维生素D治疗小鼠肺烟曲霉感染的机制研究

Study on inflammatory mechanism of vitamin D in the treatment of aspergillus fumigatus lung disease

摘要:

目的 通过检测维生素D（VitD）在小鼠肺感染烟曲霉后，对其肺组织和相关炎症因子的影响，以探究 VitD在烟曲霉肺部感染治疗中的作用机制，为临床治疗提供参考依据。方法 选择一组实验小鼠喂食含VitD的饲料（VitD+组），另一组小鼠喂食不含VitD的饲料（VitD-组），每组10只。喂养7?d后，用一定量的烟曲霉活化孢子感染小鼠。处死小鼠，取其肺组织和血清：用10%中性甲醛浸泡肺组织，行HE染色、革兰染色、糖原染色，观察肺实变情况；免疫组织化学法观察肺组织炎症因子IL-1β、IL-6和TNF-α表达；同时用酶联免疫吸附试验检测小鼠血清中炎症因子IL-1β、IL-6和TNF-α的含量；健康小鼠10只，喂食饲料，7?d后取肺巨噬细胞在体外与烟曲霉孢子共培养于6孔板，其中3孔培养基中加入VitD，另3孔培养基不加VitD，收集共培养1和3?h后的肺巨噬细胞，荧光显微镜下观察肺巨噬细胞吞噬烟曲霉孢子的变化，并用Trizol提取总RNA，逆转录聚合酶链反应（RT-PCR）检测IL-8和NF-κB的表达，同时采用Western blotting检测IL-8和NF-κB的表达。结果 活化的烟曲霉孢子感染小鼠后，VitD+组小鼠较VitD-组小鼠肺部真菌量减少、肺实变轻、炎症因子IL-1β、IL-6和TNF-α表达低（P?<0.05），相应血清中的炎症因子IL-1β、IL-6和TNF-α含量减少（P?<0.05）；肺巨噬细胞与烟曲霉孢子体外共培养后，VitD+组小鼠肺巨噬细胞吞噬烟曲霉孢子可在一个合理水平而不易引起自噬死亡；同时RT-PCR和Western blotting结果显示VitD+组小鼠的IL-8和NF-κB表达低于VitD-组小鼠（P?<0.05）。结论 烟曲霉感染小鼠后，VitD可通过抑制炎症因子的表达调控巨噬细胞抵抗烟曲霉的感染，从而提高小鼠的生存率和生存质量，在烟曲霉肺部感染治疗中起重要作用。

Abstract:

Objective To explore the mechanisms of vitamin D treatment in aspergillus fumigatus infection of lung by detecting the lung tissue and inflammatory cytokines in mice,and to provide a reference basis for clinical treatment. Methods A group of mice fed with vitamin D feed, another group of mice fed the diet without vitamin D. After seven days, the mice infected a certain amount of activing aspergillus fumigatus were killed, the serum and lung tissue were collected. The lung tissue were deal with 10% neutral formalin for pathological examination, including HE staining, Gram staining and glycogen staining to observe the changes of lung. Immunohistochemistry was performed to observe the expression of inflammatory cytokines IL-1β, IL-6 and TNF-α in lung tissue. ELISA kit was used to detect inflammatory factors IL-1β, IL-6 and TNF-α of the serum. In addition, the spores of aspergillus fumigatus and lung macrophages separated from lungs of health mice were co-cultured. Vitamin D was added to one of two medium. After a period of time, the pulmonary macrophages after co-culture were collected to observe the changes of macrophage cells by fluorescence microscope. Trizol was used to extract total RNA, and the the expressions of IL-8 and NF-κB were detected by RT-PCR. At the same time, the expression of IL-8 and NF-κB were analyzed by Western blotting. Results After mice infected by the activation of aspergillus fumigatus, compared with vitamin D deficiency, the pathological results showed that the content of pulmonary fungus and the number of pulmonary fungus of vitamin D group was less, IL-6, IL-1β and TNF-α were less in the lung tissue, and the difference was statistically significant (P?