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本研究采用SD大鼠乳鼠心肌细胞原代培养,复制心肌细胞缺氧复氧损伤模型,考察琥珀酸对心肌细胞缺氧复氧损伤的LDH漏出率的影响,并进一步采用流式细胞术及Western blot考察琥珀酸对心肌细胞凋亡,cleaved caspase-3及p-Akt的影响,探讨琥珀酸对新生大鼠原代心肌细胞缺氧复氧损伤的保护作用。研究结果发现:①琥珀酸31.25~500 mg·L-1对原代心肌细胞活力无明显影响,琥珀酸400,200,100,50 mg·L-1均可显著降低心肌细胞缺氧复氧损伤LDH漏出率(P<0.01或P<0.05);②琥珀酸400,200 mg·L-1可显著降低缺氧复氧损伤所致的心肌细胞凋亡百分数(P<0.05),抑制心肌细胞缺氧复氧所致的cleaved caspase-3蛋白表达增加(P<0.05);③琥珀酸400 mg·L-1可显著增加心肌细胞的p-Akt蛋白表达(P<0.05),而琥珀酸200 mg·L-1对p-Akt蛋白表达无明显影响。因此,本研究认为琥珀酸可通过激活Akt的磷酸化而抑制心肌细胞缺氧复氧所致的坏死和凋亡。
In this study, SD rat neonatal rat cardiomyocytes were cultured in primary culture to replicate cardiomyocyte hypoxia-reoxygenation injury model to investigate the effect of succinate on LDH leakage rate of cardiomyocytes subjected to hypoxia-reoxygenation injury. Flow cytometry and Western blot was used to investigate the effect of succinate on apoptosis, cleaved caspase-3 and p-Akt in cardiomyocytes and the protective effect of succinate on hypoxia-reoxygenation injury in neonatal rat cardiomyocytes. The results showed that: (1) 31.25-500 mg · L-1 succinic acid had no significant effect on the primary cardiomyocyte viability. 400,200,100,50 mg · L-1succinate could significantly reduce the LDH leakage of cardiomyocytes under hypoxia-reoxygenation injury P <0.01 or P <0.05). ② Succinic acid 400, 200 mg · L-1 could significantly decrease the percentage of cardiomyocyte apoptosis induced by hypoxia-reoxygenation injury (P <0.05) and inhibit the hypoxia-reoxygenation The protein expression of cleaved caspase-3 increased (P <0.05). ③ Succinic acid 400 mg · L-1 significantly increased the expression of p-Akt in cardiomyocytes (P <0.05) -Akt protein expression had no significant effect. Therefore, the present study suggests that succinate inhibits cardiomyocyte hypoxia-reoxygenation-induced necrosis and apoptosis by activating Akt phosphorylation.