雷帕霉素对肺间质纤维化大鼠辅助性T细胞亚群平衡变化的影响

Effect of rapamycin on the balance of T helper cell subsets in rats with pulmonary fibrosis

目的：探讨雷帕霉素对肺纤维化过程中辅助性T细胞（Th细胞）平衡变化的影响。方法将15只SD大鼠按随机数字表法分为对照组、模型组、雷帕霉素组，每组5只。经气管内注入博莱霉素5 mg/kg复制大鼠肺纤维化模型，对照组气管内注入生理盐水1.25 mL/kg；雷帕霉素组于制模后3 d灌胃雷帕霉素1 mg·kg^-1·d^-1，连续10 d。制模后28 d处死动物，取肺组织进行病理学观察，用Ashcroft评分评估肺纤维化程度；免疫组化法检测肺组织中γ-干扰素（IFN-γ）、白细胞介素（IL-4、IL-17）的表达；流式细胞仪检测外周血CD4＋和CD8＋调节性T细胞的比例。结果雷帕霉素组肺纤维化程度较模型组明显减轻，Ashcroft评分明显降低（分：2.92±0.64比5.76±1.76，F＝16.276，P＝0.080）。肺组织IL-4及IL-17表达量（A值）在模型组最高（4789.0±1014.6、19139.0±2433.3），其次是雷帕霉素组（3547.0±953.8、10380.0±2352.4），对照组表达量最低（1627.0±914.8、4419.0±923.6）；IFN-γ表达量（A值）在对照组最高（9956.0±1172.6），雷帕霉素组其次（7487.0±998.4），模型组最低（6054.0±1045.2），组间两两比较差异均具有统计学意义（均P＜0.01）。与对照组和模型组比较，雷帕霉素组CD4^＋CD25^＋Foxp3^＋细胞占CD4＋CD25＋、CD4＋和淋巴细胞的比例均明显升高〔分别为（57.36±8.84）%比（41.28±5.91）%、（34.52±4.56）%；（4.77±0.48）%比（3.15±0.37）%、（3.14±0.28）%；（1.97±0.22）%比（1.24±0.17）%、（1.44±0.29）%，均P＜0.05〕；CD8^＋CD25^＋Foxp3^＋细胞占CD8^＋CD25^＋、CD8^＋和淋巴细胞的比例也明显升高〔（73.92±7.69）%比（33.44±4.46）%、（49.14±11.38）%；（1.73±1.05）%比（0.46±0.15）%、（0.71±0.42）%；（0.31±0.20）%比（0.09±0.04）%、（0.14±0.09）%，均P＜0.05〕。模型组仅CD8^＋CD25^＋Foxp3^＋细胞占CD8^＋CD25^＋细胞的比例较对照组明显升高〔（49.14±11.38）%比（33.44±4.46）%， P＜0.05〕。结论肺纤维化中存在Th1/Th2、Th17/Treg失衡及向Th2、Th17偏移；雷帕霉素具有抑制肺纤维化的作用，可能通过促进Treg细胞及纠正Th1/Th2失衡实现。

Objective To study the effect of rapamycin on the balance of T helper cell subsets in rats with pulmonary fibrosis. Methods Fifteen male Sprague-Dawley （SD） rats were randomly divided into control group, model group and rapamycin group, with 5 rats in each group. Pulmonary fibrosis model was reproduced by using the method of intratracheal instillation of bleomycin （5 mg/kg）. Control group was treated by intratracheal instillation of saline （1.25 mL/kg） to obtain the negative control. The rats of the rapamycin group were given rapamycin （1 mg·kg^-1·d^-1） by gastric perfusion for consecutive 10 days beginning on the 3rd day after intratracheal instillation of bleomycin. On the 28th day all rats were sacrificed, and the peripheral blood and the lung tissues were harvested. The lung tissue was observed. And the severity of pulmonary fibrosis in rats was assessed by Ashcroft score. The lung tissues were performed using immunohistochemical staining to detect the expression ofγ-interferon （IFN-γ） and interleukins （IL-4, IL-17）. Flow cytometry was used to detect the percentages of CD4^＋ T cells and CD8^＋ T cells. Results The severity of pulmonary fibrosis was improved in rats of rapamycin group compared with that of model group, and the Ashcroft score was decreased （2.92±0.64 vs. 5.76±1.76, F = 16.276, P = 0.080）. The expression levels of IL-4 and IL-17 （A value） in model group were the highest （4 789.0±1 014.6, 19 139.0±2 433.3）, followed by those of the rapamycin group （3 547.0±953.8, 10 380.0±2 352.4）, and the least was found in the control group （1 627.0±914.8, 4 419.0±923.6）. The expression levels of IFN-γ （A value） in control group were the highest （9 956.0±1 172.6）, followed by those of the rapamycin group （7 487.0±998.4）, and the least was found in the model group （6 054.0±1 045.2）. There were significantly differences in above parameters among three groups （all P〈0.01）. Compared with the control group and model group, the percentages of CD4^＋CD25^＋Foxp3^＋T cells in CD4^＋CD25^＋, CD4^＋cells, and lymphocytes were significantly increased in rapamycin group [（57.36±8.84）% vs. （41.28±5.91）%, （34.52±4.56）%; （4.77±0.48）% vs. （3.15±0.37）%, （3.14±0.28）%;（1.97±0.22）%vs. （1.24±0.17）%, （1.44±0.21）%, all P〈0.05], and the percentages of CD8^＋CD25^＋Foxp3^＋T cells in CD8^＋CD25^＋, CD8^＋cells, and lymphocytes were also significantly increased in rapamycin group [（73.92±7.69）% vs. （33.44±4.46）%, （49.14±11.38）%; （1.73±1.05）% vs. （0.46±0.15）%, （0.71±0.42）%;（0.31±0.20）% vs. （0.09±0.04）%, （0.14±0.09）%, all P 〈 0.05]. The percentage of CD8^＋CD25^＋Foxp3^＋ T cells in CD8^＋CD25^＋in model group was significantly higher than that in control group [（49.14±11.38）% vs. （33.44±4.46）%, P 〈 0.05]. Conclusions T helper cell subsets are imbalanced in pulmonary fibrosis rats. Rapamycin can prevent bleomycin-induced pulmonary fibrosis, and its antifibrotic effect maybe the promotion of proliferation and function of regulatory T cells and imbalance regulation of T helper cell subsets.