平喘方通过上调哮喘小鼠肺泡灌洗液中miR-23b的表达改善气道重塑的研究

Pingchuan Formula Improves Airway Remodeling by Upregulating miR-23b Expression in BALF of Asthmatic Mice

研究平喘方通过上调外泌体miR-23b干预TGF-β/EMT,改善OVA诱导的哮喘小鼠气道重塑的内在机制。将40只BALB/c雄性小鼠,按照随机数字表分为空白组、哮喘模型组、平喘方组、地塞米松组,每组10只。第1 d、14 d模型组、平喘方组、地塞米松组腹腔注射10%OVA+铝镁佐剂致敏,第21~49 d隔日运用2.5%OVA雾化激发,并每日灌胃给药,连续给药28 d,末次给药24 h后取材。苏木素-伊红(HE)染色及马松(Masson)染色观察各组小鼠肺组织病理学变化,透射电镜下观察肺泡灌洗液(BALF)中的外泌体形态,免疫印迹法(Western blot)检测外泌体表面标记蛋白CD9、CD63、CD81含量,实时荧光定量聚合酶链式反应(Real-Time PCR)检测BALF外泌体中miR-23b mRNA水平,免疫印迹法(Western blot)检测转化生长因子β1(TGF-β1)、平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)、E-钙黏蛋白(E-cadherin)、紧密连接蛋白1(ZO-1)蛋白含量。各组小鼠BALF中提取外泌体均表达CD9、CD63、CD81蛋白,证明本研究成功提取外泌体。与空白组比较,病理染色见模型组小鼠支气管周围炎性细胞浸润,气道管壁肌层增厚,胶原纤维增多,外泌体中miR-23b mRNA水平显著降低,肺组织中Collagen Ⅰ、Collagen Ⅱ、TGF-β1、α-SMA、Vimentin蛋白水平显著升高(P<0.01),E-cadherin、ZO-1蛋白水平显著降低(P<0.01);与模型组比较,平喘方组与地塞米松组病理损伤较轻,未见明显炎性细胞浸润,气道胶原纤维减少,外泌体中miR-23b mRNA水平显著升高(P<0.01),Collagen Ⅰ、Collagen Ⅱ、TGF-β1、α-SMA、Vimentin蛋白水平明显下调(P<0.01,P<0.05),E-cadherin、ZO-1蛋白水平显著升高(P<0.01,P<0.05)。平喘方可以改善哮喘小鼠气道重塑,其机制可能与上调外泌体miR-23b表达,进而抑制TGF-β/EMT信号轴,缓解气道重塑有关。

To investigate the mechanism of Pingchuan formula’intervention in TGF-β/EMT by upregulating exosome miR-23b and its improvement in OVA-induced airway remodeling in asthmatic mice,40 BALB/c male mice were divided into blank group,asthma model group,Pingchuan fumula group,and dexamethasone group according to the random number table,with 10 mice in each group.The 1 d and 14 d model group,the Pinchuan formula group,and the dexamethasone group were sensitized by intraperitoneal injection of 10%OVA+aluminum-magnesium adjuvant,stimulated by atomization of 2.5%OVA every other day from the 21 d to 49 d,and were given daily intragastric administration for 28 d,and samples were taken 24 h after the last administration.Blank group and model group were given the same amount of normal saline intragastric administration,and samples were taken 24 h after the last administration.Hematoxylin-eosin(HE)and Masson staining were used to observe the lung histopathological changes of mice in each group.The morphology of exosomes in alveolar lavage fluid(BALF)was observed under transmission electron microscopy,and the contents of exosome surface marker proteins CD9,CD63 and CD81 were detected by Western blot.Real-Time fluorescence quantitative polymerase chain reaction(Real-Time PCR)was used to detect miR-23b mRNA levels in BALF exosomes.The contents of transforming growth factorβ1(TGF-β1),smooth muscle actin(α-SMA),Vimentin,E-cadherin and compact connectin 1(ZO-1)were detected by Western blot.CD9,CD63 and CD81 proteins were expressed in the exosomes extracted from BALF of mice in all groups,which proved that the exosomes were successfully extracted in this study.Compared with the blank group,pathological staining showed that the model group of mice had inflammatory cell infiltration around the bronchus,thickened muscle layer of airway wall,and increased collagen fibers.The mRNA level of miR-23b in exosomes was significantly decreased(P<0.01).The protein levels of Collagen Ⅰ,Collagen Ⅱ,TGF-β1,α-SMA and Vimentin in lung tissues were significantly increased(P<0.01),while the protein levels of E-cadherin and ZO-1 were significantly decreased(P<0.01).Compared with model group,the pathologic injury of Pingchuan formula group and dexamethasone group was mild,no obvious inflammatory cell infiltration was observed,airway collagen fibers were decreased,and the level of miR-23b mRNA in exosomes was significantly increased(P<0.01).The protein levels of Collagen Ⅰ,Collagen Ⅱ,TGF-β1,α-SMA and Vimentin were significantly decreased(P<0.01,P<0.05),while the protein levels of E-cadherin and ZO-1 were significantly increased(P<0.01,P<0.05).Pingchuan formula can improve airway remodeling in asthmatic mice,and its mechanism may be related to up-regulation of exosomal miR-23b expression,thereby inhibiting TGF-β/EMT signal axis and improving airway remodeling.