVEC)were divided into four groups(n=3):a non‑mechanical stretch group(group Nc),a low‑expression GPS2 group(group GPS2 siRNA),a mechanical stretch 4 h group(group CS 4 h)and a low‑expression GPS2 and mechanical stretch 4 h group(group CS 4 h+GPS2 siRNA).Group GPS2 siRNA and group CS 4 h+GPS2 siRNA were transfected with GPS2 small interfering RNA(siRNA).In contrast,a mechanical distraction model was established in the group CS 4 h and the group CS 4 h+GPS2 siRNA,by cyclic stretching at an amplitude of 20%and a frequency of 0.5 Hz.The levels of catenin(p120),Occludin and GPS2 were detected by Western blot,and the positioning and expression of p120 and occludin were detected by immunofluorescence.The levels of reactive ox‑ygen species(ROS)and mitochondrial membrane potential(MMP)were detected by flow cytometry.A total of 40 healthy male C56BL/6 mice were divided into 4 groups according to the random number table method(n=10):a control group(group C),a GPS2 knockdown group(group si‑GPS2),a mechanical ventilation 4 h group(group MV 4 h),and a GPS2 knockdown+mechanical ventilation 4 h group(group MV 4 h+si‑GPS2).Mice in group si‑GPS2 and group MV 4 h+si‑GPS2 were injected with GPS2 siRNA transfection complex via the tail vein,twice a week.Furthermore,mice in group MV 4 h and group MV 4 h+si‑GPS2 were mechanically ventilated through tra‑cheal intubation for 4 h.The pathological changes of the lungs were observed by H‑E staining,and malondialdehyde(MDA)and super‑oxide dismutase(SOD)levels were detected in lung tissues.Results Compared with group Nc,groups GPS2 siRNA,CS 4 h and CS 4 h+GPS2 siRNA showed decreased levels of GPS2,p120,and occludin(P<0.05),and increased ROS levels(P<0.05),decreased MMP levels(P<0.05),as well as weakened fluorescence intensity and reduced distribution of p120 and occludin,while the cell gap was en‑larged.Compared with the group CS 4 h,the group CS 4 h+GPS2 siRNA showed further decreases in the levels of GPS2,p120 and oc‑cludin(P<0.05),increases in ROS levels(P<0.05),decreases in MMP levels(P<0.05),as well as further weaken fluorescence intensity and reduced distribution of p120 and occludi,while the cell gap further was enlarged.In animal experiments:compared with group C,groups si‑GPS2,MV 4 h and MV 4 h+si‑GPS2 presented increased MDA levels in lung tissues of mice(P<0.05),and decreased SOD levels(P<0.05),with obvious injury in the lung tissues.Compared with group MV 4 h,the group MV 4 h+si‑GPS2 showed increased MDA levels in the lung tissues(P<0.05)and decreased SOD levels(P<0.05),with aggravated change in the lung tissues.Conclu⁃sion GPS2 can involve in cell mechanical stretch and VILI in mice by regulating mitochondrial function.