Effect Of Bufei Jianpi Decoction On Th1 / Treg Cells In COPD Rat Model

Objectives1. To evaluate the effects of Bufei Jianpi granules in the treatment of chronic obstructive pulmonary disease (COPD)rats.2. To explore the mechanisms of Bufei Jianpi granules regulating Thl7/Treg cells in COPD rats.MethodsSeventy-two Sprague-Dawley rats were randomized into Control+Saline group(CSG), COPD Model+Saline group (CMSG), COPD Model+high-dose Bufei Jianpi group (HDG), COPD Model+medium-dose Bufei Jianpi group (MDG), COPD+low-dose Bufei Jianpi group (LDG) and COPD+Aminophylline groups (AG). The stable COPD rat model was duplicated by using the combination of repeated cigarette smoke inhalations and bacterial infections From week 9 up to 20, rats were administrated intragastricly with normal saline in CSG and CMSG (2 ml d-1), Bufei Jianpi granules in HDG (9.68 g·kg-1·d-1), MDG (4.84 g·kg-1·d-1) and LDG (2.42 g·kg-1·d-1), aminophylline in AG (2.3 mg·kg-1·d-1). At the e nd of week 20, the animals were sacrificed and samples were harvested. The lung function tidal volume (TV), peak expiratory flow (PEF) and expiratory flow in 50% tidal volume (EF50) were measured with whole body plethysmography. Pulmonary pathological changes were observed by optical microscopy. The levels ofinterleukin (IL)-17, IL-6, IL-10 and transforming growth factor (TGF)-β in serum and bronchoalveolar lavage fluid (BALF) were determined with enzymelinkedimmunosorbentassay methods. The expressions of CD4+IL-17+/CD4+ and CD44+Foxp3+/CD4+ in peripheral blood were detected using flow cytometry. The gene expressions of IL-17 and Forkhead box P3 (Foxp3) were detected by using quantitative polymerase chain reaction. The protein expressions of retinoid-ralated orphan receptor (ROR)-yt and Foxp3 were analyzed by western blotting.Results1 General statusFrom weeks 2 to 8, rats in model group were emaciated, depressed, inactive, reduced food or water intake. The symptoms were improved after treatment of HDG, MDG LDG and AG, especially in HDG, MDG.Body weight of rats in each group increased with age, but it in CMSG was lower than CSG from week 4 (P<0.05). Body weight of HDG, MDG, LDG and AG increased quickly from week 13, but there were no significant statistical difference among the groups (P>0.05).2 Lung functionTV, EF50, PEF decreased significantly in COPD rats compared with CSG from the 8th week (P<0.05 or P<0.01). After administrated Bufei Jianpi and aminophylline 4weeks, 8weeks and 12 weeks, EF50, PEF in HDG, MDG, LDG and AG were significantly improve than CMSG (P<0.05 or P<0.01); TV in HDG, MDG were significantly improve than CMSG (P<0.05 or P<0.01). After treated COPD rats 4weeks,8weeks and 12 weeks, EF50, PEF in HDG were significantly improve than CMSG (P<0.05 or P<0.01). After treated COPD rats 12 weeks, TV in HDG, MDG were improve than LDG and AG (P<0.05). After treated COPD rats 12 weeks, PEF in MDG were improve than LDG and AG (P<0.05).3 Lung morphologyAt the end of the 20th week, lung histological impaired obvioushy in CMSG. The HDG, MDG, LDG and Aminophylline groups alleviated the pathological damage in different degree, especially in that treated with the HDG、MDG group.4 Cell count and classification in peripheral blood and BALFTotal cellular score, white blood cell and neutrophil ratio in peripheral blood and BALF, CMS were significantly higher than CSG (P<0.05 or P<0.01); HDG, MDG, LDG and AG were significantly higher than CMSG (P<0.05 or P<0.01), especially in HDG, MDG, (P<0.05 or P<0.01).Lymphocyte ratio in peripheral blood, CMSG were lower than CSG, and HDG, MDG, LDG and AG were higher than CMS but no statistically significant (P>0.05).5 Inflammatory factors associated with Th17 and Treg in serum and BALFIn serum and BALF, IL-17and IL-6 in CMSG were significantly higer than CSG (P<0.01); TGF-βand IL-10 in CMSG were significantly lower than CSG (P<0.01). In serum and BALF. IL-17 and IL-6 in HDG and MDG were significantly lower than CSG, and IL-10 and TGF-β in HDG and MDG were significantly higer than CS (P<0.05 or P<0.01). In serum, IL-6 in HDG and MDG were significantly lower than AG (P<0.05 or P<0.01). In BALF, IL-6 in HDG were lower than AG (P<0.05). In BALF, IL-10 in HDG, MDG, LDG were significantly higer than AG (P<0.05 or P<0.01).6 Expression of Th17 and Treg cells in peripheral bloodIn peripheral blood, expression of CD4+IL-17+/CD4+Tcells, CD4+Foxp3+/CD4+T cells and ratio of (CD4+IL-17+/CD4+)/(CD4+Foxp3+/CD4+T cells) in CSG were significantly higer than CMSG (P<0.01). Expression of CD4+IL-17+/CD4+T cells, CD4+Foxp3+/CD4+T cells and ratio of (CD4+IL-17+/CD4+)/(CD4+Foxp3+/CD4+T cells) in HDG and MDG were significantly lower than CMSG (P<0.05 or P<0.01), and HDG is lower than MDG, LDG and AG (P<0.05 or P<0.01). Expression of CD4+1L-17+/CD4+ Tcells, CD4+Foxp3+/CD4+T cells in HDG and MDG were significantly lower than AG (P<0.05 or P<06.01).7 Gene expressions of IL-17 and Foxp3In lung tissue, gene expression of IL-17, Foxp3 and ratio of IL-17/Foxp3 in CMSG were significantly higer than CSG (P<0.01). Gene expression of IL-17 and Foxp3 in HDG, MDG, LDG and AG were significantly lower than CSG (P<0.01). Gene expression of IL-17 and Foxp3 in HDG, MDG, LDG were significantly lower than AG (P<0.01). Gene expression of IL-17 in HD were significantly lower than MD and AG (P<0.05 or P<0.01). Ratio of IL-17/Foxp3 in HDG and MDG were significantly lower than CMS(P<0.05 or P<0.01).8 protein expression of RORyt and Foxp3In lung tissue, protein expression of Foxp3 and RORyt and ratio of RORyt/Foxp3 in CMS were significantly higer than CSG (P<0.01).Protein expression of Foxp3 and RORyt in HDG, MDG and LDG were significantly lower than CMSG (P<0.01). Protein expression of Foxp3 in HDG, MDG and LDG were significantly lower than AG (P<0.05 or P<0.01).Conclusions1 There are beneficial effect of Bufei Jianpi granules in the treatment of stable COPD, which can significantly improve symptom, and lung function, reduce the pathological damage in the lungs and airway, especially in HDG and MDG.2 Bufei Jianpi granules, which are more effective on relieving inflammation than AG and by improve the expression of IL-10 and TGF-β1 on the differentiation of Treg cells and reducing the expression of IL-17 and IL-6 on the differentiation of Th17 cells, and HDG and MDG are more effective than AG.3 Bufei Jianpi granules, especially HDG and MDG can reduce the ratio of Th17/Treg and relieving inflammation in peripheral blood and lung tissue by reducing IL-17 and Foxp3 gene, RORyt and Foxp3 protein in lung, and CD4+IL-17+/CD4+T, CD4+Foxp3+/CD4+T lymphocyte cells in blood.