【作者】 王爽；

【导师】 冯加纯；

【作者基本信息】 吉林大学 ， 神经病学， 2022， 博士

【摘要】 背景:脑缺血/再灌注时触发的炎症反应涉及神经胶质细胞激活、外周免疫细胞的招募以及炎性因子的释放,而细胞因子作为重要的效应因子一直是研究的热点。课题组在前期工作中发现脑梗死患者急性期血清中白细胞介素-2(IL-2)的水平下降,肢体远隔缺血后处理发挥保护作用的同时促进血清中IL-2升高。目前有关IL-2对脑卒中影响的研究较少,部分研究显示IL-2/IL-2抗体复合物特异性刺激Treg细胞增殖并增强其功能,通过抑制炎症反应减轻脑缺血/再灌注损伤。但IL-2抗体(JES6-1)阻断了IL-2上CD122的结合表位,CD122是IL-2受体中负责下游信号转导的关键亚基,脑组织中同样分布IL-2受体,因此,IL-2在脑缺血/再灌注损伤中是否可通过激活IL-2受体的下游信号通路而对神经细胞产生作用还不能明确。脑缺血/再灌注后自噬被激活,其可发挥抗凋亡到促凋亡的双重作用。明确自噬的变化规律及异常,阻止其从抗凋亡向促凋亡的转变,可能利于缺血性脑卒中的治疗。而IL-2下游的多个信号转导途径与自噬密切相关,且研究显示IL-2促进神经元存活并参与中枢神经系统稳态修复机制。因此,我们将IL-2作为研究对象,探究其在脑缺血/再灌注损伤中的作用及可能的机制。目的:明确IL-2在脑缺血/再灌注损伤中的作用;探究其是否通过干预自噬减少神经元凋亡,以及其是否能够调控星形胶质细胞和小胶质细胞的炎症状态从而减轻神经元损伤。方法:体内实验应用C57BL/6小鼠大脑中动脉闭塞(MCAO)模型;体外实验应用小鼠海马神经元细胞系HT22细胞、小鼠原代星形胶质细胞和小胶质细胞的糖氧剥夺/复糖复氧模型(OGD/R)。1、ELISA法检测缺血/再灌注后72 h内不同时间点脑组织中IL-2的变化情况。2、Western blot检测体内和体外脑缺血/再灌注后72 h内不同时间点自噬的变化情况,分别选定自噬发生转折的时间点进行后续实验。体内实验中,设置假手术组(sham)、MCAO组和MCAO+不同浓度IL-2组,通过TTC染色测定脑梗死体积并进行神经功能学评分,通过TUNEL染色观察细胞的凋亡。体外实验中,设置正常组(control)、OGD/R组和OGD/R+不同浓度IL-2组,采用MTT法测定HT22细胞的存活率,通过TUNEL染色观察细胞的凋亡。3、体内实验中,设置sham组、MCAO组、MCAO+IL-2组,MCAO+自噬激动剂雷帕霉素(RAPA)组,MCAO+IL-2+RAPA组,通过TTC染色测定脑梗死体积并进行神经功能学评分;设置sham组、MCAO组和MCAO+IL-2组,通过Western blot检测自噬水平及自噬相关蛋白、PI3K-Akt-m TOR通路及溶酶体功能相关蛋白,应用免疫荧光染色和透射电镜观察自噬小体和自噬溶酶体。体外实验中,设置OGD/R组、OGD/R+IL-2组、OGD/R+RAPA组、OGD/R+IL-2+RAPA组、OGD/R+自噬抑制剂巴佛洛霉素-A1(Baf-A1)组和OGD/R+IL-2+Baf-A1组,采用MTT法测定HT22细胞的存活率;设置control组、control+Baf-A1组、OGD/R组、OGD/R+Baf-A1组、OGD/R+IL-2组和OGD/R+IL-2+Baf-A1组,通过Western blot检测自噬水平,并进一步检测自噬相关蛋白、PI3K-Akt-m TOR通路及溶酶体功能相关蛋白,应用免疫荧光染色和透射电镜观察自噬小体和自噬溶酶体。4、体内实验中,设置sham组、MCAO组、MCAO+IL-2组,应用流式CBA检测脑组织中的细胞因子水平(IL-6、TNF-α、IL-4和IL-10)。体外实验中,对于星形胶质细胞,Western blot检测OGD/R后72 h内不同时间点自噬的变化情况,选定自噬发生转折的时间点进行后续实验;设置OGD/R组和OGD/R+不同浓度IL-2组,采用MTT法测定星形胶质细胞的存活率;设置control组、OGD/R组、OGD/R+IL-2组、OGD/R+RAPA组和OGD/R+Baf-A1组,Western blot检测LC3和Cx43的表达,免疫荧光染色观察LC3与Cx43共定位情况;Cx43 si RNA转染星形胶质细胞,设置control组、OGD/R组、OGD/R+IL-2组和OGD/R+IL-2+Cx43 KD组,应用流式CBA检测培养液中细胞因子的水平,并收集上述培养液作为条件培养基再灌注OGD/R后的HT22细胞并进行流式凋亡检测。对于小胶质细胞,设置control组、OGD/R组、OGD/R+IL-2组,进行流式表型检测,流式CBA检测培养液中的细胞因子,并收集上述培养液作为条件培养基再灌注OGD/R后的HT22细胞并进行流式凋亡检测。结果:1、IL-2的变化:缺血/再灌注后脑组织中IL-2水平下降(P<0.05),其中再灌注后18 h的IL-2水平最低(P<0.01)。2、IL-2的保护作用:脑缺血/再灌注后自噬先增强后减弱(P<0.001),18 h和16 h分别为体内实验和体外实验中自噬变化的关键转折点,将其定为后续实验的时间点,此时自噬水平升高且可能存在自噬溶酶体降解障碍。体内实验中,侧脑室注射IL-2(5ng/m L,1μL)可减小脑梗死体积(P<0.001)并减少神经细胞的凋亡(P<0.05)。体外实验中,IL-2(5 ng/m L)可促进OGD/R后神经元的存活(P<0.05),减少神经元的凋亡(P<0.01)。3、IL-2改善自噬障碍:体内实验中,脑缺血/再灌注后,IL-2+RAPA组较IL-2组的脑梗死体积增加(P<0.01),但较RAPA组的脑梗死体积减小(P<0.001);IL-2抑制了脑缺血/再灌注后LC3的表达(P<0.05),激活了PI3K-Akt-m TOR通路(PPI3K<0.001,PAkt<0.05,Pm TOR<0.05),IL-2虽对脑缺血/再灌注后溶酶体标记蛋白LAMP1的表达无影响,但促进了溶酶体酶中的组织蛋白酶B(CTSB)和组织蛋白酶D(CTSD)的表达(PCTSB<0.01,PCTSD<0.05),进而改善了自噬溶酶体降解障碍;此外,免疫荧光和透射电镜显示IL-2减少了脑缺血/再灌注后自噬小体和自噬溶酶体的数量。体外实验中,RAPA和Baf-A1降低OGD/R后神经元的存活率(PRAPA<0.01,PBaf-A1<0.01),而IL-2可部分逆转上述药物所致的神经元损伤(PRAPA<0.001,PBaf-A1<0.001);IL-2抑制OGD/R后神经元LC3的表达(P<0.05),激活了PI3K-Akt-m TOR通路(PPI3K<0.05,PAkt<0.01,Pm TOR<0.05),但应用Baf-A1后,显示IL-2未抑制自噬小体的生成,与此同时,IL-2虽对OGD/R后LAMP1、CTSB、CTSD和自噬小体溶酶体融合相关蛋白Rab7的表达无明显影响,但促进了Rab7下游效应因子RILP在OGD/R后的表达(P<0.05),进而改善了自噬溶酶体降解障碍;免疫荧光和透射电镜中同样观察到IL-2减少了OGD/R后神经元内自噬小体和自噬溶酶体的数量。4、IL-2调控炎症状态:体内实验中,IL-2降低了脑缺血/再灌注后促炎因子IL-6和TNF-α的表达(PIL-6<0.05,PTNF-α<0.05),增加了抗炎因子IL-10的表达(P<0.05)。体外实验中,星形胶质细胞OGD/R后自噬先增强后减弱(P<0.001),选取16 h作为后续实验的时间点;IL-2(5 ng/m L)可促进OGD/R后星形胶质细胞的存活(P<0.05);星形胶质细胞Cx43通过自噬降解(P<0.05),而IL-2抑制自噬(P<0.05)并减少Cx43的降解(P<0.01);此外,IL-2降低了OGD/R后IL-6的表达(P<0.05),此条件培养液使OGD/R后神经元的凋亡减少(P<0.01),但敲低Cx43后此结果被逆转,出现IL-6升高(P<0.05),IL-10下降(P<0.05),同时条件培养液促进OGD/R后神经元的凋亡(P<0.05)。在小胶质细胞中,IL-2促进OGD/R后的小胶质细胞向抗炎表型转化(P<0.05),使IL-6和TNF-α分泌减少(PIL-6<0.05,PTNF-α<0.05),IL-10分泌增多(P<0.05),此条件培养液使OGD/R后神经元的凋亡减少(P<0.05)。结论:1、缺血/再灌注后脑组织中IL-2水平下降。2、IL-2在脑缺血/再灌注损伤中发挥保护作用。3、IL-2可能通过促进自噬溶酶体降解改善脑缺血/再灌注损伤中的自噬障碍。4、IL-2可促进脑缺血/再灌注损伤后的炎症状态向抗炎转化,其机制可能与调控OGD/R后星形胶质细胞Cx43自噬降解及小胶质细胞极化有关。更多还原

【Abstract】 Background:The inflammatory response triggered by cerebral ischemia/reperfusion involves the activation of glial cells,the recruitment of peripheral immune cells,and the release of inflammatory factors,and cytokines as important effector factors have always been a research hotspot.In our previous work,we found that the level of serum interleukin-2(IL-2)decreased over time in patients with acute ischemic stroke,and remote ischemic postconditioning of limbs had therapeutic effects on patients while promoting the increase of serum IL-2.At present,there are few studies on the effects of IL-2 on ischemic stroke,and some studies have shown that IL-2/IL-2 antibody complex specifically stimulates Treg cell proliferation and enhances its function,alleviating cerebral ischemia/reperfusion injury by inhibiting inflammatory response.However,IL-2 antibody(JES6-1)blocks the binding epitope of CD122 on IL-2,which is a key subunit of IL-2 receptor responsible for downstream signal transduction,and IL-2 receptor is also distributed in brain tissue,therefore,it is not clear whether IL-2 can affect nerve cells in cerebral ischemia/reperfusion injury by activating the downstream signaling pathway of IL-2 receptor.Autophagy is activated after cerebral ischemia/reperfusion,which can play a dual role of anti-apoptotic and pro-apoptotic.It may be beneficial for the treatment of ischemic stroke to clarify the abnormal autophagy after cerebral ischemia/reperfusion and prevent its transformation from anti-apoptotic to pro-apoptotic.Multiple signal transduction pathways of IL-2 are closely related to autophagy,and previous studies have shown that IL-2 can promote neuronal survival and participate in the homeostasis repair mechanisms of brain.Therefore,we took IL-2 as the research object to explore its role and possible mechanism in cerebral ischemia/reperfusion injury.Object:To clarify the role of IL-2 in cerebral ischemia/reperfusion injury.To explore whether IL-2 can reduce neuronal apoptosis by interfering with autophagy,and whether it can regulate the inflammatory state of astrocytes and microglia to reduce neuronal injury.Methods:In vivo,C57BL/6 mice were used as the research object,and the middle cerebral artery occlusion(MCAO)model was used as the ischemia/reperfusion injury model.In vitro,the mouse hippocampal neuron cell line HT22 cells,primary astrocytes,and microglia were used as the research objects,and the oxygen-glucose deprivation/reoxygenation model(OGD/R)was used as the ischemia/reperfusion injury model.1.ELISA was used to detect the changes of IL-2 in brain tissues within 72 h after ischemia/reperfusion.2.Western blot was used to detect changes of autophagy within 72 h after cerebral ischemia/reperfusion in vivo and in vitro,and the time points of autophagy transition were selected for subsequent experiments.In vivo,it was divided into sham group,MCAO group,and MCAO with different concentrations of IL-2 groups.The cerebral infarction volume was measured by TTC and the neurological function score was performed,in addition,TUNEL staining was used to observe cell apoptosis.In vitro,it was divided into the control group,OGD/R group,and OGD/R with different concentrations of IL-2 groups.The survival rate of HT22 cells was determined by MTT assay,and the apoptosis of HT22 cells was observed by TUNEL staining.3.In vivo,it was divided into sham group,MCAO group,MCAO+IL-2 group,MCAO+autophagy agonist Rapamycin(RAPA)group,and MCAO+IL-2+RAPA group.The cerebral infarction volume was measured by TTC and the neurological function score was performed.And then,it was divided into sham group,MCAO group,and MCAO+IL-2group,and the level of autophagy,autophagy-related proteins,PI3K-Akt-m TOR pathway,and lysosomal functional proteins were detected by Western blot,and the autophagosomes and autolysosome were observed by immunofluorescence staining and transmission electron microscopy.In vitro,it was divided into OGD/R group,OGD/R+IL-2 group,OGD/R+RAPA group,OGD/R+IL-2+RAPA group,OGD/R+autophagy inhibitor bafilomycin-A1(Baf-A1)group,and OGD/R+IL-2+Baf-A1 group,and MTT assay was used to determine the survival rate of HT22 cells.And then,it was divided into control group,control+Baf-A1 group,OGD/R group,OGD/R+Baf-A1 group,OGD/R+IL-2 group,and OGD/R+IL-2+Baf-A1 group,and autophagy level was detected by Western blot.Autophagy-related proteins,PI3K-Akt-m TOR pathway,and lysosomal functional proteins were further detected.Autophagosomes and autolysosome were observed by immunofluorescence staining and transmission electron microscopy.4.In vivo,it was divided into sham group,MCAO group,and MCAO+IL-2 group,and the levels of cytokines(IL-6,TNF-α,IL-4,and IL-10)in brain tissues were detected by CBA.In vitro,for astrocytes,Western blot was used to detect the changes of autophagy within 72 h after OGD/R,and the time point of autophagy transition was selected for subsequent experiments.It was divided into OGD/R group and OGD/R with different concentrations of IL-2 groups,and MTT assay was used to determine the survival rate of cells.And then,it was divided into control group,OGD/R group,OGD/R+IL-2 group,OGD/R+RAPA group,and OGD/R+Baf-A1 group.Western blot was used to detect the expressions of LC3 and Cx43,and immunofluorescence staining was used to observe the co-location of LC3 and Cx43.Cx43 si RNA was transfected into astrocytes and they were divided into control group,OGD/R group,OGD/R+IL-2 group,and OGD/R+IL-2+Cx43 KD group,and cytokines in the astrocytes culture medium after OGD/R were detected by CBA,and then,the above culture medium was collected as the conditioned medium and re-perfused HT22 cells,and cell apoptosis was measured by flow cytometry.For microglia,it was divided into control group,OGD/R group,and OGD/R+IL-2 group,and the microglia were detected by flow cytometry.The cytokines in the culture medium after OGD/R were detected by CBA,and then,the above culture medium was collected as conditioned medium and re-perfused HT22 cells,and cell apoptosis was measured by flow cytometry.Results:1.Changes of IL-2: The level of IL-2 in brain tissue decreased after ischemia/reperfusion(P<0.05),and 18 h after reperfusion had the lowest level of IL-2(P<0.01).2.Protective effect of IL-2: After cerebral ischemia/reperfusion,autophagy was first enhanced and then weakened(P<0.001).In vivo and in vitro,18 h and 16 h were set as the time points of subsequent experiments respectively,at which autophagy level increased and autolysosome degradation disorder might exist.In vivo,lateral ventricle injection of IL-2(5ng/m L,1 μL)reduced cerebral infarction volume(P<0.001)and decreased the apoptosis of nerve cells(P<0.05).In vitro,IL-2(5 ng/m L)could promote the survival of neurons after OGD/R(P<0.05)and reduce the apoptosis of neurons(P<0.01).3.Effect of IL-2 on autophagy: In vivo,after cerebral ischemia/reperfusion,the cerebral infarction volume in the IL-2+RAPA group increased compared with the IL-2 group(P<0.01),but decreased compared with the RAPA group(P<0.001).IL-2 inhibited the expression of LC3 after cerebral ischemia/reperfusion(P<0.05)and activated the PI3K-Akt-m TOR pathway(PPI3K<0.001,PAkt<0.05,Pm TOR<0.05).Although IL-2 had no effect on the expression of lysosomal marker protein LAMP1 after cerebral ischemia/reperfusion,but it promoted the expression of cathepsin B(CTSB)and cathepsin D(CTSD)(PCTSB<0.01,PCTSD<0.05),and then improved autolysosome degradation disorder.In addition,immunofluorescence and transmission electron microscopy showed that IL-2reduced the number of autophagosomes and autolysosome after cerebral ischemia/reperfusion.In vitro,RAPA and Baf-A1 reduced the survival rate of neurons after OGD/R(PRAPA<0.01,PBaf-A1<0.01),while IL-2 partially reversed the neuron injury induced by these drugs(PRAPA<0.001,PBaf-A1<0.001).IL-2 inhibited the expression of LC3 in neurons after OGD/R(P<0.05)and activated the PI3K-Akt-m TOR pathway(PPI3K<0.05,PAkt<0.01,Pm TOR<0.05).However,application of Baf-A1 showed that IL-2 did not inhibit the formation of autophagosomes.Meanwhile,IL-2 did not affect the expression of LAMP1,CTSB,CTSD,and Rab7 after OGD/R,but it promoted the expression of RILP after OGD/R(P<0.05),and then improved autolysosome degradation disorder.Immunofluorescence and transmission electron microscopy also observed that IL-2 reduced the number of autophagosomes and autolysosome of neurons after OGD/R.4.Effect of IL-2 on inflammation: In vivo,after cerebral ischemia/reperfusion,IL-2decreased the expression of pro-inflammatory cytokines IL-6 and TNF-α(PIL-6<0.05,PTNF-α<0.05),and increased the expression of anti-inflammatory factor IL-10(P<0.05).In vitro,for astrocytes,autophagy was enhanced and then decreased after OGD/R(P<0.001),and 16 h was selected as the time point of subsequent experiments.IL-2(5 ng/m L)could promote the survival of astrocytes after OGD/R(P<0.05).Cx43 was degraded by autophagy(P<0.05),and IL-2 inhibited autophagy(P<0.05)and decreased Cx43 degradation(P<0.01).In addition,IL-2 decreased the expression of IL-6 after OGD/R(P<0.05),and the conditioned medium reduced the apoptosis of neurons after OGD/R(P<0.01),but this result was reversed after Cx43 KD.IL-6 was increased(P<0.05)and IL-10 was decreased(P<0.05),at the same time,the conditioned medium promoted the apoptosis of neurons after OGD/R(P<0.05).In microglia,IL-2 promoted the transformation of microglia into anti-inflammatory phenotype after OGD/R(P<0.05),and reduced the secretion of IL-6 and TNF-α(PIL-6<0.05,PTNF-α<0.05),but increased secretion of IL-10(P<0.05),and the conditioned medium reduced the apoptosis of neurons after OGD/R(P<0.05).Conclusions:1.The IL-2 in brain tissue was decreased after ischemia/reperfusion.2.IL-2 can play a protective role in cerebral ischemia/reperfusion injury.3.IL-2 may improve autophagy disorder in cerebral ischemia/reperfusion injury by promoting autolysosome degradation.4.IL-2 can promote the inflammatory state to anti-inflammatory in cerebral ischemia/reperfusion injury,and the mechanism may be related to the regulation of the astrocytes Cx43 autophagy degradation and microglia polarization after OGD/R.更多还原