运脾泻肺化痰汤通过抑制PI3K/AKT/NF-κ B 信号轴
减轻幼龄哮喘大鼠气道炎症及黏液高分泌

Yunpi Xiefei Huatan Decoction Alleviates Airway Inflammation and Mucus Hypersecretion in Young Asthmatic Rats via Inhibiting PI3K/AKT/NF-κB Signaling Pathway

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DOI：10.7661/j.cjim.20230306. 113

中文关键词: 支气管哮喘运脾泻肺化痰汤白细胞介素-17 白细胞介素-23 黏蛋白5AC 中药复方

英文关键词:asthma Yunpi Xiefei Huatan Decoction interleukin-17 interleukin-23 mucin 5AC Chinese herbal compound formula

基金项目:国家自然科学基金地区科学基金资助项目（No.81860868）;贵州省科技厅项目（No.[2020]1Y365）;贵州省“十四五”中医药、民族医药重点学科建设项目（No.黔中医药函[2021]95号）

作者	单位
王文洲;高倩倩;陈竹	1.贵州中医药大学第二临床医学院（贵阳 550002）,2.贵州中医药大学第二附属医院儿科（贵阳 550001）

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中文摘要:

目的基于白细胞介素（IL）-23/辅助性T细胞17（Th17）炎症轴与磷酯酰肌醇-3激酶（PI3K）/蛋白激酶B（AKT）/核因子κB（NF-κB）信号轴相关性探讨运脾泻肺化痰汤对幼龄哮喘大鼠气道炎症、黏液高分泌的影响。方法将幼龄SD大鼠56只随机分为7组：正常组、模型组、低剂量运脾泻肺化痰汤组（LTCM）、中剂量运脾泻肺化痰汤组（MTCM）、高剂量运脾泻肺化痰汤组（HTCM）、布地奈德组、联合组，每组各8只。除正常组外，其余各组大鼠腹腔注射卵清白蛋白与氢氧化铝凝胶混合液致敏，1%卵清蛋白雾化激发。正常组、模型组均灌胃0.9%氯化钠2 mL；LTCM、MTCM、HTCM分别灌胃运脾泻肺化痰汤灌胃10、20、40 g/kg；布地奈德组予0.01%布地奈德2 mL雾化吸入20 min；联合组给予MTCM灌胃及布地奈德组雾化，连续4周。分类计数肺泡灌洗液（BALF）中不同种类白细胞；HE、过碘酸-雪夫染色（PAS）观察肺组织病理改变；ELISA检测BALF及血清中IL-17、IL-23水平，免疫组化检测肺组织IL-17、IL-23表达；Western Blot检测肺组织PI3K/AKT/NF-κB信号通路相关蛋白及黏蛋白5AC（MUC5AC）蛋白表达，RT-PCR检测肺组织中的IL-17、IL-23、PI3K、AKT、NF-κB p65、MUC5AC mRNA相对表达量。结果与正常组比较，模型组肺组织IL-23、IL-17表达、血清及BALF中IL-17、IL-23水平、BALF白细胞计数、中性粒细胞、嗜酸性粒细胞比例、IL-17、IL-23、PI3K、AKT、NF-κB p65、MUC5AC mRNA表达、p-PI3K/PI3K、p-AKT/AKT、NF-κB p-p65/ NF-κB p65、MUC5A蛋白表达升高（P<0.05），BALF嗜碱性粒细胞、淋巴细胞及单核细胞比例下降（P<0.05）。与模型组比较，干预组肺组织IL-23、IL-17表达、BALF白细胞计数、中性粒细胞、嗜酸性粒细胞比例、IL-17、IL-23、PI3K、AKT、NF-κBp-65、MUC5AC mRNA表达、p-PI3K/PI3K、p-AKT/AKT、NF-κB p-p65/ NF-κB p65、MUC5AC蛋白表达降低（P<0.05），嗜碱性粒细胞比例升高（P<0.05）；MTCM、HTCM、布地奈德、联合组血清及BALF IL-17、IL-23水平下降（P<0.05）；LTCM、MTCM、HTCM、联合组BALF单核细胞比例升高（P<0.05）；MTCM及联合组BALF淋巴细胞比例升高（P<0.05）。联合组肺组织IL-23、IL-17表达、BALF嗜酸性粒细胞、中性粒细胞比例、p-PI3K/PI3K、MUC5AC蛋白表达较LTCM、MTCM、HTCM、布地奈德组不同程度降低（P<0.05）。结论运脾泻肺化痰汤可能通过IL-23/Th-17炎症轴抑制PI3K/AKT/NF-κB信号轴转导，通过降低血清及BALF中IL-23、IL-17水平，减少炎细胞迁移、气道炎性浸润及杯状细胞增生从而抑制MUC5AC过表达，减轻气道黏液高分泌及气道炎症。

英文摘要:

Objective To explore the effect of Yunpi Xiefei Huatan Decoction（YXHD） on airway inflammation and mucus hypersecretion in young asthmatic rats based on the correlation of interleukin（IL）-23/T helper cell（Th17） inflammation axis and phosphatidylinositol 3 kinase（PI3K）/protein kinase B（AKT）/nuclear factor kappa-B（NF-κB） signal axis. Methods Fifty-six young SD rats were randomly divided into 7 groups：normal group，model group，low-dose group of YXHD（LTCM），middle-dose group of YXHD（MTCM），high-dose group of YXHD（HTCM），budesonide group and combined group，8 rats in each group. Except for the normal group，rats in other groups were sensitized by intraperitoneal injection of a mixture of ovalbumin and aluminum hydroxide gel，and excited by nebulization of 1% ovalbumin. The rats in the normal group and the model group were given 0.9% sodium chloride 2 mL by gavage. The LTCM，MTCM and HTCM were gavaged with 10，20 and 40 g/kg YXHD，respectively. In the budesonide group，2 mL of 0.01% budesonide was atomized and inhaled for 20 min. The rats in the combined group were given intervention in MTCM and budesonide group. The intervention was performed for continuously 4 weeks. The different kind of white blood cells in bronchoalveolar lavage fluid（BALF） were counted in a classified manner. HE，periodic acid Schiff staining（PAS）were used to observe the pathological changes of lung tissues. The levels of IL-17 and IL-23 in BALF and serum were detected by Enzyme linked immunosorbent assay（ELISA）. The expression of IL-17 and IL-23 in the lung tissue was detected by immunohistochemistry（ICH）. The expressions of PI3K/AKT/NF-κB signaling pathway related proteins and mucin 5AC（MUC5AC） protein in the lung tissue were detected by Western Blot. The mRNA expression of IL-17，IL-23，PI3K，AKT，NF-κB p65 and MUC5AC in the lung tissue were detected by reverse transcription PCR（RT-PCR）. Results Compared with the normal group，the expressions of IL-23 and IL-17 in lung tissue，IL-17 and IL-23 in serum and BALF，BALF white blood cell count，proportion of neutrophils and eosinophils，IL-17，IL-23，PI3K，AKT，NF-κB p65，MUC5AC mRNA expressions，p-PI3K/PI3K，p-AKT/AKT，NF-κB p-p65/ NF-κB p65，and MUC5AC protein expressions increased（P<0.05），BALF proportion of basophils，lymphocytes and monocytes decreased in the model group（P<0.05）. Compared with model group，the expressions of IL-23，IL-17，BALF white blood cell count，proportion of neutrophils and eosinophils，IL-17，IL-23，PI3K，AKT，NF-κBp-65，MUC5AC mRNA，p-PI3K/PI3K，p-AKT/AKT，NF-κB p-p65/ NF-κB p65，MUC5AC proteins in the lung tissue decreased（P<0.05），BALF proportion of basophils increased in intervention group（P<0.05）. The levels of IL-17，and IL-23 in serum and BALF decreased in MTCM，HTCM，budesonide，and combined group（P<0.05）. BALF proportion of monocytes increased in LTCM，MTCM，HTCM and combined group（P<0.05）. BALF proportion of lymphocytes increased in MTCM and combined group（P<0.05）.The expression of IL-23 and IL-17 in lung tissue，BALF proportion of neutrophils and eosinophils，p-PI3K/PI3K，and MUC5AC protein expressions in the combined group were lower than those in the LTCM，MTCM，HTCM，and budesonide groups to different degrees （P<0.05）. Conclusions YXHD may inhibit PI3K/AKT/NF-κB signal axis transduction by IL-23/Th17 inflammatory axis. YXHD alleviated airway mucus hypersecretion and airway inflammation by reducing the levels of IL-23 and IL-17 in serum and BALF，inflammatory cell migration，airway inflammatory infiltration，goblet cell proliferation，and the overexpression of MUC5AC.

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