通脉舒络胶囊对缺血性脑卒中大鼠Treg细胞介导的免疫炎症反应的影响

Effect of Tongmai Shuluo capsule on Treg cell mediated immune inflammatory response in ischemic stroke rats

目的研究通脉舒络胶囊对缺血性脑卒中大鼠神经局部及外周免疫中Treg细胞亚群功能的影响。方法将120只SD雄性大鼠随机分为假手术组、模型组、通脉舒络低剂量组、通脉舒络中剂量组、通脉舒络高剂量组,每组24只。除假手术组外,其余组大鼠采用大脑中动脉栓塞方法制备脑缺血模型。造模成功后,通脉舒络低、中、高剂量组大鼠分别给予0.032,0.064,0.128 g/mL浓度的通脉舒络胶囊药液灌胃,假手术组和模型组大鼠给予等量生理盐水灌胃,均1次/d,连续14 d。记录各组大鼠脑缺血再灌注后24 h、3 d、7 d、14 d的神经功能缺损评分,HE染色观察各组大鼠脾脏组织病理形态,ELISA法测定海马和脾脏组织中Treg细胞特异性转录因子叉头翼状螺旋转录因子3(Foxp3)及白细胞介素-10(IL-10)含量。结果通脉舒络各组大鼠脑缺血再灌注后24 h、3 d、7 d、14 d的神经功能缺损评分均明显低于同期模型组(P均<0.05),且通脉舒络中剂量组均明显低于同期通脉舒络低、高剂量组(P均<0.05)。HE染色显示模型组大鼠脾实质内白髓、红髓分布不清,生发中心缩小且轮廓不清,淋巴细胞松散聚集;通脉舒络各组大鼠脑缺血再灌注后7 d开始,脾脏白髓体积变小、数目减少,淋巴细胞浸润明显改善,几乎无红髓堵塞充血,生发中心生成。与假手术组比较,模型组大鼠脑缺血再灌注后24 h海马和脾脏组织中Foxp3、IL-10含量均明显降低(P均<0.05),脑缺血再灌注后7 d海马和脾脏组织中Foxp3、IL-10含量均明显增高(P均<0.05);通脉舒络中剂量组大鼠脑缺血再灌注后24 h、3 d、7 d、14 d海马和脾脏组织中Foxp3、IL-10含量均明显高于同期模型组(P均<0.05)。结论通脉舒络胶囊能够抑制脑缺血后局部脑组织及脾脏的免疫炎症反应,促进抗炎细胞因子释放,通过抗炎及维持免疫耐受机制发挥脑保护作用。

Objective It is to study the effects of Tongmai Shuluo capsule on the function of Treg cell subsets in local and peripheral immunity of ischemic stroke rats.Methods One hundred and twenty male SD rats were randomly divided into sham operation group,model group,Tongmai Shuluo capsule low-dose group,medium-dose group and high-dose group,with 24 rats in each group.All the rats in each group except for sham operation group were modeled with middle cerebral artery embolization to prepare cerebral ischemia models.After successful modeling,the rats in the Tongmai Shuluo capsule low,medium and high dose groups were given 0.032,0.064 and 0.128 g/mL Tongmai Shuluo capsule by gavage,and the rats in the sham and model groups were given equal amounts of normal saline by gavage,all once per day,continuously treated for 14 days.The neurological deficit scores of the rats in each group were evaluated at 24 hours,3 days,7 days and 14 days after cerebral ischemia-reperfusion.The pathological changes of spleen tissues of the rats in each group were observed by HE staining,the levels of Treg cell-specific transcription factor forkhead box protein P3,(Foxp3)and interleukin-10(IL-10)in in hippocampal and spleen tissues were determined by ELISA.Results The neurological deficit scores of the rats in each Tongmai Shuluo capsule group were significantly lower than those in the model group at 24 h,3 d,7 d and 14 d after cerebral ischemia-reperfusion(all P<0.05),and the scores in the Tongguo Shuluo capsule medium-dose group was significantly lower than those in the low and high-dose groups(all P<0.05).HE staining showed that the distribution of white marrow and red marrow in the splenic parenchyma of rats in the model group was unclear,the germinal centers were shrunken and poorly outlined,and lymphocytes were loosely aggregated;starting from 7 d after cerebral ischemia-reperfusion in the rats of each Tongmai Shuluo capsule group,the white marrow of the spleen became smaller in volume and reduced in number,and the lymphocyte infiltration were improved significantly,with almost no red marrow blockage and congestion,and germinal centers were generated.Compared with the sham operation group,the contents of Foxp3 and IL-10 in hippocampus and spleen tissues of the rats in the model group were significantly decreased at 24 h after cerebral ischemia-reperfusion(all P<0.05),and were significantly increased at 7 d after cerebral ischemia-reperfusion(all P<0.05);the contents of Foxp3 and IL-10 in hippocampus and spleen tissues of rats in the Tongmai Shuluo capsule medium-dose group were significantly higher than those in the model group at 24 h,3 d,7 d and 14 d after cerebral ischemia-reperfusion(all P<0.05).Conclusion Tongmai Shuluo capsule can inhibit the immune inflammatory response of local brain tissue and spleen after cerebral ischemia,promote the release of anti-inflammatory cytokines,thus to play play a protective role in ischemic stroke by anti-inflammatory and maintenance of immune tolerance.