基于p38MAPK通路研究血府逐瘀汤含药血清减轻心肌细胞缺氧复氧损伤的作用

Effects of Xuefu Zhuyu Decoction containing serum on hypoxia reoxygenation injury of cardiomyocytes based on p38MAPK pathway

目的基于p38MAPK通路研究血府逐瘀汤含药血清减轻心肌细胞缺氧复氧损伤的作用。方法制备低剂量、中剂量、高含量血府逐瘀汤含药血清,培养心肌H9c2细胞并分为对照组、模型组、低剂量组、中剂量组、高剂量组。除对照组外,其余各组给予缺氧复氧处理,同时用含有10%空白血清或10%含药血清的培养基进行干预,检测培养基中乳酸脱氢酶(lactate dehydrogenase,LDH)及磷酸肌酸激酶同工酶(creatine kinase isoenzyme,CK-MB)的含量、心肌细胞的存活率及凋亡率、细胞中B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白质(Bcl-2-associated X Protein,Bax)、裂解型caspase-3(cleaved caspase-3)、磷酸化p38MAPK(phosphorylation-p38MAPK,p-p38MAPK)、磷酸化JNK(phosphorylation-JNK,p-JNK)、磷酸化ERK(phosphorylation-ERK,p-ERK)的表达水平。结果与对照组、低剂量组、中剂量组、高剂量组相比,模型组培养基中LDH、CK-MB的含量、凋亡率、细胞中Bax、cleaved caspase-3、p-p38MAPK、p-JNK的表达水平均显著升高,存活率及细胞中Bcl-2、p-ERK的表达水平均显著降低,组间差异均具有统计学意义(F值分别9.39、10.27、13.28、13.09、14.59、10.40、8.17,8.31、11.37、9.38,P值均<0.05)。与模型组比较,低剂量组、中剂量组、高剂量组心肌细胞培养基中LDH、CK-MB的含量明显减少[LDH(U/L):(185.68±25.73)比(131.22±18.29),(104.57±13.27),(78.34±10.93);CK-MB(U/L):(24.58±4.48)比(16.79±2.15),(11.32±1.46),(9.01±1.25),P值均<0.05];心肌细胞的存活率明显增加、凋亡率明显降低[存活率(%):(42.59±6.29)比(56.51±5.62),(67.32±6.11),(80.34±8.27);凋亡率(%):(27.68±5.56)比(17.03±2.84),(12.55±2.02),(8.68±1.09),P值均<0.05];心肌细胞中Bcl-2的表达水平明显增加、Bax及cleaved caspase-3的表达水平明显降低[Bcl-2:(0.24±0.05)比(0.37±0.06),(0.59±0.09),(0.77±0.12);Bax:(0.88±0.15)比(0.64±0.09),(0.49±0.08),(0.34±0.06);Cleaved caspase-3:(1.15±0.32)比(0.90±0.14),(0.67±0.10),(0.38±0.06),P值均<0.05];心肌细胞中p-p38MAPK的表达水平明显降低[(1.22±0.20)比(0.93±0.13),(0.78±0.07),(0.47±0.06),P值均<0.05];各组间p-JNK、p-ERK的表达水平无明显变化(P>0.05)。结论血府逐瘀汤含药血清能够减轻心肌细胞缺氧复氧损伤,其作用机制可能与抑制p38MAPK通路激活有关。

Objective To study the protective effect of Xuefu Zhuyu Decoction containing serum on hypoxia reoxygenation injury of cardiomyocytes based on p38MAPK pathway.Methods Low dose,medium dose and high content of Xuefu Zhuyu Decoction containing serum were prepared.H9c2 cells were cultured and divided into control group,model group,low dose group,medium dose group and high dose group.Apart from the control group,the other groups were treated with hypoxia reoxygenation,and the medium containing 10%blank serum or 10%drug containing serum was used for intervention.The content of lactate dehydrogenase(LDH)and creatine kinase isoenzyme(CK-MB)in the medium,the survival rate and apoptosis rate of cardiomyocytes,the expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved caspase-3,phosphorylation-p38MAPK(p-p38MAPK),phosphorylation-JNK(p-JNK)and phosphorylation-ERK(p-ERK)were detected.Results Compared with the control group,low dose group,medium dose group and high dose group,the contents of LDH,CK-MB,the apoptosis rate,the expression levels of Bax,cleaved caspase-3,p-p38MAPK and p-JNK in the model group were significantly increased,and the survival rate and the expression levels of Bcl-2,p-ERK were significantly lower in the model group(F values were 9.39,10.27,13.28,13.09,14.59,10.40,8.17,8.31,11.37 and 9.38 respectively,all P values<0.05).Compared with model group,the contents of LDH,CK-MB significantly decreased[LDH(U/L):(185.68±25.73)vs(131.22±18.29),(104.57±13.27),(78.34±10.93);CK-MB(U/L):(24.58±4.48)vs(16.79±2.15),(11.32±1.46),(9.01±1.25),all P values<0.05];the survival rate significantly increased,and the apoptosis rate significantly decreased[survival rate(%):(42.59±6.29)vs(56.51±5.62),(67.32±6.11),(80.34±8.27);the apoptosis rate(%):(27.68±5.56)vs(17.03±2.84),(12.55±2.02),(8.68±1.09),all P values<0.05];the expression levels of Bcl-2,Bax,cleaved caspase-3 significantly decreased[Bcl-2:(0.24±0.05)vs(0.37±0.06),(0.59±0.09),(0.77±0.12);Bax:(0.88±0.15)vs(0.64±0.09),(0.49±0.08),(0.34±0.06);Cleaved caspase-3:(1.15±0.32)vs(0.90±0.14),(0.67±0.10),(0.38±0.06),all P values<0.05];the expression levels of p-p38MAPK significantly decreased[(1.22±0.20)vs(0.93±0.13),(0.78±0.07),(0.47±0.06),all P values<0.05];no significant changes have been found in the expression levels of p-JNK and p-ERK between the groups(P>0.05).Conclusion Xuefu Zhuyu Decoction containing serum can alleviate hypoxia reoxygenation injury of cardiomyocytes,and its mechanism may be related to the inhibition of p38MAPK pathway activation.