参附注射液对LPS诱导的RAW264.7细胞HMGB1核转位的干预作用
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篇名: | 参附注射液对LPS诱导的RAW264.7细胞HMGB1核转位的干预作用 |
TITLE: | Intervention Effect of Shenfu Injection on the Nuclear Translocation of HMGB 1 in LPS-induced RAW 264.7 Cells |
摘要: | 目的:探讨参附注射液(SFI)对脂多糖(LPS)诱导的巨噬细胞中高迁移率族蛋白B1(HMGB1)核转位的干预作用。方法:以经LPS诱导的小鼠单核巨嗜细胞RAW264.7为对象,以组蛋白去乙酰化酶抑制剂RGFP966为阳性对照,在CCK-8法筛选给药剂量的基础上,采用免疫荧光法观察低、中、高剂量(3、6、12μL/mL)SFI对细胞中HMGB1核转位的影响,采用实时荧光聚合酶链式反应法检测细胞中HMGB1mRNA的表达情况;采用Westernblotting法检测细胞中HMGB1、Toll样受体4(TLR4)的表达情况,并比较细胞胞核、胞浆中HMGB1的表达情况;采用酶联免疫吸附测定法检测细胞上清液中HMGB1、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)水平。结果:在空白对照组中,HMGB1主要定位于细胞核;经LPS诱导后,HMGB1由细胞核向胞浆迁移。与空白对照组比较,LPS组细胞中HMGB1mRNA及其蛋白、TLR4的蛋白表达量以及上清液中HMGB1、IL-1β、TNF-α水平均显著升高(P<0.01);细胞胞核中HMGB1的蛋白表达量显著降低,而胞浆中HMGB1的蛋白表达量显著升高(P<0.01)。经SFI作用后,各给药组细胞HMGB1的核移位及分泌均受到不同程度的抑制;与LPS组比较,各给药组细胞中HMGB1mRNA及其蛋白的表达量,阳性对照组和SFI中、高剂量组细胞中TLR4的蛋白表达量以及各给药组细胞上清液中HMGB1、IL-1β、TNF-α水平均显著降低(P<0.01);阳性对照组和SFI中、高剂量组细胞胞核中HMGB1的蛋白表达量均显著升高,而各给药组细胞胞浆中HMGB1的蛋白表达量均显著降低(P<0.01)。结论:SFI可能通过抑制RAW264.7细胞中HMGB1的核移位及分泌出胞,避免炎症通路的激活和炎症因子的产生,从而发挥减轻LPS致炎症反应的作用。 |
ABSTRACT: | OBJECTIVE:To investigate the intervention effect of Shenfu i njection(SFI)on the nuclear translocation of high mobility group box 1(HMGB1) in lipopolysaccharide (LPS)-induced RAW 264.7 cells. METHODS : Using LPS-induced RAW264.7 cells as objects ,the histone deacetylase inhibitor RGFP 966 as positive control ,CCK-8 assay was used to screen drug dosage,and the effects of low ,medium and high doses (3,6,12 μL/mL)of SFI on HMGB 1 nuclear translocation in RAW 264.7 cells were observed by immunofluorescence method ;mRNA expression of HMGB 1 in RAW 264.7 cells were detected by real time fluorescent PCR. Western blotting assay was used to determine protein expression of HMGB 1 and Toll-like receptor 4(TLR4);the expression of HMGB 1 were compared between nucleus and cytoplasm. The levels of HMGB 1,IL-1β and TNF-α in supernatant of cells were detected by ELISA. RESULTS :In blank control group ,HMGB1 was mainly located in the nucleus ;after LPS induction, HMGB1 migrated from nucleus to cytoplasm. Compared with blank control group , mRNA and protein (No.81760738) expression of HMGB 1, protein expression of TLR 4 in RAW264.7 cells as well as the levels of HMGB 1,IL-1β and TNF-α in supernatant of cells were increased significantly in LPS group (P<0.01). The protein expression of HMGB 1 was decreased significantly in nucleus while was in creased significantly in cytoplasm (P<0.01). After SFI treatment ,the nuclear translocation and secretion of HMGB 1 were inhibited in different degrees ;compared with LPS group ,mRNA and protein expression of HMGB 1 in administration groups ,protein expression of TLR 4 in RAW 264.7 cells of positive control group ,SFI medium- and high-dose groups as well as the levels of HMGB 1,IL-1β and TNF-α in supernatant of cells in administration groups were decreased significantly (P<0.01). In positive control group ,SFI medium- and high-dose groups ,the protein expressions of HMGB1 in nucleus were increased significantly ,while protein expressions of HMGB 1 in cytoplasm were decreased significantly (P<0.01). CONCLUSIONS :SFI may inhibit the nuclear translocation and secretion of HMGB 1 in RAW 264.7 cells,thus avoiding the activation of inflammatory pathways and the production of inflammatory factors ,so as to reduce the inflammatory response induced by LPS. |
期刊: | 2020年第31卷第21期 |
作者: | 艾飞,刘霞,黎晖,褚春薇,陈向云,郭俊峰,杨毅,梅丽艳,苗纪飞,温泉,叶森 |
AUTHORS: | AI Fei,LIU Xia, LI Hui,CHU Chunwei,CHEN Xiangyun,GUO Junfeng,YANG Yi,MEI Liyan,MIAO Jifei,WEN Quan,YE Sen |
关键字: | 参附注射液;RAW264.7细胞;高迁移率族蛋白B1;炎症因子;核转位 |
KEYWORDS: | Shenfu injection ;RAW264.7 cells;HMGB1;Inflammatory factors ;Nuclear translocation |
阅读数: | 466 次 |
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