摘要
研究苦参碱对肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)炎症反应的影响,探讨其机制是否与miR-25-3p介导Krüppel样转录因子4(Krüppel like transcription factor 4,Klf4)途径有关。以TNF-α诱导建立HUVECs炎症模型。不同浓度苦参碱(0.625、1.25、2.5 mmol·L^(-1))作用24、48 h后,CCK-8方法检测细胞增殖。2.5 mmol·L^(-1)苦参碱处理48 h后采用实时荧光定量PCR检测TNF-α、白细胞介素-6(interleukin-6,IL-6)和白细胞介素-1β(interleukin-1β,IL-1β)、Klf4 mRNA表达和miR-25-3p表达,免疫印迹法检测TNF-α、IL-6、IL-1β、Klf4蛋白表达。转染anti-miR-25-3p至HUVECs,上述方法检测miR-25-3p对TNF-α诱导的细胞增殖及相关炎症因子的表达。进一步转染miR-25-3p至细胞,与苦参碱共同孵育,检测增殖及炎症因子、miR-25-3p和Klf4表达的变化。生物信息学分析和双荧光素酶报告基因实验验证miR-25-3p与Klf4靶向关系。结果显示,苦参碱能抑制TNF-α诱导的HUVECs增殖,降低TNF-α、IL-6、IL-1βmRNA和蛋白表达,提高Klf4 mRNA和蛋白表达、降低miR-25-3p表达;生物信息学分析显示,miR-25-3p与Klf4的序列存在特异性互补结合位点,双荧光素酶活性报告实验证实miR-25-3p在HUVECs中靶向负调控Klf4表达;抑制miR-25-3p表达可降低TNF-α诱导的细胞增殖、TNF-α、IL-6、IL-1βmRNA和蛋白表达;过表达miR-25-3p能够逆转苦参碱对TNF-α诱导的细胞增殖及TNF-α、IL-6、IL-1β、Klf4 mRNA和蛋白表达的作用。实验结果表明苦参碱通过miR-25-3p介导Klf4途径抑制TNF-α诱导的HUVECs炎症反应。
This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs)and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4)pathway.The HUVEC cell inflammation model was induced by TNF-αstimulation.After 24 or 48 hours of incubation with different concentrations of matrine(0.625,1.25,and 2.5 mmol·L^(-1)),CCK-8 assay was used to detect cell proliferation.After treatment with 2.5 mmol·L^(-1)matrine for 48 h,the expression of TNF-α,interleukin-6(IL-6),interleukin-1β(IL-1β),and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR,and the protein expression of TNF-α,IL-6,IL-1β,and Klf4 was detected by Western blot.The anti-miR-25-3p was transfected into HUVECs,and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method.The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors,miR-25-3p,and Klf4.The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay.The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation,decrease the mRNA and protein expression of TNF-α,IL-6,and IL-1β,increase the mRNA and protein expression of Klf4,and reduce the expression of miR-25-3p.Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences.Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting.The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α,IL-6,and IL-1β.MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α,IL-6,IL-1β,and Klf4.This study shows that matrine inhibits the inflammatory response induced by TNF-αin HUVECs through miR-25-3p-mediated Klf4 pathway.
作者
向紫萍
李妍洁
马欢
王杏
张会欣
王超
XIANG Zi-ping;LI Yan-jie;MA Huan;WANG Xing;ZHANG Hui-xin;WANG Chao(Graduate School,North China University of Science and Technology,Tangshan 063210,China;Key Laboratory of Metabolic Disease,Hebei General Hospital,Shjiazhuang 050051,China;Chemical Engineering Institute,Shijazhuang University,Shijiazhuang 050035,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2023年第17期4731-4737,共7页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81370900)
河北省中医药科研计划项目(2023005)
石家庄学院博士科研启动基金项目(22BS002)。
作者简介
向紫萍,硕士,研究方向为代谢病,E-mail:2831370747@qq.com;通信作者:张会欣,正高级工程师,研究方向为心血管药理,E-mail:hxzhang76@163.com;通信作者:王超,研究员,研究方向为代谢病,E-mail:cwyx163@163.com。
