类叶升麻苷通过上调miR-204对缺氧/复氧诱导H9C2细胞凋亡的抑制作用

Acteoside inhibites hypoxia/reoxygenation-induced apoptosis by up-regulating miR-204 in H9C2 cells

目的探讨类叶升麻苷对缺氧/复氧(H/R)处理大鼠心肌细胞(H9C2)损伤的影响及其分子机制。方法体外培养H9C2细胞,H/R(4 h/20 h)建立心肌细胞损伤模型,并采用1、10、100μmol/L类叶升麻苷,转染miR-204模拟物阴性对照(miR-NC)、转染miR-204模拟物(miR-24),100μmol/L类叶升麻苷干预+转染miR-204抑制剂阴性对照、100μmol/L类叶升麻苷+转染miR-204抑制剂干预H/R细胞。分别进行RT-qPCR、MTT、流式细胞术、Western blot检测miR-204表达水平、细胞活力、细胞凋亡率和相关蛋白表达,利用相应试剂盒检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)水平,酶联免疫吸附试验(ELISA)检测白细胞介素-6(IL-6)、白细胞介素-β(IL-β)和肿瘤坏死因子-α(TNF-α)的含量。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果与心肌损伤模型比较,10、100μmol/L类叶升麻苷的细胞凋亡率[(25.62±1.96)%比(18.17±1.27)%,(11.24±0.57)%]、Bax(0.71±0.05比0.51±0.04、0.29±0.03)、LDH[(243.16±11.31)比(121.22±4.52),(94.39±2.82)U/g]、MDA[(1.82±0.07)比(1.13±0.04),(0.92±0.04)nmol/mg]、IL-6[(121.45±6.18)比(87.16±4.53),(47.11±2.24)pg/mL]、IL-1β[(229.82±8.48)比(175.32±8.73),(113.14±5.63)pg/mL]和TNF-α表达水平[(138.18±6.60)比(92.24±4.04),(61.53±4.17)pg/mL]降低,Bcl-2(0.18±0.01比0.35±0.03、0.52±0.04)、SOD[(18.72±1.26)比(38.81±1.51),(45.43±1.29)U/mg]和GSH-Px表达水平[(58.74±2.28)比(89.24±2.82),(94.66±3.05)U/mg]升高(P均<0.05);与miR-NC比较,转染miR-204的细胞凋亡率[(24.12±1.12)%比(9.26±0.49)%]、Bax(0.62±0.04比0.25±0.02)、LDH[(229.11±8.47)比(86.32±5.92)U/g]、MDA[(1.75±0.08)比(0.85±0.05)nmol/mg]、IL-6[(134.47±7.31)比(55.26±2.13)pg/mL]、IL-1β[(211.14±9.70)比(98.11±3.18)pg/mL]和TNF-α表达水平[(152.92±3.49)比(51.34±2.66)pg/mL]降低,Bcl-2(0.22±0.01比0.57±0.03)、SOD[(20.92±1.38)比(47.68±1.76)U/mg]和GSH-Px表达水平[(62.65±2.76)比(91.13±3.80)U/mg]升高(P<0.05);10、100μmol/L类叶升麻苷可提高H/R诱导H9C2细胞中miR-204的表达水平(P<0.05);且下调miR-204逆转了类叶升麻苷对H/R处理H9C2细胞凋亡、氧化应激和炎症因子的影响。结论类叶升麻苷可能通过上调miR-204表达缓解H/R诱导的H9C2细胞损伤。

Objective To investigate the effect and molecular mechanism of acteoside on injury of rat myocardial cells(H9C2)treated with hypoxia/reoxygenation(H/R).Methods H9C2 cells were cultured in vitro,and H/R(4 h/20 h)was used to establish cardiomyocytes injury model.The H/R-induced cells were treated with 1,10,100μmol/L acteoside,transfected with miR-204 mimics negative control(miR-NC),miR-204 mimics(miR-204),treated with 100μmol/L acteoside plus transfected with miR-204 inhibitor negative control or miR-204 inhibitor.RT-qPCR,MTT,flow cytometry and Western blot were used to detect miR-204 expression level,cell activity,apoptosis rate and the expression of related proteins,respectively.The corresponding kits were used to detect the expression levels of lactate dehydrogenase(LDH),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX).ELISA was used to evaluate the levels of interleukin-6(IL-6),interleukin-β(IL-β)and tumor necrosis factor-α(TNF-α).Independent sample t test was used for comparison between two groups,one-way analysis of variance(ANOVA)was used for comparison between multiple groups,and SNK-q test was used for pair wise comparison between groups.Results Compared with cardiomyocytes injury model,the apoptosis rate[(25.62±1.96)%vs(18.17±1.27)%,(11.24±0.57)%],the expression levels of Bax(0.71±0.05 vs 0.51±0.04,0.29±0.03),LDH[(243.16±11.31)vs(121.22±4.52),(94.39±2.82)U/g],MDA[(1.82±0.07)vs(1.13±0.04),(0.92±0.04)nmol/mg],IL-6[(121.45±6.18)vs(87.16±4.53),(47.11±2.24)pg/mL],IL-1β[(229.82±8.48)vs(175.32±8.73),(113.14±5.63)pg/mL]and TNF-α[(138.18±6.60)vs(92.24±4.04),(61.53±4.17)pg/mL]were significantly decreased in 10 and 100μmol/L acteoside groups,and the expression levels of Bcl-2(0.18±0.01 vs 0.35±0.03,0.52±0.04),SOD[(18.72±1.26)vs(38.81±1.51),(45.43±1.29)U/mg]and GSH-Px[(58.74±2.28)vs(89.24±2.82),(94.66±3.05)U/mg]were significantly increased(P<0.05).Compared with miR-NC group,apoptosis rate[(24.12±1.12)%vs(9.26±0.49)%],the expression levels of Bax(0.62±0.04 vs 0.25±0.02),LDH[(229.11±8.47)vs(86.32±5.92)U/g],MDA[(1.75±0.08)vs(0.85±0.05)nmol/mg],IL-6[(134.47±7.31)vs(55.26±2.13)pg/mL],IL-1β[(211.14±9.70)vs(98.11±3.18)pg/mL]and TNF-α[(152.92±3.49 vs 51.34±2.66)pg/mL]were significantly decreased,and the expression levels of Bcl-2(0.22±0.01vs 0.57±0.03),SOD[(20.92±1.38)vs(47.68±1.76)U/mg]and GSH-Px[(62.65±2.76)vs(91.13±3.80)U/mg]were significantly increased in miR-204 group(P<0.05).10,100μmol/L acteoside,significantly increased the expression level of miR-204 in H/R-induced cells.In addition,down-regulation of miR-204 reversed the effects of acteoside on the apoptosis,oxidative stress and inflammatory factor of H9C2 cells treated with H/R(P<0.05).Conclusion Acteoside may alleviate H/R-induced H9C2 cell injury by up-regulating the expression of miR-204.