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目的探讨硫化氢后处理对原代培养的新生大鼠心肌细胞缺氧复氧损伤的保护作用及可能机制。方法以硫氢化钠(NaHS)作为外源性硫化氢供体。原代培养新生大鼠的心肌细胞随机分为5组:①正常对照组;②缺氧复氧组(hypoxia-reoxygenation,HR组);③二甲基亚砜组(dimethyl sulfoxide,DMSO组);④硫化氢后处理组(hydrogen sulfide post-con-ditioning,H2S组);⑤LY294002+H2S后处理组(LY+H2S组)。分别于缺氧前和复氧2 h检测各组的心肌细胞存活率、培养液中CK和LDH的活性;复氧末,用流式细胞学技术检测各组心肌细胞凋亡情况;Western blot检测HIF-1α、p-Akt与Akt蛋白的表达情况。结果通过比较可知,复氧2 h时,H2S组心肌细胞存活率较HR组升高显著[(71.55±1.46)%vs(62.03±1.29)%,P<0.01],CK、LDH活性以及心肌细胞凋亡率明显降低[(244.99±21.38)U/L vs(329.53±26.14)U/L,(371.64±18.62)U/L vs(427.69±13.25)U/L,(10.37±1.51)%vs(17.38±2.04)%,P<0.01];同时,HIF-1α和p-Akt蛋白表达水平明显升高[(63.93±4.64)%vs(41.82±6.14)%,(62.59±2.11)%vs(41.04±4.01)%,P<0.01]。然而,在H2S后处理同时加入PI3K/Akt信号通路特异性抑制剂LY294002后,与H2S组相比,LY+H2S组心肌细胞存活率降低明显(63.65±2.01)%,CK、LDH活性以及心肌细胞凋亡率升高显著[(307.80±21.68)U/L,(421.51±15.95)U/L,(15.17±1.56)%,P<0.01];HIF-1α和p-Akt蛋白表达水平亦降低明显[(51.79±4.27)%,(47.42±3.15)%,P<0.05和P<0.01]。结论 H2S后处理可通过PI3K/Akt信号通路促进HIF-1α表达,从而拮抗心肌细胞的缺氧复氧损伤。
Objective To investigate the protective effect and possible mechanism of hydrogen sulfide (H2SO4) postconditioning on primary cultured neonatal rat cardiomyocytes subjected to hypoxia-reoxygenation injury. Methods Sodium hydrogen sulfide (NaHS) was used as exogenous hydrogen sulfide donor. Primary cultured neonatal rat cardiomyocytes were randomly divided into 5 groups: ① normal control group; ② hypoxia-reoxygenation group (HR group); ③ dimethyl sulfoxide group (DMSO group); ④ hydrogen sulfide post-con-ditioning group (H2S group); ⑤ LY294002 + H2S group (LY + H2S group). The survival rate of cardiomyocytes and the activities of CK and LDH in each group were detected before hypoxia and 2 h after reoxygenation respectively. At the end of reoxygenation, the apoptosis of cardiomyocytes was detected by flow cytometry. Western blot HIF-1α, p-Akt and Akt protein expression. Results Compared with HR group, the survival rate of cardiomyocytes in H2S group increased significantly ([(71.55 ± 1.46)% vs (62.03 ± 1.29)%, P <0.01], CK and LDH activity and myocardial cells The apoptotic rate was significantly lower than that of the control group [(244.99 ± 21.38) U / L vs (329.53 ± 26.14) U / L, (371.64 ± 18.62) U / L vs (427.69 ± 13.25) U / L vs (63.33 ± 4.64)% vs (41.82 ± 6.14)%, (62.59 ± 2.11)% vs (41.04 ± 4.11)%, respectively (P <0.05), while the expression of HIF-1α and p- ± 4.01)%, P <0.01]. However, compared with H2S group, the viability of cardiomyocytes in LY + H2S group decreased significantly (63.65 ± 2.01%), the activities of CK and LDH and the activities of myocardial cells and cardiomyocytes increased after H 2 S reprocessing and the PI3K / Akt signaling pathway specific inhibitor LY294002 (P <0.01). The expression of HIF-1α and p-Akt also decreased significantly ([(307.80 ± 21.68) U / L, (421.51 ± 15.95) U / L, (15.17 ± 1.56)%, P <0.01] [(51.79 ± 4.27)%, (47.42 ± 3.15)%, P <0.05 and P <0.01]. Conclusion H2S postconditioning can promote HIF-1α expression through PI3K / Akt signaling pathway and thus antagonize hypoxia-reoxygenation injury of cardiomyocytes.