桔梗总皂苷调控Th17/Treg免疫失衡改善哮喘模型小鼠气道炎症

Platycodonis Radix Total Saponins Regulates Th17/Treg Immune Imbalance to Alleviate Airway Inflammation in Asthmatic Mice

关键词:  桔梗总皂苷  Notch通路  哮喘  Th17/Treg

Key words:  Platycodonis Radix total saponins  Notch pathway  asthma  Th17/Treg

摘要:

目的 探究桔梗总皂苷是否通过介导Notch通路对哮喘Th17/Treg平衡进行调控。方法 48只BALB/c小鼠随机分为正常对照组，模型组，桔梗总皂苷低、中、高剂量组(15，30，60 mg·kg^-1)和地塞米松组，每组8只。雾化吸入卵清蛋白建立小鼠哮喘模型，药物干预8周后，测定小鼠气道高反应性。取材支气管肺泡灌洗液(bronchoalveolar lavage fluid，BALF)细胞计数、ELISA检测、HE、PAS及Masson染色，流式细胞术检测小鼠肺组织Th17、Treg细胞百分比及Th17/Treg比值变化情况，qPCR检测小鼠肺组织Notch1、Jagged1、RORγt、Foxp3表达水平，Western blotting检测小鼠肺组织中Notch1、Jagged1、Hes1及RORγt、Foxp3、IL-10、IL-17蛋白表达情况。结果 与模型组相比，桔梗总皂苷中、高剂量组小鼠气道阻力减小，BALF中炎性细胞数量及炎性因子IL-4、IL-5、IL-13含量降低，血清IL-17、IL-6、IgE含量降低，BALF中INF-γ及血清中IL-10含量水平显著升高(P<0.05或P<0.01)。HE、PAS及Masson结果显示，桔梗总皂苷高剂量组及地塞米松组能够显著缓解炎性细胞浸润及支气管膜破坏情况，减少杯状细胞增生，同时减轻组织胶原纤维散出程度。流式细胞术结果显示，桔梗总皂苷中、高剂量组Th17细胞数量及Th17/Treg占比均显著减少，Treg细胞数量显著增加(P<0.05或P<0.01)。qPCR及Western blotting结果显示，桔梗总皂苷给药处理后小鼠肺组织Notch1、Jagged1、RORγt mRNA及Notch1、Jagged1、Hes1、RORγt、IL-10、IL-17蛋白的表达水平均显著降低，Foxp3 mRNA及Foxp3蛋白的表达水平显著升高(P<0.05或P<0.01)。结论 桔梗总皂苷能够介导Notch通路对哮喘Th17/Treg平衡进行调控，从而起到减轻气道炎症，抑制哮喘发作的作用。

Abstract:

OBJECTIVE To explore whether Platycodonis Radix total saponins regulate Th17/Treg balance in asthma by mediating Notch pathway. METHODS Forty-eight BALB/c mice were randomly divided into namely normal control group, model group, Platycodonis Radix total saponins low dose, medium dose and high dose groups(15, 30, 60 mg·kg^-1) and dexamethasone group, 8 mice in each group. Mice asthma model was established by ovalbumin sensitization, and airway hyperreactivity was measured after 8 weeks of corresponding drug intervention. The samples were collected and used for bronchoalveolar lavage fluid(BALF) cell count, ELISA, HE, PAS and Masson staining. Flow cytometry was used to detect the percentage of Th17 and Treg cells and the change of Th17/Treg ratio in the lung tissues of mice. The expression levels of Notch1, Jagged1, RORγt and Foxp3 were detected by qPCR, and the expression levels of Notch1, Jagged1, Hes1 and RORγt, Foxp3, IL-10 and IL-17 were detected by Western blotting. RESULTS Compared with model group, Platycodonis Radix total saponins medium and high dose groups airway resistance significantly reduced, the number of inflammatory cells and the content of inflammatory factors IL-4, IL-5 and IL-13 in BALF decreased, and the contents of IL-17, IL-6 and IgE in serum decreased. The levels of INF-γ in BALF and IL-10 in serum were significantly increased(P<0.05 or P<0.01). The results of HE, PAS and Masson experiments showed that the high dose group of Platycodonis Radix total saponins and dexamethasone group could significantly alleviate inflammatory cell infiltration and bronchial membrane destruction, reduce goblet cell proliferation, and reduce the degree of collagen fiber release from tissues. Flow cytometry results showed that the number of Th17 cells and Th17/Treg ratio in medium and high dose groups of Platycodonis Radix total saponins were significantly decreased, and the number of Treg cells was significantly increased(P<0.05 or P<0.01). qPCR and Western blotting results showed that the mRNA levels of Notch1, Jagged1, RORγt and the protein expression levels of Notch1, Jagged1, Hes1, RORγt, IL-10 and IL-17 in lung tissues of mice treated with Platycodonis Radix total saponins were significantly decreased, the expression levels of Foxp3 mRNA and Foxp3 protein were significantly increased(P<0.05 or P<0.01). CONCLUSION Platycodonis Radix total saponins can mediate the Notch pathway to regulate Th17/Treg balance in asthma, thus reducing airway inflammation and inhibiting asthma attack.