参附注射液对LPS诱导的RAW264.7细胞HMGB1核转位的干预作用

Intervention Effect of Shenfu Injection on the Nuclear Translocation of HMGB1 in LPS-induced RAW264.7 Cells

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/m L)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HMGB1 mRNA的表达情况;采用Western blotting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HMGB1 m RNA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HMGB1 m RNA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。

OBJECTIVE:To investigate the intervention effect of Shenfu injection(SFI)on the nuclear translocation of high mobility group box 1(HMGB1)in lipopolysaccharide(LPS)-induced RAW264.7 cells.METHODS:Using LPS-induced RAW264.7 cells as objects,the histone deacetylase inhibitor RGFP966 as positive control,CCK-8 assay was used to screen drug dosage,and the effects of low,medium and high doses(3,6,12μL/m L)of SFI on HMGB1 nuclear translocation in RAW264.7 cells were observed by immunofluorescence method;mRNA expression of HMGB1 in RAW264.7 cells were detected by real time fluorescent PCR.Western blotting assay was used to determine protein expression of HMGB1 and Toll-like receptor 4(TLR4);the expression of HMGB1 were compared between nucleus and cytoplasm.The levels of HMGB1,IL-1βand TNF-αin 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 expression of HMGB1,protein expression of TLR4 in RAW264.7 cells as well as the levels of HMGB1,IL-1βand TNF-αin supernatant of cells were increased significantly in LPS group(P<0.01).The protein expression of HMGB1 was decreased significantly in nucleus while was increased significantly in cytoplasm(P<0.01).After SFI treatment,the nuclear translocation and secretion of HMGB1 were inhibited in different degrees;compared with LPS group,mRNA and protein expression of HMGB1 in administration groups,protein expression of TLR4 in RAW264.7 cells of positive control group,SFI medium-and high-dose groups as well as the levels of HMGB1,IL-1βand TNF-αin 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 HMGB1 in cytoplasm were decreased significantly(P<0.01).CONCLUSIONS:SFI may inhibit the nuclear translocation and secretion of HMGB1 in RAW264.7 cells,thus avoiding the activation of inflammatory pathways and the production of inflammatory factors,so as to reduce the inflammatory response induced by LPS.