升阳益胃汤对慢性阻塞性肺疾病肺脾气虚证模型大鼠组织病理学及血清炎症因子、外周血Th17/Treg平衡的影响

Effect of Shengyang Yiwei Decoction(升阳益胃汤) on Histopathology,Serum Inflammatory Factors and Peripheral Blood Th17/Treg Balance in Chronic Obstructive Pulmonary Disease Model Rats with Lung-spleen Qi Deficiency Syndrome

目的 探讨升阳益胃汤治疗慢性阻塞性肺疾病(COPD)肺脾气虚证的可能作用机制。方法 将84只SD大鼠随机分为空白组、模型组、瑞巴派特组、地塞米松组和升阳益胃汤高、中、低剂量组,每组12只。除空白组外,其余各组大鼠采用香烟烟熏+脂多糖气管内滴注+大黄混悬液灌胃的方法建立COPD肺脾气虚证大鼠模型,共计造模28天。在造模第15天开始,升阳益胃汤高、中、低剂量组大鼠分别给予升阳益胃汤24.930、12.465、6.233 g/(kg·d)灌胃,地塞米松组给予醋酸地塞米松片溶液0.0675 mg/(kg·d)灌胃,瑞巴派特组给予瑞巴派特片溶液9 mg/(kg·d)灌胃,空白组、模型组给予生理盐水10 ml/(kg·d)灌胃,各组均每日灌胃1次,连续14天。给药结束后采用HE染色观察大鼠肺、气管、胃、小肠组织的病理变化;流式细胞术检测外周血辅助性T细胞17 (Th17)、调节性T细胞(Treg)水平,计算Th17/Treg值;采用ELISA法检测大鼠肺组织髓过氧化物酶(MPO)及血清中二胺氧化酶(DAO)、白细胞介素6 (IL-6)、白细胞介素10(IL-10)、白细胞介素17 (IL-17)、转化生长因子β (TGF-β)水平;实时荧光定量PCR测定肺组织叉状头/翅膀状螺旋转录因子(FoxP3) mRNA、维甲酸相关核孤儿受体γ (RORγt) mRNA表达。结果 与空白组比较,模型组大鼠肺组织肺泡结构不完整并有大量炎症细胞浸润,气管及胃肠黏膜均有损伤,Th17、Th17/Treg值、MPO、DAO、IL-6、IL-17、TGF-β水平及RORγt mRNA升高,Treg、IL-10水平、FoxP3 mRNA降低(P<0.05)。与模型组比较,升阳益胃汤高剂量组可明显改善肺、气管、胃、小肠组织病理损伤,而地塞米松组仅能改善肺和气管组织病理损伤,瑞巴派特组仅能改善胃肠组织病理损伤;除地塞米松组FoxP3 mRNA表达无差异外,升阳益胃汤高剂量组及地塞米松组、瑞巴派特组Th17、Th17/Treg值、MPO、DAO、IL-6、IL-17、TGF-β水平及RORγt mRNA降低,Treg、IL-10水平及FoxP3 mRNA升高(P<0.05)。升阳益胃汤高剂量组较升阳益胃汤中、低剂量组更能降低Th17、Th17/Treg值及肺组织MPO和血清炎症因子水平,改善RORγt、FoxP3 mRNA表达(P<0.05)。结论 升阳益胃汤治疗COPD肺脾气虚证的作用机制可能与同时改善肺、气管、胃、小肠病理损伤,调控Th17/Treg平衡,降低相关炎症因子水平有关。

Objective To explore the mechanism of Shengyang Yiwei Decoction(升阳益胃汤,SYD)in the treatment of chronic obstructive pulmonary disease(COPD)with lung-spleen qi deficiency syndrome.Methods Eightyfour SD rats were randomly divided into 7 groups,namely blank group,model group,rebamipide group,dexamethasone group,high-,medium-and low-dose SYD group,with 12 rats in each group.Except for the blank control group,rats were given cigarette smoke plus intratracheal instillation of lipopolysaccharide plus intragastric administration of Dahuang(Radix et Rhizoma Rhei)suspension to establish the model of COPD with lung-spleen qi deficiency syndrome for a total of 28 days.Since the 15th day of modeling,the rats in the high-,medium-and low-dose SYD groups were given 24.930,12.465,and 6.233 g/(kg·d)of SYD by gavage,respectively.The rats in the dexamethasone group were given 0.0675 mg/(kg·d)of dexamethasone acetate tablet solution,while those in the rebamipide group were given 9 mg/(kg·d)of rebamipide tablet solution by gavage.The blank group and model group were given 10 ml/(kg·d)of normal saline by gavage.All groups were administered once a day for 14 consecutive days.After treatment,the pathological changes of lung,trachea,and gastrointestinal tissues were observed by HE staining.The levels of T helper 17(Th17)and regulatory T cells(Treg)in the peripheral blood were detected by flow cytometry,and the Th17/Treg ratio was calculated.The changes of diamine oxidase(DAO),interleukin 6(IL-6),interleukin 10(IL-10),interleukin 17(IL-17)and transforming growth factor beta(TGF-β)in serum and myeloperoxidase(MPO)in lung tissue of rats were detected by ELISA.The expression of forkhead/winged helix transcription factor(FoxP3)mRNA and retinoic acid-related nuclear orphan receptorγ(RORγt)mRNA in lung tissue were determined by realtime fluorescence quantitative PCR.Results Compared to those in the blank group,the alveolar structure of the rats in the model group was incomplete with a large number of inflammatory cells infiltration,and damaged airway and gastrointestinal mucosa;the values of Th17,Th17/Treg ratio,MPO,DAO,IL-6,IL-17,TGF-βlevels and RORγt mRNA in the model group significantly increased,while the levels of Treg,IL-10 and FoxP3 mRNA were reduced(P<0.05).Compared to those in the model group,the histopathological damage of the lung,trachea,and gastrointestinal tissues were improved in the high-dose SYD group,while only the damage of lung and trachea in the dexamethasone group,and the damage of gastrointestinal tissues in the rebamipide group improved;the values of Th17,Th17/Treg ratio,MPO,DAO,IL-6,IL-17,TGF-β,and RORγt mRNA were significantly reduced in high-dose SYD group,dexamethasone group and rebamipide group,while Treg,IL-10 levels and FoxP3 mRNA increased(P<0.05),but there was no difference in expression of FoxP3 mRNA in dexamethasone group.The high-dose SYD group was better than the medium-and low-dose groups in decreasing Th17,Th17/Treg ratios,MPO in lung tissue and serum inflammatory factor levels,and improving RORγt and FoxP3 mRNA expressions(P<0.05).Conclusion SYD is effective in the treatment of COPD with lung-spleen qi deficiency,which may be related to its function of improving the pathological damage of the lung,trachea,gastrointestinal tissues simultaneously,regulating Th17/Treg balance,and reducing the levels of related inflammatory factors.