白藜芦醇对脑出血后小胶质细胞功能的影响及其机制

Effect and mechnism of resveratrol on the functional of microglia after intracerebral hemorrhage

目的探讨白藜芦醇(Res)对脑出血(ICH)后小胶质细胞功能的影响及其可能机制。方法(1)动物实验:将12周龄SD大鼠随机分为对照组、ICH组和Res组(n=9);每组随机分为术后6、24、72 h三个亚组(n=3)。ICH组采用自体血造模法建立ICH模型,对照组只进针而不注入自体血;Res组在ICH组的基础上腹腔注入50 mg/(kg.d)白藜芦醇。造模成功后6、24、72 h对大鼠行改良神经功能缺损评分(mNSS),采用组织切片和HE染色、尼氏染色、TUNEL染色及免疫荧光法观察大鼠脑组织Toll样受体4(TRL4)、CD36、血红素加氧酶1(HO-1)及抗核转录因子红系2相关因子2(Nrf2)蛋白表达情况。(2)细胞实验:取BV2小鼠小胶质细胞,分为对照组、Fe^(2+)(FeSO410μmol/L)组、Fe^(2+)+Res低剂量(25μmol/L)组及Fe^(2+)+Res高剂量(50μmol/L)组,培养至24、72 h时行Westernblotting检测TLR4、CD36、Nrf2、p-Nrf2和HO-1蛋白表达水平,行细胞免疫荧光定位观察Nrf2蛋白。结果(1)mNSS评分显示,对照组大鼠神经功能正常,ICH组有明显的神经功能障碍;ICH组大鼠mNSS分值明显高于对照组(P<0.01);Res组大鼠24、72 h mNSS分值明显低于ICH组(P<0.05,P<0.01)。HE染色结果显示,ICH组大鼠脑组织中红细胞浸润、炎性细胞浸润增多,Res组脑组织中红细胞和炎性细胞浸润较ICH组改善。尼氏染色结果显示,ICH组大鼠脑组织尼氏小体溶解较对照组增多,Res组尼氏小体溶解较ICH组减少。TUNEL染色结果显示,ICH组大鼠脑组织神经细胞凋亡指数较对照组上升(P<0.05),而Res组大鼠脑组织神经细胞凋亡指数较ICH组降低(P<0.05)。免疫荧光染色结果显示,ICH组大鼠脑组织中TLR4、CD36、Nrf2及HO-1蛋白表达均高于对照组(P<0.05);与同一时间点ICH组比较,Res组大鼠脑组织中TLR4表达下降(P<0.05),CD36、Nrf2和HO-1蛋白表达均上升(P<0.05)。(2)与对照组比较,Fe^(2+)组BV2细胞TLR4、CD36、HO-1蛋白相对表达量增高(P<0.05),72 h时Nrf2和p-Nrf2表达量增高(P<0.05),细胞核内Nrf2蛋白表达量增高(P<0.01);与Fe^(2+)组比较,Fe^(2+)+Res低剂量组和Fe^(2+)+Res高剂量组TLR4表达量降低(P<0.001),CD36、HO-1、Nrf2、p-Nrf2和细胞核内Nrf2蛋白表达量增高(P<0.05);与Fe^(2+)+R es低剂量组比较,Fe^(2+)+R es高剂量组HO-1、Nr f2、p-Nr f2和细胞核内Nr f2蛋白表达量均增高(P<0.05)。结论白藜芦醇可改善脑出血大鼠的神经功能;脑出血后72 h内Fe^(2+)激活的小胶质细胞主要表现为促炎功能;白藜芦醇可能通过调控Nrf2/HO-1信号通路,促进脑出血后小胶质细胞的功能向抗炎转变。

Objective To investigate the effect of resveratrol(Res)on microglia function after intracerebral hemorrhage(ICH)and its possible mechanism.Methods(1)Animal experiment:27 SD rats(12-week-old)were randomly divided into 3 groups(9 each):control group,ICH group and Res group;each group was randomly divided into three subgroups(3 each)at 6 h,24 h and 72 h after operation.Rats in ICH group were modeled by autologous blood modeling method,while in control group were only injected with needles without autologous blood injection.Rats in Res group were intraperitoneally injected with 50 mg/(kg.d)resveratrol on the basis of ICH group,and 2%dimethyl sulfoxide(DMSO)solvent of the same volume were injected in control group and ICH group.6 h,24 h and 72 h after successful modeling,the corresponding rats were subjected to modified neurological severity score(mNSS).Then the rats in each subgroup were sacrificed and their brain tissues were taken from the same area and embedded in wax blocks.The expression of TRL4,CD36,HO-1 and Nrf2 protein in rat brain tissue was observed by tissue section,HE staining,Nissl staining,TUNEL staining and immunofluorescence method.(2)Cell experiment:BV2 mouse microglia cells were divided into control group,Fe^(2+)group(FeSO410μmol/L)and Fe^(2+)+low dose resveratrol(25μmol/L)group and Fe^(2+)+high dose resveratrol(50μmol/L)group.The expression levels of TLR4,CD36,Nrf2,p-Nrf2,and HO-1 proteins were detected by Western blotting after incubation for 24 h and 72 h,respectively,and the localization of Nrf2 protein was observed by cell immunofluorescence.Results(1)mNSS score indicated that rats in ICH group had obvious neurological dysfunction while normal in control group.mNSS score of rats was significantly higher in ICH group than in control group(P<0.01).As time went by 24 h or 72 h,mNSS score of rats reduced significantly in Res group than in ICH group(P<0.05,P<0.01).HE staining of rat brain tissue indicated that increased infiltration of red blood cells and inflammatory cells were in ICH group,and the infiltration of red blood cells and inflammatory cells in the brain tissue of rats in Res group was improved compared with those in ICH group.Nissl staining of rat brain tissue showed that,compared with the control group,the dissolution of nissl corpuscles in brain tissue of ICH group increased,and in Res group decreased.TUNEL staining of rat brain tissue showed that the neurocyte apoptosis index in brain tissue of rats in ICH group increased significantly compared with that in control group(P<0.05),and it was significantly lower in Res group compared with ICH group(P<0.05).The immunofluorescence of proteins indicated that the expressions of TLR4,CD36,Nrf2 and HO-1 protein in rats'brain tissue in ICH group increased significantly compared with rats in control group(P<0.05).Compared with rats in ICH group,the expression of TLR4 protein in brain tissue of the rats in Res group decreased at the same time point(P<0.05),while the protein expressions of CD36,Nrf2 and HO-1 increased significantly(P<0.05).(2)Compared with the control group,the relative expression of TLR4,CD36 and HO-1 protein in BV2 cells of Fe^(2+)group increased(P<0.05),the expressions of Nrf2 and p-Nrf2 increased at 72 h(P<0.05),the expression of Nrf2 protein in the nucleus increased(P<0.01);Compared with the Fe^(2+)group,the expression of TLR4 in the low dose group of Fe^(2+)+Res and the high dose group of Fe^(2+)+Res decreased(P<0.001),and the expression of CD36,HO-1,Nrf2,p-Nrf2 and nuclear Nrf2 protein increased(P<0.05).Conclusions Intraperitoneal injection of resveratrol can improve the neurological function of rats after ICH.The microglia activated by Fe^(2+)within 72 hours after intracerebral hemorrhage mainly showed pro-inflammatory function.Resveratrol may regulate Nrf2/HO-1 signal pathway and promote the transformation of microglia function to anti-inflammatory after ICH.