商陆皂苷甲对哮喘小鼠气道炎症的缓解作用及对肺组织中IL-6和STAT3表达水平的影响

Alleviation of esculentoside A on airway inflammation of asthmatic mice and its effect on expression levels of IL-6 and STAT3 in lung tissue

目的:探讨商陆皂苷甲(EsA)对卵清蛋白(OVA)诱导哮喘小鼠气道炎症的药理作用,并阐明其作用机制。方法:40只清洁级BALB/c雌性小鼠随机分为空白对照组、OVA组、低剂量EsA组和高剂量EsA组,每组10只;除空白对照组外,其他组小鼠用腹腔注射致敏,分别在第1、7和14天致敏,从第21天开始采用1%OVA激发,每1 d为1个激发周期,每个激发周期激发1次,共进行3次;空白对照组和OVA组小鼠应用生理盐水0.2 mL灌胃给药,低剂量EsA组和高剂量EsA组小鼠分别按照10和20 mg·kg-1灌胃给药,从第17天开始连续给药7 d,每天1次。采用HE染色法观察各组小鼠肺组织形态表现,直接计数法计算各组小鼠肺泡灌洗液(BALF)中总炎症细胞、嗜酸性粒细胞、中性粒细胞和淋巴细胞数量,流式细胞术检测BALF中嗜酸性粒细胞、白细胞介素4(IL-4)和干扰素γ(IFN-γ)水平,酶联免疫吸附测定(ELISA)法检测各组小鼠BALF中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)水平,Western blotting法检测各组小鼠肺组织中白细胞介素6(IL-6)和信号传导及转录激活蛋白-3(STAT3)蛋白表达水平,免疫组织化学法和免疫荧光法检测各组小鼠肺组织中IL-6和STAT3蛋白表达量。结果:与空白对照组比较,OVA组小鼠肺组织支气管和血管周围炎性细胞浸润,支气管部分损伤;与OVA组比较,低和高剂量EsA组小鼠肺组织中炎性细胞浸润明显减轻,气道损伤缓解。与空白对照组比较,OVA组小鼠BALF中总炎症细胞、嗜酸性粒细胞、中性粒细胞和淋巴细胞数量增加(P<0.05),IL-4、IL-1β和TNF-α水平升高(P<0.05或P<0.01),而IFN-γ水平降低(P<0.05);与OVA组比较,低和高剂量EsA组小鼠BALF中总炎症细胞、嗜酸性粒细胞、中性粒细胞和淋巴细胞数量降低(P<0.05),IL-4、IL-1β和TNF-α水平降低(P<0.05或P<0.01),而IFN-γ水平升高(P<0.05)。与空白对照组比较,OVA组小鼠肺组织中IL-6和STAT3蛋白表达水平明显升高(P<0.05);与OVA组比较,低和高剂量EsA组小鼠肺组织中IL-6和STAT3蛋白表达水平降低(P<0.05)。结论:EsA可缓解哮喘小鼠气道炎症,其作用机制与调节IL-6和STAT3信号通路有关。

Objective:To explore the pharmacological effect of esculentoside A(EsA)on the ovalbumin(OVA)-induced airway inflammation in the asthmatic mice,and to clarify its mechanism of action.Methods:Forty clean BALB/c female mice were randomly divided into blank control group,OVA group,low dose of EsA group and high dose of EsA group,with 10 mice in each group.They were sensitized by intraperitoneal injection and sensitization occurred on days 1,7,and 14,respectively.Starting from the 21st day with 1%OVA,and one excitation period was perfomed every day,with each excitation period being excited once for three times.The mice in blank control group and OVA group were given 0.2 mL of normal saline by gavage.The mice in low dose of EsA group and high dose of EsA group received 10 and20 mg·kg-1EsA by gavage,starting from the 17th day,for 7 consecutive days,once a day.HE staining method was used to observe the morphological performance of lung tissue of the mice in various groups,and direct counting method was used to calculate the number of total inflammatory cells,eosinophils,neutrophils and lymphocytes in BALF of the mice in various groups.Cytometry method was used to detect the expressions of eosinophils,IL-4 and IFN-γin BALF,and ELISA method was used to detect the levels of IL-1βand TNF-α;Western blotting method was used to determine the expression levels of IL-6 and STAT3 proteins in lung tissue of the mice in various groups;immunohistochemistry and immunofluorescence methods were used to detect the expressions of IL-6 and STAT3 proteins in lung tissue of the mice in various groups.Results:Compared with blank control group,the bronchi and perivascular inflammatory cells in lung tissue of the mice in OVA group were infiltrated with partial bronchial damage;compared with OVA group,the infiltration of inflammatory cells in lung tissue of the mice in low and high doses of EsA groups were reduced and airway injury was alleviated;compared with blank control group,the number of total inflammatory cells,eosinophils,neutrophils and lymphocytes in BALF of the mice in OVA group was increased(P<0.05),the expression levels of IL-4,IL-1βand TNF-αwere increased(P<0.01),while the expression level of IFN-γwas decreased(P<0.05);compared with OVA group,the number of total inflammatory cells,eosinophils,neutrophils and lymphocytes in BALF of the mice in low and high doses of EsA groups were decreased(P<0.05),the levels of IL-4,IL-1βand TNF-αwere decreased(P<0.01),and the levels of IFN-γwere increased(P<0.05).Compared with blank control group,the expression levels of IL-6 and STAT3 proteins in lung tissue of the mice in OVA group were significantly increased(P<0.05);compared with OVA group,the expression levels of IL-6and STAT3 proteins in lung tissue of the mice in low and high doses of EsA groups were decreased(P<0.05).Conclusions:EsA may alleviate the airway inflammation in the asthmatic mice,and its mechanism may be related to regulating the IL-6 and STAT3 signaling pathway.