Intervention Effect of Shenfu Injection on the Nuclear Translocation of HMGB 1 in LPS-induced RAW 264.7 Cells / 中国药房

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.