基于miR-34调控KLF4/VEGF传导轴探讨活血荣络方促脑梗死后血管新生的机制

Mechanism of Huoxue Rongluo Recipe on promoting angiogenesis after cerebral infarction based on miR-34 regulating KLF4/VEGF conduction axis

目的基于miR-34家族基因与Kruppel样因子4(Kruppel-like factor 4,KLF4)/血管内皮生长因子(vascular endothelial growth factor,VEGF)传导轴的相关性探讨活血荣络方促脑梗死后血管新生的机制。方法将SD大鼠随机分为对照组、模型组、活血荣络方(11.7 g/kg)组和丁苯酞(60 mg/kg)组,每组10只,建立大脑中动脉闭塞/再灌注(middle cerebral artery occlusion/reperfusion,MCAO/R)模型,连续ig药物7 d。采用改良神经功能缺损评分(modified neurological severity score,mNSS)评价大鼠神经功能缺损症状;采用免疫荧光染色观察海马区CD34及皮质区VEGF表达;采用Western blotting法检测各组大鼠梗死侧脑组织中KLF4和VEGF蛋白表达情况;采用qRT-PCR法检测各组大鼠脑组织中miR-34家族基因的表达;采用Pearson相关分析探讨miR-34家族基因与mNSS、KLF4、VEGF的相关性。结果活血荣络方能够显著改善大鼠神经功能缺损症状(P<0.01),增加海马区CD34阳性细胞及皮质区VEGF表达(P<0.01),增强脑组织中KLF4和VEGF蛋白表达(P<0.01),抑制miR-34家族基因miR-34a、miR-34b、miR-34c mRNA表达(P<0.01);且mNSS与miR-34a、miR-34b、miR-34c呈高度正相关(P<0.05);VEGF与miR-34a、miR-34b、miR-34c呈高度负相关(P<0.05);KLF4与miR-34b呈高度负相关(P<0.05),与miR-34c呈高度负相关(P<0.01)。结论活血荣络方能够抑制miR-34a、miR-34b、miR-34c mRNA表达,激活KLF4/VEGF传导轴,促进脑梗死后血管新生,进而改善神经功能缺损症状。

Objective Based on the correlation between miR-34 family genes and Kruppel-like factor 4(KLF4)/vascular endothelial growth factor(VEGF)conduction axis to investigate the mechanism of Huoxue Rongluo Recipe(活血荣络方)on promoting angiogenesis after cerebral infarction.Methods SD rats were randomly divided into control group,model group,Huoxue Rongluo Recipe(11.7 g/kg)group and butylphthalide(60 mg/kg)group,10 rats in each group.Middle cerebral artery occlusion/reperfusion(MCAO/R)rats model were established,and rats were ig drugs for 7 d.Modified neurological severity score(mNSS)was used to evaluate the symptoms of neurological deficit in rats;Immunofluorescence staining was used to observe the expression of CD34 in hippocampus and VEGF in cortex;Western blotting was used to detect the expression of KLF4 and VEGF in infarcted brain tissue;qRT-PCR was used to detect the expression of miR-34 family genes in brain tissue;Pearson correlation analysis was used to observe the correlation between miR-34 family genes and m NSS,KLF4 and VEGF.Results Huoxue Rongluo Recipe significantly improved the symptoms of neurological deficits in rats(P<0.01),increased expression of CD34 positive cells in hippocampus and expression of VEGF in cortex(P<0.01),enhanced expression of KLF4 and VEGF protein in brain tissue(P<0.01),inhibited m RNA expressions of miR-34 a,miR-34 b and miR-34 c(P<0.01);mNSS was highly positively correlated with miR-34 a,miR-34 b and miR-34 c(P<0.05);VEGF was highly negatively correlated with miR-34 a,miR-34 b and miR-34 c(P<0.05);KLF4 was highly negatively correlated with miR-34 b(P<0.05),highly negatively correlated with miR-34 c(P<0.01).Conclusion Huoxue Rongluo Recipe can inhibit expressions of miR-34 a,miR-34 b and miR-34 c,activate KLF4/VEGF conduction axis,promote angiogenesis after cerebral infarction,and then improve the symptoms of neurological deficits.