扶正透邪解毒方调控Th17/Treg平衡改善铜绿假单胞菌肺炎大鼠肺损伤的机制研究  

Study on the mechanism of regulating Th17/Treg balance by Fuzheng Touxie Jiedu Decoction to improve lung injury in rats with Pseudomonas aeruginosa pneumonia

作　　者：李奕璇 晏军 孔令博[1] 柴立民[1] 张迪 LI Yixuan;YAN Jun;KONG Lingbo;CHAI Limin;ZHANG Di(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China;The Third Affiliated Hospital,Beijing University of Chinese Medicine,Beijing 100029,China;Shandong Provincial Third Hospital,Jinan 250031,China)

机构地区：[1]北京中医药大学东直门医院,北京100700 [2]北京中医药大学第三附属医院 [3]山东省立第三医院

出　　处：《北京中医药大学学报》2024年第12期1681-1692,共12页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金青年科学基金项目(No.81703900),北京中医药大学东直门医院2020年度科技创新专项(No.DZMKJCX-2020-027)。

摘　　要：目的探讨扶正透邪解毒方对铜绿假单胞菌肺炎(PAP)大鼠肺损伤的影响并探究其可能的作用机制。方法经口气管插管后滴入3×109CFU/mL铜绿假单胞菌悬液0.1 mL以复制PAP大鼠模型。将36只SD大鼠按照随机数字表法分为正常组,模型组,扶正透邪解毒方低(3.18 g/kg)、中(6.36 g/kg)、高(12.72 g/kg)剂量组,哌拉西林组(0.27 g/kg),每组6只。各给药组大鼠给予相应药物,正常组和模型组大鼠灌胃等体积蒸馏水,1次/d,连续7 d。采用实时荧光PCR法检测各组大鼠肺组织铜绿假单胞菌核酸值;苏木素-伊红染色法观察各组大鼠肺组织病理形态变化;流式细胞术检测各组大鼠外周血中辅助性T细胞(Th17)和调节性T细胞(Treg)的分化情况;酶联免疫吸附测定检测各组大鼠血清肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、白细胞介素-17(IL-17)含量;实时荧光PCR法检测各组大鼠肺组织核孤儿受体γt(RORγt)、叉头蛋白3(FoxP3)及相关炎症因子的mRNA表达;蛋白质印迹法检测各组大鼠肺组织RORγt、FoxP3及相关炎症因子的蛋白表达。结果造模大鼠血清铜绿假单胞菌均为阳性且病理组织切片有明显炎症病变,表明造模成功。模型组大鼠肺组织可见大量炎性细胞浸润、肺组织结构破坏、上皮细胞脱落。与正常组比较,模型组大鼠外周血Th17占CD4+T细胞比例升高,Treg占比下降,Th17/Treg升高(P<0.01);血清中TNF-α、TGF-β、IL-6、IL-17含量升高,IL-10含量下降(P<0.01);模型组大鼠RORγt、TNF-α、TGF-β、IL-6、IL-17 mRNA表达升高,FoxP3、IL-10 mRNA表达下降(P<0.01);RORγt、TNF-α、TGF-β、IL-6、IL-17蛋白表达水平升高,FoxP3、IL-10蛋白表达水平下降(P<0.05,P<0.01)。与模型组比较,扶正透邪解毒方低、中、高各剂量组及哌拉西林组大鼠肺组织结构破坏、上皮细胞脱落、炎性细胞浸润等情况均有所改善;扶正透邪解毒方�Objective To investigate the effect of Fuzheng Touxie Jiedu Decoction on lung injury in Pseudomonas aeruginosa pneumonia(PAP)rats and explore its possible mechanism.Methods The suspension of Pseudomonas aeruginosa(3×109 CFU/mL)0.1 mL was injected by orotracheal cannula to replicate the PAP rat model.A total of 36 SD rats were divided into the normal group,the model group,the Fuzheng Touxie Jiedu Decoction low-(3.18 g/kg),medium-(6.36 g/kg),high-(12.72 g/kg)dose groups and the piperacillin group(0.27 g/kg)according to random number table method,with six rats in each group.Rats in each drug administration group received the corresponding drugs,while rats in the normal and model groups received an equal volume of distilled water by gavage once a day for seven consecutive days.Nucleic acid values of Pseudomonas aeruginosa were detected in the lung tissues of rats in each group by real-time fluorescence PCR.Hematoxylin-eosin staining was used to observe pathological morphological changes in lung tissues in rats in each group.Flow cytometry assay was used to detect the differentiation of T helper 17(Th17)cells and regulatory T(Treg)cells in the peripheral blood of rats in each group.Serum tumor necrosis factor-α(TNF-α),transforming growth factor-β(TGF-β),interleukin-6(IL-6),interleukin-10(IL-10),and interleukin-17(IL-17)levels were detected in rats of each group with an enzyme-linked immunosorbent assay.The mRNA level of retinoidrelated orphan receptorγt(RORγt),forkhead box protein 3(FoxP3),and related inflammatory factors were detected by real-time fluorescence PCR.The protein expressions of RORγt,FoxP3,and related inflammatory factors were detected by Western blotting.Results The positive detection of Pseudomonas aeruginosa in the serum of model rats and the obvious inflammatory lesions observed in the pathological tissue sections indicated successful modeling.A large number of inflammatory cell infiltration,structural damage and epithelial cell shedding were observed in the lung issue of the model group rats

关 键 词：扶正透邪解毒方 铜绿假单胞菌 细菌性肺炎 辅助性T细胞/调节性T细胞 炎性肺损伤 大鼠 

分 类 号：R285.5[医药卫生—中药学]