摘要
基于体内动物实验、体外细胞实验和分子对接探讨牛蒡子苷元(arctigenin,ARG)对脂多糖(lipopolysaccharide,LPS)滴鼻致小鼠急性肺损伤(acute lung injury,ALI)模型及LPS诱导NP-69细胞损伤中的保护作用及机制。采用LPS滴鼻致小鼠鼻黏膜损伤,Western blot法检测小鼠鼻黏膜闭合蛋白(Occludin)、闭锁小带蛋白1(zonula occludens-1,ZO-1)的蛋白表达,检测小鼠肺泡灌洗液中炎症因子的含量,测定小鼠肺组织湿/干质量比评估肺水肿程度,HE染色法观察小鼠肺组织病理改变;采用LPS诱导NP-69细胞炎性损伤模型,检测细胞上清液中炎症因子的含量考察其抗炎作用,RT-PCR法检测ZO-1、人β-防御素3(human beta-defensin 3,HBD3)、白介素22受体亚基a1(interleukin-22 receptor subunit alpha-1,IL-22Ra1)、Janus激酶1(Janus kinase 1,JAK1)、酪氨酸激酶2(tyrosine kinase 2,Tyk2)、信号转导和转录激活因子1(signal transducer and activator of transcription 1,STAT1)mRNA的表达水平,Western blot法检测ZO-1、HBD3、IL-22Ral、JAK1、p-JAK1、Tyk2、STAT1、p-STAT1的蛋白表达情况;分子对接技术预测ARG与相关靶蛋白的结合能力。结果显示,ARG可显著减轻LPS诱导的小鼠ALI模型肺水肿和病理改变,降低炎症因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和白细胞介素-1β(interleukin-1β,IL-1β)的水平,上调小鼠鼻黏膜Occludin、ZO-1的蛋白表达。RT-PCR结果显示,ARG处理后,LPS诱导的体外上皮屏障破坏得到改善。在机制上,ARG上调了ZO-1、HBD3、IL-22Ra1、JAK1和TYK2的mRNA表达,上调ZO-1、HBD3、IL-22Ra1、JAK1、p-JAK1、p-STAT1、Tyk2的蛋白表达水平。分子对接实验表明,ARG的化学成分对TNF-α、JAK1和STAT1具有良好的亲和力。以上结果表明,ARG可以通过减轻炎症,促进鼻黏膜上皮屏障修复,减轻急性肺损伤,其机制与激活IL-22/JAK1/STAT1信号通路有关。
The effects of arctigenin(ARG)in acute lung injury(ALI)were investigated based on animal experiments in vivo,cell experiments in vitro,and molecular docking.The model of ALI in mice and NP-69 cell injury induced by lipopolysaccharide(LPS)were used.Western blot was used to detect the protein expression of Occludin and zonula occludens-1(ZO-1)in the nasal mucosa of mice after nasal injury induced by LPS.The levels of inflammatory factors in the mouse alveolar lavage fluid were detected by ELISA.The wet/dry mass ratio of mouse lung tissue was measured to evaluate the degree of pulmonary edema.Lung histopathological changes in mice were observed by HE staining.The inflammatory injury model of NP-69 cells induced by LPS was used to detect the content of inflammatory factors in the cell supernatant to investigate the anti-inflammatory effects of ARG.The mRNA expression levels of ZO-1,human beta-defensin 3(HBD3),interleukin-22 receptor subunit alpha-1(IL-22Ra1),Janus kinase 1(JAK1),tyrosine kinase 2(Tyk2),and signal transducer and activator of transcription 1(STAT1)were determined by RT-PCR.The protein expression of ZO-1,HBD3,IL-22Ra1,JAK1,p-JAK1,JAK1,Tyk2,STAT1 and p-STAT1 were detected by Western blot.Molecular docking techniques were used to predict the binding ability of ARG with related target proteins.The results showed that ARG significantly reduced the pulmonary edema and pathological changes in mice,reduced the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β),and upregulated the protein expression of Occludin and ZO-1 in the mouse nasal mucosa.RT-PCR results showed that the epithelial barrier disrupted by LPS was alleviated after ARG treatment in vitro.Mechanistically,ARG upregulated the mRNA ex pression of ZO-1,HBD3,IL-22Ra1,JAK1,and TYK2,identically upregulated the protein expression levels of ZO-1,HBD3,IL-22Ra1,JAK1,p-JAK1,p-STAT1,and Tyk2.Molecular docking experiments showed that ARG has good affinity with TNF-α,JAK1,and STAT1.The above results indicate that ARG cou
作者
朱紫陌
崔白梅
聂发龙
肖纯
李秀芳
ZHU Zi-mo;CUI Bai-mei;NIE Fa-long;XIAO Chun;LI Xiu-fang(Department of Pharmacology,Yunnan University of Chinese Medicine,Kunming 650500,China;Yunnan Institute of Traditional Chinese Medicine and Materia Medica,Kunming 650021,China)
出处
《天然产物研究与开发》
CAS
CSCD
北大核心
2024年第8期1298-1306,1287,共10页
Natural Product Research and Development
基金
国家自然科学基金(81860724)
云南省科技厅中医药基础研究联合专项(202001AZ070001-001)
云南省科技厅中医药基础研究联合专项(202101AZ070001-287)
云南省科技厅科技人才与平台计划(202305AC160043)。
关键词
急性肺损伤
牛蒡子苷元
鼻上皮屏障
分子机制
acute lung injury
arctigenin
nasal epithelial barrier
molecular mechanism
