从TLR4信号通路探讨电针减轻小鼠脑缺血再灌注损伤的炎症反应机制

Study on the mechanism of electroacupuncture in alleviating inflammatory reaction of cerebral ischemia reperfusion injury mice through TLR4 signaling pathway

目的:通过TLR4信号通路探讨电针减轻脑缺血再灌注损伤小鼠的炎症反应机制。方法:选择健康成年野生型C57BL/6n小鼠30只(体质量20~25 g),随机分为3组:假手术组、大脑中动脉栓塞(MCAO)缺血再灌注组及电针组。所有小鼠均采用腹腔注射麻醉,复制Longa式线栓法并在此基础上完善建立小鼠右侧MCAO缺血再灌注模型。造模成功后所有小鼠行尾静脉超微超顺磁氧化铁颗粒(USPIO)注射,电针组行电针患侧足三里穴及百会穴透刺患侧太阳穴治疗。造模成功小鼠48 h行MRI,扫描结束后取血、取脑。图像上传至后处理工作站,测量T2WI及SWI冠状位梗死侧与正常侧信号值比值。运用实时定量聚合酶链式反应(PCR)法及酶联免疫(ELISA)法测量脑组织梗死区域TLR4的mRNA表达量及血清中IL-1β、TNF-α等炎性因子的含量,并采用HE染色法、免疫组化染色法观察病灶脑组织神经元结构破坏情况及梗死区域铁粒子的吞噬情况。结果:①小鼠右侧MCAO缺血再灌注模型采用改良Longa式线栓法成功复制,且梗死面积稳定。②电针组小鼠T2WI右边梗死侧最低信号与左边正常侧间比值与MCAO缺血再灌注组相比,差异有统计学意义(P<0.05)。③假手术组小鼠炎性因子(IL-1β、TNF-α)、通路中TLR4 mRNA表达量与MCAO缺血再灌注组、电针组比较,差异均有统计学意义(均P<0.05)。④相较于假手术组,MCAO缺血再灌注组及电针组可见小鼠缺血脑组织区域神经细胞结构破坏,以及小胶质细胞增加明显、散在分布于细胞间隙的铁颗粒与少量被小胶质细胞吞噬的铁颗粒。结论:借助USPIO MRI增强扫描监测,与病理组织学对照发现,针刺能够改善小鼠神经功能缺损症状、减少活化的小胶质细胞数量,提高冠状位T2WI信号值,缩小脑梗死面积,减轻炎症反应;可能机制为下调了TLR4信号通路中TLR4 mRNA及信号相关下游促炎因子IL-1β、TNF-α的表达。

Objective:To explore the mechanism of electroacupuncture in alleviating the inflammatory response of cerebral ischemia reperfusion injury mice through TLR4 signaling pathway.Methods:30 healthy adult C57BL/6n mice(body weight at 20~25 g)were randomly divided into three groups:sham operation group,middle cerebral artery occlusion(MCAO)ischemia reperfusion group and electroacupuncture(EA)group.Anesthetize all mice by intraperitoneal injection,copy Longa type line plug method and perfect to establish on the basis of the change of right middle cerebral artery embolism ischemia-reperfusion mice model,after the success of the building all lines of USPIO tail vein injection in mice,including the EA group of Zusanli,Baihui point and“temples”treatment.The mouse was successfully modeled for 48 h,and the blood and brain were taken after the MRI scan.Images were uploaded to the post-processing workstation,T2WI and SWI coronary infarction side with normal side signal value ratio.Real time PCR and ELISA were used to measure the expression of TLR4 mRNA and the content of IL-1β,TNF-αand other inflammatory factors in the cerebral infarction area,and HE staining and immunohistochemical staining were used to observe the destruction of neuron structure in the focal brain tissue and the phagocytosis of iron particles in the infarction area.Results:①The model of right middle cerebral artery ischemia-reperfusion in mice was successfully replicated by modified Longa method with stable infarct area.②Compared with the MCAO ischemia reperfusion group,the ratio of the lowest signal on the right infarct side of T2WI and the normal interlateral ratio on the left side of T2WI in the EA group was significantly different(P<0.05).③Inflammatory factors(IL-1β,TNF-α)and the expression of TLR4 mRNA in the pathway in the sham operation group were different from those in the MCAO ischemia reperfusion group and the EA group(P<0.05).④Compared with the sham operation group,the structural damage of nerve cells in the ischemic brain tissues of mice,a significant increase in microglia cells,scattered iron particles in the intercellular space and a small amount of iron particles engulfed by microglia cells were observed in the MCAO ischemia reperfusion group and the EA group.Conclusions:With the aid of USPIO enhanced MRI method of monitoring,and pathological histology,acupuncture can improve the symptoms of nerve function defect in mice,decrease the number of activated microglia and increased coronal T2WI signal value,cerebral infarction area is narrowed,inflammation is reduced,cut TLR4 mRNA in TLR4 signaling pathways and the possible mechanism for the signal related downstream proinflammatory factor IL-1β,the expression of TNF-α.