益气通络方通过PI3K/Akt/HIF-1α通路减轻脑缺血再灌注损伤的氧化应激及凋亡的机制研究

Study on the mechanism of Yiqi Tongluo Fang reducing oxidative stress and apoptosis in cerebral ischemia-reperfusion injury through PI3K/Akt/HIF-1αpathway

目的探讨益气通络方通过磷脂酰肌醇-3激酶/丝氨酸—苏氨酸蛋白激酶/低氧诱导因子-1α(phosphoinositide 3-kinase/serine threo-nine kinase/hypoxia inducible factor-1α,PI3K/Akt/HIF-1α)信号通路对脑缺血/再灌注大鼠的神经保护作用。方法40只大鼠随机分为假手术组、模型组、益气组、尼莫益气组,每组10只,采用大脑中动脉栓塞再灌注法进行造模,连续灌胃相应药物14天,采用Zea-longa评分法检测大鼠神经肌肉功能,病理染色观察脑组织形态。ELISA检测乳酸脱氢酶(lactate dehydrogenase,LDH)含量和细胞内超氧化物歧化酶(superoxide dismutase,SOD)活力及活性氧(reactive oxygen species,ROS)分布水平,血清肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、超敏C反应蛋白(hypersensitive C-reactive protein,hs-CRP)、血管内皮生长因子(vascular endothelial growth factor,VEGF)的含量。Western blot法检测脑组织PI3K、p-Akt、HIF-1α、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)蛋白表达。结果与假手术组比较,模型组大鼠脑组织明显损伤,血清中TNF-α、IL-6、hs-CRP、LDH、ROS含量升高,SOD含量降低,脑组织中Bax表达水平升高,PI3K、p-Akt、HIF-1α、Bcl-2表达水平降低(P<0.05);与模型组比较,益气组及尼莫益气组大鼠脑组织损伤程度较轻,血清中TNF-α、IL-6、hs-CRP、LDH、ROS含量降低,SOD、VEGF含量升高,脑组织中Bax表达水平降低,PI3K、p-Akt、HIF-1α、Bcl-2表达水平升高(P<0.05)。结论益气通络方能够抑制大鼠神经细胞凋亡、降低炎症损伤,其机制可能与激活PI3K/Akt、上调HIF-1α信号通路及Bax/Bcl-2比率有关。

Objective To investigate the neuroprotective effect of Yiqi Tongluo Fang on cerebral ischemia/reperfusion rats through PI3K/Akt/HIF-1αsignaling pathway.Methods Forty rats were randomly divided into sham operation group,model group,Yiqi group and Nimo Yiqi group,with 10 rats in each group.The model was established by middle cerebral artery occlusion and reperfusion.The corresponding drugs were continuously administered for 14 days.The neuromuscular function of rats was detected by Zea-longa scoring method,and the morphology of brain tissue was observed by pathological staining.Lactate dehydrogenase(LDH)content,intracellular superoxide dismutase(SOD)activity and reactive oxygen species(ROS)distribution level,serum tumor necrosis factorα(TNF-α),interleukin-6(IL-6),high-sensitivity C-reactive protein(hs-CRP)and vascular endothelial growth factor(VEGF)content were detected by the method of ELISA.The expressions of PI3K,p-Akt,HIF-1α,Bax and Bcl-2 in brain tissue were detected by Western blot.Results Compared with the sham operation group,the brain tissue of the model group was significantly damaged,the contents of TNF-α,IL-6,hs-CRP,LDH and ROS in serum were increased,the content of SOD was decreased,the expression of Bax in brain tissue was increased,and the expression of PI3K,p-Akt,HIF-1αand Bcl-2 were decreased(P<0.05).Compared with the model group,the degree of brain tissue injury in the Yiqi group and the Nimo Yiqi group was mild,the contents of TNF-α,IL-6,hs-CRP,LDH and ROS in serum were decreased,the contents of SOD and VEGF were increased,the expression level of Bax in brain tissue was decreased,and the expressions of PI3K,p-Akt,HIF-1αand Bcl-2 were increased(P<0.05).Conclusion Yiqi Tongluo Fang can inhibit neuronal apoptosis and reduce inflammatory injury in rats.The mechanism may be related to the activation of PI3K/Akt,up-regulation of HIF-1αsignaling pathway and Bax/Bcl-2 ratio.