The Role And Mechanism Of NLRC4 In Intracerebral Hemorrhage

Background:Intracerebral hemorrhage is a common disease in neurosurgery with high mortality and high disability.However,current treatment methods are very limited,which imposes a burden on society and families.The NLR family CARD domain-containing protein 4(NLRC4)inflammasome is a complex composed of a variety of proteins.After recognition of the injury-associated pattern,NLRC4 phosphorylates,and NLRC4 inflammasome is activated,causing the maturation and secretion of downstream inflammatory factors and aggravating the inflammation after ICH.However,the role of NLRC4 in aseptic inflammation after cerebral hemorrhage and upstream regulation mechanisms are not known.Objective:To investigate the role of NLRC4 in rat intracerebral hemorrhage and whether NLRC4 is regulated by LRRK2 in the inflammatory response following intracerebral hemorrhage in rats.Methods:? A rat model of intracerebral hemorrhage was established by autologous blood injection into the basal ganglia.(2)Western blot was used to detect the expression of NLRC4 and pNLRC4 at various time points after intracerebral hemorrhage.CO-IP was used to detect the binding of NLRC4 to pro-caspase-1.(3)NLRC4 siRNA was synthesized.After 24 hours of rat intracerebral hemorrhage model,NLRC4 siRNA(1 OD)was injected into the right lateral ventricle and Western blot was used to detect the best interference effecive NLRC4 siRNA.(4)Modified neurological severity score(mNSS)were used to detect the neurological functions ofSham,ICH,ICH+NC,and ICH+NLRC4 siRNA groups;intracerebral edema content was detected by dry and wet weight method;brain histomorphology was detected by HE staining and Nissl staining;pNLRC4,caspase-1,IL-1? and IL-18 were detected by Western blot.The expression of myeloperoxidase MPO was detected by immunofluorescence.(5)Western blot was used to detect the expression of pNLRC4,caspase1,IL-1? and IL-18 in Sham,ICH,ICH+DMSO,ICH+GNE7915 groups.Results:(1)After intracerebral hemorrhage,NLRC4 is phosphorylated in brain tissue,and the phosphorylation level of NLRC4 peaks at 72 h after ICH and then decreases.Furthermore,NLRC4 binds to Pro-caspase1 in brain tissue 72 h after ICH,indicating the activation of NLRC4 inflammasome.(2)After being synthesized,NLRC4 siRNA(sense chain:GCUGAGGCCCACGUAUAAATT antisense strand:UUUAUACGUGGGCCUCAGCTT)has the best interference effect on NLRC4.(3)The interfere with NLRC4 of NLRC4 siRNA improved neurological function scores,reduced brain edema,improved brain histomorphology,and decreased expression of downstream inflammatory factors IL-1?,IL-18 and MPO.(4)After using the LRRK2 inhibitor GNE7915,the expression of pNLRC4,caspase1,IL-1? and IL-18 was decreased,It indicates that NLRC4 is regulated by upstream regulation of LRRK2 in the inflammatory response after intracerebral hemorrhage in rats.Conclusion : NLRC4 may activate NLRC4 inflammasome by phosphorylation to promoting inflammatory response after intracerebral hemorrhage in rats,and this effect may be regulated by LRRK2.