木通皂苷D对神经酰胺诱导的小鼠急性肺损伤的保护作用研究

Protective effects of Akebia saponin D on ceramide-induced acute lung injury

目的探讨木通皂苷D(ASD)对神经酰胺诱导的肺上皮细胞凋亡及小鼠急性肺损伤的影响。方法将BEAS-2B细胞接种于96孔板,培养12 h后将细胞分为空白对照组、模型组和低、中、高剂量实验组。空白对照组加入等量二甲亚砜(DMSO);模型组加入40μmol·L^(-1)C6-神经酰胺;低、中、高剂量实验组分别加入50,100和200μmol·L^(-1)ASD,2 h后加入40μmol·L^(-1)C6-神经酰胺。用CCK-8检测细胞活力。实验小鼠随机分为对照组、模型组和实验组,每组5只。对照组和模型组小鼠腹腔注射给予0.9%NaCl;实验组小鼠每天腹腔注射给予300 mg·kg-1 ASD,连续给药7 d。末次给药后2 h,模型组和实验组直接气管滴入C6-神经酰胺建立急性肺损伤小鼠模型。用Bradford法检测肺泡灌洗液(BALF)蛋白含量。结果在细胞实验中,低、中、高剂量实验组和模型组、空白对照组的细胞活力分别为(72.21±4.14)%,(77.20±3.33)%,(88.61±1.30)%,(62.95±5.16)%和(100.00±0)%,低、中、高剂量实验组细胞活力与模型组比较,差异均有统计学意义(均P<0.05)。在动物实验中,实验组、模型组和对照组BALF中蛋白浓度分别为(0.46±0.13),(0.71±0.12)和(0.32±0.15)mg·mL^(-1),实验组BALF中蛋白浓度与模型组比较,差异有统计学意义(P<0.05)。结论ASD能够抑制肺上皮细胞凋亡,缓解神经酰胺诱导的急性肺损伤的发生发展。

Objective To investigate the effects of Akebia saponin D(ASD)on ceramide-induced apoptosis of lung epithelial cells and acute lung injury.Methods BEAS-2 B cells were exposed to 40μmol·L^(-1)of C6-ceramide to establish an in vitro airway epithelium injury model.BEAS-2 B cells were divided into five groups:the blank control group,the model group,the experimental-L group,the experimental-M group,and the experimental-H group.The model group was treated with 40μmol·L^(-1)of C6-ceramide.The experimental-L,-M,and-H groups were treated with 50μmol·L^(-1),100μmol·L^(-1),and 200μmol·L^(-1)of ASD,respectively,and then stimulated with 40μmol·L^(-1)of C6-ceramide.The blank control group cells were treated with an equal volume of dimethyl sulfoxide(DMSO).Cell viability was determined by cell counting Kit-8(CCK-8)assay.Apoptosis was determined by nuclear staining with Hoechst 33258.The acute lung injury murine model was established by using C6-ceramide intratracheal instillation.BALB/c mice were randomly divided into 3 groups:the control group,the model group,and the experimental group.The mice in the control group and the model group were treated with narmal saline.The mice in the experimental group were pretreated with 300 mg·kg-1 of ASD for 7 days.Protein concentrations in bronchoalveolar lavage fluid(BALF)were determined by the Bradford protein assay.Histological changes in lung tissues of the mice were observed by hematoxylin and eosin(HE)staining.Results For in vitro experiments,the cell viability in experimental-L,-M,and-H groups,the model group,and the blank control group were(72.21±4.14)%,(77.20±3.33)%,(88.61±1.30)%,(62.95±5.16)%,and(100.00±0)%,respectively.Compared between the three concentrations experimental groups and model group,the difference of the factors were significant(P<0.05).Morphological changes of nuclear condensation were attenuated in the three concentrations experimental groups compared with the model group.For in vivo experiments,the protein concentrations in bronchoalveolar lavage fluid(BALF)of the experimental group,the model group,and the control group were(0.46±0.13),(0.71±0.12)and(0.32±0.15)mg·m L^(-1),respectively.Compared between the experimental group and the model group,the difference of the factors were significant(P<0.05).Lung histopathological changes were also ameliorated in the experimental group compared with the model group.Conclusion ASD protects bronchial epithelial cells from ceramide-induced apoptosis and attenuates the development of acute lung injury.