The Role Of CARD9-mediated NF-?B Signaling Pathway In Ischemic Brain Injury

Aim:To establish a model of ischemic brain injury in mice,and to observe the relevance between trend variability of caspase recruitment domain protein 9(CARD9)and nuclear factor-?B(NF-?B)and the effect on expression of inflammatory cytokines in cerebral hemisphere after ischemic brain injury.To investigate the the effect of siRNA silencing CARD9 expression on ischemic brain injury,NF-?B and inflammatory cytokines,and to verify its mechanism.Methods:SPF male Balb/c mice were randomly divided into sham operation group(group A),ischemia 3h group(group B),ischemia 6h group(group C),and ischemia12 h group(group D).In the ischemic group,permanent middle cerebral artery occlusion(pMCAO)model was established in mice with an intraluminal silicon-coated filament.The brain tissue in each group were taken at 3h,6h and 12 h after operation,and the Western blot and RT-PCR were used to analyze the expression of CARD9,NF-?Bp65,p-NF-?Bp65 and the mRNA of inflammatory cytokines.The next step was to divide mice into sham operation group,ischemia 12 h group and siCARD9 group,and to verify the regulation of CARD9 siRNA technology to eurological symptom score,degree of cerebral edema,NF-?B signaling pathway and inflammatory cytokines TNF-?,IL-l?,IL-6.Results:Western blot results showed that the expression of CARD9 and NF-?B increased with the time of ischemia,and the expression differences between the ischemic group and the sham group were statistically significant(P<0.05),suggesting that the expression of CARD9 and NF-?B may be related to ischemic brain injury.RT-PCR results showed that the expression of inflammatory cytokines TNF-?,IL-l?,IL-6 mRNA in the ischemic group increased with time,compared with the sham operation group(P<0.01);Correlation analysis showed that CARD9 expression and the NF-?B activation levels,inflammatory cytokines TNF-?,IL-6,IL-l? mRNA expression were positively correlated.After the pretreatment with CARD9 siRNA,the expression of CARD9 mRNA was significantly decreased in siCARD9 group compared with ischemia 12 h group(P<0.05),the neurological symptom scores and brain tissue water content were significantly lower in the siCARD9 group(P<0.05),The expression levels of CARD9 and NF-?B and inflammatory cytokines TNF-?,IL-l?,IL-6 mRNA were also decreased(P<0.05).Conclusion:CARD9 may mediate the NF-?B signaling pathway to regulate the inflammatory response and promote the formation of ischemic brain injury.