| 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-?,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 ?L)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-?(PIL-6<0.05,PTNF-?<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-?(PIL-6<0.05,PTNF-?<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. |
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