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.
ABSTRACT
OBJECTIVE： To study the improvement and anti-inflammation mechanism of Shenfu injection on lung tissue of endotoxin shock model rats. METHODS： Totally 48 rats were randomized into control group，model group，dexamethasone group （positive control，1 mg/kg） and Shenfu injection low-dose，medium-dose and high-dose groups （5，10，15 mL/kg），with 8 rats in each group. Except for normal group， other groups were given intraperitoneal injection of lipopolysaccharide （LPS） to induce endotoxin shock model. After modeling， each group was given relevant medicine once intraperitoneally. 24 h after medication， HE staining was used to observe pathological changes of lung tissue in rats and pathological scoring was conducted. RT-PCR was used to determine mRNA levels of P65 and P50 proteins related to NF-κB signaling pathway. Western blot assay was used to determine the expression levels of P65 and P50 proteins in lung tissue， and the expression levels of P65 protein in nucleus and cytoplasm of lung tissue were also determined. The level of TNF-α in plasma in rats were determined by ELISA. RESULTS： Compared with control group， alveolar septum became thicker， obvious vascular engorgement was found， and a large number of neutrophils infiltrated the interstitium in model group. Histopathological score， mRNA and protein expression levels of P65 and P50 in lung tissues were increased significantly （P＜0.01 or P＜0.001）； the protein expression of levels P65 in nucleus and cytoplasm and level of TNF-α in plasma were increased significantly （P＜0.001）. Compared with model group， alveolar structure of rats in dexamethasone group and Shenfu injection medium-dose and high-dose groups was complete， no obvious bleeding was observed， and the degree of inflammatory cell infiltration was improved significantly. Histopathological score， mRNA and protein expression levels of P65 and P50 in lung tissue and level of TNF-α in plasma were decreased significantly （P＜0.05 or P＜0.01 or P＜0.001）. The protein expression level of P65 in nucleus and cytoplasm of lung tissue were decreased significantly in dexamethasone group and Shenfu injection low-dose and medium-dose groups were decreased significantly （P＜0.05 or P＜0.01 or P＜0.001）. CONCLUSIONS： Shenfu injection can decrease mRNA and protein expression levels of P65 and P50 in lung tissue， level of TNF-α in plasma， and protect lung tissue of endotoxin shock rats.