丹参川芎嗪注射液改善缺血性卒中大鼠Th17/Treg平衡减轻神经缺血损伤的研究

Effect of Danshen and Ligustrazine Injection on Th17/Treg balance in rats with ischemic stroke

关键词:  丹参川芎嗪注射液  缺血性卒中  Th17  Treg  神经损伤

Key words:  Danshen and Ligustrazine Injection  ischemic stroke  Th17  Treg  nerve injury

摘要:

[目的] 探讨丹参川芎嗪注射液改善缺血性卒中大鼠Th17/Treg平衡减轻神经缺血损伤的效果。[方法] 制作大脑中动脉阻塞（MCAO）大鼠模型，并将实验大鼠随机分为假手术组、模型组和参芎组，每组有24 h、3 d、7 d、14 d共4个时相，每时相各10只动物。参芎组于造模后按体质量腹腔注射0.9 mL/kg的丹参川芎嗪注射液，每日1次；假手术组和模型组给予等量生理盐水。治疗结束后，采用NSS评分对动物的神经功能进行评分；TTC（氯化三苯基四氮唑）染色法检测脑梗死体积；并分离大脑中动脉供血区的脑组织，流式细胞术检测其中Th17和Treg细胞的比例，ELISA法检测炎性细胞因子水平。[结果] 造模后，模型组和参芎组NSS评分和梗死体积均随时间逐渐降低，组内比较均差异显著（P<0.01）；且从第3 d开始，各时间点模型组和参芎组NSS评分和梗死体积组间比较差异显著（P<0.05，P<0.01）。模型组脑内Th17细胞比例在造模后逐渐升高，于第7 d达峰；且第3、7、14 d的比例均显著高于假手术组（P<0.01）。模型组脑内Treg细胞比例在24 h和3 d有所降低，但至7 d时较假手术组大量增加（P<0.01）。参芎组能降低各时间点Th17细胞比例、提高Treg细胞比例，且第7 d和14 d尤为明显，与模型组比较差异显著（P<0.05，P<0.01）。模型组各时间点Th17/Treg比值均显著高于假手术组（P<0.05，P<0.01）；而参芎组能降低Th17/Treg比值，与模型组比较差异显著（P<0.05，P<0.01）。模型组脑内IL-6和TNF-α水平逐渐升高，于第7 d达到峰值；且各时间点的水平均高于假手术组，组间比较差异显著（P<0.01）。模型组脑内IL-4和IL-10水平较假手术组有所降低，且IL-4降低更为明显（P<0.05）。与模型组相比，参芎组能降低各时间点IL-6和TNF-α水平（P<0.05，P<0.01）；且能升高IL-4和IL-10水平，特别是IL-4升高更为明显（P<0.05）。[结论] 丹参川芎嗪注射液能够调节缺血性卒中大鼠Th17/Treg平衡及相关炎症因子水平，进而改善神经元存活微环境，减轻神经缺血损伤。

Abstract:

[Objective] To investigate the effect of Danshen and Ligustrazine Injection on Th17/Treg balance in rats with ischemic stroke.[Methods] The middle cerebral artery occlusion (MCAO) rat model was made,and the experimental rats were randomly divided into three groups:sham operation group,model group and Shenxiong group. Each group was divided into four phases:24 hours,3 days,7 days and 14 days,10 animals in each phase. The rats in Shenxiong group were injected with 0.9 ml/kg Danshen and Ligustrazine Injection once daily,and the rats in sham operation group and model group were given the same amount of normal saline. At the end of treatment,the neural function of the animals was scored by NSS score,TTC (triphenyltetrazolium chloride) staining method was used to detect the brain infarction volume,and the brain tissue of the blood supply area of the middle cerebral artery was separated. The proportion of Th17 and Treg cells was detected by flow cytometry,and the level of inflammatory cytokines was detected by ELISA.[Results] The NSS scores and the brain infarction volume of model group and Shenxiong group decreased gradually with time,and there were significant differences between them (P<0.01);and from the third day,significant differences were detected in the NSS scores and the brain infarction volume between the model and Shenxiong groups at each time point (P<0.05,P<0.01). The proportion of Th17 cells in the brain of the model group increased gradually after the model was established,and reached the peak on the 7th day,and the proportion on the 3rd,7th and 14th day was significantly higher than that of the sham operation group (P<0.01). The ratio of Treg cells in the model group decreased at 24h and 3D,but increased significantly at 7d (P<0.01). Compared with the model group,the ratio of Th17 cells and Treg cells in Shenxiong group decreased significantly (P<0.05,P<0.01). The ratio of Th17/Treg in the model group was significantly higher than that in the sham operation group (P<0.05,P<0.01),while that in the Shenxiong group was significantly lower than that in the model group (P<0.05,P<0.01). The level of IL-6 and TNF-α in the brain of the model group increased gradually and reached the peak on the 7th day,and the level of each time point was higher than that of the sham operation group,the difference between the groups was significant (P<0.01). The level of IL-4 and IL-10 in model group was lower than that in sham operation group,and the decrease of IL-4 was more obvious (P<0.05). Compared with the model group,Shenxiong group can reduce the level of IL-6 and TNF-α at each time point (P<0.05,P<0.01),and can increase the level of IL-4 and IL-10,especially the level of IL-4 (P<0.05).[Conclusion] Salvia miltiorrhiza and Ligustrazine Injection can regulate the Th17/Treg balance and the level of related inflammatory factors in rats with ischemic stroke,thus improving the microenvironment of neuron survival and alleviating the injury of nerve ischemia.