针刺对大鼠脑缺血后小胶质细胞极化和炎性反应的影响

Acupuncture ameliorates neurological function by suppressing microglia polarization and inflammatory response after cerebral ischemia in rats

目的:观察针刺对脑缺血再灌注(I/R)损伤大鼠小胶质细胞极化和炎性反应的影响,探讨其潜在的作用机制。方法:雄性SD大鼠随机分为假手术组、模型组、针刺组,每组10只。采用大脑中动脉栓塞法制备大鼠脑I/R损伤模型。针刺组大鼠在造模后于“大椎”“百会”“水沟”及双侧“足三里”“风池”进行针刺,20 min/次,1次/d,连续7 d。采用神经功能评分法评估各组大鼠神经功能;尼氏染色法观察脑梗死侧皮质区神经元损伤情况;免疫荧光染色法检测缺血侧皮质区神经元特异性核蛋白(NeuN)、离子钙结合蛋白1(Iba-1)、诱导型一氧化氮合酶(iNOS)、精氨酸酶1(Arg1)、脑源性神经营养因子(BDNF)和胶质细胞源性神经营养因子(GDNF)的表达;ELISA法检测缺血脑组织中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6和IL-10的含量;Western blot法检测缺血脑组织中BDNF、GDNF、Toll样受体4(TLR4)、髓样分化因子88(MyD88)和核因子κB(NF-κB)的表达水平。结果:与假手术组比较,模型组大鼠神经功能评分升高(P<0.01),脑缺血侧皮质区神经元数量及尼氏体减少,NeuN阳性细胞数、Arg1阳性表达减少(P<0.01),Iba-1、iNOS阳性表达增加(P<0.01),BDNF、GDNF阳性表达和蛋白表达水平及IL-10含量降低(P<0.01),TNF-α、IL-6含量升高(P<0.01),TLR4、MyD88和NF-κB蛋白表达水平升高(P<0.01)。与模型组比较,针刺组大鼠神经功能评分降低(P<0.01),缺血侧皮质区神经元数量及尼氏体增多,NeuN阳性细胞数、Arg1阳性表达增多(P<0.05),Iba-1、iNOS阳性表达减少(P<0.01),BDNF、GDNF阳性表达和蛋白表达水平及IL-10含量升高(P<0.01,P<0.05),TNF-α、IL-6含量降低(P<0.01),TLR4、MyD88和NF-κB蛋白表达水平降低(P<0.05,P<0.01)。结论:针刺干预可以通过调节脑I/R后小胶质细胞极化、减轻炎性反应和增加神经营养因子的分泌,对I/R后脑组织起神经保护作用,其机制可能与抑制TLR4/MyD88/NF-κB信号通路有关。

Objective To observe the effect of acupuncture on microglia polarization and inflammatory reaction in rats with cerebral ischemia-reperfusion injury(CIRI),so as to explore its mechanisms underlying improvement of CIRI.Methods Thirty male SD rats were randomly divided into sham operation,model,and acupuncture groups,with 10 rats in each group.The CIRI model was established by occlusion of the middle cerebral artery(MCAO)for 1 h,followed by reperfusion.After modeling,rats in the acupuncture group received manual acupuncture stimulation of“Dazhui”(GV14),“Baihui”(GV20),“Shuigou”(GV26),bilateral“Zusanli”(ST36)and“Fengchi”(GB20)by twirling the needles rapidly for 10 s/acupoint every 10 min,with the needles retained for 20 min.The treatment was conducted once daily for successive 7 days.The neurological function was evaluated according to Longa’s method.The state of CIRI was observed after Nissl staining,and the expression levels of Iba-1,iNOS,Arg1,BDNF,GDNF and NeuN in the ischemic cortex tissue were detected by immunofluorescence staining.The contents of TNF-α,IL-6 and IL-10 in the ischemic tissue were assayed by ELISA.The protein expression levels of BDNF,GDNF,TLR4,MyD88 and NF-κB in the ischemic tissues were detected by Western blot.Results The neurological deficit score on the 24 h and 7^(th) day was considerably higher in the model group than in the sham operation group(P<0.01),and evidently lower on the 7^(th) day in the acupuncture group than in the model group(P<0.01).The number of NeuN positive cells,the area of immunofluorescence dual labelling of Arg1,BDNF and GDNF positive staining,IL-10 content,BDNF and GDNF protein expressions were significantly decreased(P<0.01),and the immunofluorescence dual labelling area of Iba-1 and iNOS,TNF-αand IL-6 contents,the pretein expression levels of TLR4,MyD88 and NF-κB considerably increased(P<0.01)in the model group relevant to the sham operation group.In contrast to the model group,the acupuncture group had a significant increase in the number of NeuN positive cells,the immunofluorescence dual labelling area of Arg1,BDNF and GDNF positive staining,IL-10 content,and BDNF and GDNF protein expressions(P<0.05,P<0.01),and an evident decrease in Iba-1 and iNOS positive staining,contents of TNF-αand IL-6,and the protein expression levels of TLR4,MyD88 and NF-κB(P<0.01,P<0.05).Nissl staining showed a marked reduction in the number of neurons,the nucleus pyknosis and nissl bodies and loose arrangement of the neuronal cells in the model group,which was relatively milder in the acupuncture group.Conclusion Acupuncture intervention can improve neurological function in CIRI rats,which may be related to its effects in regulating the polarization of microglia,reducing inflammatory reaction and increasing the secretion of neurotrophic factors in the brain,inhibiting TLR4/MyD88/NF-κB signaling pathway.