本研究旨在分析苦参碱对肿瘤坏死因子-α(TNF-α)诱导的人脐静脉内皮细胞(HUVECs)炎症反应的影响,并探讨其机制是否与miR-25-3p介导的Krüppel样因子4(Klf4)通路有关。TNF-α刺激诱导HUVEC细胞炎症模型。用不同浓度的苦参碱(0.625、1.25和2.5mmol·L~(-1))孵育24或48小时后,CCK-8检测细胞增殖。用2.5mmol·L~(-1)苦参碱处理48h,TNF-α的表达,白细胞介素-6(IL-6),白细胞介素-1β(IL-1β),实时荧光定量PCR检测Klf4mRNA和miR-25-3p,和TNF-α的蛋白表达,IL-6,IL-1β,通过Westernblot检测Klf4。抗miR-25-3p转染HUVECs,用上述方法检测抗miR-25-3p对TNF-α诱导的细胞增殖和炎症因子的影响。再转染miR-25-3p,与苦参碱孵育,检测细胞增殖及相关炎症因子表达的变化,miR-25-3p,Klf4通过生物信息学分析和双荧光素酶报告基因检测验证miR-25-3p与Klf4的靶向关系。结果表明苦参碱可以抑制TNF-α诱导的HUVEC增殖,降低TNF-α的mRNA和蛋白表达,IL-6和IL-1β,增加Klf4的mRNA和蛋白表达,降低miR-25-3p的表达。生物信息学分析显示miR-25-3p与Klf4序列存在特异性互补结合位点。双荧光素酶报告基因测定证实miR-25-3p通过靶向负调控HUVECs中Klf4的表达。抑制miR-25-3p表达可降低TNF-α诱导的细胞增殖及TNF-αmRNA和蛋白表达,IL-6和IL-1β。MiR-25-3p过表达可逆转苦参碱对TNF-α诱导的细胞增殖及TNF-αmRNA和蛋白表达的影响,IL-6,IL-1β,Klf4本研究表明苦参碱通过miR-25-3p介导的Klf4通路抑制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-α stimulation. 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-α in HUVECs through miR-25-3p-mediated Klf4 pathway.