尼古丁对哮喘大鼠树突状细胞髓样分化因子88及白介素-10、白介素-12表达的影响

Effects of nicotine on the expression of myeloid differentiation factor 88,IL-10 and IL-12 in bone marrow-derived dendritic cells of asthmatic rat

目的通过研究尼古丁对哮喘大鼠模型树突状细胞(DCs)Toll受体转导途径中重要接头蛋白髓样分化因子88(MyD88)表达影响及其与白介素-10(IL-10)、白介素-12(IL-12)的关系,从细胞水平探讨尼古丁加重哮喘的免疫学机制。方法制备哮喘大鼠模型,培养骨髓来源的DCs及脾脏来源的淋巴细胞,实验随机分为4组:对照组、哮喘组、尼古丁组和哮喘加尼古丁组,其中对照组和哮喘组大鼠DCs在培养5 d后在培养液中加入一定体积的PBS继续培养72 h,尼古丁组和哮喘加尼古丁组大鼠DCs在培养5 d后加入质量浓度为400μg/ml尼古丁作用72 h。收集各组细胞及细胞上清,采用酶联免疫吸附双抗体夹心法(ELISA)测定各组IL-10和IL-12的浓度。采用免疫印迹(Western blot)法测定细胞内MyD88的含量。采用MTT法测定DCs刺激同种异体淋巴细胞增殖的能力。结果①哮喘加尼古丁组和哮喘组大鼠DCs IL-10的表达均高于对照组,IL-12的表达均低于对照组,差异均具有统计学意义(P<0.01)。哮喘加尼古丁组大鼠DCs IL-10和IL-12的表达均低于哮喘组大鼠,差异均具有统计学意义(P<0.05)。②尼古丁组、哮喘组和哮喘加尼古丁组大鼠DCs MyD88的表达低于对照组,差异均具有统计学意义(P<0.05)。哮喘加尼古丁组大鼠DCs MyD88的表达低于哮喘组,差异均具有统计学意义(P<0.01)。③哮喘组大鼠DCs刺激同种异体淋巴细胞增殖的能力与对照组相比明显增强,差异具有统计学意义(P<0.01)。哮喘加尼古丁组大鼠DCs刺激同种异体淋巴细胞增殖的能力与哮喘组和对照组相比明显减弱,差异均具有统计学意义(P<0.05)。④DCs MyD88的表达与IL-12呈显著的正相关(r=0.644,P<0.01),与IL-10的表达无明显相关性。IL-12与IL-10的表达呈显著的负相关(r=-0.388,P<0.05)。DCs中IL-10的表达与淋巴细胞增殖活性呈显著的正相关(r=0.524,P<0.01);IL-12的表达与淋巴细胞增殖活性无明显相关性。结论尼古丁可以抑制哮喘大鼠DCs MyD88以及IL-10、IL-12的表达,并降低DCs刺激淋巴细胞的增殖能力。这可能是吸烟加重哮喘的免疫学机制之一。其中MyD88依赖的Toll受体转导途径可能在尼古丁下调IL-12的表达中发挥了一定作用。

To investigate the mechanism of nicotine aggravating asthma on cellular level,we study the effect of nicotine on the expression of myeloid differentiation factor 88（MyD88） and interleukin-10（IL-10）,interleukin-12（IL-12） in bone marrow-derived dendritic cells（DCs） of asthmatic rat.The rats are sensitized and the bone marrow-derived DCs were cultured,which were then randomly divided into 4 groups as follows： control group,asthma group,nicotine group,and asthma ＋ nicotine group.In both control and asthma groups,the DCs were cultured for 5 days,then added with a certain volume of PBS for 72 h,while in both nicotine and Asthma ＋ nicotine groups,the DCs were cultured for 5 days and then added with liquid nicotine（400 μg/ml） for 72 h.Then we collected cells and cell supernatant of all 4 groups.Simultaneously,rat spleen lymphocytes and bone marrow derived DCs were cultured together.ELISA and Western blot showed the expression levels of IL-10,IL-12 and MyD88 decreased significantly in asthma ＋ nicotine group,while MTT showed the stimulating activity of DCs on allogeneic lymphocytes decreased significantly in asthma ＋ nicotine group.In addition,the expression level of IL-12 was positively correlated with the expression of MyD88,but negatively correlated with the expression of IL-10.Furthermore,the stimulating activity of DCs on allogeneic lymphocytes was positively correlated with the expression of IL-10.The result suggested that nicotine can inhibit DCs to express IL-10,IL-12,MyD88 and reduce the stimulating activity of DCs on allogeneic lymphocytes in the asthma rats,which might be the immunological mechanisms of smoking aggravating asthma.The further study shown the effects on IL-12 may caused by the expression of MyD88 protein,which mediated by MyD88-dependent toll receptor signaling pathways.