补阳还五汤通过甲酰肽受体2减轻大鼠脑缺血再灌注后氧化应激损伤

Buyang Huanwutang Alleviated Oxidative Stress Following Cerebral Ischemia/Reperfusion in Rats by Formyl Peptide Receptor 2

目的:探讨补阳还五汤通过甲酰肽受体2(FPR2)对脑缺血再灌注模型大鼠氧化应激反应的抑制作用及神经保护作用。方法:将48只雄性SD大鼠随机分为假手术组、模型组、补阳还五汤组和补阳还五汤联合FPR2抑制剂(Boc-2)组,假手术组仅游离血管,不做插线处理。其他各组运用改良Longa法构建大脑中动脉栓塞(MCAO)模型,缺血2 h后再灌注;再灌注后补阳还五汤组和补阳还五汤联合Boc-2组给予补阳还五汤(16 g·kg^(-1))灌胃,每日2次;补阳还五汤联合Boc-2组术前30 min腹腔注射Boc-2(0.4 mg·kg^(-1));假手术组与模型组给予等体积生理盐水。再灌注24 h后,退化神经元染色(FJC)观察FJC阳性细胞数目的变化;蛋白免疫印迹法(Western blot)检测缺血侧脑组织凋亡相关蛋白B细胞淋巴瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax)和活化型半胱氨酸天冬氨酸蛋白水解酶-3(cleaved Caspase-3)蛋白表达;生化试剂盒检测缺血侧脑组织中超氧化物歧化酶(SOD),丙二醛(MDA),谷胱甘肽(GSH),一氧化氮(NO)水平;免疫荧光检测还原型辅酶Ⅱ(NADPH)氧化酶2(NOX2)平均荧光强度。结果:与假手术组比较,模型组的FJC阳性细胞数目增加(P<0.01),Bcl-2表达减少(P<0.01),Bax和cleaved Caspase-3表达增加(P<0.01),NO和MDA含量增加(P<0.05,P<0.01),GSH和SOD活性下降(P<0.05,P<0.01),NOX2表达增加(P<0.01);与模型组比较,补阳还五汤组FJC阳性细胞数目减少(P<0.01),Bcl-2表达增加(P<0.01),cleaved Caspase-3和Bax明显减少(P<0.05,P<0.01),NO和MDA减少(P<0.05,P<0.01),GSH和SOD增加(P<0.01),NOX2表达减少(P<0.01)。给予Boc-2后,补阳还五汤的作用部分被抑制。结论:补阳还五汤可以减轻脑缺血再灌注大鼠氧化应激损伤,抑制细胞凋亡,其作用机制可能与FPR2调控NOX2的表达有关。

Objective:To investigate the role of formyl peptide receptor 2(FPR2)in the inhibitory effects of Buyang Huanwutang(BYHWT)on the oxidative stress and its protective effects on cerebral ischemiareperfusion in rats.Method:Forty-eight male SD rats were randomly divided into sham group,model group,BYHWT group and BYHWT combined with FPR2 inhibitor(Boc-2)group.In the sham group,only the vessels were isolated.In other groups,the middle cerebral artery occlusion(MCAO)model was constructed using the modified Longa method and reperfused after 2 h of ischemia.BYHWT(16 g·kg^(-1))was given by gavaged twice daily after reperfusion in BYHWT group and BYHWT+Boc-2 group.Boc-2(0.4 mg·kg^(-1))was injected intraperitoneally 30 min before surgery.Equal volume of saline were given instead in sham and model group.After 24 h of reperfusion,Fluoro-Jade C(FJC)staining was performed to observe the changes in the number of FJC-positive cells.Western blot was performed to detect the expression of apoptosis-related B-cell lymphoma-2(Bcl-2),Bcl-2 associated X(Bax),and cleaved aspartic acid cysteine proteolytic enzyme-3(Caspase-3).Besides,superoxide dismutase(SOD),malondialdehyde(MDA),glutathione(GSH),and nitric oxide(NO)was measured.The mean fluorescence intensity of nicotinamide adenine dinucleotide phosphateⅡ(NADPH)oxidase 2(NOX2)was examined by immunofluorescence.Result:Compared with sham group,the model group showed increased number of FJC-positive cells(P<0.01),decreased Bcl-2 expression(P<0.01),increased Bax and cleaved Caspase-3 expression(P<0.01),increased NO and MDA content(P<0.05,P<0.01),decreased GSH and SOD activities(P<0.05,P<0.01),and increased NOX2 expression(P<0.01).Compared with model group,there were decreased FJC-positive cells(P<0.01),up-regulated Bcl-2 expression(P<0.01)with down-regulated cleaved Caspase-3 and Bax(P<0.05,P<0.01),decreased NO and MDA(P<0.05,P<0.01)with increased GSH and SOD(P<0.01),and decreased NOX2 expression(P<0.01)in the BYHWT group.All the above effects were partially blocked by Boc-2.Conclusion:BYHWT can reduce oxidative stress injury and inhibit apoptosis in cerebral ischemia/reperfusion rats,which may be related with the down-regulation of NOX2 expression by FPR2.