摘要
目的有研究表明硫化氢(hydrogen sulfide,H2S)能减少缺血再灌注损伤心肌细胞的凋亡,但确切机制不清。文中旨在研究心肌细胞缺氧/复氧(hypoxia/reoxygenation,HR)损伤中,H2S能否通过调节miR-455的表达和减少内质网应激(endoplasmic reticulum stress,ERS)介导细胞凋亡发挥心肌保护作用。方法原代培养新生SD大鼠心肌细胞,建立心肌细胞HR模型。实验分组:对照组(心肌细胞正常培养27 h);HR组(将心肌细胞更换不含血清的DMEM液后进行HR处理);H2S保护组[心肌细胞缺氧前30 min给予40μmol/L的Na HS(H2S前体)预处理,其余处理同HR组]。采用MTT法检测细胞活力,全自动化学分析法检测培养液LDH漏出量,流式细胞仪检测心肌细胞凋亡率,RT-PCR法、蛋白印记法分别检测miR-455及ERS介导细胞凋亡信号通路的标志物葡萄糖调节蛋白78(glucose regulated protein 78,Grp78)和caspase-12的mRNA、蛋白的表达。为检测miR-455是否参与H2S抑制ERS介导的细胞凋亡,将心肌细胞又分为3组,阴性对照组(转染miR-455阴性对照片段24 h);miR-455模拟剂组(转染miR-455模拟剂24 h),miR-455拮抗剂组(转染miR-455拮抗剂24 h),分别给予40μmol/L的Na HS预处理,再进行HR处理,检测Grp78、caspase-12的表达。结果与HR组比较,H2S保护组可增加HR损伤的心肌细胞活力[(67.02±6.90)%vs(29.27±5.66)%,P<0.05],减少LDH漏出量[(91.33±10.63)U/L vs(168.17±15.38)U/L,P<0.05],同时降低细胞凋亡率[(13.98±1.90)%vs(24.31±2.79)%,P<0.05]。与HR组比较,H2S保护组可降低HR损伤后细胞Grp78、Grp78 mRNA、caspase-12、caspase-12 mRNA的表达(P<0.05)。与阴性对照组比较,miR-455模拟剂组Grp78、Grp78 mRNA、caspase-12、caspase-12 mRNA表达显著升高,而miR-455拮抗剂组表达则显著下降(P<0.05)。结论 H2S可通过调节miR-455的表达水平,减少HR损伤心肌细胞的ERS介导的细胞凋亡,发挥其对心肌的保护作用。
Objective The protective effect of hydrogen sulfide （ H： S） against myocardial isehemia/reperfusion （IR） injury via anti-apoptotic signaling is well established, but the underlying mechanism remains unclear. This study was to investigate whether H2 S could protect cardiomyocytes from endoplasmic reticulum stress （ERS）-mediated apoptosis in hypoxia/reoxygenation （HR） injury by regulating the expression of microRNA-455 （ miR-455 ）. Methods Cardiomyocytes from neonatal SD rats were primarily cultured and the model of HR injury was established. The cardiomyocytes were divided into a control group （normally cultured for 27 hours）, an HR group （subjected to HR injury）, and an H2S protection group （pretreated with the precursor of H2S NariS at 40 p, mol/L at 30 min before HR treatment followed by the same procedure as in the HR group）. The cell viability was monitored by MTT, the release of lactate dehydrogenase （LDH） in the culture supernatant measured by full-automatic chemical analysis, and the apoptosis rate of the cardiomyoeytes detected by flow eytometry. The mRNA and protein expressions of Grp78 and easpase-12 were determined by real-time RT-PCR and Western bot. To verify whether miR-455 was involved in the ERS-mediated apoptosis of the cardiomyoeytes, the cells were subjected to HR after transfeeted with miR-455 mimic or anti-miR-455 oligonueleotide （AMO） for 24 hours, followed by detection of the expressions of Grp78 and caspase-12. Results After HR injury, the H2S protection group showed an enhanced viability of the cardiomyoeytes in comparison with the control group （ [ 67.02 ± 6.901 vs [ 29.27 ± 5.66 ] % ）, an decreased LDH release （ [ 91.33 ± 10.63] vs [ 168.17 ± 15.38] U/L）, and a reduced rate of cell apoptosis （ [ 13.98 ± 1.90] vs （24.31±2.79] % ）. H2S pretreat- ment significantly downregulated the mRNA and protein expressions of Grp78 and caspase-12 （ 1.66 ± 0.39 vs 2.56 ± 0.34 ; 1.75 ± 0.32 vs 2.54 ± 0.48 ; 2.01 ± 0.45 vs 3.26 ± 0.34
出处
《医学研究生学报》
CAS
北大核心
2014年第12期1245-1249,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金(81100139)
第二军医大学博士研究生创新基金(BC201227)
