重组白细胞介素-35对支气管哮喘模型小鼠T淋巴细胞亚群及细胞因子的影响*

Effect of recombinant IL-35 on T lymphocyte subsets and cytokines in mice with bronchial asthma*

摘要:

目的 探究重组白细胞介素-35（rIL-35）对支气管哮喘模型小鼠T淋巴细胞亚群和细胞因子的影 响及其作用机制。方法 100只SPF级雄性BALB/c小鼠随机分为对照组、哮喘组、rIL-35低剂量组、rIL-35中 剂量组和rIL-35高剂量组，每组20只。哮喘组和rIL-35各剂量组小鼠采用卵白蛋白（OVA）复制支气管哮喘模 型，对照组小鼠则以生理盐水代替OVA进行处理。rIL-35各剂量组小鼠在OVA激发结束后腹腔注射不同剂量 rIL-35，连续3 d，对照组和哮喘组小鼠注射等剂量的生理盐水。比较各组小鼠的气道反应性、肺泡灌洗液 （BALF）中总细胞、淋巴细胞、中性粒细胞和嗜酸性粒细胞的数量；采用流式细胞仪检测各组小鼠BALF中T淋 巴细胞亚群的水平；采用酶联免疫吸附实验（ELISA）检测小鼠BALF中IgE、白细胞介素-2（IL-2）、干扰素-γ （IFN-γ）、白细胞介素-4（IL-4）、白细胞介素-5（IL-5）、白细胞介素-17（IL-17）、白细胞介素-22（IL-22）、 白细胞介素-10（IL-10）和转化生长因子-β（TGF-β）水平。结果 对照组小鼠未见明显异常状态。哮喘组小 鼠致敏后活动减少，雾化激发时出现不同程度的咳嗽、呼吸急促、烦躁不安等症状。rIL-35低、中和高剂量组小 鼠雾化激发时上述反应症状减轻。不同组激发时肺阻力（RL） 和肺顺应性（Cdyn） 比较，差异有统计学意义 （P <0.05），不同剂量乙酰甲胆碱激发时RL和Cdyn比较，差异有统计学意义（P <0.05）。乙酰甲胆碱与rIL-35存 在交互作用，随着乙酰甲胆碱剂量增大，rIL-35剂量减小，激发时RL越大（F =202.320，P =0.000），Cdyn越小（F = 5.623，P =0.000）。与对照组比较，哮喘组小鼠BALF中的细胞总数、淋巴细胞、中性粒细胞和嗜酸性粒细胞等炎 症细胞的数量均增加（P <0.05），Th2、Th17、Th17/Treg、IgE、IL-4、IL-5、IL-17、IL-22 水平升高（P < 0.05），而Th1、Treg、Th1/Th2、IL-2、IFN-γ、IL-10 和TGF-β水平降低（P <0.05）。与哮喘组小鼠比较， rIL-35低、中和高剂量组小鼠BALF中的细胞总数、淋巴细胞、中性粒细胞和嗜酸性粒细胞等炎症细胞的数量降 低（P <0.05），均呈随rIL-35剂量增加而减少的趋势，Th2、Th17、Th17/Treg、IgE、IL-4、IL-5、IL-17、IL- 22水平降低（P <0.05），均呈随rIL-35剂量增加而降低的趋势，而Th1、Treg、Th1/Th2、IL-2、IFN-γ、IL-10 和TGF-β水平升高（P <0.05），且呈随rIL-35蛋白剂量增加而升高的趋势。结论 rIL-35可通过调控Th1/Th2 和Th17/Treg细胞的失衡及炎症细胞因子的分泌，抑制支气管哮喘小鼠体内的炎症反应。

Abstract:

Objective To investigate the effects of recombinant interleukin-35 (rIL-35) on T lymphocyte subsets and cytokines in bronchial asthma mice and its possible mechanism. Methods A toal of 100 male BALB/c mice were randomly divided into control group, asthma group and rIL-35 group. In the asthma group and the rIL-35 group, the bronchial asthma model was established by ovalbumin (OVA), and the control group was treated with normal saline instead of OVA, 20 mice in per group. The rIL-35 group were intraperitoneally injected with rIL-35 protein at the end of OVA treatment for 3 consecutive days, while the control group and asthma group were injected with the same dose of normal saline. The total cells, lymphocytes, neutrophils and eosinophils in bronchoalveolar lavage fluid (BALF), and the airway reactivity from each group were compared. The levels of T lymphocyte subsets in each group were detected by flow cytometry. The IgE, interleukin-2 (IL-2), interferon-γ (IFN-γ), interleukin-4 (IL- 4), interleukin-5 (IL-5), interleukin-17 (IL-17), interleukin-22 (IL-22), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) in BALF were measured with enzyme linked immunosorbent assay. Results The mice in the control group were in normal state. The activities in the asthma group had reduced, and symptoms, such as coughing, shortness of breath, and irritability, emerged. The above-mentioned symptoms were alleviated in the rIL- 35 low, medium, and high dose groups. There were statistically significant differences in lung resistance (RL) and dynamiccompliance (Cdyn) in different groups (F = 986.310 and 31.160, P = 0.000). The differences in RL and Cdyn when stimulated with different doses of methacholine were statistically significant (F = 2765.867 and 207.625, P = 0.000). There is an interaction between methacholine and rIL-35. As the dose of methacholine increases, the dose of rIL-35 decreases. The greater the RL during challenge (F = 202.320, P = 0.000), the smaller the Cdyn (F = 5.623, P = 0.000). Compared with the control group, the total number of cells, lymphocytes, neutrophils, eosinophils, and other inflammatory cells in the BALF of the asthma group increased (P < 0.05), Th2, Th17, Th17/Treg, IgE, IL- 4, IL-5, IL-17, IL-22 increased (P < 0.05), while Th1, Treg, Th1/Th2, IL-2, IFN-γ, IL-10, and TGF-β decreased (P < 0.05). Compared with the asthma group, the total number of cells, lymphocytes, neutrophils, eosinophils, and other inflammatory cells in the BALF of in the rIL-35 low, medium and high dose groups decreased (P < 0.05), in a way of rIL-35 protein dose-dependent decreased tendency; Th2, Th17, Th17/Treg, IgE, IL-4, IL-5, IL-17, IL-22 decreased (P < 0.05), in a way of rIL-35 protein dose-dependent decreased tendency; while Th1, Treg, Th1/Th2, IL-2, IFN-γ, IL-10, and TGF-β increased (P < 0.05), in a way of rIL-35 protein dose-dependent increased tendency. Conclusion RIL-35 inhibits the inflammatory response in bronchial asthma mice by regulating the imbalance of Th1/Th2, Th17/ Treg cells and the secretion of inflammatory cytokines.