阿魏酸通过磷酸肌醇3激酶/丝氨酸/苏氨酸蛋白激酶途径保护大鼠免受心肌缺血/再灌注损伤

Ferulic acid protects rats from myocardial ischemia/reperfusion injury via the phosphoinositide 3-kinase/serine/threonine protein kinase pathway

目的研究阿魏酸对大鼠心肌缺血/再灌注(I/R)损伤的保护作用及其可能的机制。方法将雄性SD大鼠采用随机数字表法分为四组(n=12):假手术组(Sham组),I/R模型组(I/R组);阿魏酸+I/R组(Fer+I/R组);阿魏酸+蛋白激酶抑制剂(LY294002)+I/R组(Fer+LY+I/R)。通过结扎左冠状动脉前降支30 min,然后再灌注2 h,建立I/R损伤大鼠模型。通过苏木精-伊红(HE)染色分析大鼠的左心室病理学变化,结合透射电子显微镜观察其超微结构;通过TUNEL/DAPI双重染色检测大鼠心肌细胞凋亡率。通过蛋白质印迹法(Western blotting)检测PI3K/Akt信号通路关键调控因子的蛋白表达水平[包括PI3K、p-Akt(Ser 473)、Akt、eNOS、p-eNOS(Ser1177)和p-mTOR(Ser2448)],以及血清超氧化物歧化酶(SOD)、丙二醛(MDA)、肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)和IL-1β水平。结果HE染色结果显示,相较于假手术组,I/R组大鼠心肌中表现出明显的炎性细胞浸润、细胞膜损伤、细胞坏死和水肿;而Fer+I/R组大鼠表现心肌细胞坏死、炎性细胞浸润,细胞水肿均明显低于I/R组,并且心肌纤维结构清晰完整。TUNEL/DAPI双重染色结果显示,Fer+I/R组大鼠[(18.73±1.01)%]心肌细胞凋亡率明显低于I/R组[(55.45±1.14)%](P<0.001)。Western blotting检测结果表明,与I/R组相比,Fer+I/R组大鼠Cleaved caspase-3蛋白表达水平显著降低(P<0.05),PI3K、p-Akt、Akt、p-eNOS(Ser1177)和p-mTOR(Ser2448)蛋白表达明显上调(P<0.05)。相较于I/R组,阿魏酸显著抑制MDA5、TNF-α、IL-6和IL-1β的表达(P<0.05),并上调SOD1蛋白表达水平(P<0.05);而LY294002处理逆转了阿魏酸在I/R模型大鼠中对上述细胞因子的调控作用。结论阿魏酸通过激活PI3K/Akt信号通路抑制心肌细胞凋亡,减轻炎症反应和氧化应激水平,保护大鼠免受缺血/再灌注诱导的心肌损伤。

Objective To study the protective effect of ferulic acid on myocardial ischemia/reperfusion(I/R)injury in rats and its potential mechanism.Methods Male Sprague-Dawley(SD)rats were randomly divided into 4 groups(n=12):sham operation(sham)group,I/R group;ferulic acid+I/R(FER+I/R)group;and ferulic acid+LY294002(protein kinase inhibitors)+I/R(FER+LY+I/R)group.A rat model of I/R injury was established by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for 2 h.The pathological changes in the left ventricle of rats were analyzed by hematoxylin-eosin(HE)staining,and the ultrastructure was observed by transmission electron microscopy.The apoptosis rate of rat cardiomyocytes was detected by TUNEL/DAPI dual staining.Western blotting was used to detect the protein expression levels of key regulators of the PI3K/Akt signaling pathway,including PI3K,p-Akt(Ser473),Akt,e NOS,p-e NOS(Ser1177)and p-m TOR(Ser2448),and serum levels of superoxide dismutase(SOD),malondialdehyde(MDA),tumor necrosis factor-α(TNF-α),interleukin 6(IL-6)and IL-1β.Results The results of HE staining showed that compared with the sham-operation group,the rats in the I/R group showed obvious inflammatory cell infiltration,cell membrane damage,cell necrosis and edema in the myocardium,while the rats in the Fer+I/R group showed myocardial cell necrosis,inflammatory cell infiltration and cell edema that were significantly lower than those in the I/R group,and the myocardial fiber structure was clear and complete.The results of TUNEL/DAPI dual staining showed that the apoptosis rate of cardiomyocytes in the Fer+I/R group was(18.73±1.01)%,which was significantly lower than that in the I/R group(55.45±1.14)%(P<0.001).Western blotting results showed that compared with the I/R group,the expression level of cleaved caspase-3 protein in the Fer+I/R group was significantly decreased(P<0.05),PI3K,p-Akt,Akt,p-e NOS(Ser1177)and p-m TOR(Ser2448)protein expression was significantly upregulated(P<0.05).Compared with the I/R group,ferulic acid significantly inhibited the expression of MDA,TNF-α,IL-6 and IL-1β(P<0.05)and upregulated the expression level of SOD protein(P<0.05).LY294002 treatment reversed the regulatory effect of ferulic acid on the aforementioned cytokines in I/R model rats.Conclusion Ferulic acid inhibited cardiomyocyte apoptosis by activating the PI3K/Akt signaling pathway,attenuated the level of the inflammatory response and oxidative stress,and protected rats from I/R-induced myocardial injury.