昼夜环境紊乱对缺血性中风后血管新生的影响及活血荣络方的干预作用

Effects of environmental circadian disruption on post-ischemic stroke vascular neovascularization and interventional effect of Huoxue Rongluo Recipe

目的观察昼夜环境紊乱对缺血性中风后血管新生的影响及活血荣络方的干预作用。方法SD大鼠随机分假手术组、模型组(MCAO)、活血荣络方组(MCAO+HXRLF)、模型+生物节律紊乱组(MCAO-ECD)及模型+生物节律紊乱+活血荣络方组(MCAO-ECD+HXRLF),建立大脑中动脉闭塞再灌注模型,给药10 d。采用神经功能缺损评分评价神经功能缺损;HE染色观察病理变化;免疫荧光观察海马及皮质CD34数;Western blot法检测血管新生及生物节律关键蛋白;ELISA法检测血清血管内皮生长因子(vascular endothelial growth factor,VEGF)、褪黑素(melatonine,MT)及炎症因子。结果昼夜环境紊乱加重梗死后神经缺损(P<0.01),减少皮质及海马CD34阳性细胞数(P<0.05),降低脑内血管新生蛋白(P<0.05)及血清中VEGF(P<0.01)、MT(P<0.05)水平,升高血清IL-1β(P<0.01)与TNF-α(P<0.05)水平,活血荣络方改善昼夜环境紊乱加重的神经功能缺损(P<0.01),增加皮质及海马区CD34阳性细胞数表达(P<0.05),增加脑组织血管新生蛋白表达(P<0.05),升高血清中VEGF(P<0.01)、MT(P<0.05)水平,降低炎症因子水平(P<0.05)。结论昼夜环境紊乱加重脑梗死后神经功能损伤,抑制血管新生,活血荣络方可逆转该进程,促进血管新生,减轻炎症反应,改善神经功能缺损。

Aim To observe the effect of circadian environmental disorder on post-ischemic stroke vascular neovascularization and the intervention effect of blood activation and Rongluo formula.Methods SD rats were randomly divided into control group,model group,Huoxue Rongluo Recipe group,model+environmental circadian disruption group and model+environmental circadian disruption+Huoxue Rongluo Recipe group,with 10 rats in each group.The rats were evaluated by the modified neurological severity score(mNSS),and the histopathological changes in each group were observed by HE staining;the CD34 expression in hippocampal and cortical areas was observed by immunofluorescence staining;and the brain expression in each group was detected by Western blot.The expression of VEGF,MMP-2,MMP-9,Bmal1 and Per1 in brain tissues of the infarcted side of the rats in each group was detected by Western blot;the expression of VEGF,melatonin,IL-1β,IL-18 and TNF-αin serum of each group was detected by ELISA.Results Disturbance of circadian environment aggravated the symptoms of neurological deficits after cerebral infarction(P<0.01),decreased the expression of CD34 positive cells in cortical and hippocampal areas(P<0.05),decreased the expression of VEGF,MMP-2 and MMP-9 proteins in brain tissues of infarct area(P<0.05),decreased the serum levels of VEGF(P<0.01),melato-nin levels(P<0.05),and elevated serum levels of IL-1β(P<0.01)and TNF-α(P<0.05).Rongluo formula significantly improved the symptoms of neurological deficits(P<0.01),increased the expression of CD34-positive cells in cortical and hippocampal areas(P<0.05),increased the expression of VEGF,MMP-2 and MMP-9 in the infarcted brain tissue(P<0.05),increased serum levels of VEGF(P<0.01)and melatonin(P<0.05),and decreased levels of inflammatory factors(P<0.05).Conclusions The process of ischemic stroke is aggravated by circadian environmental disorder,inflammatory reaction and inhibition of vascular neovascularization.Huoxue Rongluo Recipe can effectively reverse the process and promote vascular neovascularization after cerebral infarction,thus improving the symptoms of neurological deficits.