基于HIF-VEGF-Notch通路探讨通脉活血汤对局灶性脑缺血再灌注大鼠的保护作用

Protective effect of Tongmai Huoxue decoction on focal cerebral ischemia-reperfusion in rats based on HIF-VEGF-Notch pathway

目的探讨通脉活血汤通过影响大鼠低氧诱导因子(HIF)-血管内皮生长因子(VEGF)-Notch通路对局灶性脑缺血再灌注损伤(FCIRI)的保护作用。方法将54只雄性SD大鼠随机分为假手术组、模型组、抑制剂组[HIF-1α抑制剂YC-1,2.5 mg/(kg·d)]和通脉活血汤低、中、高剂量组[20、40、80 mg/(kg·d)]。线栓法构建大鼠脑中动脉阻塞(MACO)模型,模拟局灶性脑缺血再灌注。建模后假手术组和模型组腹膜注射2.5 ml/d 5%葡萄糖注射液,抑制剂组和通脉活血汤组分别注射对应剂量YC-1和通脉活血汤。对大鼠神经功能进行评分、检测大鼠脑脊液中VEGF水平、检测冰冻切片中胚胎干细胞关键蛋白(Sox2)和胶质酸性蛋白(GAFP)细胞数、RT-PCR检测HIF-1、VEGF、Notch1 mRNA水平、Western印迹检测HIF-1α、Notch1蛋白水平。结果与假手术组相比,模型组脑组织中Sox2、GFAP染色细胞数显著降低(P<0.05);神经功能评分、脑组织中Notch1、VEGF和HIF-1 mRNA水平、脑脊液中VEGF水平、脑组织中HIF-1α和Notch1蛋白水平显著升高(P<0.05)。与模型组相比,抑制剂组和通脉活血汤低、中、高剂量组脑组织中Sox2、GFAP染色细胞数显著升高(P<0.05),且通脉活血汤各剂量组升高水平呈剂量依赖性(P<0.05);神经功能评分、脑组织中Notch1、VEGF和HIF-1 mRNA、脑脊液中VEGF水平、脑组织中HIF-1α和Notch1蛋白水平显著降低(P<0.05),且通脉活血汤各剂量组降低水平呈剂量依赖性(P<0.05)。与抑制剂组相比,通脉活血汤高剂量组神经功能评分、Sox2、GFAP染色细胞数、脑组织中Notch1、VEGF和HIF-1 mRNA、脑脊液中VEGF水平、脑组织中HIF-1α和Notch1蛋白水平差异无统计学意义(P>0.05)。结论在局灶性脑缺血模型大鼠的治疗过程中,通脉活血汤可能通过影响HIF-VEGF-Notch通路来促进神经干细胞增殖分化,从而起到神经保护的作用。

Objective To investigate the protective effect of Tongmai Huoxue decoction on focal cerebral ischemia reperfusion injury by influencing HIF-VEGF-Notch pathway in rats.Methods 54 male Sprague Dawley rats were divided into sham,model,inhibitor[YC-1,2.5 mg/(kg·d)]and Tongmai Huoxue decoction low,medium and high dose groups[20,40,80 mg/(kg·d)].The rat model of middle cerebral artery occlusion(MACO)was created by thread embolization to simulate focal cerebral ischemia reperfusion.The sham group and model group were injected with 2.5 ml/d 5%glucose injection,and the inhibitor group and each dose groups were injected with corresponding doses of YC-1 and Tongmai Huoxue decoction respectively.Rat neurological function score,the level of vascular endothelial growth factor(VEGF)in cerebrospinal fluid,the amount of embryonic stem cell key protein(Sox2)and glial acidic protein(GAFP)cells in freezing tissue slices,the level of hypoxia inducible factor(HIF)-1,VEGF,Notch 1 mRNA were detected,and Western blot was used to detect the levels of HIF-1αand Notch1 protein.Results Compared with sham group,the number of Sox2 and GFAP stained cells in model group was significantly decreased(P<0.05);Neurological function score,levels of Notch1,VEGF and HIF-1 mRNA in brain tissue,level of VEGF in cerebrospinal fluid,and levels of HIF-1αand Notch1 protein in brain tissue were significantly increased(P<0.05).Compared with model group,the number of Sox2 and GFAP staining cells in the brain tissues of the inhibitor group and the low-dose,medium-dose and high-dose Tongmai Huoxue decoction groups were significantly increased(P<0.05),and the increase level in the Tongmai Huoxuxue decoction group was dose-dependent(P<0.05);Neurofunctional scores,mRNA levels of Notch1,VEGF and HIF-1 in brain tissues,levels of VEGF,HIF-1αand Notch1 protein in cerebrospinal fluid were significantly decreased,and the decreased levels in Tongmai Huoxue decoction dose group were dose-dependent(P<0.05).There were no significant differences in neurological function score,Sox2,GFAP staining cell number,Notch1,VEGF and HIF-1 mRNA in brain tissue,levels of vascular endothelial growth factor in cerebrospinal fluid,levels of HIF-1αand Notch1 protein in brain tissue between high-dose Tongmai Huoxue decoction group and inhibitor group(P>0.05).Conclusions Tongmai Huoxue decoction might promote the proliferation and differentiation of neural stem cells by influencing HIF-VEGF-Notch pathway in the treatment of rats with focal cerebral ischemia,thus playing a neuroprotective role.