基于miRNA测序探究重楼皂苷Ⅰ抗炎的关键因子  

Probing the key anti-inflammatory factors of polyphyllinⅠbased on miRNA sequencing

作　　者：郑星宇 黄慧敏[2] 李志浩 黄慧琳[2] 武伦 刘经健 杨旺 柯昌虎 李鹏[1,2] ZHENG Xingyu;HUANG Huimin;LI Zhihao;HUANG Huilin;WU Lun;LIU Jingjian;YANG Wang;KE Changhu;LI Peng(School of Pharmaceutical Sciences,Hubei University of Medicine,Hubei Shiyan 442000,China;Department of Pharmacy,Sinopharm Dongfeng General Hospital,Hubei University of Medicine,HubeiShiyan 442008,China;Hubei Key Laboratory for Wudang Local Chinese Medicine Research,Hubei Shiyan 442000,China)

机构地区：[1]湖北医药学院药学院,湖北十堰442000 [2]湖北医药学院附属国药东风总医院药学部,湖北十堰442008 [3]武当特色中药研究湖北省重点实验室,湖北十堰442000

出　　处：《中国医院药学杂志》2025年第5期554-560,575,共8页Chinese Journal of Hospital Pharmacy

基　　金：湖北省中医药管理局2023~2024年度中医药科研项目(编号:ZY2023F075);“十四五”湖北省高等学校优势特色学科群(生物与医药)项目(编号:2023BMXKQT6);武当特色中药研究湖北省重点实验室(湖北医药学院)开放课题(编号:WDCM2022012)。

摘　　要：目的:基于微小RNA(microRNA,miRNA)高通量测序技术,旨在探索重楼皂苷Ⅰ(polyphyllinⅠ,PPⅠ)对脂多糖(lipopolysaccharide,LPS)诱导的人髓系白血病单核细胞(THP-1)巨噬细胞炎症响应中关键miRNA的作用和调控机制。方法:LPS刺激构建THP-1巨噬细胞炎症模型;CCK-8法检测PPⅠ对THP-1巨噬细胞活性的影响;ELISA试剂盒检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的释放。对PPⅠ给药组和未给药模型组进行miRNA测序;实时荧光定量PCR(qRT-PCR)对6个差异miRNA进行验证;加权共基因表达网络分析确定最相关的共表达模块;LASSO回归分析进一步确定关键miRNAs;通过GO和KEGG分析差异miRNAs的功能和其潜在抗炎意义。结果:PPⅠ1~8μmol·L^(-1)质量浓度范围内对THP-1巨噬细胞无毒性,2、4、6μmol·L^(-1)PPⅠ处理LPS诱导的THP-1细胞后,TNF-α、IL-6的分泌量显著下降。共鉴定出836个差异表达miRNAs,其中398个miRNA上调,438个miRNA下调;qRT-PCR结果显示,miR-11401、miR-372-3p、miR-4494、miR-8084、miR-445-3p、miR-6744-3p表达与miRNA测序结果一致。加权共基因表达网络分析共获得16个共表达模块,其中暗橘色共表达模块与PPⅠ干预治疗LPS诱导的巨噬细胞炎症呈显著负相关(r=-0.99,P<0.01);通过LASSO回归分析筛选到4个miRNA(miR-6881-3p、miR-5583-3p、miR-570-3p和miR-4757-3p)。差异miRNA靶基因主要与炎症代谢通路相关。结论:重楼皂苷Ⅰ通过调节多种miRNA在治疗LPS诱导的THP-1巨噬细胞炎症中发挥作用。OBJECTIVE To investigate the key miRNAs of polyphyllinⅠ(PPⅠ)involved in lipopolysaccharide(LPS)-induced inflammation in human myeloid leukemia monocytic(THP-1)macrophages based on high-throughput sequencing of microRNAs(miRNAs).METHODS An in vitro LPS-induced macrophage inflammation model in THP-1 cells was con⁃structed.The influence of PPⅠon the activity of THP-1 macrophages was examined by cell counting kit-8(CCK-8)assay.Enzyme-linked immunosorbent assay(ELISA)was performed to detect the release of tumor necrosis factor-alpha(TNF-α),and interleukin-6(IL-6).MiRNA sequencing was performed in LPS-induced THP-1 cells either treated with PPⅠor not,and the identified six differentially expressed miRNAs were validated by quantitative real-time polymerase chain reaction(qRT-PCR).The weighted gene co-expression network analysis(WGCNA)identified the most relevant co-expressed modules.LASSO regression analysis further identified key miRNAs.The biological function and anti-inflammatory property of differentially expressed miRNAs were predicted by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrich⁃ment analyses.RESULTS PPⅠtreatment at the concentrations of 1-8μmol·L^(-1) was non-toxic to THP-1 macrophages.Secretion of TNF-αand IL-6 significantly decreased after the treatment of 2,4 and 6μmol·L^(-1) PPⅠtreatment in LPS-induced THP-1 cells.A total of 836 differentially expressed miRNAs were identified,of which 398 miRNAs were up-regulated and 438 were down-regulated.The qRT-PCR further validated the differential expressions of miR-11401,miR-372-3p,miR-4494,miR-8084,miR-445-3p and miR-6744-3p screened by miRNA sequencing.WGCNA yielded a total of 16 co-expressed modules,of which the dark orange module showed a significant negative correlation with the anti-inflammatory property of PPⅠagainst LPS-induced macrophage inflammation(r=-0.99,P<0.01).Four miRNAs,including miR-6881-3p,miR-5583-3p,miR-570-3p,analysis.Target genes of differentially expressed miRNAs were mainly asso⁃ciated wi

关 键 词：重楼皂苷Ⅰ THP-1细胞 miRNA测序 炎症 

分 类 号：R932[医药卫生—生药学]