注射用丹参多酚酸配伍注射用血栓通(冻干)通过抑制MMPs对脑缺血再灌注损伤大鼠血脑屏障通透性的影响及机制

Effect of Salvianolate Lyophilized Injection and Xueshuantong Injection(Lyophilized)on Permeability of Blood-brain Barrier via Inhibition of MMPs in Cerebral Ischemia/Reperfusion Injury Rats

目的:探究注射用丹参多酚酸配伍注射用血栓通(冻干)通过抑制金属基质蛋白酶(MMPs)对脑缺血再灌注损伤大鼠血脑屏障(BBB)通透性的影响及机制。方法:采用线栓法建立大脑中动脉栓塞/再灌注(MCAO/R)模型。选取雄性Wistar大鼠60只,随机分为假手术组,模型组,依达拉奉组(6 mg·kg^-1),丹参多酚酸组(21 mg·kg^-1),血栓通组(100 mg·kg^-1),丹参多酚酸配伍血栓通组(21 mg·kg^-1+100 mg·kg^-1),尾静脉注射给药每日1次,连续2 d。采用神经功能(mNSS)评分法观察大鼠神经功能缺损情况;苏木素-伊红(HE)染色观察大鼠脑缺血半暗带组织病理学变化;Nissl染色及神经元特异核蛋白(NeuN)荧光染色检测缺血半暗带神经元损伤情况;免疫球蛋白G(IgG)/CD31免疫荧光双染观察缺血半暗带IgG渗漏情况;免疫荧光法观察紧密连接(TJs)相关蛋Occludin,Claudin-5及基底膜(BM)相关蛋白Ⅳ型胶原(Col-Ⅳ),层粘连蛋白(LN),纤维连接蛋白(FN)表达情况;蛋白免疫印迹法(Western blot)检测金属基质蛋白酶MMP-2,MMP-9蛋白表达情况。结果:与模型组比较,丹参多酚酸组、血栓通组、丹参多酚酸配伍血栓通组可显著改善大鼠脑缺血再灌注损伤神经功能缺损、组织形态学变化,并可抑制神经元损伤,减轻缺血半暗带IgG渗漏,明显升高TJs蛋白Claudin-5,Occludin及基底膜相关蛋白Col-Ⅳ,LN,FN表达(P<0.05,P<0.01),明显降低MMP-2,MMP-9蛋白表达(P<0.05,P<0.01);且配伍用药组疗效最佳,对各项指标均有明显的改善,改善程度优于丹参多酚酸或血栓通单独干预。结论:注射用丹参多酚酸、注射用血栓通(冻干)及两者配伍应用可减轻大鼠脑缺血再灌注损伤,且配伍应用效果优于单独应用,其机制可能与通过抑制MMPs的表达减轻脑微血管内皮紧密连蛋白及基底膜损伤,进而抑制缺血后BBB损伤有关。

Objective:To investigate the effect of salvianolate lyophilized injection and Xueshuantong injection(lyophilized)on the permeability of blood-brain barrier(BBB)via inhibition of metallomatrix protease(MMPs)in cerebral ischemia/reperfusion injury rats.Method:The focal cerebral ischemia/reperfusion model in rats was built by middle cerebral artery occlusion/reperfusion(MCAO/R)technique.Male Wistar rats were randomly divided into sham operation group,ischemia/reperfusion(I/R)group,edaravone(Eda,6 mg·kg^-1)group,salvianolate lyophilized injection(SLI,21 mg·kg^-1)group,Xueshuantong(XST,100 mg·kg^-1)group and SLI combined with XST(SLI+XST,21 mg·kg^-1+100 mg·kg^-1)group.Drugs were injected via tail vein for 2 d,while sham group and I/R group were injected with the same amount of normal saline.Neurological deficit score,hematoxylin-eosin(HE)staining and Nissl staining were assessed 2 d after MCAO/R.The permeability of BBB was observed by the leakage of IgG/CD31.The expressions of Claudin-5,Occludin,collagen-Ⅳ(Col-Ⅳ),Laminin,Fibronectin were observed by immunofluorescence staining,and MMP-2 and MMP-9 were observed by Western blot.Result:Compared with the I/R group,SLI group,XST group and SLI+XST group showed improvements in neurological deficit score,HE staining and Nissl staining.The leakage of IgG was alleviated;The positive expressions of Claudin-5,Occludin,Col-Ⅳ,Laminin,Fibronectin in ischemic penumbra were significantly up-regulated,while the expressions of MMP-2 and MMP-9 were down-regulated.The effect in improving SLI combined with XST was much better than a single factor.Conclusion:Salvianolate lyophilized injection and Xueshuantong injection(lyophilized)can alleviate cerebral ischemia/reperfusion injury and exert the synergistic effect when they are used in combination.The mechanisms might be associated with the improvement in the permeability of blood-brain barrier by inhibiting MMPs in cerebral ischemia/reperfusion injury rats.