摘要
目的探讨槲皮素(quercetin, QUE)减轻H9c2心肌细胞缺氧/复氧(hypoxia/reoxygenation, H/R)损伤的可能机制及叉头框蛋白O3(forkhead box O3, FOXO3)在其中的作用。方法以大鼠心肌细胞株H9c2为研究对象, 按随机数字表法分为5组(每组6个复孔):正常对照组(CON组)、缺氧/复氧组(H/R组)、QUE组、QUE+FOXO3干扰小RNA(small interfering RNA, siRNA)组(QUE+si组)、QUE+阴性对照组(QUE+NC组)。采用缺氧6 h复氧6 h制备H/R损伤模型。CON组细胞正常培养;H/R组细胞制备H/R损伤模型;QUE组给予终浓度为20 μmol/L的QUE孵育24 h后制备H/R损伤模型;QUE+si组和QUE+NC组分别用FOXO3 siRNA和FOXO3 siRNA阴性对照转染细胞, 24 h后给予终浓度为20 μmol/L的QUE再孵育24 h, 然后制备H/R损伤模型。采用细胞计数试剂盒(cell counting kit-8, CCK-8)法检测各组细胞活力, DCFH-DA法检测各组细胞活性氧(reactive oxygen species, ROS)水平, ELISA法检测各组细胞中丙二醛(malondialdehyde, MDA)含量和超氧化物歧化酶(superoxide dismutase, SOD)活力, Western blot法检测FOXO3、超氧化物歧化酶2(superoxide dismutase, SOD2)蛋白水平。Hoechst33258染色法检测各组H9c2心肌细胞凋亡情况。结果与CON组比较:H/R组、QUE+NC组、QUE+si组细胞活力下降(P<0.05), H/R组、QUE组、QUE+NC组、QUE+si组ROS水平升高(P<0.05);H/R组、QUE+si组MDA含量升高(P<0.05), SOD活力下降(P<0.05);H/R组FOXO3、SOD2表达下调(P<0.05);H/R组、QUE组、QUE+NC组、QUE+si组细胞凋亡率增加(P<0.05)。与H/R组比较:QUE组细胞活力升高(P<0.05), FOXO3、SOD2表达上调(P<0.05);QUE组、QUE+NC组ROS水平下降(P<0.05), MDA含量减少(P<0.05), SOD活力升高(P<0.05), 细胞凋亡率下降(P<0.05)。与QUE+NC组比较, QUE+si组细胞活力下降(P<0.05), ROS水平升高(P<0.05), MDA含量升高(P<0.05), SOD活力下降(P<0.05), FOXO3、SOD2表达下调(P<0.05), 细胞凋亡率增加(P<0.05)。QUE+NC组与QUE组细胞活力、ROS水平、MDA含量、SOD活力和细胞凋亡
Objective To investigate the possible mechanism of quercetin(QUE)in alleviating hypoxia/reoxygenation(H/R)injury of H9c2 cardiomyocytes and the role of forkhead box O3(FOXO3)plays in it.Methods Rat cardiomyoblast cell line(H9c2 cells)were used as the research object and were divided into five groups according to the random number table method(n=6):control group(CON group),hypoxia/reoxygenation group(H/R group),QUE group,quercetin+FOXO3 small interfering RNA(siRNA)group(QUE+si group)and quercetin+negative control group(QUE+NC group).The H/R injury model was established by hypoxia for 6 h followed by reoxygenation for 6 h.Cells in the CON group were cultured normally,and cells in the H/R group were subjected to the H/R injury model.In the QUE group,the cells were incubated with quercetin at a concentration of 20μmol/L and incubated for 24 h,and then subjected to the H/R injury model.The cells in the QUE+si group and QUE+NC group were respectively transfected with FOXO3 siRNA and FOXO3 siRNA negative control,and after 24 h,incubated with quercetin at a concentration of 20μmol/L for another 24 h;then these cells were established in a H/R injury model.The cell viability was assayed by cell counting kit-8(CCK-8).The level of reactive oxygen species(ROS)was detected by the DCFH-DA method.The content of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)were detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of FOXO3 and SOD2 were analyzed by Western blot analysis.The apoptosis of H9c2 cardiomyocytes in each group was detected by Hoechst 33258 staining.Results Compared with the CON group,the cell viability decreased in the H/R group,QUE+NC group and QUE+si group(P<0.05);the levels of ROS increased in the H/R group,QUE group,QUE+NC group and QUE+si group(P<0.05);the content of MDA increased and the activity of SOD decreased in the H/R group and QUE+si group(P<0.05);the protein expression of FOXO3 and SOD2 decreased in the H/R group(P<0.05);the cell apoptosis rate increased in the H/
作者
李林桂
胡俊凯
张超
胡晶辉
孟晓文
嵇富海
Li Lingui;Hu Junkai;Zhang Chao;Hu Jinghui;Meng Xiaowen;Ji Fuhai(Department of Anesthesiology,the First Affiliated Hospital of Soochow University,Suzhou 215006,China;Institute of Anesthesiology,Soochow University,Suzhou 215006,China;Department of Anesthesiology,Suzhou Ninth People's Hospital,Suzhou 215299,China)
出处
《国际麻醉学与复苏杂志》
CAS
2023年第9期897-903,共7页
International Journal of Anesthesiology and Resuscitation
基金
国家自然科学基金(82072130)
江苏省自然科学基金(BK20200195)
江苏省"333"高层次人才培养工程项目(BRA2020089)
江苏省医学科研重点项目(ZD2022021)
江苏省高等学校自然科学研究面上项目(22KJD320002)
苏州市麻醉学临床医学中心项目(Szlcyxzxj202102)。
