健脾防哮方抑制哮喘小鼠气道上皮间质转化的研究

Inhibition of airway epithelial-mesenchymal transition by Jianpi Fangxiao decoction in asthmatic mice

目的探讨健脾防哮方对卵蛋白诱导的哮喘小鼠气道上皮间质转化的影响。方法将60只雄性Balb/c小鼠按照随机数字表法分为正常组、模型组、健脾防哮方低剂量组、健脾防哮方中剂量组、健脾防哮方高剂量组和地塞米松组,每组10只。适应饲养后,除正常组外,其余组均于第1,7,14天腹腔注射0.1 mL致敏液(生理盐水0.1 mL+卵蛋白0.05 mg+氢氧化铝1 mg),第21天开始予1%的卵蛋白雾化液(卵蛋白0.1 g+生理盐水10 mL)加压雾化激发4周建立哮喘模型。雾化激发前,正常组及模型组开始给予25 mL/(kg·d)蒸馏水灌胃,健脾防哮方低、中、高剂量组分别给予5.233 g/(kg·d)、10.465 g/(kg·d)、20.93 g/(kg·d)健脾防哮方灌胃,地塞米松组给予地塞米松0.5 mg/(kg·d)灌胃,均持续4周。实验结束后,苏木素-伊红(HE)染色观察肺组织病理形态学变化,Masson染色观察肺组织胶原沉积与纤维增生情况,免疫组化和Western blot法检测各组小鼠肺组织中E-钙黏素(E-cadherin)、N-钙黏素(N-cadherin)和α平滑肌肌动蛋白(α-SMA)表达情况,Real-time PCR法检测各组小鼠肺组织中E-cadherin、N-cadherin、α-SMA mRNA表达情况。结果与正常组比较,模型组小鼠支气管上皮细胞不完整,存在坏死且炎性细胞浸润明显,气道周围存在明显胶原纤维沉积;与模型组比较,各药物组小鼠病理改变均有不同程度减轻,胶原蛋白沉积明显减少,其中健脾防哮方高、中剂量组和地塞米松组改善更明显。与正常组比较,模型组小鼠肺组织中N-cadherin和α-SMA蛋白及mRNA表达量均明显增加(P均<0.05),E-cadherin蛋白及mRNA表达量均明显降低(P均<0.05);与模型组比较,各药物组小鼠肺组织中N-cadherin和α-SMA蛋白及mRNA表达量均明显降低(P均<0.05),健脾防哮方高、中剂量组和地塞米松组E-cadherin蛋白及mRNA表达量均明显增高(P均<0.05),且健脾防哮方高、中剂量组各指标表达变化较健脾防哮方低剂量组更明显(P均<0.05)。结论健脾防哮方可以减轻卵蛋白诱导的哮喘气道上皮损伤,抑制气道上皮间质转化。

Objective It is to explore the effect of Jianpi Fangxiao decoction on ovalbumin-induced airway epithelial-mesenchymal transition in asthmatic mice.Methods Sixty male Balb/c mice were randomly divided into normal group,model group,low-dose Jianpi Fangxiao decoction group,medium-dose Jianpi Fangxiao Decoction group,high-dose Jianpi Fangxiao Decoction group and dexamethasone group,10 mice in each group.After getting used to feeding,all the groups except for the normal group were given 0.1 mL sensitization solution(0.1 mL normal saline+0.05 mg egg albumin+1 mg aluminum hydroxide)by intraperitoneal injection on the 1st,7th,and 14th days,and were stimulated by 1%ovalbumin atomization solution(0.1 g ovalbumin+10 mL normal saline)by pressure atomization for 4 weeks to establish asthma models.Before the aerosol stimulation,the normal group and the model group were given distilled water 25 mL/(kg·d)by gavage,and the low-dose,medium-dose and high-dose groups of Jianpi Fangxiao decoction were respectively given Jianpi Fangxiao decoction 5.233 g/(kg·d),10.465 g/(kg·d)and 20.93 g/(kg·d)by gavage,and the dexamethasone group was given dexamethasone 0.5 mg/(kg·d)by gavage,all the groups were continuously treated for 4 weeks.After the experiment,the pathological changes of lung tissue was observed by hematoxylin-eosin(HE)staining,the collagen deposition and fibrosis in lung tissue was observed by Masson staining,and the expressions of E-cadherin,N-cadherin andα-smooth muscle actin(α-SMA)in lung tissue of the mice in each group was detected by immunohistochemistry and Western blot,the expression of E-cadherin and N-cadherin,α-SMA mRNA in lung tissue of the mice in each group was detected by Real-time PCR method.Results Compared with the normal group,the bronchial epithelial cells of the mice in the model group were incomplete,with necrosis and obvious inflammatory cell infiltration,and there was obvious collagen fiber deposition around the airway;compared with the model group,the pathological changes of the mice in each treatment group were alleviated to varying degrees,and the collagen deposition was significantly reduced,and the improvement was more obvious in the high-dose and medium-dose groups of Jianpi Fangxiao decoction and the dexamethasone group.Compared with the normal group,the protein and mRNA expressions of N-cadherin andα-SMA in the lung tissue of the mice in the model group were significantly increased(all P<0.05),while the protein and mRNA expressions of E-cadherin were significantly decreased(all P<0.05);compared with the model group,the protein and mRNA expressions of N-cadherin andα-SMA in the lung tissue of the mice in each treatment group were significantly decreased(all P<0.05),the expression of E-cadherin protein and mRNA in the high-dose and middle-dose groups of Jianpi Fangxiao decoction and the dexamethasone group were significantly increased(all P<0.05),and the expression changes of each index in the high-dose and middle-dose groups of Jianpi Fangxiao decoction were more obvious than those in the low-dose Jianpi Fangxiao decoction group(P<0.05).Conclusion Jianpi Fangxiao decoction can alleviate ovalbumin-induced airway epithelial damage and inhibit airway epithelial-mesenchymal transition in asthma.