摘要
目的 探究补肺活血胶囊对小鼠肺纤维化的影响及作用机制.方法 90 只C57BL/6J小鼠随机分为对照组、模型组、NLRP3 抑制剂(MCC950,10 mg/kg)组、补肺活血胶囊(0.32、0.63、1.62 g/kg、)组,每组 15 只小鼠.通过对小鼠气管内单次注射 2.5 mg/kg的博来霉素来诱导肺纤维化,对照组小鼠接受单次气管注射等体积的磷酸缓冲盐溶液(PBS).造模成功后第 1 天开始给药,持续给药 21 d.统计小鼠每天的存活率和体质量,采集小鼠肺泡灌洗液(BALF)并测定总细胞与总蛋白,测定小鼠肺组织湿干质量比(W/D)与羟脯氨酸(HYP);采用苏木精-伊红(HE)染色和Masson染色观察肺组织病理学变化,采用qRT-PCR检测上皮间质转化(EMT)相关蛋白[E-钙黏蛋白(E-cad)、波形蛋白(Vim)、α-平滑肌肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)]的mRNA表达;采用Western blotting检测EMT相关蛋白以及Nod样受体蛋白 3(NLRP3)通路相关蛋白[NLRP3、凋亡相关斑点样蛋白(ASC)、mature-半胱氨酸天冬氨酸蛋白酶(Caspase-1)、mature-白细胞介素(IL)-1β、mature-IL-18]表达.结果 相较于模型组,MCC950 与补肺活血胶囊干预后肺纤维化小鼠存活率明显提高,体质量明显增加,肺组织W/D、BALF总细胞与总蛋白、HYP含量均明显降低(P＜0.05、0.01);肺组织病理形态明显改善;肺组织上皮细胞标志物E-cad mRNA及蛋白的表达增加,间质细胞标志物Vim、α-SMA、TGF-β1 mRNA和蛋白的表达下降,NLRP3 信号通路中相关蛋白表达水平降低(P＜0.05、0.01).结论 补肺活血胶囊能通过抑制NLRP3 通路介导的EMT改善博来霉素诱导的肺纤维化程度.
Abstract
Objective To investigate the effect and mechanism of Bufei Huoxue Capsules on pulmonary fibrosis in mice.Methods A total of 90 C57BL/6J mice was randomly divided into control group,model group,NLRP3 inhibitor(MCC950,10 mg/kg),and Bufei Huoxue Capsules(0.32,0.63,and 1.62 g/kg)group,each group had 15 mice.Mice were given a single intratracheal injection of 2.5 mg/kg bleomycin to induce pulmonary fibrosis,mice in the control group received a single intratracheal injection of an equal volume of PBS.The drug was administered first day after successful modeling,and the whole administration process lasted for 21 d.The survival rate and body weight of mice were counted.Bronchoalveolar lavage fluid(BALF)was collected and the total cell count and total protein were determined.The wet/dry weight ratio(W/D)and hydroxyproline(HYP)of lung tissue were measured.The pathological changes of lung tissue were observed by hematoxylin and eosin(HE)staining and Masson trichrome staining.qRT-PCR was used to detect the mRNA expression of EMT-related proteins(E-cad,VIM,α-SMA,and TGF-β1).Western blotting was used to detect the expression of EMT-related proteins and NLRP3 pathway-related proteins(NLRP3,ASC,mature-Caspase-1,mature-IL-1β,mature-IL-18).Results Compared with the model group,the survival rate of pulmonary fibrosis mice after MCC950 and Bufei Huoxue Capsules intervention was significantly improved,the body weight was significantly increased,the W/D of lung tissue,the total cell count and total protein and HYP content of BALF were significantly decreased(P<0.05,0.01),the pathological morphology of lung tissue was significantly improved,the expression of epithelial cell marker E-cad mRNA and protein in lung tissue was increased,the expression of mesenchymal cell marker Vim,α-SMA,TGF-β1 mRNA and protein was decreased,and the expression of related proteins in NLRP3 signaling pathway was decreased(P<0.05,0.01).Conclusion Bufei Huoxue Capsules can improve bleomycin-induced pulmonary fibrosis in mice by inhibiting NLRP3 pathway-mediated EMT.
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