基于JAK/STAT通路探讨益气温阳护卫法干预支气管哮喘大鼠防御素水平的机制

Study on the Mechanism of Method of Replenishing Qi and Warming Yang and Protecting Defense Qi Intervening the Level of Defensin in Asthmatic Rats Based on JAK/STAT Pathway

目的:基于Janus激酶(Janus kinase,JAK)/信号转导子和转录激活子(signal transducer and activator of transcription,STAT)通路探讨益气温阳护卫法干预支气管哮喘大鼠防御素水平的机制。方法:SPF级雄性SD大鼠50只,随机分为正常组、模型组及益气温阳护卫汤低剂量组(5 g·kg^(-1))、中剂量组(10 g·kg^(-1))、高剂量组(20 g·kg^(-1)),每组10只。除正常组外,其余各组均采用腹腔注射抗原液(卵清蛋白+氢氧化铝)结合雾化(卵清蛋白)激发的方式建立支气管哮喘大鼠模型。造模第8天,各药物组开始灌胃相应剂量的益气温阳护卫汤,正常组、模型组灌胃等体积生理盐水,每天2次,持续4周。RT-PCR检测肺组织β-防御素基因表达水平;ELISA检测血清干扰素-γ(interferon gamma,IFN-γ)、白细胞介素-4(interleukin-4,IL-4)水平;RT-PCR检测肺组织JAK mRNA、STAT6 mRNA表达水平;瑞氏染色法检测肺泡灌洗液中免疫细胞水平。结果:(1)与正常组比较,模型组和各药物组大鼠肺组织β-防御素基因表达水平升高(P<0.05);与模型组比较,各药物组β-防御素基因表达水平降低(P<0.05);各药物组β-防御素基因表达水平比较,差异无统计学意义(P>0.05)。(2)与正常组比较,模型组和各药物组大鼠血清IFN-γ水平降低(P<0.05);与模型组比较,各药物组血清IFN-γ水平升高(P<0.05);与正常组比较,模型组和各药物组大鼠血清IL-4水平升高(P<0.05);与模型组比较,各药物组血清IL-4水平降低(P<0.05);各药物组血清IFN-γ、IL-4水平比较,差异无统计学意义(P>0.05)。(3)与正常组比较,模型组和各药物组大鼠肺组织JAK mRNA、STAT6 mRNA表达水平升高(P<0.05);与模型组比较,各药物组肺组织JAK mRNA、STAT6 mRNA表达水平降低(P<0.05);各药物组肺组织JAK mRNA、STAT6 mRNA表达水平比较,差异无统计学意义(P>0.05)。(4)与正常组比较,模型组和各药物组大鼠肺泡灌洗液中,中性粒细胞、巨噬细胞、嗜酸性粒细胞和淋巴细胞水平升高(P<0.05);与模型组比较,各药物组中性粒细胞、嗜酸性粒细胞、巨噬细胞和淋巴细胞水平降低(P<0.05);各药物组中性粒细胞、嗜酸性粒细胞、巨噬细胞和淋巴细胞水平比较,差异无统计学意义(P>0.05)。结论:益气温阳护卫法可减轻支气管哮喘大鼠气道炎症反应,降低β-防御素基因表达水平,其机制可能与调节JAK/STAT信号通路有关。

Objective:To study on the mechanism of method of replenishing qi and warming yang and protecting defense qi intervening the level of defensin in asthmatic rats based on Janus kinase(JAK)/signal transducer and activator of transcription(STAT) pathway.Methods:A total of 50 SPF male SD rats were randomly divided into the normal group(group A),the model group(group B),low-dose group of Decoction for Replenishing Qi and Warming Yang and Protecting Defense Qi(group C,5 g·kg^(-1)),the medium-dose group of Decoction for Replenishing Qi and Warming Yang and Protecting Defense Qi,(group D,10 g·kg^(-1)) and the high-dose group of Decoction for Replenishing Qi and Warming Yang and Protecting Defense Qi,(group E,20 g·kg^(-1)),with 10 rats in each group.Except the group A,the other groups were given by intraperitoneal injection of Antigen Solution(ovalbumin+aluminum hydroxide) combined with atomization(Ovalbumin) excitation to establish the model of asthmatic rats.On the 8th day of modeling,the group C-E began to gavage the corresponding dose of Decoction for Replenishing Qi and Warming Yang and Protecting Defense Qi,while the group A and group B were gavaged with the same amount of saline,twice a day,for 4 weeks.The expression level of serum β-defensins was detected by RT-PCR;the levels of serum interferon gamma(IFN-γ) and interleukin-4(IL-4) were detected by ELISA;the expression levels of JAK mRNA and STAT6 mRNA in lung tissue were detected by RT-PCR;and the level of immune cells in bronchoalveolar lavage fluid was detected by Wright′s staining method.Results:(1) Compared with the group A,the serum β-defensins expression level of rats in group B-E was significantly increased.Compared with the group B,the serum β-defensins expression level of rats in group C-E was significantly decreased(P<0.05);there was no significant difference in the expression level of β-defensins in group C-E.(2) Compared with the group A,the serum IFN-γ level of rats in group B-E was significantly decreased.Compared with the group B,the serum IFN-γ level of rats in group C-E was significantly increased.Compared with the group A,the serum IL-4 level of rats in group B-E was significantly increased.Compared with the group B,the serum IL-4 level of rats in group C-E was significantly decreased(P<0.05);there were no significant differences in the serum IFN-γ and IL-4 levels of rats in group C-E.(3) Compared with the group A,the JAK mRNA and STAT6 mRNA expression levels of lung tissue of rats in group B-E were significantly increased.Compared with the group B,the JAK mRNA and STAT6 mRNA expression levels of lung tissue of rats in group C-E were significantly decreased(P<0.05);there were no significant differences in the JAK mRNA and STAT6 mRNA expression levels of lung tissue of rats in group C-E.(4) Compared with the group A,the levels of neutrophils,macrophages,eosinophils and lymphocytes in BALF of rats in group B-E were significantly increased.Compared with the group B,the levels of neutrophils,eosinophils,macrophages and lymphocytes in group C-E were significantly decreased(P<0.05);there were no significant differences in the levels of neutrophils,eosinophils,macrophages and lymphocytes in group C-E.Conclusion:Method of replenishing qi and warming yang and protecting defense qi can reduce the inflammatory response and increase the expression level of β-defensins in asthmatic rats,and its mechanism may be related to the regulation of JAK/STAT signaling pathway.