FUNDC1 Inhibits NLRP3-Mediated In Flammatory After Intracerebral Hemorrhage By Promoting Mitophagy

Background:Inflammation is an important factor of secondary brain injury after Intracerebral hemorrhage(ICH).NLR family Pyrin domain-containing 3(NLRP3),a member of the nod-like receptor family,plays an important role in the inflammatory process that occurs in ICH induced injury.Fun14 domain containing 1(FUNDC1)is a protein located in the outer membrane of mitochondria and mitochondria-associated er-membrane(MAM).It is a mitochondrial receptor that can eliminate mitochondrial dysfunction following hypoxia and mitochondrial stress.It recruits and interacts with light chain kinase 3(LC3)and performs mitochondrial autophagy in mitochondria.Mitochondrial autophagy can inhibit the activation of inflammasomes by clearing damaged mitochondria.However,the effect of FundC1 on post-ICH inflammatory response remains unclear.Objective:To investigate whether FUNDC1 can inhibit the activation of NLRP3 inflammasome by regulating mitophagy and reduce the inflammatory response after intracerebral hemorrhage.Methods:(1)The model of intracerebral hemorrhage was established by injecting tail venous blood into the basal ganglia of mice.Western blot was used to detect the expression of FUNDC1 at 6h,12h,24h,48h and 72h after intracerebral hemorrhage.(2)Chemical synthesis of FUNDC1 siRNA interfered with FUNDC1,and Western blot was used to detect the interference effect and screen the best interference effect.(3)Modified neurological deficit score(mNSS)was used to evaluate the neurological deficits of mice in sham group,ICH group,ICH+NC group,ICH+FUNDC1 siRNA group.Brain water content was measured by dry and wet weight method.Blood brain barrier permeability was measured by Evans Blue(EB).HE staining and Nissl staining were used to detect the morphological changes of brain tissue in each group.The expression level of myeloperoxidase(MPO)around hematoma in each group was detected by immunofluorescence staining.(4)Western blot was used to detect the expression of TOM20,COX4I1,NLRP3,Pro-caspase-1,cleaved caspase-1,Pro-IL-1 β,cleaved IL-1 β,Pro-IL-18,and cleaved IL-18 around hematoma in the brain tissues of mice in the sham group,ICH group,ICH+NC group,and FUNDC1 siRNA group.The expression of IL-1 β and IL-18 was detected by ELISA.The autophagosomes were observed by transmission electron microscopy.The expression of TOM20 around hematoma in mice was detected by immunofluorescence staining.(5)Mitophagy inhibitor(mdivi-1)was intraperitoneally injected before ICH model was established.The expressions of TOM20,NLRP3,Pro-caspase-1,cleaved-caspase-1,Pro-IL-1 β,cleaved IL-1 β,Pro-IL-18 and cleaved IL-18 in the brain tissues of mice around the hematoma in the sham group,ICH group,ICH+DMSO group,and ICH+mdivi-1 group were detected by Western blot.(6)The mice model of intracerebral hemorrhage was established after the injection of FUNDC1 adenovirus into the lateral ventricle to increase the expression of FUNDC1.Meanwhile,the intraperitoneal injection of mdivi-1 inhibited the activity of mitophagy.Western blot was used to detect the expression of FUNDC1,TOM20,NLRP3,Pro-caspase-1,cleaved-caspase-1,Pro-IL-1 β,cleaved-IL-1 β,Pro-IL-18,and cleaved IL-18 in brain tissues of mice in ICH group,ICH+mdivi-1 group,ICH+DMSO group,ICH+FUNDC1 AAV group,ICH+AAV(NC)group,ICH+FUNDC1(AAV)+mdivi-1 group;The expression of IL-1 βand IL-18 was detected by ELISA.Results:(1)The expression level of FUNDC1 in brain tissue of mice increased and reached the peak at 12h after ICH.(2)The siRNA with the best interference effect on FUNDC1 was screened out(Sense:UCUUCAGGCAACAGACUUUTT;Antisense;AAAGUCUGUUGCCUGAAG ATT).(3)Interfering with FUNDC1 increased neurological deficit scores,brain water content,Blood Brain Barrier damage,neuronal degeneration and necrosis after intracerebral hemorrhage in mice,and aggravated brain tissue damage.(4)Interference with FUNDC1 increased the expression of TOM20,COX4I1,NLRP3,cleaved-caspase-1,cleaved-IL-1β and cleaved-IL-18 around the hematoma in mice after intracerebral hemorrhage,and decreased the formation of autophagosomes.(5)Inhibition of mitochondrial autophagy increased expression of TOM20,NLRP3,cleaved-caspase-1,cleaved-IL-1β and cleaved-IL-18 around hematoma in mice after intracerebral hemorrhage.(6)Overexpression of FUNDC1 promoted mitophagy,thereby inhibited the activation of NLRP3 inflammasome,and alleviated inflammatory injury after intracerebral hemorrhage.Using mitophagy inhibitor significantly reversed this effect.Conclusion:Interfering FUNDC1 may aggravates intracerebral hemorrhage injury by inhibiting mitophagy to promote the activation of NLRP3 inflammasome.