摘要
目的探讨不同半夏炮制品体外抗肺纤维化的作用及筛选出的半夏制品对肺纤维化小鼠的干预作用。方法用10μg/L转化生长因子β1(TGF-β1)诱导人胚肺成纤维细胞系(MRC-5)建立肺纤维化体外模型,经水提生半夏(2.1、4.2、8.4、16.7 g/L)、清半夏(2.1、4.2、8.4、16.7 g/L)、法半夏(2.1、4.2、8.4、16.7 g/L)和姜半夏(2.1、4.2、8.4、16.7 g/L)分别处理48 h后,通过CCK-8法检测细胞活性、天狼星红染色法测定胶原含量进行抗纤维化筛选。用Western blot分析筛选出的生半夏干预MRC-5细胞表达α-平滑肌肌动蛋白(α-SMA)的情况。C57BL/6N小鼠经口腔进行气管滴注博来霉素(2 mg/kg)建立肺纤维化模型,第14天开始灌胃生半夏水提液(2.5、5.0、10.0 g/kg),第28天取肺组织经HE和Masson染色观察,并测定羟脯氨酸(HYP)含量。结果与对照组比较,TGF-β1诱导后的MRC-5细胞对生半夏(8.4 g/L)的细胞活性抑制作用更敏感(P<0.05),而未经TGF-β1诱导的MRC-5细胞则需要更高浓度的生半夏或清半夏(16.7 g/L)抑制细胞活性(P<0.05)。与模型对照组比较,生半夏(8.4、16.7 g/L)能够抑制TGF-β1诱导的胶原合成(P<0.05),清半夏、法半夏、姜半夏各组未见抑制效应。与模型对照组比较,生半夏(2.1、4.2、8.4 g/L)能够抑制TGF-β1诱导的MRC-5细胞中α-SMA蛋白的表达(P<0.05)。生半夏(10 g/kg)能够缓解肺纤维化模型小鼠肺间质中的炎性浸润,恢复肺泡结构,减少胶原沉积,降低肺组织中的HYP含量(P<0.05)。结论生半夏对TGF-β1诱导后的病理性肌成纤维细胞具有活性抑制作用,并能阻止成纤维细胞向肌成纤维细胞分化,抑制其胶原合成,缓解模型小鼠的肺纤维化进展,为临床应用半夏治疗肺纤维化提供新的思路。
Objective To explore the effects of different Pinelliae Rhizoma products on the intervention of pulmonary fibrosis.Methods Using transforming growth factorβ1(10μg/L)induced human embryonic lung fibroblast cell line MRC-5 to establish an in vitro model of pulmonary fibrosis then treated with waterextracts of Pinelliae Rhizoma(PR)or Pinelliae Rhizoma Praeparatum cum Alumine(PRPCA)or Pinelliae Rhizoma Praeparatum cum Zingibereet Alumine(PRPCZEA)or Pinelliae Rhizoma Praeparatum(PRP).The working concentration(PR,PRPCA,PRPCZEA,PRP)equivalent tothe crude herb was 2.1,4.2,8.4,16.7 g/L respectively.After 48 hours,the cell activity was detected by CCK-8 method and the collagen content was determined by Sirius red staining method.After screening,the expression ofα-SMA protein inmodeltreated with PRwas assessed using Western blot.In vivo,C57 BL/6 N mice were exposed to bleomycin then treated with water extract of PR(2.5,5.0,10.0 g/kg)on day 14.On the 28 th day,the lung tissue was observed by he and Masson staining,and the content of hydroxyproline(Hyp)was measured.Results Compared with the control group,16.7 g/L of PR or PRPCA inhibited the cell viability of MRC-5(P<0.05),while 8.4 g/L of PR can selectively suppress the cell viability of MRC-5 after TGF-β1 stimulation(P<0.05).Compared with the model group,PR(8.4,16.7 g/L)reduced the synthesis of collagen in MRC-5 cells induced by TGF-β1(P<0.05),but PRPCA,PRPCZEA and PRP groups had no statistically significant difference in inhibitory effects on collagen.Western blotanalysis showed that PR(2.1、4.2、8.4 g/L)decreased the expression ofα-SMA in MRC-5 cells induced by TGF-β1 compared with the model group(P<0.05).In vivo experiments,the bleomycin-treated mice receiving PR(10 g/kg)displayed less collagen deposition in the lung interstitium,less inflammatory infiltration,and more restored alveolar structure compared with mice only receiving bleomycin.PR of 10 g/kg reversed HYP level in lung tissue after bleomycin challenge(P<0.05).Conclusion PR reduces cell viability of pa
作者
贺双双
李彧
张彬彬
魏树全
申萌萌
林家伊
万熙
张澜
李天罡
史渊源
李亚东
HE Shuangshuang;LI Yu;ZHANG Binbin;WEI Shuquan;SHEN Mengmeng;LIN Jiayi;WAN Xi;ZHANG Lan;LI Tiangang;SHI Yuanyuan;LI Yadong(School of Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China;School of Life Sciences,Beijing University of Chinese Medicine,Beijing 100029,China;Department of Pulmonary and Critical Care Medicine,Guangzhou First People’s Hospital,Guangzhou 510180,China)
出处
《北京中医药大学学报》
CAS
CSCD
北大核心
2022年第3期275-283,共9页
Journal of Beijing University of Traditional Chinese Medicine
基金
国家重点研发计划项目(No.2018YFC1705502)
国家自然科学基金项目(No.81202787)
国家中医药管理局中医药创新团队及人才支持计划项目(No.ZYYCXTD-C-202006)
北京市自然科学基金会项目(No.7212177)
北京中医药大学科研创新团队项目(No.2019-JYB-TD-006)。
