胱天蛋白酶募集域蛋白9与小鼠缺血性脑损伤炎症反应的相关性研究

Correlation between caspase recruitment domain protein 9 and infl ammatory responses in ischemic brain injury in mice

目的研究缺血性脑损伤小鼠脑组织中胱天蛋白酶募集域蛋白9(caspase recruitment domain protein 9,CARD9)与炎症反应的关系。方法取24只SPF级BALB/c雄性小鼠随机(随机数字法)分成4组,即假手术组、缺血3 h组、缺血6 h组、缺血12 h组,每组6只。缺血组小鼠采用线栓栓塞法构建永久性大脑中动脉阻塞(permanant middle cerebral artery occlusion,pMCAO)模型,各组于术后预定时间点处死小鼠。通过Western blot检测患侧脑组织中CARD9水平和NF-κB的活化,RT-PCR和ELISA分别检测患侧脑组织中炎症因子TNF-α、IL-lβ、IL-6 mRNA及蛋白表达量。采用SPSS 21.0软件进行统计分析,每两组间计量资料比较采用独立样本t检验,采用Pearson相关分析比较CARD9与炎症因子之间的相关性。结果与假手术组相比,缺血3 h、6 h和12 h组脑组织CARD9表达均增加[(0.325±0.011)vs(0.462±0.019),P=0.036;(0.735±0.036),P=0.003;(0.903±0.024),P=0.001];与假手术组相比,缺血3 h、6 h和12 h组缺血脑组织NF-κB的活化均升高[(0.227±0.016)vs(0.316±0.017),P=0.041;(0.445±0.021),P=0.016;(0.671±0.039),P=0.008]。与假手术组相比,缺血3 h、6 h和12 h组脑组织TNF-ɑ表达均增加[(0.53±0.06)vs(1.06±0.10),P=0.009;(1.47±0.15),P=0.004;(2.78±0.18),P=0.001];与假手术组相比,缺血3 h、6 h和12 h组脑组织IL-lβ表达均增加[(0.55±0.07)vs(1.01±0.11),P=0.009;(2.13±0.16),P=0.003;(3.09±0.18),P=0.001];与假手术组相比,缺血3 h、6 h和12 h组脑组织IL-6表达均增加[(1.99±0.18)vs(4.10±0.41),P=0.006;(8.54±0.84),P=0.002;(11.56±0.96),P=0.001]。Pearson相关分析显示CARD9与NF-κB活性、TNF-α、IL-1β、IL-6均存在正相关(r=0.894,P=0.001;r=0.747,P=0.008;r=0.810,P=0.001,r=0.773,P=0.007)。结论小鼠缺血性脑损伤早期脑组织中CARD9与炎症反应存在正相关。

Objective To investigate the relationship between caspase recruitment domain protein 9(CARD9)level and infl ammatory response in cerebral tissue of ischemic brain injury mice.Methods Totally 24 SPF BALB/c male mice were randomly(random number)divided into 4 groups:sham operated group,ischemia 3 h group,ischemia 6 h group,and ischemia 12 h group,6 mice in each group.The permanant middle cerebral artery occlusion(pMCAO)model in the ischemia groups was established by using line embolism to block blood fl ow.Mice in each group were sacrifi ced at the predetermined time point after operation.CARD9 and p-p65NF-κB levels were detected by Western blot,and the inflammatory factors mRNA and protein including TNF-ɑ,IL-lβand IL-6 were detected by RT-PCR and ELISA,respectively.The data were analyzed by SPSS 21.0 software,the comparison of measurement data between each two groups was analyzed by independent sample t test,and the correlations between CARD9 and infl ammatory factors were analyzed by Pearson analysis.Results Compared with the sham operated group,the CARD9 levels in the ischemia 3 h,6 h and 12 h groups were increased signifi cantly[(0.325±0.011)vs.(0.462±0.019),P=0.036;(0.735±0.036),P=0.003;(0.903±0.024),P=0.001],the p-p65NF-κB levels in the ischemia 3 h,6 h and 12 h groups were increased signifi cantly[(0.227±0.016)vs.(0.316±0.017),P=0.041;(0.445±0.021),P=0.016;(0.671±0.039),P=0.008],the TNF-ɑlevels in the ischemia 3 h,6 h and 12 h groups were signifi cantly increased[(0.53±0.06)vs.(1.06±0.10),P=0.009;(1.47±0.15),P=0.004;(2.78±0.18),P=0.001],the IL-lβlevels in the ischemia 3 h,6 h and 12 h groups were signifi cantly increased[(0.55±0.07)vs.(1.01±0.11),P=0.009;(2.13±0.16),P=0.003;(3.09±0.18),P=0.001],and the IL-6 levels in the ischemia 3 h,6 h and 12 h groups were signifi cantly increased[(1.99±0.18)vs.(4.10±0.41),P=0.006;(8.54±0.84),P=0.002;(11.56±0.96),P=0.001].Pearson analysis showed that CARD9 was positively correlated with the p-p65NF-κB and TNF-ɑ,IL-lβ,IL-6(r=0.894,P=0.001;r=0.747,P=0.008;r=0.810,P=0.001;r=0.773,P=0.007).Conclusions A positive correlation exists between CARD9 and infl ammatory responses in the early stage of ischemic brain injury in mice.