| Part ?:Correlation between Tim-3 protein and related inflammatory factors and ICH in ratObjective:To study the expression changes of Tim-3 protein at different time points after intracranial hematoma(ICH),and Tim-3 mainly expressed cells in brain tissue.Methods:Thirty-six SD rats were randomly divided into Sham group and 6 groups of 12h,1d,3d,5d and 7d after ICH,with 6 rats in each group.All SD rats were sacrificed at the corresponding time points.The brain tissue in the range of 0.15-0.2 cm around the hematoma of each group of SD rats was taken.The corresponding area was taken from the Sham group.Western blotting was used to detect the expression of Tim-3 protein in brain tissue of this range.In addition,the expression levels of inflammatory factors IL-17 and IL-1? in this region were detected by enzyme-linked immunosorbent assay.The main expression cells of Tim-3 protein were detected by immunofluorescence technique at the peak time point of Tim-3 expression.Results:1,Western blot analysis showed that the expression of Tim-3 protein in the brain tissue of rats after ICH increased first and then decreased.The expression of Tim-3 was the most significant in the 1st day after ICH(P<0.001).2,The detection of pro-inflammatory factors IL-17 and IL-1? by rat brain tissue enzyme-linked immunosorbent assay showed that the expression of two pro-inflammatory factors and Tim-3 was similar after ICH.3,At the peak time of Tim-3 protein expression(1d),immunofluorescence assay was used to detect the expression of Tim-3 protein in astrocytes,microglia and neurons,indicating that Tim-3 protein was mainly expressed in microglia(P<0.001).Conclusion:1,The expression of Tim-3 in the perihematomal brain tissue of rats after ICH increased first and then recovered,and the expression of Tim-3 protein reached the peak at 1d.2,The expression of related pro-inflammatory factors IL-17 and IL-1? is similar to the expression trend of Tim-3 after ICH.3,Tim-3 protein is mainly expressed in microglia,but not astrocytes or neurons,1 day after ICH.Part ?:Up-regulation and down-regulation of Tim-3 protein expression in behavioral and secondary brain injury after intracerebral hemorrhage in rats.Objective:To explore the effects of rhTim-3 and siRNA injected into the lateral ventricle of rats on behavior and secondary brain injury after intracerebral hemorrhage in rats.Methods:In the rat experiment,120 subjects were randomly assigned to sham,intracerebral hemorrhage+vehicle group(ICH+vehicle),intracerebral hemorrhage+human recombinant TIM-3 group(ICH+rhTIM-3),intracerebral hemorrhage+non-specific small interfering RNA group(ICH+Ctr siRNA),intracerebral hemorrhage+small interfering RNA group(ICH+siTim3),a total of 5 groups(n=24/group).Intervention was performed by injecting rhTIM-3,CtrsiNRA and siTim3 into the lateral ventricle.Six rats in each group were analyzed by Western Blot and Elisa;six rats in each group were tested for behavior;six rats were divided into TUNEL staining and FLuoro-Jade B staining;six rats in each group were evaluated for cerebral edema.Results:1,Western Blot analysis showed that the expression of Tim-3 was up-regulated and down-regulated after ICH by rhTIM-3 and siTim3(P<0.01).2,In the 3-day short-term behavioral score test,rhTIM-3 intervention aggravated the behavioral score after intracerebral hemorrhage(P<0.05),while siTim3 relieved the symptoms of behavioral damage after intracerebral hemorrhage(P<0.05);similar results were obtained in the 28-day long-term behavioral Adhesive removal test and the Foot-fault test(P<0.05).3,In the FJB staining test,FJB-positive cells showed a significant increase in intracerebral hemorrhage compared with Sham group,and rhTIM-3 significantly up-regulated the number of FJB-positive cells(P<0.05),while the number of positive cells in Ctr-siRNA group decreased after siTim3 intervention(P<0.05).4,TUNEL staining results showed that the intracerebral hemorrhage significantly increased the apoptotic index compared with Sham group and rhTIM-3 aggravated apoptotic index(P<0.01),while the apoptosis index of CtrsiRNA group decreased after siTim3 intervention(P<0.05).5,Cerebral edema experimental results confirmed that rhTIM-3 intervention aggravated hemorrhagic brain tissue edema(P<0.05),while siTim3 intervention relieved brain edema compared with CtrsiRNA group(P<0.05).Conclusion:RhTim-3 and siTim3 injection into the lateral ventricle of rats can affect the behavioral and secondary brain injury after ICH in rats.Among them,rhTIM-3 aggravated the behavioral symptoms after ICH and aggravated the secondary brain injury;while siRNA can alleviate the behavioral symptoms after ICH and relieve the secondary brain injury.Part ?:Experimental study on the effect of Tim-3 signaling pathway on secondary brain injury after ICH in rats.Objective:The potential mechanism of Tim-3 signaling pathway was studied by microglia typing,inflammatory factor detection and co-immunoprecipitation.Methods:1,The changes of microglia to M1 and M2 after rhTIM-3 and siRNA intervention were analyzed by immunofluorescence staining after ICH.2,The expression of proinflammatory and anti-inflammatory factors in brain tissue was detected by Western Blot.3,The interaction between Tim-3 protein and Gal-9 and TLR-4 was detected by immunoprecipitation.4,Immunofluorescence staining was performed to detect the interaction between Tim-3 protein and Gal-9 and TLR-4 after interference.5,The expression of HIF-la in the cytoplasm and nucleus of brain tissue was detected by Western Blot assay after interference with rhTIM-3 and siRNA.Results:1,M1 and M2 microglia were activated after ICH(P<0.01).rhTIM-3 intervention promoted the transformation of microglia into M1 type,preventing microglia from transforming into M2,and siRNA intervention was the opposite.2,After ICH,pro-inflammatory and anti-inflammatory factors were activated,rhTIM-3 intervention promoted the activation of pro-inflammatory factors iNOS,TNF-?,IL-1?,and inhibited the expression of anti-inflammatory factors arginasel,IL-4,IL-10;siRNA Intervention inhibits the activation of pro-inflammatory cytokines iNOS,TNF-?,IL-1?,and promotes the expression of anti-inflammatory factors arginasel,IL-4,IL-10.3,Tim-3 protein interacts with Gal-9,Gal-9 and TLR-4 after ICH;rhTIM-3 enhances the interaction between TIM-3 protein and Gal-9,but weakens the interaction between Gal-9 and TLR-4 relationship.4,Double-labeled fluorescent co-staining of microglia in vitro further confirmed that rhTIM-3 enhanced the interaction between TIM-3 protein and Gal-9,but weakened the interaction between Gal-9 and TLR-4.5,HIF-la in the cytoplasm and nucleus of ICH was significantly increased.After rhTIM-3 intervention,HIF-1? in the cytoplasm decreased significantly,and the expression in the nucleus increased significantly.Conclusion:Human recombinant TIM-3 interferes with the conversion of microglia to Ml after ICH,and the pro-inflammatory factor is significantly increased.Tim-3 protein promotes TLR-4 exposure and promotes inflammatory response by competitively binding to Gal-9.The mechanism of action of Tim-3 after cerebral hemorrhage is closely related to the spatial distribution of HIF-1? in the cytoplasm and nucleus. |
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