白藜芦醇在PD小鼠模型中对TLR3/TRIF信号通路及脑内炎症微环境的影响

The Effect of Resveratrol on TLR3/TRIF Signaling Pathway and Cerebral Inflammatory Microenvironment in the Brain of PD Mice

目的建立1-甲基-4苯基-1,2,3,6-四氢吡啶(MPTP)诱导的小鼠PD模型,探究白藜芦醇(resveratrol,Res)对PD小鼠TLR3/TRIF信号通路及中脑黑质炎症微环境的影响。方法雄性C57/BL6小鼠30只,按照随机数字表法分为对照组、MPTP组和MPTP+Res组,每组各10只。对照组腹腔注射相同体积生理盐水;MPTP组腹腔注射生理盐水新鲜配制的MPTP 30mg/(kg·d),连续7d,结束注射7d后取材;MPTP+Res组:Res用乙醇溶解为50mg/mL,用PBS以1∶4的体积比进行稀释,连续注射MPTP 7d后,腹腔注射Res 28d,结束注射7d后取材。采用免疫组化及免疫荧光分别观察小鼠中脑黑质中多巴胺能神经元的变化及小胶质细胞、星形胶质细胞的活化状态。采用实时荧光定量PCR技术检测中脑黑质相关炎症因子mRNA的表达。结果免疫组化结果显示:与MPTP组相比,MPTP+Res组小鼠中脑黑质TH标记的多巴胺能神经元的数量及形态未发生明显变化;CD11b标记的小胶质细胞胞体相对变小,呈圆形及椭圆形,突起减少,变细、变长;而GFAP标记的星形胶质细胞胞体变小,胞体呈圆形,突起变短。qPCR实验结果表明:与MPTP组相比,MPTP+Res组小鼠中脑黑质部位TLR3、IFN-β的mRNA表达量明显增加,差异有统计学意义(P<0.05),但TRIF的mRNA表达量未见明显变化,促炎因子iNOS、TNF-α的mRNA表达量明显下降,差异有统计学意义(P<0.05)。而抗炎因子Arg^(-1)的mRNA表达量明显增加。结论 Res可能通过抑制小胶质细胞及星形胶质细胞活化及激活TLR3/TRIF信号通路,降低中脑黑质的促炎因子的释放,从而改善MPTP诱导的小鼠PD模型中的炎症微环境。

Objective To construct the animal model of PD mice induced by MPTP and explore the effect of resveratrol on the TLR3/TRIF signaling pathway and the inflammatory microenvironment of the midbrain substantia nigra in PD mice.Methods 30 male C57/BL6 mice were divided into the control group,MPTP group and MPTP＋Res group in accordance with the random number table,and each group consists of 10 mice.The rats of the control group received 30mg/kg normal saline daily by intraperitoneal injection,those of MPTP group were administered with the same amount of MPTP daily in the same way for 7days in succession,and those of MPTP＋Res group were injected with MPTP for 7days in succession and then they were injected with resveratrol for 28 days in succession.Immunohistochemistry and immunofluorescence were adopted to observe the changes of dopaminergic neurons in the substantia nigra,the microglial cells and the activation state of astrocytes.Real-time fluorescence quantitative PCR technique was used to detect the mRNA expression of inflammatory factors in the substantia nigra.Results Firstly,the immunohistochemistry results showed that the number and morphology of dopamine neurons in the substantia nigra TH in MPTP＋Res group did not change significantly,the CD11 blabeled microglia cells were relatively small,round and oval while the projections became less,thinner,and longer.The GFAP labeled astrocytes were smaller,the cells were round,and the projections became shorter.Secondly,the results of the qPCR experiment showed that TLR3,IFNβmRNA expressions of midbrain substantia nigra parts in the mice of MPTP＋Res group increased significantly（P〈0.05）,but TRIF mRNA expression was not significantly changed.Proinflammatory factors of iNOS and TNF-α mRNA expression reduced significantly（P〈0.05）.The mRNA expression increased significantly in the anti-inflammatory factor Arg-1.Conclusion Resveratrol may decrease the release of proinflammatory factors in the midbrain substantia nigra and improve the inflammatory microenvironment in MPTP-induced mouse model by inhibiting the activation of microglia and astrocytes and activating TLR3/TRIF signaling pathway.