Detrimental Effect Of EMMPRIN And Intervention Of Minocycline Of EMMPRIN In Intracerebral Hemorrhage

BackgroundIntracerebral hemorrhage(ICH)is a common and devastating disease,causing severe neurological dysfunction and even death of patients,and bringing unduly burdensome to individuals,families and society.The inflammatory reaction after intracerebral hemorrhage is the main factor leading to the progression of the disease.In this process,matrix metalloproteinases(MMPs)and pro-inflammatory cytokines released by microglia play a key role.MMPs can directly disruption of the blood-brain barrier and cause fatal brain edema.In addition,peripheral inflammatory cells,such as white blood cells,can penetrate the disruption of blood-brain barrier and further expand the inflammatory injury,leading to the deterioration of the disease.Therefore,finding the key signaling pathways that mediate the activation of microglia and induce the expression of MMPs and the release of pro-inflammatory cytokines will help to find new methods and strategies for the treatment of intracerebral hemorrhage,which is of great clinical and social significance.Recent studies have shown that myocardial infarction and cerebral infarction can promote the expression of Extracellular matrix metalloproteinase inducer(EMMPRIN/CD147).Studies on ischemia model suggest that EMMPRIN may induce the production of MMP-9.However,it is not known whether EMMPRIN is involved in brain injury after ICH.Minocycline is a semi-synthetic second-generation tetracycline that can penetrate the blood-brain barrier and inhibit the expression of MMPs and reduce the inflammatory cascade in intracerebral hemorrhage models.However,the related molecular mechanisms are still not fully understood.ObjectiveTo explore the role of EMMPRIN/CD147 in the pathophysiological process of ICH,and to observe whether minocycline can play a neuroprotective role after ICH by inhibiting EMMPRIN/CD147,so as to find a new target and a new method for the treatment of ICH.MethodsPart Ⅰ:Effect of EMMPRIN/CD147 on brain injury after intracerebral hemorrhage in mice and its molecular mechanism(1)Using collagenase type Ⅶ stereotaxic injection method to establish in a mouse model of ICH,the animals can be divided into saline group,ICH-ld,ICH-3d,ICH-7d group.Brain tissue slices calculating the change of hematoma volume and Fluoro Jade C staining was used to observe the degeneration of neurons to evaluate the success of the model.Then,the expression of EMMPRIN around perihematomal was observed by Western blot and immunofluorescence staining.MMP-9,Iba1 staining(microglia activation)and MPO staining(neutrophils infiltration)were performed at the corresponding time points.Finally,the cell origin of EMMPRIN and its consistency with MMP-9 expression were determined by double immunofluorescence staining.(2)The use of anti-CD 147 antibodies blocked EMMPRIN signal,then uses the immune staining and Western blot technique to observe a blocked EMMPRIN signal the influence of the expression of EMMPRIN and MMP-9,at the same time,adopt the method of immunohistochemical observation of neutrophil infiltration,Fluoro Jade C evaluation degeneration of neurons,brain tissue slices calculating hematoma volume,corner test and forelimbs test assess neurological function scores.Part Ⅱ:In vitro study on the role of EMMPRIN/CD147 signal in microglia activation and the inhibitory effect of minocycline(1)Experiment 1:BV2 microglia were stimulated with thrombin to establish the model of intracerebral hemorrhage in vitro.Model validation in vitro:divided into control group and thrombin group.Control group:no treatment was done;thrombin group was treated with thrombin(20 U/mL)for 24 hours.The activation of microglia was observed by immunofluorescence staining with Iba1,and the expression of EMMPRIN,MMP-9 and NF-κB was detected by Western blot.(2)Experiment 2:The expression of MMP-9 and NF-κB in BV2 cells induced by thrombin was observed after EMMPRIN knockdown.They were divided into control group:no treatment was done,siRNA negative control group:cells were transfected with negative control sequence,siRNA-EMMPRIN-477,siRNA-EMMPRIN-548,siRNA-EMMPRIN-618.Western blot was used to detect the expression efficiency of EMMPRIN.At the same time,siRNA with high efficiency of inhibiting the expression of EMMPRIN after transfection was selected for the next experiment,and the expression of MMP-9 and NF-κB in thrombin induced microglia was observed by Western blot.(3)Experiment 3:To verify the effect of NF-κB:BV2 cells were pretreated with NF-κB inhibitor(Bay 11-7082,10μmol/L)for 60 min,and then treated with 20 U/mL thrombin.After 24h,the expression of EMMPRIN,MMP-9 and NF-κB was observed by Western blot.(4)Experiment 4:To verify the effect of anti-CD 147 antibody and minocycline in vitro intervention:divided into control group,thrombin group,the anti-CD147 group and minocycline group,anti-CD 147 antibody group and minocycline group respectively added anti-CD147 antibody(10 μg/mL)and minocycline(50 μg/mL)preprocessing BV-2 cells after 60 minutes added 20 U/mL thrombin,after 24 hours using Iba1 immunofluorescence staining microglia observation of cell morphology,and Western blot technique to observe the expression of EMMPRIN and MMP-9.Part Ⅲ:Minocycline exerts neuroprotective effects after intracerebral hemorrhage by regulating EMMPRIN/CD147 signalsThis section using minocycline intervention right striatum bleeding in mice model,the experimental animals were divided into saline+vehicle group,saline+minocycline group,ICH+vehicle group,ICH+minocycline,3 days after treatment by immune staining and Western blot technique to observe the expression of EMMPRIN and MMP-9 change,using Western blot,measured the extravasation of Evans blue and the brain water content to observe the integrity of the blood-brain barrier,as well as microglia activation and inflammatory cells infiltration and the change of inflammatory cytokines release,TUNEL staining was used to evaluate apoptosis,Fluoro Jade C staining was used to observe neuronal degeneration,HE staining was used to evaluate brain injury,and neurological function scores were performed.Statistical analysisThe data were expressed as mean ± standard deviation.All data analyses were performed using a blind method.T-test was used to compare the mean values between the two groups.One-way ANOVA was used to compare the mean values between multiple groups.Bonferroni’s post hoc test or Dunnett’s test was used to analyze the pairwise comparison.A difference of P<0.05 indicated statistical significance.ResultsPart Ⅰ:1 Intracerebral hemorrhage induced increased expression of EMMPRIN and MMP-9.(1)Successfully established a mouse intracerebral hemorrhage modelBrain sections were used to calculate the volume of hematoma.One day after ICH,definite hematoma was visible(11.80±2.83 mm3).On the third day,the hematoma volume(16.46±2.82 mm3)increased further,and on the seventh day,the hematoma volume(6.16±1.02mm3)decreased significantly.FJC staining indicated that a large number of FJC positive cells could be found on the first day after ICH,and the number of FJC positive cells was slightly decreased on the third day after ICH,and the number of FJC positive cells was further decreased on the seventh day after ICH,but the number of the first two time points was significantly higher than that of the saline group(P<0.01).The above two parts of the experiment indicated that the modeling was successful.(2)The expression of EMMPRIN around hematoma after intracerebral hemorrhage is related to the expression of MMP-9 and neuroinflammationImmunofluorescence staining showed that there were only a few EMMPRIN positive cells in the brain slices of mice in saline group.The expression of EMMPRIN positive cells was significantly increased on day 1 after ICH,and the number of EMMPRIN positive cells was the highest on day 3,and the number of EMMPRIN positive cells decreased on day 7 compared with the previous time point.The differences of EMMPRIN positive cells at the above three time points compared with the saline group were statistically significant(P<0.05).The results of Western blot showed that intracerebral hemorrhage could significantly induce the expression of EMMPRIN,and the trend of change was consistent with the results of immunofluorescence staining.Immunohistochemical staining showed that there were no MMP-9 positive cells in the saline group,but a large number of MMP-9 positive cells could be detected on the brain slices of the saline group for 1 day.The number of MMP-9 positive cells further increased in the 3 day group of intracerebral hemorrhage.The number of MMP-9 positive cells decreased but was still higher than that in the saline group on the seventh day after ICH(P<0.05).The results suggested that the expression trend of EMMPRIN was consistent with that of MMP-9.MPO staining indicated that the number of MPO positive cells could not be detected in the brain of mice in the saline group,but the number of MPO positive cells was significantly increased on the first day after hemorrhage,and decreased on the third and seventh day after ICH,compared with that in the saline group(P<0.01).It suggested that the temporal variation trend of EMMPRIN and brain injury was consistent.(3)Co-localization of EMMPRIN with various cell typesEMMPRIN was only colocalized with CD31(endothelial cells)in saline group.EMMPRIN was co-localized with Iba1(microglia),GFAP(astrocytes),CD31(endothelial cells)and MMP-9 in 3 days after ICH,and microglia had the highest number of colocalization,suggesting that intracerebral hemorrhage can promote the upregulation of EMMPRIN in microglia,astrocytes and endothelial cells,and microglia was the dominant expression.Immunofluorescence staining showed that the majority of Iba1(microglia)positive cells responded similarly to the expression of MMP-9 and EMMPRIN after ICH.2 Blocking EMMPRIN signal inhibits MMP-9 expression,reduces neutrophil infiltration and cerebral hematoma volume,and improves neurological function.Western blot confirmed that the protein expression levels of EMMPRIN and MMP-9 in the ICH+anti-CD147 group were significantly decreased compared with the vehicle group(P<0.001)after 3 days of treatment with anti-CD147 antibody.Immunofluorescence staining also found that the number of EMMPRIN positive cells in the anti-CD 147 injection group was significantly decreased,and the difference was statistically significant compared with the ICH+vehicle group(P<0.05).MPO staining indicated that the number of MPO positive cells in ICH+anti-CD147 group was significantly lower than that in ICH+vehicle group(P<0.001).Compared with the ICH+vehicle group,the expressions of IL-1β and TNF-α in ICH+anti-CD 147 group were significantly decreased(P<0.01).FJC staining assay showed that anti-CD147 injection significantly reduced the number of FJC positive cells,and the difference was statistically significant compared with the ICH+vehicle group(P<0.01).Meanwhile,the volume of cerebral hematoma in ICH+anti-CD 147 group(13.35±1.01mm3)was significantly smaller than that in ICH+vehicle group(15.46±1.46mm3)(P<0.01).The degree of neurological impairment in anti-CD 147 injection group was significantly lower than that in ICH+vehicle group(P<0.05).Part Ⅱ 1 Thrombin activates microglia and promotes the expression of EMMPRIN and MMP-9The results of Ibal immunofluorescence staining showed that the microglia in the thrombin group were larger and rounded than those in the control group.The elongated projections turned into fine pseudopods,and branched into amoeba shape.The ratio of the number of activated BV2 cells to the total BV2 cells in thrombin group was significantly higher than that in control group(P<0.01).Western blot analysis showed that the expressions of EMMPRIN,MMP-9 and nucleic NF-κB p65 in BV2 microglia in thrombin group were significantly increased compared with those in control group(P<0.01).In the experiment of RNA interference with EMMPRIN,siRNA-EMMPRIN 618 transfection inhibited the expression of EMMPRIN with the highest efficiency(P<0.05).Western blot analysis showed that the expressions of EMMPRIN,MMP-9 and nucleic NF-κB p65 in siRNA-EMMPRIN group were significantly decreased compared with thrombin group(P<0.05).There was no significant difference between thrombin group and siRNA-NC group(P>0.05).These results suggest that EMMPRIN is the upstream signal of MMP-9 and NF-κB.2 EMMPRIN signal mediated the expression of MMP-9 in microglia through NF-κB pathway.Western blot analysis showed that thrombin stimulation promoted the high expression of EMMPRIN,nucleic NF-κB p65 and MMP-9 in microglia.Adding NF-κB inhibitor significantly decreased the expression of MMP-9(P<0.05);However,the expression of EMMPRIN could not be changed(P>0.05),suggesting that thrombin activated EMMPRIN signal may mediate the expression of MMP-9 through NF-κB pathway.3 Anti-CD147 antibody or minocycline inhibited the expression of EMMPRIN and MMP-9 in thrombin induced microgliaAfter anti-CD 147 was added,the overall morphology of microglia was similar to that of the control group.The microglia cells in the minocycline group were small and surrounded by tiny projections,which were similar to those in the control group and anti-CD147 antibody group.Western blot results showed that the expressions of EMMPRIN and MMP-9 in microglia in the thrombin group were significantly increased compared with those in the control group(P<0.05),and the expressions of EMMPRIN and MMP-9 were significantly inhibited by the addition of anti-CD 147 antibody or minocycline(P<0.05).In conclusion,the results suggest that thrombin activates microglia through the Emmprin signaling pathway.Part Ⅲ 1 Minocycline decreased the expression of EMMPRIN/CD147.Immunofluorescence and Western blot were used to observe the expression of EMMPRIN in mice with cerebral hemorrhage after 3 days intervention with minocycline.A small amount of EMMPRIN positive cells and protein expression were observed in the normal saline group,and the number and protein of EMMPRIN positive cells and protein were significantly increased in the ICH+vehicle group compared with the normal saline group.Minocycline injection significantly decreased the number of EMMPRIN positive cells and protein expression(P<0.05);Meanwhile,we found that MMP-9 positive cells and protein expression could not be detected in the normal saline group,while MMP-9 positive cells and protein expression was significantly increased in the ICH+vehicle group.Minocycline group significantly decreased the expression of MMP-9(P<0.05),suggesting that minocycline can reduce the expression of EMMPRIN and MMP-9.2 Minocycline exerts neuroprotective effects by decreasing the expression of EMMPRIN/MMP-9.Compared with the saline group,the expression of ZO-1 and Occludin decreased significantly after intracerebral hemorrhage(P<0.05),and minocycline injection significantly inhibited the decrease of ZO-1 and Occludin(P<0.05),but the overall level of ZO-1 and Occludin was still lower than that of the saline group(P<0.05).Minocycline treatment group Evans blue extravases in ipsilateral hemisphere were significantly decreased compared with ICH+Vehicle group(P<0.05),and brain water content in minocycline treatment group was significantly decreased compared with ICH+Vehicle group(P<0.05).Compared with ICH+vehicle group,the number of activated Iba1 cells and MPO positive cells in ICH+Minocycline group were significantly decreased(P<0.01).Western blot results showed that compared with saline+vehicle group,the expressions of TNF-α and IL-1β in ICH+vehicle group were significantly increased(P<0.05),and minocycline injection significantly inhibited the expression of inflammatory cytokines(P<0.01).TUNEL assay results showed that there were only a small number of TUNEL positive cells in the saline group and a large number of TUNEL positive cells in and around the hematoma area in the ICH group.Compared with ICH+vehicle group,the number of TUNEL positive cells in ICH+Minocycline group was significantly decreased(P<0.01).Fluoro-Jade C staining showed a significant decrease in FJC positive cells in ICH+Minocycline group compared with ICH+vehicle group(P<0.01).Based on assessment of the volume of hematoma brain injury,saline group damage exists only a needle,compared with ICH+vehicle group,ICH hematoma volume ICH+Minocycline group decreased(P<0.01),for example ICH+Minocycline group can be in 7 days after treatment can reduce the nerve function defect(P<0.01),and neural function in the first and the third days did not see obvious improvement,prompt Minocycline has nerve protective effect in acute stage.Conclusions:1.EMMPRIN can mediate the upregulation of MMP-9 and exacerbate neurological dysfunction in a mouse model of ICH.2.EMMPRIN signal mediates the expression of MMP-9 in microglia through NF-κB pathway.3.Minocycline plays a neuroprotective role by regulating EMMPRIN/CD147 signals after intracerebral hemorrhage.