CARD9基因敲除对C57BL/6小鼠激素抵抗型哮喘模型气道炎症的影响

Effects of caspase recruitment domain protein 9 gene knockout on airway inflammation in C57BL/6 mice with steroid resistant asthma

目的探讨胱天蛋白酶募集域蛋白9(CARD9)在C57BL/6小鼠激素抵抗型哮喘气道损伤和炎症中的作用。方法采用随机数字表法,将6~8周龄无特定病原体(SPF)级C57BL/6雌性小鼠分为A组(对照组)、B组(模型组)和C组(地塞米松治疗组),每组6只,B组及C组利用卵清白蛋白(OVA)/完全弗氏佐剂(CFA)腹部皮下注射,OVA雾化激发构建小鼠模型,C组每次致敏前30 min给予地塞米松,末次激发24 h后检测其病理变化及支气管肺泡灌洗液(BALF)细胞计数,进行肺组织炎症浸润情况评分,Western blot检测造模前后CARD9蛋白的变化;然后将雌性6~8周龄C57BL/6野生型小鼠12只和CARD9基因敲除型小鼠12只分为4组,每组6只,其中D组为野生型对照组,E组为野生型模型组,F组为CARD9基因敲除对照组,G组为CARD9基因敲除模型组,造模如上述对照组和模型组。苏木精-伊红染色观察肺组织病理变化,ELISA法检测BALF中白细胞介素(IL)-4、IL-5和IL-17,RT-PCR法检测CXC趋化因子配体10(CXCL-10)和IL-17 mRNA水平。结果B组炎症浸润评分[(3.33±0.82)比(0.67±0.52)分]和BALF总细胞计数[(10.13±4.83)×10^(5)个/ml比(3.76±0.84)×10^(5)个/ml]均高于A组(均P<0.05);C组炎症浸润评分[(2.83±0.75)分]和BALF总细胞计数[(9.80±3.19)×10^(5)个/ml]与B组差异无统计学意义(P>0.05);B组CARD9蛋白表达高于A组(0.245±0.090比0.047±0.014,P=0.004);肺组织病理结果显示,与E组及F组相比,G组的肺组织炎症细胞浸润及组织结构破坏程度加重。与E组及F组相比,G组炎症细胞总数、中性粒细胞数量和嗜酸粒细胞数量均升高(均P<0.05);IL-4、IL-5及IL-17表达水平均升高(均P<0.05);支气管肺组织中IL-17及CXCL-10 mRNA表达水平亦均升高(均P<0.05)。结论CARD9基因的缺失可能通过升高IL-17及CXCL-10等中性粒细胞趋化因子,增加中性粒细胞的浸润,从而加重C57BL/6小鼠激素抵抗型哮喘。

Objective To investigate the role of caspase recruitment domain protein 9(CARD9)in airway injury and inflammation of steroid resistant asthma in C57BL/6 mice.Methods C57BL/6 mice were divided into A group(control group),B group(model group)and C group(dexamethasone treatment group),with 6 mouse in each group using random number table.The mouse asthma model was established in B and C group by subcutaneous injection of ovalbumin(OVA)/complete Freund adjuvant(CFA)in the abdomen and OVA aerosol challenge,the pathological change and cell count in broncho alveolar lavage fluid(BALF)were detected in order to confirm the model as steroid resistant asthma,and the lung tissue inflammatory infiltration was scored.Western blot was used to detect the changes of CARD9 protein between the group A and B;then wild-type and CARD9 knockout mice were divided into D group(wild-type control group),E group(wild-type model group),F group(CARD9 knockout control group)and G group(CARD9 knockout model group),the following indicators were observed and compared after establishing steroid resistant asthma model separately:HE staining was used to observe the pathological changes of lung tissue,ELISA was used to detect the protein levels of interleukin-4(IL-4),interleukin-5(IL-5)and interleukin-17(IL-17)in BALF,and RT-PCR was used to detect the mRNA levels of CXC motif chemokine ligand-10(CXCL-10)and IL-17 in lung.Results The inflammatory score(3.33±0.82 vs 0.67±0.52)and BALF total cell count[(10.13±4.83)×10^(5)/ml vs(3.76±0.84)×10^(5)/ml]in B group were higher than those in the A group with statistical significance(P<0.05).There was no significant difference between group C and group B in inflammatory infiltration score(2.83±0.75 vs 3.33±0.82)and BALF total cell count[(9.80±3.19)×10^(5)/ml vs(10.13±4.83)×10^(5)/ml](P>0.05).Moreover the protein level of CARD9 was increased in the B group than A group(0.245±0.090 vs 0.047±0.014,P=0.004).Compared to E group and F group,more obviously inflammatory cells,neutrophils,eosinophils infiltration and tissue injury were observed in G group(P<0.05),so did the expression of IL-4(P<0.05),IL-5 and IL-17.Meanwhile the mRNA expression levels of IL-17 and CXCL-10 also increased in lung tissue(P<0.05)of G group.Conclusion CARD9 gene deletion may aggravate the steroid resistant of asthma by increasing neutrophil chemokines,such as IL-17 and CXCL-10,therefore increasing infiltration of neutrophils in C57BL/6 mice asthma model.