首页|山楂有机酸通过PI3K/AKT和MAPK信号通路保护心肌缺血再灌注损伤的缺血后适应作用

山楂有机酸通过PI3K/AKT和MAPK信号通路保护心肌缺血再灌注损伤的缺血后适应作用

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目的:探讨山楂有机酸通过缺血后适应保护心肌缺血再灌注(I/R)损伤的作用机制。方法:采用Langendorff离体心脏灌流装置对SD大鼠心脏进行缺血再灌注并记录心脏动力学改变，从再灌注期开始全程在灌流液中给予不同浓度的山楂有机酸。采用三苯四唑氯(TTC)染色评估心肌梗死面积。采用CellTiter 96 ®溶液细胞增殖法测定心肌细胞存活率;流式细胞仪检测H2O2(过氧化氢)诱导的心肌细胞凋亡;采用试剂盒检测乳酸脱氢酶(LDH)释放、细胞内丙二醛(MDA)生成、抗氧化酶[超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)]活力;以试剂盒检测半胱氨酸天冬氨酸酶-3(Caspase-3)和Caspase-9活力;Western blot法检测线粒体凋亡调控蛋白(BAX和BCL-2)、磷酸化丝氨酸-苏氨酸蛋白激酶(p-AKT)、磷酸化细胞外信号调节激酶(p-ERK)、磷酸化c-Jun氨基末端激酶(p-JNK)和磷酸化丝裂原活化蛋白激酶(p-P38)的表达变化。结果:与空白对照组相比，模型对照组的左心室舒张压显著降低，心肌梗死面积显著增加，细胞活力显著下降(P＜0。01)，LDH释放和细胞内MDA生成显著增加(P＜0。01)，SOD和GSH-Px的活力显著降低(P＜0。01)，Caspase-3和Caspase-9的活力显著增加(P＜0。01)，BAX的表达上调，BCL-2的表达显著下调(P＜0。01)，AKT和ERK的磷酸化被抑制、P38和JNK的磷酸化被激活(P＜0。05或P＜0。01);与模型对照组比较，山楂有机酸25、100 µg/mL组显著改善了小鼠离体心脏左心室舒张压(P＜0。01)，降低了心肌I/R损伤引起的梗死面积(P＜0。01)，明显提高了心肌细胞存活率，降低了过氧化氢致心肌细胞损伤的LDH泄漏和MDA生成、提高了 SOD和GSH-Px活力(P＜0。05或P＜0。01)，明显降低过氧化氢诱导的心肌细胞凋亡以及Caspase-3和Caspase-9活力，下调BAX、BCL-2蛋白表达(P＜0。05或P＜0。01)，上调p-AKT和p-ERK蛋白表达(P＜0。05)，下调p-JNK和p-P38的蛋白表达(P＜0。01)，PI3K特异性抑制剂LY294002、JNK和P38特异性激动剂Anisomycin以及ERK特异性抑制剂PD98059降低山楂有机酸100 μg/mL对过氧化氢引起的细胞存活率升高和Caspase-3活力的降低(P＜0。05或P＜0。01)。结论:PI3K/AKT和丝裂原活化蛋白激酶(MAPK)信号通路可能介导了山楂有机酸通过缺血后适应提高抗氧化酶活力、抑制心肌细胞凋亡，保护心肌I/R损伤的作用。
Protective Effects of Organic Acid-rich Fraction of CRATAEGI FRUCTUS PostConditioning on Myocardial Ischemia-Reperfusion Injury through PI3K/AKT and MAPK Signaling Pathways
Objective:To investigate the mechanism of protective effects of organic acid-rich fraction of CRATAEGI FRUCTUS(OACP)against myocardial ischemia-reperfusion(I/R)injury by postconditioning.Methods:Hearts of Sprague-Dawley rats were perfused on Langendorff'apparatus and cardiac dynamic changes were recorded.Different concentrations of OACP were given in the perfusion solution from the reper-fusion stage.Myocardial infarct size was evaluated by TTC staining.Cell viability was measured through CellTiter 96 ® AQueous solution cell proliferation assay.H2O2-induced apoptosis was measured by flow cytometer.LDH release,intracellular MDA production,and antioxidant enzymes(SOD and GSH-Px)were determined by the corresponding kits.Caspase-3 and caspase-9 activity were measured by the assay kits.Expression levels of apoptosis-related proteins(Bax and Bcl-2),p-Akt,p-ERK,p-JNK and p-p38 were analyzed by Western blot.Results:Compared with control group,left ventricular diastolic pressure(LVDP)was significantly decreased in model group,myocardial infarct size was increased,and cell viability was significantly reduced(P＜0.01).LDH release and intracellular MDA level was significantly increased(P＜0.01).The activities of SOD and GSH-Px were significantly decreased(P＜0.01),while the activities of caspase-3 and caspase-9 were significantly increased(P＜0.01).The expressions of Bax was up regulated,while the expression of Bcl-2 was significantly decreased(P＜0.01).Akt and ERK phosphorylation was inhibited,while JNK and p38 phosphorylation was activated(P＜0.05 or P＜0.01).Compared with model group,OACP at 100 µg/mL markedly improved LVDP(P＜0.01),and decreased the infarct size(P＜0.01).OACP at 100 μg/mL significantly increased myocardial cell survival rate It markedly reduced LDH leakage and MDA production,and enhanced the ac-tivity of SOD and GSH-Px in H2O2-induced cardiomyocyte injury(P＜0.05 or P＜0.01).OACP significantly inhibited cardiomyocyte apopto-sis and the activity of caspase-3 and caspase-9.It down regulated the protein expressions of Bax and Bcl-2(P＜0.05 or P＜0.01).OACP up regulated the protein expressions of p-Akt and p-ERK(P＜0.05),while down regulated the protein expressions of p-JNK and p-p38(P＜0.01).The PI3K-specific inhibitor LY294002,JNK and p38-specific agonist Anisomycin,and the ERK-specific inhibitor PD98059 blocked the effect of OACP on cell viability and activity of caspase-3 induced by H2O2(P＜0.05 or P＜0.01).Conclusion:Effect of OACP against myocardial I/R injury may be related to enhancing antioxidant enzymes and suppressing apoptosis by postconditioning through PI3K/Akt and MAPK signaling pathways.

Organic acid-rich fraction of Crataegus pinnatifida fruitMyocardial ischemia-reperfusion injuryPostconditioningOxidative stressAntioxidant enzymeCell apoptosisPI3K/AKT and MAPK signaling pathways

权赫秀、龚铭、罗涛、席俊丽、韩英、李永鑫、杨明、邵峰、唐芳瑞

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江西中医药大学中医学院生命科学学院生理学教研室,南昌 330004

南昌大学第一附属医院血液净化中心,南昌 330004

江西中医药大学中医心理学与脑科学重点实验室,江西省中医药管理局,南昌 330004

江西中医药大学现代中药制剂教育部重点实验室,南昌 330004

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山楂有机酸 心肌缺血再灌注损伤 缺血后适应 氧化应激 抗氧化酶 细胞凋亡 磷脂酰肌醇3-羟激酶/丝氨酸-苏氨酸蛋白激酶和丝裂原活化蛋白激酶信号通路

2024

中药药理与临床
中国药理学会 四川省中医药科学院

中药药理与临床

北大核心
影响因子：0.996
ISSN：1001-859X
年,卷(期)：2024.40(7)