电针调节小胶质细胞TLR4/MyD88/NF-κB信号通路改善小鼠脑缺血再灌注损伤炎性反应的实验研究

Experimental study on electroacupuncture regulating microglia TLR4/MyD88/NF-κB signal pathway to improve inflammatory response of mice with cerebral ischemia-reperfusion injury

目的探讨电针对小鼠缺血性脑卒中急性期炎性反应及TLR4信号通路的影响,为电针治疗脑缺血的炎性反应提供更为深入的理论依据。方法将C57BL/6N小鼠按体重随机分为假手术组、模型组及电针组,每组24只。除假手术组外,模型组及电针组小鼠均行右侧大脑中动脉闭塞术,并在30 min后拔除线栓实现再灌注。电针组小鼠手术清醒后1 h开始给予百会穴、太阳穴、足三里穴电刺激治疗,每12 h 1次。每组分别于术后12 h、24 h、48 h、72 h各取6只小鼠进行神经功能缺损评分,磁共振扫描小鼠脑组织并在后处理站上计算脑水肿体积百分比,ELISA法检测血清炎性因子肿瘤坏死因子-α(TNF-α)及白细胞介素-1β(IL-1β)水平,HE染色及IBA1免疫组化染色观察脑组织病理改变,定量聚合酶链反应(q-PCR)法测定脑组织中TLR4 mRNA表达量,Western blot法测定脑组织中TLR4、MyD88及NF-κB p65蛋白表达量。结果模型组和电针组小鼠在造模后和磁共振扫描前均出现了不同程度的神经功能缺损。术后48 h和72 h,电针组神经功能评分及血清TNF-α、IL-1β水平均明显低于模型组(P均<0.05),IBA1免疫组化染色活化小胶质细胞数目明显少于模型组(P<0.05);术后72 h,电针组脑水肿体积百分比、病灶侧TLR4 mRNA表达量及TLR4、MyD88、NF-κB p65蛋白表达量均明显低于模型组(P均<0.05),HE染色显示神经元受损程度明显较模型组轻。结论电针治疗能改善缺血再灌注小鼠神经功能缺损,减轻脑组织水肿,其可能是通过抑制小胶质细胞的活化,下调脑组织TLR4 mRNA和TLR4蛋白表达,进而控制其下游MyD88、NF-κB蛋白的表达,减少血清炎性因子的释放,从而发挥脑缺血再灌注保护作用。

Objective It is to explore the effects of electroacupuncture on the inflammatory response and TLR4 signaling pathway in the acute phase of ischemic stroke in mice,and provide a more in-depth theoretical basis for electroacupuncture to treat the inflammatory response of cerebral ischemia.Methods C57BL/6N mice were randomly divided into sham operation group,model group and electroacupuncture group according to their body weight,with 24 mice in each group.Except for the sham operation group,the mice in the model group and the electroacupuncture group underwent right middle cerebral artery occlusion,and the thread plug was removed 30 minutes later to achieve reperfusion.The mice in the electroacupuncture group were given electrical stimulation at Baihui,Taiyang,and Zusanli 1 hour after operation,once every 12 hours.6 mice in each group were taken at 12 h,24 h,48 h,72 h after operation to evaluate their neurological deficits scores and scan their brain tissues by magnetic resonance,and the percentage of brain edema volume was calculated on the post-processing station,the levels of serum inflammation indicators tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were detected by ELISA.The pathological changes of brain tissue was observed by HE staining and IBA1 immunohistochemical staining,and the expression of TLR4 mRNA in brain tissue was detected by quantitative polymerase chain reaction(q-PCR)method,the expression of TLR4,MyD88 and NF-κB p65 protein in brain tissue was determined by Western blot method.Results The mice in the model group and the electroacupuncture group had different degrees of neurological deficits after the modeling and before the MRI scan.At 48 h and 72 h after operation,the neurological score and the levels of serum TNF-αand IL-1βin the electroacupuncture group were significantly lower than those in the model group(all P<0.05),and the number of activated microglia by IBA1 immunohistochemical staining was significantly less than that in the model group group(P<0.05);at 72 hours after operation,the percentage of brain edema volume,TLR4mRNA expression and TLR4,MyD88,and NF-κB p65 protein expression in the lesion side of the electroacupuncture group were significantly lower than those in the model group(all P<0.05),HE staining showed that the degree of neuron damage was significantly less than that of the model group.Conclusion Electroacupuncture can improve the neurological deficit and reduce brain tissue edema in mice with ischemia-reperfusion,which may be through inhibiting the activation of microglia,down-regulating the expression of TLR4mRNA and TLR4 protein in brain tissue,and then controlling its downstream MyD88 and NF-κB protein expression and reducing the release of serum inflammatory factors,thereby plays a protective effect on cerebral ischemia and reperfusion.