肉桂醛预处理对H9C2心肌细胞缺氧复氧损伤后自噬的影响

Effect of Cinnamaldehyde Pretreatment on Autophagy After Hypoxia/Reoxygenation Injury in H9C2 Cardiomyocytes

目的:探讨肉桂醛处理能否通过自噬途径减轻缺氧复氧(H/R)导致的H9C2心肌细胞损伤。方法:选取浓度0μmol/L~100μmol/L肉桂醛分别对H9C2心肌细胞进行2 h、4 h、6 h预处理,将细胞缺氧2 h/复氧4 h,建立H/R损伤模型。CCK-8法检测细胞活力,确定最佳给药浓度和给药时间;酶标法检测肉桂醛预处理对H/R心肌细胞培氧液中乳酸脱氢酶(LDH)漏出率的影响;以3-甲基腺嘌呤(3-MA)为自噬抑制剂和雷帕霉素(Rapa)为自噬增强剂,试验随机分为:正常对照组、模型对照组、肉桂醛60μmol/L组、3-甲基腺嘌呤5 mmol/L组、雷帕霉素100 nmol/L组,以单丹磺酰戊二胺(MDC)自噬荧光染色检测细胞自噬体的聚集,Westernblot检测自噬相关蛋白Beclin-1、LC3II/LC3I、P62的表达。结果:与正常对照组相比0μmol/L~100μmol/L肉桂醛处理2 h、4 h、6 h后,细胞活力均有不同程度提高,且在60μmol/L、6 h达到峰值;与正常对照组相比,模型对照组LDH水平显著升高(P<0.01),自噬荧光强度显著增加,P62蛋白表达显著下调,Beclin蛋白表达显著上调,LC3II/LC3I比值显著增加(P<0.01)。与模型对照组比较,肉桂醛60μmol/L组、3-甲基腺嘌呤5 mmol/L组自噬荧光强度明显降低(P<0.05或P<0.01),Beclin-1蛋白明显下调、LC3II/LC3I比值显著降低,P62蛋白表达显著上调(P<0.01),而雷帕霉素作用相反(P<0.05或P<0.01)。肉桂醛60μmol/L组LDH释放显著降低(P<0.01)。结论:肉桂醛可通过抑制缺氧复氧后的自噬水平,增加细胞活力,减轻心肌细胞损伤。

Objective: To investigate the effect of cinnamaldehyde(CA)pretreatment on hypoxia/reoxygenation(H/R)-induced autophagy in H9 C2 cardiomyocytes. Methods: After being pre-treated with CA at 0 μmol/L-100 μmol/L for 2 h, 4 h and 6 h, respectively, H9 C2 cardiomyocytes were exposed to 2-h hypoxia and 4-h reoxygenation for establishing the H/R injury model. Cell activity was detected by CCK-8 method to determine the optimal concentration and administration time. The leakage rate of lactate dehydrogenase(LDH)in cell culture supernatant after CA pretreatment was detected by ELISA. Following the selection of 3-methyladenine(3-MA) as autophagy inhibitor and rapamycin(Rapa) as autophagy enhancer, the cells were randomly divided into five groups: normal control group, model group, 60 μmol/L CA group, 5 mmol/L 3-MA group, and 100 nmol/L Rapa group. MDC fluorescence staining was conducted to detect autophagosome aggregation in cells, and Western blot assay was used to measure the protein expression levels of Beclin-1 and P62 L as well as C3 II/LC3 I. Results: Compared with the normal control group, pretreatment with CA at 0 μmol/L-100 μmol/L for 2 h, 4 h and 6 h increased the cell viability to different degrees, with the peak noticed at 60 μm and 6 h. Compared with the normal control group, the model group exhibited elevated LDH(P<0.01),enhanced fluorescence intensity, down-regulated P62 protein expression, up-regulated Beclin protein expression, and increased LC3 II/LC3 I(P<0.01). The comparison with the model group revealed that 60 μmol/L CA and 5 mmol/L 3-MA both obviously weakened the average fluorescence intensity(P<0.05 or P<0.01), decreased Beclin-1 protein expression and LC3 II/LC3 I, and increased P62 protein expression(P<0.01), which were significantly reversed by Rapa(P<0.05 or P<0.01). In the 60 μmol/L CA group, the release of LDH was significantly diminished(P<0.01). Conclusion: CA alleviates cardiomyocyte injury by inhibiting their autophagy after H/R and increasing viability.