硫化氢对去甲肾上腺素诱导的心肌细胞肥大SD乳鼠的心肌保护作用机制研究

Protective effect of hydrogen sulfide on norepinephrine-induced hypertrophic myocardium in neonatal rats

目的观察硫化氢对去甲肾上腺素(NE)诱导的心肌细胞肥大SD乳鼠的心肌保护作用,并探讨其机制。方法利用胶原酶消化分离培养SD大鼠乳鼠的心肌细胞,设置硫化氢组(100μmol/L NE+10μmol/L硫氢化钠)、模型组(100μmol/L NE+等量生理盐水)、对照组(等量生理盐水+等量生理盐水)、硫化氢对照组(等量生理盐水+10μmol/L硫氢化钠)。观察乳鼠原代心肌细胞光镜下变化;培养3 d后观察各组心肌细胞形态学变化,检测心肌细胞表面积、增殖和凋亡率,检测并对比丙二醛(MDA)和超氧化物歧化酶(SOD)的含量,对比鞘氨醇激酶1(Sph K1)、1-磷酸鞘氨醇受体1(S1P1)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B (AKT) mRNA和Sph K1、S1P1蛋白的相对表达量、磷酸化PI3K (p-PI3K)/PI3K、磷酸化AKT (p-AKT)/AKT蛋白表达相对值。结果培养期间细胞逐渐趋于心肌细胞的特性;对照组心肌细胞均正常生长,硫化氢对照组与对照组相近,模型组严重异常;心肌细胞表面积、凋亡率、MDA含量、Sph K1、S1P1 mRNA的相对表达量、Sph K1、S1P1蛋白的相对表达量比较,模型组最高,硫化氢组次之,对照组和硫化氢对照组均最低; OD值、SOD含量、p-PI3K/PI3K、p-AKT/AKT蛋白表达相对值比较,对照组最高,硫化氢组次之,模型组最低,除对照组和硫化氢对照组差异无统计学意义(P> 0. 05),上述指标其余两组间对比差异均有统计学意义(P <0. 05);各组PI3K、AKT mRNA的相对表达量差异均无统计学意义(P> 0. 05)。结论硫化氢能够控制NE诱导的心肌细胞肥大病变,促进增殖、抑制凋亡,减轻心肌细胞氧化应激损伤,推测与通过S1P/S1P1信号通路降低Sph K1、S1P1表达,上调p-PI3K/PI3K、p-AKT/AKT蛋白表达相对值有关。

Objective To observe the effect of myocardial protection of hydrogen sulfide on norepinephrine(NE)induced cardiomyocyte hypertrophy rats,and to explore the mechanism.Methods Cardiomyocytes from SD rats were digested and isolated by collagenase,and cardiomyocyte hypertrophy models were induced by NE.Hydrogen sulfide group(100μmol/L NE+10μmol/L sodium hydrosulfide hydrate),model group(100μmol/L NE+equivalent saline),control group(equivalent saline+equivalent saline)and hydrogen sulfide control group(equivalent saline+10μmol/L sodium hydrosulfide hydrate)were set up.The changes of primary cardiomyocytes in neonatal rats under light microscope were observed.After 3 d,morphological changes of myocardial cells were observed.The myocardial cell surface area,proliferation and apoptosis rates were compared.Levels of MDA and SOD were detected and compared.Expressions of SphK1,S1P1,PI3K,AKT mRNA,SphK1,S1P1 proteins and relative expressions of p-PI3K/PI3K,p-AKT/AKT were compared.Results The cultured cells gradually displayed cardiomyocytes characteristics.Cells of control group showed normal growth.Hydrogen sulfide control group was similar to control group.Model group showed severe abnormalities.Regarding cell surface area,apoptosis rates,MDA contents,expressions of SphK1,S1P1 mRNA and proteins,model group were the highest,followed by hydrogen sulfide group,while control group and hydrogen sulfide control group were the lowest.Regarding OD values,SOD contents and relative expressions of p-PI3K/PI3K,p-AKT/AKT,control group were the highest,followed by hydrogen sulfide group,while model group was the lowest.Regarding the above indices,control group and hydrogen sulfide control group showed no significant differences(P>0.05),while the other two groups had significant differences(P<0.05).There was no significant difference in PI3K and AKT mRNA expressions among all groups(P>0.05).Conclusion Hydrogen sulfide can control hypertrophic cardiomyocytes induced by NE,reduce oxidative stress injury,promote proliferation and inhibit apoptosis.Underlying mechanism might be reduction of SphK1,S1P1 expressions and up-regulation of p-PI3K/PI3K,p-AKT/AKT relative expressions through S1P/S1P1 signaling pathway.