The Neuroprotective Effect Of IL-4 On Modulating Microglia M2 Polarization Through The JAK1/STAT6 Pathway After ICH In Mice

Objective: To investigate whether IL-4 mediates the process that the JAK1/STAT6 signaling pathway induces the microglia polarization of M2 subtype to alleviate inflammation and mitigate neuronal apoptosis to ameliorate the recovery of neurological injury following ICH.Methods: 1.We established an in vitro model of ICH by culturing BV2 cells induced by thrombin.The expression and costaining of Iba-1+CD16/32,Iba-1+CD206 was detected by immunofluorescence assay.Whether BV2 cells were activated after thrombin treatment,the relative fluorescence intensity of the two costaining and expressed markers was tested.The BV2 cells induced by thrombin were divided into four groups:(1)Control group: Model group induced by thrombin;(2)Thr+PBS group: BV2 polarization model induced by thrombin was administrated by PBS;(3)Thr+IL-4 group: BV2 polarization model induced by thrombin was administrated by IL-4;(4)IL-4+AG group: BV2 polarization model induced by thrombin was administrated by IL-4 and AG-490.Then Western blot was used to detect the expression levels of p-JAK1,JAK1,p-STAT6 and STAT6.Immunofluorescence was used to detect the relative fluorescence intensity of Iba-1+CD16/32 and Iba-1+CD206 co-staining of BV2 cells in each group.2.The collagenase VII was used to establish a ICH model in mice,C57BL/6 mice were randomly divided into four groups:(1)Control group: normal control group;(2)Sham group: sham operation group;(3)ICH-PBS group: ICH model injected by PBS;(4)ICH-IL-4 group: ICH model injected by IL-4.Behavioral tests,cerebral edema,and cerebral hematoma volume measurements were performed on days 1,3 and 7 after surgery in mice of each group.At the same time,Western blot was used to detect the expression levels of p-JAK1,JAK1,STAT6 and p-STAT6 pathway proteins and apoptosis-related proteins(cleaved-caspase-3,BAX and Bcl-2)in different groups at different time points.The costaining levels of NeuN+caspase-3,Iba-1+CD16/32 and Iba-1+CD206 were detected.Statistical analysis of each data was used to observe whether there were statistically significant differences between the research indicators.Results: 1.In vitro,CD16/32 was used as a marker of M1 type microglia,and CD206 was used as a marker for M2 type after BV2 cells induced with Thrombin.It was detected that BV2 cells were significantly induced to M1/M2 subtype.Then BV2 cells treated in different ways,the expression levels of JAK1/STAT6 pathway proteins were significantly increased,and the fluorescence intensity of Iba-1+CD206 costaining was significantly enhanced,while the fluorescence intensity of Iba-1+CD16/32 costaining was significantly weakened,but compared with the Thr+IL-4 group,the above results were significantly reversed—the levels of p-JAK1/JAK1 and p-STAT6/STAT6 were significantly decreased in the IL-4-AG group.The fluorescence intensity of Iba-1+CD206 costaining was significantly decreased in Thr+PBS group,but the fluorescence intensity of Iba-1+CD16/32 costaining was significantly enhanced.2.In vivo,the ICH model of C57/BL6 mouse was successfully established with collagenase VII.After IL-4 treatment,the neurological damage score of mice was improved more obviously,the brain edema in ipsilateral hemisphere were significantly decreased,and the hematoma volume were significantly reduced.At the same time,Iba-1+CD206 costaining cells were significantly reduced in ICH-IL-4 group compared with ICH-PBS group,while the number of Iba-1+CD16/32 costaining cells increased in ICH-IL-4 group significantly compared with ICH-PBS group.The levels of pro-apoptosis proteins(BAX and cleaved-caspase-3)were significantly decreased in the ICH-IL-4 group,while the anti-apoptotic protein,Bcl-2,was significantly increased,and the ratios of p-JAK1/JAK1 and p-STAT6/STAT6 were also significantly higher.Conclusion: In this study,IL-4 mediates JAK1/STAT6 pathway to promote polarization of M2 microglia and alleviate secondary neurological injury in mice following ICH.