The Role And Mechanism Of Tyrosine Kinase Fyn In Inflammatory And Apoptosis After Intracerebral Hemorrhage In Rats

Background: Apoptosis and inflammatory are important factors causing secondary brain injury after intracerebral hemorrhage(ICH).Tyrosine Kinase Fyn-mediated apoptosis and inflammatory are involved in experimental models of various neurological diseases,such as Parkinson's,Alzheimer,depression,etc.,but its role in ICH is still unknown.Objective: To investigate the role of tyrosine kinase Fyn in inflammatory response and apoptosis after ICH in rats and its potential mechanism.Methods:(1)An experimental method of ICH was established by injecting 50 ?l of autologous blood into the basal ganglia.Western blot was used to detect the expression time distribution of Tyrosine Kinase Fyn at 3h,6h,12 h,24h,48 h and 72 h after ICH in rats.(2)Fyn expression was inhibited by injecting small interfering fragments(si-RNA)into the lateral ventricle at 24 h before surgery.And then the effects of Fyn-specific si-RNA on Modified Neurological Severity Score(mNss),cerebral edema(brain fluid content),morphological staining of brain tissue(H&E staining),and blood Effect of brain barrier permeability(Evans Blue)were observed.(3)Western blot was used to detect inflammatory related proteins of TNF-?,NF-?B,Caspase1,IL-1? and IL-18 in brain tissue.Immunofluorescence was used to detect the expression of myeloperoxidase(MPO).(4)Western blot was used to detect the protein levels of Pro-caspase3,Caspase3,AIF,Cyt.c,Bax and anti-apoptosis related protein Bcl-2 in the brain tissues of ICH.TUNEL method was used to detect the apoptosis of brain tissues around the hematoma.(5)Co-immunoprecipitation(CO-IP)was used to detect the interaction between Fyn and Drp1.Intraperitoneal injection of Mdivi-1 inhibited the expression of Drp1;AAV transfection over-expressed Fyn 5 weeks before surgery;P-Fyn,Fyn,P-Drp1,Drp1 Pro-caspase3,Caspase3,AIF,Cyt.c,Bax and Bcl-2 were detected by Western blot.Results:(1)Compared with the Sham group,Fyn started to increase at 3 hours after ICH in rats and reached the highest expression peak at 24 hours.(2)Compared with the ICH group,inhibiting Fyn can reduce brain edema,improve neurological function score,reduce blood-brain barrier permeability,and improve brain tissue H&E staining results.(3)Compared with the NC group,the inhibition of Fyn on the inflammation-related proteins TNF-?,NF-?B,Caspase1,IL-1? and IL-18 has no statistical significance.And MPO staining results showed that inhibition of Fyn had little effect on neutrophils infiltrating around the hematoma.(4)Compared with the NC group,inhibiting Fyn can reduce the apoptosis-related proteins Caspase3,AIF,Cyt.c,Bax,and increase the protein levels of anti-apoptosis-related protein Bcl-2;TUNEL shows that inhibiting Fyn can reduce Apoptosis of brain tissue around hematoma.(5)Compared with the NC group,CO-IP showed the interaction between Fyn and Drp1,and phosphorylation activated the Drp1 Serine 616 site.Inhibition of Fyn could reduce this interaction and reduce Ser616 phosphorylation.(6)Compared with NC group,over-expressing Fyn can increase P-Fyn,Fyn,P-Drp1 and apoptosis-related proteins Caspase3,AIF,Cyt.c,Bax and reduce anti-apoptosis related proteins bcl-2 protein levels,and the use of Mdivi-1 inhibitors can reverse the increase in apoptosis-related proteins Caspase3,AIF,Cyt.c,Bax and anti-apoptosis-related protein Bcl-2 caused by Fyn overexpression Reduced without affecting P-Fyn and Fyn expression.Conclusion: Fyn expression is increased after ICH and activates the Drp1 signaling pathway through interaction with Drp1 and phosphorylation of the Drp1 Serine 616 site,aggravating apoptosis after intracerebral hemorrhage in rats.However,Fyn had no statistically significant effect on the inflammatory response in rats after intracerebral hemorrhage.