胸腺素β4在RAW264.7巨噬细胞炎症模型中的抗炎作用机制  

The anti-inflammatory mechanism of thymosinβ4 in a RAW 264.7 macrophage inflammation model

作　　者：吴天羽 林昱君 林笑宇 朱轶 余慕雪[1] 刘王凯[1,2] WU Tianyu;LIN Yujun;LIN Xiaoyu;ZHU Yi;YU Muxue;LIU Wangkai(Department of Neonatology,the First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080,China;Department of Pediatrics,Guangdong Maternal and Child Health Hospital,Guangzhou 511400,China)

机构地区：[1]中山大学附属第一医院新生儿科,广东广州510080 [2]广东省妇幼保健院儿科,广东广州511400

出　　处：《新医学》2025年第4期345-353,共9页Journal of New Medicine

基　　金：广东省自然科学基金(2022A1515010031)。

摘　　要：目的 探讨胸腺素β4(Tβ4)对脂多糖(LPS)诱导的小鼠单核-巨噬细胞RAW264.7极化趋向,以及其对炎症反应的影响。方法 用LPS诱导RAW264.7细胞建立炎症模型。采用CCK-8法检测细胞活力,酶联免疫吸附法检测细胞因子[肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)]的质量浓度;Griess法检测细胞培养上清液中一氧化氮(NO)的分泌情况;实时荧光定量聚合酶链反应(RT-qPCR)检测核因子-κB(NF-κB)、环氧酶-2(COX-2)、TNF-α、IL-6 mRNA表达水平;蛋白免疫印迹法检测诱导型一氧化氮合酶(iNOS)、NF-κB、磷酸化NF-κB(p-NF-κB)、NF-κB抑制蛋白α(IκB-α)和磷酸化IκBα(p-IκB-α)蛋白水平;荧光染色双标法观察巨噬细胞的极化状态;免疫荧光染色检测NF-κB定位及表达。结果 1 000μg/L的胸腺素β4作用于RAW264.7细胞24 h后,细胞存活率为90.2%,与空白对照组比较差异有统计学意义(P <0.05)。各质量浓度的Tβ4均可有效抑制NO的产生(均P <0.000 1)。Tβ4降低LPS诱导的炎症模型RAW264.7细胞中促炎因子(TNF-α和IL-6)质量浓度及NO浓度,下调NF-κB、COX-2、TNF-α、IL-6 mRNA表达水平和iNOS、p-NF-κB和p-IκBα蛋白表达水平,抑制NF-κB的核转移,降低CD80的表达(均P <0.05)。结论 Tβ4具有抑制炎症反应的作用,其机制可能与抑制NF-κB信号通路和巨噬细胞M1极化有关。Objective To explore the effects of Thymosinβ4(Tβ4)on the lipopolysaccharide(LPS)-induced polarization tendency of murine monocyte-macrophage RAW264.7 cells and its influence on inflammatory responses.Methods An inflammation model was established by LPS induction in RAW264.7 cells.Cell viability was assessed using the CCK-8 assay.The concentrations of cytokines[tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)]were detected by enzyme-linked immunosorbent assay(ELISA).Nitric oxide(NO)secretion in the cell culture supernatant was detected using the Griess method.Real-time quantitative polymerase chain reaction(RT-qPCR)was employed to detect mRNA expression levels of nuclear factor-κB(NF-κB),cyclooxygenase-2(COX-2),TNF-α,and IL-6.Western blotting was used to analyze protein levels of inducible nitric oxide synthase(iNOS),NF-κB,phosphorylated NF-κB(p-NF-κB),NF-κB inhibitorα(IκB-α),and phosphorylated IκBα(p-IκB-α).The polarization status of macrophages was observed using doublefluorescence staining.Immunofluorescence staining was performed to examine NF-κB localization and expression.Results After treatment with 1000μg/L Tβ4 for 24 hours,the cell viability of RAW 264.7 cells was 90.2%,showing a statistically significant difference compared to the blank control group(P<0.05).Tβ4 at various concentrations effectively inhibited NO production(all P<0.0001).Tβ4 decreased the concentrations of pro-inflammatory cytokines(TNF-αand IL-6)and NO in the LPS-induced RAW264.7 inflammatory model.It also downregulated mRNA expression of NF-κB,COX-2,TNF-α,and IL-6,as well as protein expression of iNOS,p-NF-κB,and p-IκBα.Additionally,Tβ4 inhibited NF-κB nuclear translocation and decreased CD80 expression(all P<0.05).Conclusion Tβ4 exhibits anti-inflammatory effects,the mechanisms of which may be associated with the inhibition of the NF-κB signaling pathway and suppression of macrophage M1 polarization.

关 键 词：胸腺素Β4 脓毒症 巨噬细胞 感染 炎症 NF-ΚB通路 

分 类 号：R392[医药卫生—免疫学]