清宣通络方通过外泌体miRNA-146a靶向TRAF6基因抑制肺炎支原体感染诱导肺泡上皮细胞炎性损伤的研究

Qingxuan Tongluo Formula regulates TRAF6 gene through exosomal miRNA-146a regulating inflammatory injury induced by mycoplasma pneumoniae infection in alveolar epithelial cells

关键词:  清宣通络方  肺炎支原体感染  外泌体miRNA-146a  肿瘤坏死因子受体相关因子6  炎性损伤

Key words:  ingxuan Tongluo Formula  Mycoplasma Pneumoniae infection  exosomal miRNA-146a  Tumor necrosis factor receptor-related factor-6  inflammatory injury

摘要:

[目的] 基于外泌体miRNA-146a调控肿瘤坏死因子受体相关因子6（TRAF6）基因探讨清宣通络方抑制肺炎支原体（MP）感染诱导肺泡上皮细胞炎性损伤的作用机制。[方法] 24只实验小鼠随机分为空白组、肺炎支原体肺炎（MPP）组、阿奇霉素组、清宣通络方组，给药结束后观察小鼠体温、咳嗽情况，进行肺组织苏木精-伊红（HE）染色、病理评分及聚合酶链式反应（PCR）鉴定。肺泡上皮细胞分为正常对照组、MP感染组、miRNA-146a过表达/抑制剂组和miRNA-NC阴性对照组；采用实时荧光定量聚合酶链式反应（RT-qPCR）技术检测肺泡上皮细胞来源外泌体、肺组织中miRNA-146a及肺泡上皮细胞中TRAF6 mRNA表达水平；ELISA法检测培养液上清中白介素-17（IL-17）、白介素（IL-6）和肿瘤坏死因子-α（TNF-α）含量。[结果] 与空白组相比，MPP组小鼠出现体温升高、咳嗽次数增加、咳嗽潜伏期缩短、肺组织病理评分增高（P均<0.01）；与MPP组相比，清宣通络方组小鼠体温降低、咳嗽次数减少、咳嗽潜伏期增加、肺组织病理评分下降（P均<0.01）。与正常对照组相比，MP感染组外泌体miRNA-146a水平下降，肺泡上皮细胞TRAF6 mRNA及培养液上清中IL-17、IL-6、TNF-α含量上升（P均<0.05）。与MP感染组相比，miRNA-146a过表达组外泌体miRNA-146a水平上调，肺泡上皮细胞TRAF6 mRNA及培养液上清IL-17、IL-6、TNF-α水平下降（P均<0.05）；miRNA-146a抑制组外泌体miRNA-146a下降，肺泡上皮细胞TRAF6 mRNA及培养液上清中IL-17、IL-6、TNF-α水平上升（P均<0.05）。与miRNA-146a抑制剂组相比，清宣通络方外泌体干预组培养液上清中炎性因子水平降低（P均<0.01）；与MPP组相比，清宣通络方组小鼠肺组织中miRNA-146a含量升高（P<0.01）。[结论] 清宣通络方可能通过上调外泌体miRNA-146a，靶向调控TRAF6基因，进而影响IL-17、IL-6和TNF-α等炎性因子表达，抑制MP感染诱导的炎性损伤。

Abstract:

[Objective] To investigate the mechanism of Qingxuan Tongluo Formula in regulating Mycoplasma Pneumoniae(MP)infection in alveolar epithelial cells induced inflammatory injury based on the regulation of Tumor necrosis factor receptor-related factor-6(TRAF6) gene by exosomal miRNA-146a.[Methods] Total 24 experimental mice were randomly divided into blank group,Mycoplasma pneumoniae pneumonia(MPP) group,azithromycin group,and Qingxuan Tongluo Formula group. After the end of administration,the body temperature and cough of the mice were observed,and the HE staining,pathological score and PCR identification of lung tissue were performed. Alveolar epithelial cells are divided into normal control group,MP infection group,miRNA-146a overexpression/inhibitor group,miRNA-NC negative control group. RT-qPCR was used to detect miRNA-146a in exosomes,lung tissues of animal experimental mice,and TRAF6 mRNA expression levels in alveolar epithelial cells. Elisa was used to detect Interleukin-17(IL-17),Interleukin-6(IL-6) and Tumor necrosis factor-α(TNF-α) contents in culture medium supernatant.[Results] Compared with the blank group,tthe MPP group had an increase in body temperature,an increase in the number of coughs,a shortened cough latency,and an increase in lung histopathological scores(all P<0.01). Compared with the MPP group,the body temperature of the mice in the Qingxuan Tongluo Formula group decreased,the number of coughs decreased,the cough latency increased,and the lung histopathological score decreased(all P<0.01). Compared with normal control group,the expression level of miRNA-146a in exosomes decreased in MP infection group,and the contents of inflammatory factors as IL-17,IL-6 and TNF-α in alveolar epithelial cells and TRAF6 mRNA in culture medium supernatant were increased(all P<0.05). Compared with the MP infection group,the level of miRNA-146a in the miRNA-146a overexpression group was significantly up-regulated in exosomes,and the expression levels of TRAF6 mRNA in alveolar epithelial cells and IL-17,IL-6 and TNF-α in culture medium supernatant were decreased(all P<0.05). In the miRNA-146a inhibition group,the expression levels of miRNA-146a in exosomes were significantly decreased,and the expression levels of IL-17,IL-6 and TNF-α in culture medium supernatant and TRAF6 mRNA in culture medium supernatant were significantly increased(all P<0.05). Compared with the miRNA-146a inhibitor group,the level of inflammatory factors in the supernatant of the exosome intervention group in the Qingxuan tongluo Formula group was significantly reduced(all P<0.01). Compared with the MPP group,the content of miRNA-146a in the lung tissue of mice in the Qingxuan Tongluo Formula group was increased(P<0.01).[Conclusion] Qingxuan Tongluo formula may regulate the inflammatory injury induced by MP infection by up-regulating exosomal miRNA-146a and targeting the TRAF6 gene,which in turn affects the expression of inflammatory factors such as IL-17,IL-6 and TNF-α.