麻杏石甘汤及拆方对RSV加重型哮喘气道炎症的保护作用及调控TRPV1的机制研究

Effect of Maxing Shigan Decoction and dissembled prescriptions against airway inflammation in RSV-aggravated asthma and mechanism of regulating TRPV1

探讨麻杏石甘汤及拆方干预呼吸道合胞病毒(respiratory syncytial virus, RSV)加重型哮喘气道炎症的效应及对瞬时受体电位香草酸亚型1(transient receptor potential vanilloid-1, TRPV1)的调控作用。采用卵清蛋白复合RSV复制加重型哮喘小鼠模型(雌性C57BL/6小鼠),麻杏石甘汤及拆方进行干预。观察各组外周血嗜酸性粒细胞(eosinophil, EOS)和支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)中炎细胞水平、呼吸间歇(Penh)值变化、肺病理损伤,酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)法测定BALF中白细胞介素(interleukin, IL)-4、IL-13、P物质(substance P,SP)、前列腺素E2(prostaglandin E2, PGE2)等水平变化,定量实时聚合酶连锁反应(quantitative real time polymerase chain reaction, qPCR)、蛋白免疫印迹法(Western blot)检测小鼠肺组织中TRPV1 mRNA和蛋白表达。体外以IL-4联合RSV刺激16HBE细胞,观察麻杏石甘汤及拆方含药血清干预后TRPV1表达及经TRPV1激动剂刺激后的胞内Ca2+水平变化。结果显示,与空白组相比,模型组小鼠出现明显的哮喘表型,外周血和BALF中各类炎细胞水平、Penh值显著升高(P<0.05,P<0.01),肺组织损伤严重。与模型组相比,全方组、石膏组及麻杏草组外周血和BALF中EOS水平显著下降(P<0.01,P<0.05);全方组和石膏组可显著降低BALF中白细胞、中性粒细胞水平(P<0.01),麻杏草组可下降中性粒细胞水平(P<0.05);全方对外周血和BALF中炎细胞水平的改善作用优于拆方组(P<0.05,P<0.01);50 mg·mL-1氯化乙酰胆碱激发后,全方组和麻杏草组Penh值显著降低(P<0.01),石膏组Penh值下降(P<0.05);麻杏石甘汤及拆方可不同程度减轻肺组织病理损伤;全方组可显著降低BLAF中IL-4、IL-13、PGE2、SP水平(P<0.05,P<0.01),石膏组可下降BLAF中IL-13、PGE2、SP水平(P<0.05,P<0.01),麻杏草组可下降BALF中IL-13、PGE2水平(P<0.05,P<0.01);全方可下调肺组织中TRPV1的蛋白和mRNA表达(P<0.05,P<0.01)。麻杏石甘汤及拆方含药血清干预可下调经IL-4联合RSV刺激的16HBE细胞中TRPV1表达,抑制TRPV1激动剂引起的Ca2+内流,全方组显著优于拆方组(P<0.05)。体内、外实验结果表明,麻杏石甘汤及拆方对RSV加重型哮喘气道炎症具有保护作用,全方药物协同增效,干预作用优于拆方部分,不同组方部分在改善气道高反应性、抗过敏、抗炎方面各有贡献,其机制与调控TRPV1通道和相关炎症介质水平有关。

This study investigated the effect of Maxing Shigan Decoction(MXSGD) and its disassembled prescriptions against the airway inflammation in respiratory syncytial virus(RSV)-aggravated asthma and the regulation of transient receptor potential vanilloid-1(TRPV1). To be specific, ovalbumin(OVA) and RSV were used to induce aggravated asthma in mice(female, C57BL/6). Then the model mice were intervened by MXSGD and the disassembled prescriptions. The eosinophil(EOS) in peripheral blood, inflammatory cells in bronchoalveolar lavage fluid(BALF), enhanced pause(Penh) variation, and lung pathological damage in each group were observed, and the changes of interleukin(IL)-4, IL-13, substance P(SP), and prostaglandin E2(PGE2) in BALF were mea-sured by enzyme-linked immunosorbent assay(ELISA). Quantitative real time polymerase chain reaction(qPCR) and Western blot were used to detect mRNA and protein of TRPV1 in mouse lung tissue. In the in vitro experiment, 16 HBE cells were stimulated with IL-4 and RSV. Then the changes of TRPV1 expression after the intervention with the serum containing MXSGD and its disassembled prescriptions were observed. Besides, the intracellular Calevel after the stimulation with TRPV1 agonist was evaluated. The results showed that the mice in the model group had obvious asthma phenotype, the levels of various inflammatory cells in the peripheral blood and BALF and Penh were significantly increased(P<0.05, P<0.01), and the lung tissue was severely damaged compared with the control group. Compared with the model group, the levels of EOS in the peripheral blood and BALF were significantly decreased in the MXSGD group, the SG group and the MXC group(P<0.05, P<0.01). The levels of WBC and neutrophils in BALF were significantly decreased in the MXSGD group and SG group(P<0.01), the levels of neutrophils in BALF were decreased in the MXC group(P<0.05). The improvement effect of the MXGSD on the level of inflammatory cells in peripheral blood and BALF was better than that of two disassembled groups(P<0.05, P<0.01). After 50 mg·mLacetylcholine chloride stimulation, the Penh values of the MXSGD group and the MXC group significantly decreased(P<0.01), and the Penh value of the SG group decreased(P<0.05). The levels of IL-4, IL-13, PGE2 and SP in BALF could be significantly decreased in the MXSGD group(P<0.05, P<0.01), the levels of IL-13 and PGE2 in BALF could be decreased in the MXC group(P<0.05, P<0.01), and the levels of IL-13, PGE2 and SP in BALF could be decreased in the SG group(P<0.05, P<0.01). MXSGD could down-regulate the protein and mRNA expression of TRPV1 in lung tissue(P<0.05, P<0.01). The serum containing MXSGD and its disassembled prescriptions could down-regulate TRPV1 expression in 16 HBE cells stimulated by IL-4 combined with RSV and inhibit the inward flow of Cainduced by TRPV1 agonist, especially the serum containing MXSGD which showed better effect than the serum containing disassembled ones(P<0.05). In vivo and in vitro experiments verified the protective effect of MXSGD and its disassembled prescriptions against airway inflammation in RSV-exacerbated asthma, the whole decoction thus possessed synergy in treating asthma, with better performance than the dissembled prescriptions. Different groups of prescription had made contributions in improving airway hyperresponsiveness, anti-allergy and anti-inflammation. The mechanism is the likelihood that it regulates TRPV1 channel and levels of related inflammatory mediators.