GSTM1表达在亚低温减轻大鼠脑缺血再灌注损伤中的作用:与小胶质细胞极化的关系

Role of GSTM1 expression in mild hypothermia-induced mitigation of cerebral ischemia-reperfusion injury: relationship with microglial polarization

目的评价谷胱甘肽S转移酶μ1(GSTM1)表达在亚低温减轻大鼠脑缺血再灌注损伤中的作用及其与小胶质细胞极化的关系。方法清洁级健康雄性SD大鼠80只,8周龄,体质量260~280 g,采用随机数字表法分为4组(n=20):假手术组(S组)、脑缺血再灌注组(I/R组)、亚低温组(H组)和GSTM1抑制剂+亚低温组(IH组)。采用线栓法制备大鼠脑缺血再灌注损伤模型,阻断大鼠左侧大脑中动脉2 h时拔出线栓恢复血流灌注,期间维持大鼠脑温及肛温36~37℃;S组仅分离结扎血管,不进行阻闭;H组在拔出线栓即刻用75%酒精擦拭全身,维持脑温及肛温32~33℃3 h,其余同I/R组;IH组分别于造模前24和1 h时腹腔注射GSTM1抑制剂8.6 mg/kg,其余同H组。于再灌注24 h时对大鼠行改良神经功能缺损评分(mNSS),处死大鼠后取脑,采用TTC染色法观察大脑梗死情况并计算梗死体积百分比;Western blot法检测GSTM1、M1型小胶质细胞标记物诱导型一氧化氮合酶(iNOS)和M2型小胶质细胞标记物精氨酸1(Arg-1)的表达;qRT-PCR法检测GSTM1、iNOS和Arg-1 mRNA的表达;ELISA法检测IL-6、IL-10、TNF-α和转化生长因子β(TGF-β)的含量。结果与S组比较,其余3组mNSS、脑梗死体积百分比、iNOS、Arg-1及其mRNA表达上调,GSTM1及其mRNA表达下调,IL-6、TNF-α及IL-10和TGF-β含量升高(P<0.05);与I/R组和IH组比较,H组mNSS、脑梗死体积百分比、iNOS及其mRNA表达下调,Arg-1及其mRNA和GSTM1表达上调,TNF-α和IL-6含量下降,TGF-β和IL-10含量升高(P<0.05)。结论GSTM1表达上调参与了亚低温减轻大鼠脑缺血再灌注损伤的过程,与抑制小胶质细胞向M1型极化,促进其向M2型极化有关。

Objective To evaluate the role of glutathione S-transferaseμ1(GSTM1)expression in mild hypothermia-induced mitigation of cerebral ischemia-reperfusion(I/R)injury and the relationship with microglial polarization.Methods Eighty clean-grade healthy male Sprague-Dawley rats,aged 8 weeks,weighing 260-280 g,were divided into 4 groups(n=20 each)using a random number table method:sham operation group(S group),cerebral I/R group(I/R group),mild hypothermia group(H group),and GSTM1 inhibitor+mild hypothermia group(IH group).The rat model of cerebral I/R injury was prepared using the filament occlusion method.The filament was removed to restore blood flow after the left middle cerebral artery was blocked for 2 h,and the rats′brain and rectal temperature were maintained at 36-37℃during the period.The vessels were only isolated and ligated without occlusion in S group.In H group,the entire body was wiped with 75%ethanol immediately after removing the filament,and the brain and rectal temperatures were maintained at 32-33℃for 3 h,and the other procedures were the same as those previously described in I/R group.In IH group,GSTM1 inhibitor itaconic acid 8.6 mg/kg was intraperitoneally injected at 24 and 1 h before developing the model,and the other procedures were the same as those previously described in H group.Neurological deficits were evaluated using a modified neurological severity score(mNSS)at 24 h of reperfusion,and then the animals were sacrificed and the brains were removed for observation of cerebral infarction(by TTC staining)and for determination of the expression of GSTM1,M1-type microglial marker inducible nitric oxide synthase(iNOS),and M2-type microglial marker arginase-1(Arg-1)(by Western blot),expression of GSTM1,iNOS and Arg-1 mRNA(quantitative real-time polymerase chain reaction)and contents of interleukin-6(IL-6),IL-10,tumor necrosis factor-alpha(TNF-α)and transforming growth factor-beta(TGF-β)(by enzyme-linked immunosorbent assay).Results Compared with S group,the mNSS and percentage of cerebral infarct size were significantly increased,and the expression of iNOS and Arg-1 protein and mRNA was up-regulated,the expression of GSTM1 and mRNA was down-regulated,and the contents of IL-6,TNF-α,IL-10 and TGF-βwere increased in the other three groups(P<0.05).Compared with I/R group and IH group,the mNSS and percentage of cerebral infarct size were significantly decreased,and the expression of iNOS protein and mRNA was down-regulated,the expression of Arg-1 protein and mRNA and GSTM1 was up-regulated,the contents of TNF-αand IL-6 were decreased,and the contents of TGF-βand IL-10 were increased in H group(P<0.05).Conclusions Up-regulated expression of GSTM1 is involved in mild hypothermia-induced mitigation of cerebral I/R injury,which is associated with inhibition of microglial polarization toward the M1 phenotype and promotion of polarization toward the M2 phenotype.