基于TRPV1/NRF-1/mtTFA通路研究射干麻黄汤对寒性哮喘大鼠的改善作用

Improvement effect of Shegan Mahuang Decoction on rats with cold-induced asthma based on TRPV1/NRF-1/mtTFA pathway

研究射干麻黄汤对寒性哮喘大鼠的改善作用及其作用机制。将72只无特定病原体(specific pathogen free,SPF)的健康雄性SD大鼠随机分为空白组、模型组、阳性药(地塞米松,0.4 mg·kg-1)组以及射干麻黄汤低、中、高剂量组(3.2、6.4、12.8 g·kg-1)。除空白组予生理盐水,其他组均采用卵清蛋白(ovalbumin,OVA)制成的溶液腹腔注射致敏;之后将大鼠放入0~2℃可调节的寒冷箱中,同时予OVA溶液超声雾化激发,建立寒性哮喘大鼠模型。给药3周后,观察大鼠的一般行为学,苏木素-伊红(hematoxylin-eosin,HE)染色观察大鼠肺组织的病理变化,过碘酸希夫(periodic acid-Schiff,PAS)染色观察大鼠肺组织的黏液变化,马松(Masson)染色观察大鼠肺组织的胶原沉积变化,酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测血清及支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中炎症因子白细胞介素-4(interleukin-4,IL-4)、血管内皮生长因子(vascular endothelial growth factor,VEGF)的水平,实时荧光定量聚合酶链式反应(real-time quantitative PCR,RTPCR)检测大鼠肺组织瞬时受体电位香草素1(transient receptor potential vanilloid subfamily member 1,TRPV1)、核呼吸因子1(nuclear respiratory factor 1,NRF-1)、线粒体转录因子A(mitochondrial transcription factor A,mtTFA)mRNA的表达水平,蛋白免疫印迹法(Western blot)检测大鼠肺组织TRPV1、NRF-1、mtTFA的蛋白表达。结果显示,与空白组相比,模型组大鼠出现呼吸急促,大便次数增多、偏稀,毛发脏乱无光泽等行为学表现;病理结果显示肺组织炎症细胞浸润明显,支气管管腔变窄,黏液分泌增多,胶原沉积增多;血清及BALF中IL-4、VEGF水平显著升高;肺组织TRPV1、NRF-1、mtTFA mRNA及蛋白表达显著升高。与模型组相比,射干麻黄汤可改善大鼠的行为学表现,减轻肺组织病理变化、黏液生成及胶原沉积的变化;显著降低血清及BALF中IL-4、VEGF的水平;不同程度降低肺组织TRPV1、NRF-1、mtTFA mRNA的表达,以射干麻黄汤中剂量组最为显著;不同程度降低肺组织TRPV1、NRF-1、mtTFA蛋白的表达,以射干麻黄汤中剂量组最为显著。上述研究表明,射干麻黄汤可减轻寒性哮喘大鼠的气道炎症,抑制气道重塑,其机制可能与调节TRPV1/NRF-1/mtTFA信号通路相关。

This study investigated the therapeutic effect of Shegan Mahuang Decoction(SGMHD)on cold-induced asthma in rats and explored its underlying mechanism.Seventy-two healthy male SD rats of specific pathogen free(SPF)grade were randomly divided into a blank group,a model group,a positive control group(dexamethasone,0.4 mg·kg-1),and low-,medium-,and high-dose SGMHD groups(3.2,6.4,and 12.8 g·kg-1).The blank group received saline,while the other groups were sensitized by intraperitoneal injection of ovalbumin(OVA)solution.Subsequently,the rats were placed in a cold chamber adjustable to 0-2℃,and OVA solution was ultrasonically nebulized to induce cold-induced asthma in rats.After three weeks of treatment,the general behaviors of rats were observed.Hematoxylin-eosin(HE)staining was used to evaluate pathological changes in lung tissues,periodic acid-Schiff(PAS)staining assessed mucin changes,and Masson staining was performed to examine collagen deposition.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of the inflammatory factors interleukin-4(IL-4)and vascular endothelial growth factor(VEGF)in serum and bronchoalveolar lavage fluid(BALF).Real-time quantitative polymerase chain reaction(RTPCR)was employed to assess the mRNA expression levels of transient receptor potential vanilloid subfamily member 1(TRPV1),nuclear respiratory factor 1(NRF-1),and mitochondrial transcription factor A(mtTFA)in lung tissues.Western blot was used to measure the protein expression levels of TRPV1,NRF-1,and mtTFA in lung tissues.Compared with the blank group,the model group exhibited signs of rapid respiration,increased frequency of defecation with looser stools,and disheveled and dull fur.Pathological results showed significant infiltration of inflammatory cells in lung tissues,narrowing of bronchial lumens,increased mucin secretion,and enhanced collagen deposition in the model group.Additionally,the levels of IL-4 and VEGF in serum and BALF were significantly elevated,and the mRNA and protein expression levels of TRPV1,NRF-1,and mtTFA in lung tissues were significantly increased.Compared with the model group,SGMHD improved the behaviors of rats,alleviated pathological changes in lung tissues,mucin production,and collagen deposition,significantly decreased the levels of IL-4 and VEGF in serum and BALF,and reduced the mRNA expression levels of TRPV1,NRF-1,and mtTFA in lung tissues,with the medium-dose SGMHD group showing the most significant effect.Moreover,the protein expression levels of TRPV1,NRF-1,and mtTFA in lung tissues were also reduced,with the mediumdose SGMHD group exhibiting the most significant effect.In conclusion,this study demonstrates that SGMHD can alleviate airway inflammation and inhibit airway remodeling in cold-induced asthma rats.These effects may be associated with the modulation of the TRPV1/NRF-1/mtTFA signaling pathway.