健脾化痰通腑方通过调节Th17/Treg平衡抑制慢性阻塞性大鼠黏液过度分泌的影响及机制研究

Study on the effect and mechanism of Jianpi Huatan Tongfu prescription in inhibiting excessive mucus secretion in chronic obstructive rats by regulating Th17/Treg balance

关键词:  慢性阻塞性肺疾病、黏液分泌、健脾化痰通腑方、Th17/Treg失衡

Key words:  Chronic  obstructive pulmonary  disease, mucus  secretion, spleen-huatan  decoction, Th17/Treg  imbalance

摘要:

目的:探讨JPHT治疗COPD的可能机制。方法:本研究先用网络药理学预测JPHT治疗COPD可能作用的靶点和通路,后进一步用动物实验验证。将大鼠分为4组:空白组、COPD组、JPHT组、沙丁胺醇(ALB)组。采用在Wister大鼠气管内滴注脂多糖(LPS)联合烟熏(CS)4周建立COPD大鼠模型,分别给药JPHT、ALB两周后。观察大鼠的一般状况；检测肺功能；HE染色观察肺组织形态学变化；免疫荧光法检测肺组织中MUC5AC、AQP5和IL-17RA的表达；流氏细胞术检测脾脏组织中Th17/Treg的表达；Western blot检测肺组织中IL-17RA及NF-κB信号通路相关蛋白和AQP5表达。结果:网络药理学发现JPHT治疗COPD和IL-17A通路有关,与正常对照组相比,模型组大鼠一般状况较差,肺功能下降,肺组织出现炎症细胞浸润和肺气肿的表现,流式细胞术显示模型组Th17/Treg失衡,免疫荧光显示模型组IL-17A、MUC5AC的表达上调,AQP5表达下调,WB显示,模型组NF-κB p65、IKKβ表达上调,IκBα、AQP5表达下调,与模型组相比,健脾化痰通腑方组能可改善COPD大鼠一般状况、肺功能和病理损伤,恢复Th17/Treg失衡,抑制IL-17RA及NF-κB通路相关蛋白的表达,上调AQP5的表达,下调MUC5AC的表达,减轻肺部粘液。结论:JPHT可以恢复Th17/Treg失衡,抑制IL-17A/NF-κB通路的异常激活,调节MUC5AC、AQP5的表达,减轻肺部的粘液,对COPD有治疗作用。

Abstract:

:Objective: To investigate the possible mechanism of JPHT in the treatment of COPD. Methods: In this study, the potential targets and pathways of JPHT in the treatment of COPD were predicted by network pharmacology, and then verified by animal experiments. The rats were divided into 4 groups: blank group, COPD group, JPHT group and salbutamol (ALB) group. The COPD rat model was established by intravatracheal infusion of lipopolysaccharide (LPS) combined with smoking (CS) for 4 weeks in Wister rats, and JPHT and ALB were administered for 2 weeks. The general condition of rats was observed. To detect lung function; The morphological changes of lung were observed by HE staining. The expression of MUC5AC, AQP5 and IL-17RA in lung tissue was detected by immunofluorescence method. The expression of Th17/Treg in spleen was detected by Fluidic cell assay. Western blot analysis of IL-17RA, NF-κB signaling pathway related proteins and AQP5 expression in lung tissue. Result: Network pharmacology found that JPHT treatment of COPD was related to IL-17A pathway. Compared with the normal control group, the rats in the model group were generally in worse condition, with decreased lung function, inflammatory cell infiltration and emphysema in lung tissue. Flow cytometry showed Th17/Treg imbalance in the model group. Immunofluorescence showed up-regulated expression of IL-17A and MUC5AC and down-regulated expression of AQP5 in the model group. WB showed up-regulated expression of NF-κB p65 and IKKβ and down-regulated expression of IκBα and AQP5 in the model group. Compared with the model group, Jianpi Huatangfu decoction group could improve the general condition, lung function and pathological injury of COPD rats. Restore Th17/Treg imbalance, inhibit the expression of IL-17RA and NF-κB pathway related proteins, up-regulate the expression of AQP5, down-regulate the expression of MUC5AC, and relieve lung mucus. Conclusion: JPHT can restore the imbalance of Th17/Treg, inhibit the abnormal activation of IL-17A/NF-κB pathway, regulate the expression of MUC5AC and AQP5, relieve the pulmonary mucus, and have therapeutic effect on COPD.