平喘宁通过UPR信号通路改善寒哮大鼠气道炎症机制研究

Study on the Mechanism of Pingchuanning(平喘宁)Improving Airway Inflammation in Cold Asthma Rats Through UPR Signal Pathway

目的探讨平喘宁通过非折叠蛋白反应(unfolded protein response,UPR)信号通路抑制内质网应激(endoplasmic reticulum stress,ERS)“细胞毒性”的机制以及对寒哮大鼠气道形态学及肺组织中蛋白二硫键异构酶(PDIA2)、核因子E2相关因子2(Nrf2)、葡萄糖调节蛋白78(GRP78)mRNA和激活转录因子6(ATF6)mRNA表达水平的影响。方法将105只无特定病原体(SPF)级雄性SD健康大鼠随机分为空白组,模型组,平喘宁低、中、高剂量组,地塞米松组,桂龙咳喘宁组。以卵清蛋白及寒冷箱刺激实验大鼠复制大鼠寒哮模型,造模21 d后,空白组、模型组予以等量0.9%氯化钠溶液灌服,地塞米松组予以地塞米松片溶于0.9%氯化钠溶液的药液0.405 mg/(kg·d)灌胃给药,桂龙咳喘宁组予以桂龙咳喘宁片溶于0.9%氯化钠溶液的药液0.405 g/(kg·d)灌胃给药,平喘宁高、中、低剂量组分别予以平喘宁汤剂14.578、7.289、3.645 g/(kg·d)不同剂量灌胃给药。灌胃28 d后,在末次卵清蛋白激发后,予以解剖大鼠取出肺组织。取材后行苏木精-伊红(HE)染色法观察大鼠肺组织的病理改变;应用反转录聚合酶链式反应(RT-PCR)法、蛋白质免疫印迹(Western blot)法、酶联免疫吸附测定法(ELISA)分别检测ATF6 mRNA、GRP78 mRNA和PDIA2、Nrf2以及白细胞介素-17A(IL-17A)、白细胞介素-1β(IL-1β)的表达水平。结果HE染色:模型组大鼠的支气管黏膜褶皱增多,管腔狭窄;其他5组药物干预治疗组大鼠的病理形态学参照模型组均有不同改善。ELISA检测结果:参照模型组,其他5组药物干预治疗组大鼠肺泡灌洗液中IL-17A、IL-1β的表达水平明显降低(P<0.05);RT-PCR法检测:参照空白组,肺组织的GRP78 mRNA和ATF6 mRNA蛋白表达以模型组升高更为显著(P<0.01);参照模型组,药物干预治疗组的5组大鼠肺组织中的GRP78 mRNA和ATF6 mRNA蛋白表达均有明显下调(P<0.01)。Western blot检测:参照空白组,肺组织中的PDIA2蛋白表达以模型组升高更为显著(P<0.01),Nrf2蛋白表达以模型组下降更为显著(P<0.01);参照模型组,药物干预治疗组的5组大鼠肺组织中的PDIA2蛋白表达均有明显下调(P<0.01),Nrf2蛋白表达均有明显升高(P<0.01)。结论平喘宁通过UPR信号通路抑制ERS的“细胞毒性”从而调节寒哮大鼠肺组织中PDIA2、Nrf2、ATF6 mRNA及GRP78 mRNA表达,抑制气道炎症,缓解气道重塑,使寒哮大鼠哮喘症状趋于好转。

Objective To investigate the mechanism of Pingchuanning(平喘宁)inhibiting endoplasmic reticulum stress(ERS)cytotoxicity through unfolded protein response(UPR)signaling pathway and its effects on airway morphology and expression levels of protein disulfide isomerase(PDIA2),nuclear factor E2-related factor 2(Nrf2),glucose-regulated protein 78(GRP78)mRNA and activated transcription factor 6(ATF6)mRNA in lung tissue of cold asthmatic rats.Methods A total of 105 specific pathogen free male SD rats were randomly divided into blank group,model group,Low,middle and high doses of Pingchuanning group,dexamethasone group and Guilong Kechuanning(桂龙咳喘宁)group.The model of cold asthma in rats was established by ovalbumin and cold box stimulation.Twenty-one days after modeling,0.9%sodium chloride solution was given to the blank group and the model group,and dexamethasone tablets dissolved in 0.9%sodium chloride solution was given to the dexamethasone group at a dose of 0.405 mg/(kg·d)by gavage,Guilong Kechuanning group was treated with 0.405 g/(kg·d)of Guilong Kechuanning Tablets dissolved in 0.9%sodium chloride solution by gavage,the high,middle and low dose groups were given Pingchuanning Decoction 14.578,7.289 and 3.645 g/(kg·d)respectively.After 28 days,the rats were dissected and the lung tissues were taken out after the last ovalbumin challenge.The pathological changes of lung tissue were observed by Caesalpinia sappan-eosin(HE)staining.The expression levels of ATF6 mRNA,GRP78 mRNA,PDIA2,NRF2,interleukin-17A(IL-17A)and interleukin-1β(IL-1β)were detected by reverse transcription and polymerase chain reaction polymerase chain reaction(RT-PCR),Western blot and enzyme-linked immunosorbent assay(ELISA),respectively.Results HE staining showed that the bronchial mucosal folds were increased and the lumen was narrowed in the model group,and the pathomorphological changes were improved in the other five groups.The results of ELISA showed that the expression levels of IL-17A and IL-1βin balf of rats in the model group and the other five groups were significantly decreased(P<0.05),the expression of GRP78 mRNA and ATF6 mRNA in lung tissue in model group was higher than that in control group(P<0.01),the expression of GRP78 mRNA and ATF6 mRNA in the lung tissue of the rats in the drug intervention group were significantly down-regulated(P<0.01).Western blot showed that the expression of PDIA2 protein in lung tissue was significantly increased in model group(P<0.01),and the expression of NRF2 protein was significantly decreased in model group(P<0.01),the expression of PDIA2 protein was down-regulated(P<0.01)and the expression of NRF2 protein was up-regulated(P<0.01).Conclusion Pingchuanning can inhibit the“cytotoxicity”of ERS through UPR signal pathway and regulate the expression of PDIA2,NRF2,ATF6 mRNA and GRP78 mRNA in the lung tissue of cold asthmatic rats,inhibit airway inflammation and alleviate airway remodeling,the asthma symptoms of cold asthma rats tended to improve.