肌苷单磷酸脱氢酶在卵清蛋白诱导过敏性哮喘中的作用

The Role of Inosine Monophosphate Dehydrogenase in Ovalbumin-induced Allergic Asthma

目的探讨肌苷单磷酸脱氢酶(inosine monophosphate dehydrogenase,IMPDH)在卵清蛋白(ovalbumin,OVA)诱导过敏性哮喘发生发展中的作用。方法将36只BALB/c雌性小鼠随机分为健康对照组、OVA哮喘组和IMPDH抑制剂(利巴韦林)干预组,每组12只。收集每只小鼠的肺泡灌洗液(bronchoalveolar lavage fluid,BALF)、肺组织、外周血及脾脏,采用苏木精-伊红染色分析肺组织病理改变,免疫组织化学法检测肺组织内CD3的表达情况,血球分析仪检测BALF和外周血中白细胞总数及分类计数,qRT-PCR和Western blot分别检测肺组织IMPDH mRNA和蛋白表达水平,免疫荧光法检测肺组织IMPDH蛋白定位和表达情况,流式细胞术检测外周血和脾脏淋巴细胞亚群及其细胞内IMPDH蛋白表达水平。结果与健康对照组相比,OVA哮喘组小鼠肺内小气道周围及肺泡间隔大量炎症细胞浸润,BALF和外周血中白细胞总数均显著增加,以中性粒细胞、嗜酸性粒细胞和单核细胞的数量增多为主(均P<0.05),且肺组织内IMDPH蛋白水平显著升高(P<0.05),炎症细胞胞质内明显增多的IMPDH蛋白呈分散模式。给予利巴韦林干预后哮喘小鼠肺部炎症明显减轻,尤以BALF中嗜酸性粒细胞和中性粒细胞显著减少为特征(均P<0.05),同时肺部IMPDH蛋白水平降低(P<0.05),炎症细胞胞质内IMPDH表达减少,但细胞核内出现较多IMPDH丝状结构。此外,OVA哮喘组小鼠脾脏中B细胞占比明显增多(P<0.05),但利巴韦林干预后哮喘小鼠脾脏中B细胞占比显著减少,而NK、CD3^(+)T和CD8^(+)T细胞占比较OVA哮喘组明显增高(均P<0.05)。同时利巴韦林干预组脾脏NK细胞内IMPDH蛋白表达水平较健康对照组升高,而CD8^(+)T细胞内IMPDH蛋白表达水平降低。结论抑制IMPDH可有效缓解以啫酸性粒细胞为主的气道及全身炎症反应,同时还能降低脾脏B细胞免疫应答,增强NK及CD8^(+)T细胞等杀伤细胞的免疫应答效应,其潜在的机制可能与炎症细胞内IMPDH表达量及其形成的丝状结构有关。

Objective To investigate the role of inosine monophosphate dehydrogenase(IMPDH)in the development of ovalbumin(OVA)-induced allergic asthma.Methods Thirty-six BALB/c female mice were randomly divided into a healthy control group,an OVA asthma group,and an IMPDH inhibitor(ribavirin)intervention group,with 12 mice in each group.The bronchoalveolar lavage fluid(BALF),lung,peripheral blood and spleen were collected from each mouse.HE staining was used to analyze the pathological changes in the lung tissue,and immunohistochemistry was used to detect the expression of CD3 in the lung tissue.A hemocyte analyzer was used to determine the total number and classified counts of leukocytes in the BALF and peripheral blood.RT-PCR and Western blotting were used to detect IMPDH mRNA and protein levels,respectively,in the lung tissue.Immunofluorescence was used to detect IMPDH protein localization and expression in lung tissue,and flow cytometry was used to analyze lymphocyte subpopulations and their intracellular IMPDH protein levels in peripheral blood and spleen tissue.Results Compared with those in the healthy control group,the number of inflammatory cells around small airways and in alveolar spaces in the lungs of the OVA asthma group was greater,and the total number of leukocytes in both the BALF and peripheral blood was significantly higher,with significant increases in the numbers of neutrophils,eosinophils,and monocytes(P<0.05).There was also a significant increase in the level of IMPDH protein in lung tissue(P<0.05),and immunostaining revealed increased expression of IMPDH protein in the cytoplasm of inflammatory cells in a dispersed pattern.Subsequently,lung inflammation was significantly reduced in OVA-induced asthmatic mice after ribavirin intervention,as characterized specifically by a significant reduction in eosinophils and neutrophils in the BALF(P<0.05)and a decrease in the level of IMPDH protein in the lungs(P<0.05).Furthermore,there was also a decrease in the expression of IMPDH in the cytoplasm of inflammatory cells,whereas an increased number of IMPDH-positive filamentous structures were observed in the nuclei of inflammatory cells.Additionally,the percentage of B cells in the spleens of the mice in the OVA asthma group was significantly greater than that in the control group(P<0.05).After ribavirin intervention,the percentage of B cells in the spleens of asthmatic mice significantly decreased,whereas the percentages of NK,CD3~+and CD8~+T cells significantly increased(P<0.05).Moreover,the IMPDH protein levels in NK cells increased,whereas the IMPDH protein levels in CD8~+T cells decreased in the spleen.Conclusion Inhibition of IMPDH can effectively alleviate airway and systemic inflammation dominated by eosinophils.Moreover,it can also reduce the immune response of B cells and enhance the immune response effect of killer cells,such as NK and CD8~+T cells,in the spleen.The underlying mechanism may be related to the level of IMPDH expression in inflammatory cells and the formation of filamentous structures.