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  

Protective Effects of Organic Acid-rich Fraction of CRATAEGI FRUCTUS Post Conditioning on Myocardial Ischemia-Reperfusion Injury through PI3K/AKT and MAPK Signaling Pathways

作　　者：权赫秀[1] 龚铭 罗涛[4] 席俊丽 韩英[3] 李永鑫 杨明 邵峰 唐芳瑞 QUAN Hexiu;GONG Ming;LUO Tao;XI Junli;HAN Ying;LI Yongxin;YANG Ming;SHAO Feng;TANG Fangrui(Department of Physiology,College of Chinese Medicine and Life Science,Jiangxi University of Chinese Medicine,Nanchang 330004;Key Laboratory of Modern Preparation of Traditional Chinese Medicine,Ministry of Education,Jiangxi University of Chinese Medicine,Nanchang 330004;Key Laboratory of Psychology of TCM and Brain Science,Jiangxi Administration of Traditional Chinese Medicine,Jiangxi University of Chinese Medicine,Nanchang330004;Blood Purification Center of the First Affiliated Hospital of Nanchang University,Nanchang 330004)

机构地区：[1]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 [2]江西中医药大学现代中药制剂教育部重点实验室,南昌330004 [3]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 [4]南昌大学第一附属医院血液净化中心,南昌330004

出　　处：《中药药理与临床》2024年第7期42-48,共7页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金(编号:81260638);江西省重大科技研发项目(编号:20194ABC28009);江西省卫健委科技项目(编号:202310474,202211410);江西中医药大学中西医结合一级学科(江西省双一流学科)(编号:zxyylxk20220103);江西省研究生创新专项基金项目(编号:YC2021-S485);中药炮制技术的传承与创新(编号:CXTD22003);企业横向课题(编号:20220370)。

摘　　要：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H_(2)O_(2)(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、100μg/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�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 reperfusion stage. Myocardial infarct size was evaluated by TTC staining. Cell viability was measured through CellTiter 96■ AQueous solution cell proliferation assay. H_(2)O_(2)-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 activity of SOD and GSH-Px in H_(2)O_(2)-induced cardiomyocyte injury(P<0.05 or P<0.01). OACP significantly inhibited cardiomyocyte apoptosis and the activity of caspase-3 and caspase-9.

关 键 词：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 

分 类 号：R285.5[医药卫生—中药学]