摘要
目的探究敬钊缨毛蛛肽类毒素JZTX-V中的多肽片段对巨噬细胞炎症反应的抑制作用,并探究相关机制。方法通过免疫印迹法检测来自JZTX-V的3个多肽片段对脂多糖(lipopolysaccharide,LPS)刺激的RAW 264.7细胞中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达的作用;通过Griess反应检测细胞上清液中一氧化氮(nitric oxide,NO)浓度,通过异鲁米诺化学发光检测超氧化物的产生量;通过实时荧光定量PCR检测细胞中促炎细胞因子mRNA的表达量;通过免疫印迹法检测JZ-VF3对丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和核因子-κB(nuclear factor kappa-B,NF-κB)信号通路的影响,并用免疫荧光检测JZ-VF3对p65的核转位的影响。结果与LPS刺激组相比,用JZ-VF3处理RAW 264.7细胞可以显著抑制iNOS的激活及NO和超氧化物的产生,还可以抑制细胞中促炎细胞因子的产生。蛋白免疫印迹和免疫荧光实验揭示,JZ-VF3可以抑制MAPK和NF-κB信号通路的激活,以及p65的核转位。结论JZTX-V中的JZ-VF3多肽片段通过抑制MAPK和NF-κB信号通路的激活,从而抑制LPS诱导的RAW 264.7细胞中的炎症反应,具有成为多肽抗炎药的潜力。
Objective To investigate the inhibitory effects of the JZTX-V peptide toxin fragments on inflammatory responses in macrophages and explore the underlying mechanisms.Methods Western blot was used to examine the effects of three JZTX-V peptide fragments on lipopolysaccharide(LPS)-induced inducible nitric oxide synthase(iNOS)expression in RAW 264.7 cells.The nitric oxide(NO)concentration in the cell culture supernatant was measured using the Griess reaction,and the production of superoxide was detected by chemiluminescence assay using luminol.Real-time quantitative PCR was employed to measure the expression levels of pro-inflammatory cytokine mRNA in the cells.Western blot was also used to investigate the effects of JZ-VF3 on the mitogen-activated protein kinase(MAPK)and nuclear factor kappa-B(NF-κB)signaling pathways,and immunofluorescence was used to assess the impact of JZ-VF3 on the nuclear translocation of p65.Results Compared to the LPS-stimulated group,treatment with JZ-VF3 significantly inhibited iNOS activation and the production of NO and superoxide in RAW 264.7 cells.It also suppressed the production of pro-inflammatory cytokines in the cells.Western blot and immunofluorescence experiments revealed that JZ-VF3 could inhibit the activation of the MAPK and NF-κB signaling pathways,as well as the nuclear translocation of p65.Conclusion The JZ-VF3 peptide fragment in JZTXV inhibits inflammatory responses in LPS-induced RAW 264.7 cells by suppressing the activation of the MAPK and NF-κB signaling pathways,demonstrating potential as a peptide anti-inflammatory agent.
作者
刘欣悦
钱峰
LIU Xinyue;QIAN Feng(School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240,China)
出处
《石河子大学学报(自然科学版)》
CAS
北大核心
2024年第5期614-620,共7页
Journal of Shihezi University(Natural Science)
基金
国家自然科学基金项目(82373875)。