Study On The Role And Mechanism Of FERMT1 In Secondary Inflammation Of Cerebral Hemorrhage

Spontaneous intracerebral hemorrhage is a central nervous system disease caused by intracranial parenchymal hemorrhage caused by nontraumatic intracranial blood vessel rupture,it is characterized by high fatality rate and high disability rate.The injury types of cerebral hemorrhage can be segmented into primary injury and secondary injury.In clinical treatment,some cases of primary injury can be relieved by surgical removal,but secondary injury can not be removed and will exist for a long time,and more and more studies have found that inflammation plays a crucial role in secondary brain injury.Therefore,it is urgent to find resultful neuroprotective agents for cerebral hemorrhage to antagonize injury and relieve inflammation.As the most important inflammatory effector cells in the CNS,microglia cells can secrete inflammatory mediators,cytokines and other substances when over-activated and inducing an inflammatory response in the brain.FERM domain containing kindlin 1(FERMT1)is widely expressed in various human tissues and plays an important part in various diseases.However,the role and mechanism of Fermt1 in intracerebral hemorrhage are still unclear.Overexpression of FERMT1 has been shown to block the activation of anti-inflammatory glial cells,thereby reducing the therapeutic effect of hyperbaric oxygen therapy on neuropathic pain.In addition,FERMT1 can regulate the nuclear factor κB(NF-κB)signaling pathway and NLRP3 inflammasome.Combined with previous experiments and literature search,we speculated that FERMT1 plays a crucial part in microglia-mediated cerebral hemorrhage inflammation,and its mechanism may be related to NLRP3/NF-κB pathway signaling pathway.Therefore,this study will use three parts of the experiment to carry on the relevant elaboration.Part One Study on the differential expression of FERMT1 in patients with cerebral hemorrhageObjective: This part of the study is a clinical study.The differential expression of Fermt1 mRNA in patients with intracerebral hemorrhage and healthy controls were determined by Real-time PCR detection,and related elements were analyzed.Methods: Blood samples were collected from 40 patients with cerebral hemorrhage(ICH group)and 40 healthy participants(Control group).The detection indicators were as follows: 1.Basic information,risk factors and clinical blood test information of all subjects were collected.The volume of hematoma,whether it was accompanied by intraventricular hematoma,and whether it was lobar hemorrhage were collected for subjects in the ICH group.2.Fermt1 mRNA expression of serum obtained by centrifugation of blood samples were investigated by Real-time PCR.3.Correlation between Fermt1 mRNA expression and clinical data was analyzed.Results:1.Comparison results of clinical data: The age and gender composition of the two groups were matched(P>0.05);There were striking differences in hypertension,drinking and drug intervention history among high-risk factors(P<0.05),but no statistical differences in diabetes,hyperlipidemia,smoking,coronary heart disease and chronic kidney disease(P>0.05).There were statistically striking differences in plasma glucose and white blood cell count between the two groups(P<0.05),but no striking differences in platelet count between the two groups(P>0.05).。2.Results of Real-time PCR detection of Fermt1 mRNA expression in human serum: Compared with Control group,Fermt1 mRNA level in serum of ICH group was significantly increased,with statistical significance(P<0.05).。3.Correlation analysis between Fermt1 mRNA expression level and clinical data results: Fermt1 mRNA expression level had no significant correlation with hematoma volume,whether there was intraventricular hematoma,whether there was lobular hemorrhage,white blood cell count,platelet count and blood glucose changes(P>0.05).Summary: Elevated white blood cells and blood glucose suggest inflammation after ICH.Fermt1 mRNA levels were elevated in ICH patients and may play an important role in inflammation.The Fermt1 mRNA level in ICH patients was not affected by clinical factors such as the amount of hematoma.Part Two The regulatory effect of FERMT1 on the inflammatory response of the tissue around hematoma in ratsObjective: This part of the study was an in vivo animal experiment,which established a rat model of cerebral hemorrhage.FERMT1 expression was knocked down by transfection of siRNA,so as to clarify the role of FERMT1 in vivo environment on the secondary inflammatory response after cerebral hemorrhage.Methods:Seventy-two healthy male SD rats aged about 8 weeks were randomized to one of four groups,namely Sham group,ICH group,NC siRNA+ICH group and FermtlsiRNA+ICH group.50 u L of non-anticoagulant autologous blood was injected into the right caudate nucleus to establish an ICH model.The Sham group was not injected.At 24 hours before ICH modeling,NC siRNA+ICH group and FermtlsiRNA+ICH group were injected with NC siRNA and Fermt1 siRNA,respectively。The detection indicators were as follows:1.Forelimb placing test and Corner turn test were used to assess neurological impairment;2.Determination of brain water content by standard wet and dry method;3.3.Application of HE staining to detect the pathological changes of brain tissue around hematoma.4.The Fermt1 mRNA level in the tissue around hematoma was determined by Real-time PCR.5.The expressions of Fermt1,NLRP3,ASC,cleaved caspase-1,IκBα,p-IκBα,NF-κB p65,and p-NF-κB p65 were detected by Western blot.6.The level of microglia marker Iba1 was determined by immunofluorescence.7.The ELISA kitswere used to determine the levels of the inflammatory cytokines IL-1β and IL-18.Results:1.Fermt1 mRNA and protein levels in tissues around hematoma: Realtime PCR and Western blot showed that the mRNA and protein levels of FERMT1 in ICH group were significantly higher than those in Sham group(P<0.05).FERMT1 levels were significantly reduced after FERMT1 knockdown(P<0.05)。2.Evaluation results of brain tissue injury after ICH in rats: Rats after ICH had obvious forelimb placement defects compared with the shamoperated rats,and the forelimb placement defects were significantly alleviated in FERMT1 knockdown rats(P<0.05).For the corner test,the percentage of turns was significantly increased in rats after ICH compared with shamoperated rats,and the score of FERMT1 knockdown rats was significantly lower(P<0.05).Moreover,FERMT1 knockdown significantly reversed the elevated brain water content induced by ICH in rats(P<0.05).HE staining suggested that there was obvious hematoma in the striatum of ICH rat brain tissue,and knockdown of FERMT1 could relieve the hematoma.Meanwhile,cells were neatly arranged,with dense cytoplasm and intact nuclei in the sham-operated group.Cells were intricately arranged,with loose cytoplasm and smaller nuclei in the ICH and NC siRNA+ICH groups.In contrast,less edema and diffuse vacuoles in the interstitial region were observed when FERMT1 was knocked down.3.Activity of microglia and NLRP3 inflammasome and expression of inflammatory cytokines in rats: Immunofluorescence staining showed that,knockdown of FERMT1 reduced Iba1 expression induced by ICH.IL-1β and IL-18 levels were dramaticallyelevated in brain tissue of rats after ICH compared with sham-operated rats,and FERMT1 knockdown observably decreased IL-1β and IL-18 levels(P<0.05).The results of the western blot revealed that NLRP3,ASC and cleaved-caspase-1 protein levels were significantly increased in rats after ICH compared with sham-operated rats,and knockdown of FERMT1 prevented ICH-induced increases in these three protein levels(P<0.05).This indicated that knockdown of FERMT1 inhibited the activity of NLRP3 inflammasome in rat brain tissue.4.Expression of NF-κB signaling pathway protein in rat peripheral hematoma: The effect of FERMT1 on the NF-κB pathway was studied by western blotting.ICH significantly increased the protein levels of p-IκBa and p-NF-κB p65 in the nucleus,and knockdown of FERMT1 significantly suppressed these changes(P<0.05).ICH and FERMT1 knockdown had no significant effect on the protein levels of IκBa and NF-κB p65(P>0.05).Summary: FERMT1 expression increased in ICH rats surrounding hematoma.ERMT1 knockdown can improve the neurohistomorphologic changes,relieve brain edema and reduce neurological dysfunction in ICH rats.FERMT1 knockdown could reduce the activity of ICH microglia,the release of inflammatory cytokines and the activity of NLRP3 inflammasome.FERMT1 knockdown inhibits the activation of NF-κB signaling pathway in ICH rats.Part Three Regulation mechanism of FERMT1 on hemin induced inflammation in microglia cellsObjective: This part of the study was an in vitro cell experiment.BV2 microglia cells were transfected with Fermt1 siRNA and treated with hemin to construct an inflammatory response model of microglia cells,so as to study the relationship between FERMT1 and the inflammatory response of microglia cells in vitro and the mechanism of action.Methods: BV2 microglia cells were randomized to one of four groups:Control group,Hemin group,NC siRNA+Hemin group and Fermtl siRNA+Hemin group.The microglia inflammatory response model was constructed after hemin treatment.Before modeling,NC siRNA+Hemin group and Fermtl siRNA+Hemin group were transfected with NC siRNA and Fermt1 siRNA,respectively.The detection indexes were as follows: 1.The expressions of Fermt1,NLRP3,ASC,NF-κB p65 and p-NF-κB p65 in microglia were detected by Western blot.2.The expression levels of inflammatory cytokines IL-1β and IL-18 were detected by ELISA kit.3.Nuclear entry of NF-κB p65 in cells was detected by immunofluorescence.Results:1.FERMT1 protein expression in microglia cells: The protein level of FERMT1 was significantly increased in cells after hemin treatment,knockdown of FERMT1 resulted in a significant decrease in the protein level of FERMT1(P<0.05);2.Activity of NLRP3 inflammasome and expression of inflammatory cytokines in microglia: Hemin treatment dramatically elevated IL-1β and IL-18 levels in cell supernatant,while knockdown of FERMT1 prevented hemin-induced increases in the levels of these inflammatory factors(P<0.05).Western blot results showed that hemin treatment significantly increased the protein levels of NLRP3 and ASC in cells,and knockdown of FERMT1 decreased the protein levels of NLRP3 and ASC(P<0.05).This revealed that knockdown of FERMT1 might inhibit the activity of NLRP3 inflammasome in BV2 microglial cells.3.Expression of NF-κB protein in microglia cells: Hemin treatment increased p-NF-κB p65 levels,while FERMT1 knockdown inhibited p-NF-κB p65 levels(P<0.05).The protein level of total NF-κB p65 did not change significantly(P>0.05).4.Nuclear entry of NF-κB p65 in microglia cytoplasm:Nuclear entry of NF-κB p65 in the cytoplasm of control cells was detected by immunofluorescence staining.Hemin treatment led to an increase in NF-κB p65 entry into the nucleus,and FERMT1 knockdown inhibited it and thereby inhibiting the activation of the NF-κB signaling pathwaySummary: FERMT1 expression in microglia cells increased after hemin treatment.FERMT1 knockdown could inhibit the release of hemin-induced microglial inflammatory cytokines and the activity of NLRP3 inflammasome,which inhibits the inflammatory response.FERMT1 knockdown prevents NF-κB p65 from entering the nucleus and inhibits the activation of the NF-κB signaling pathway.Conclusions: After ICH,the high expression of FERMT1 can cause inflammatory response of microglia cells,and inhibition of FERMT1 expression can reduce nerve injury.The molecular mechanism of this process may be that FERMT1 knockdown reduces the levels of inflammatory factors and the activity of NLRP3 inflammasome by inhibiting the activation of NF-κB signaling pathway.FERMT1 can be used as a potential therapeutic target for intracerebral hemorrhage.