Levobupivacaine inhibits lipopolysaccharide-induced high mobility group box 1 release in vitro and in vivo.

The aim of the study was to investigate whether levobupivacaine (LB) suppressed lipopolysaccharide (LPS)-induced high mobility group box 1 (HMGB1) release in vitro and in vivo, and to determin its molecular mechanisms of action. RAW264.7 cells were treated with LPS and LB for 24 h. Levels of HMGB1, nuclear factor-kappa B (NF-κB) and phosphorylated p38 mitogen-activated protein kinase (MAPK) were measured by Enzyme-linked immunosorbent assay and Western blotting; the levels of HMGB1 messenger RNA were measured by real-time polymerase chain reaction. In addition, cecal ligation and puncture-induced septic C57BL/6 received LB infusion, and the levels of HMGB1 and functional parameters of multiple organs determined using several detection kits. LB inhibited HMGB1 release in vitro and in vivo. Furthermore, LB inhibited the translocation of NF-κB and phosphorylation of p38 MAPK in vitro. Mice treated with LB infusion improved survival in mice and significantly reduced cecal ligation and puncture-induced dysfunction of organs. LB suppresses LPS-induced HMGB1 release in vitro and in vivo by partially inhibiting NF-κB/p38 MAPK pathways. LB can rescue mice from sepsis and protect against organ dysfunction in septic mice.

Ge Y ，Hu S ，Zhang Y ，Wang W ，Xu Q ，Zhou L ，Mao H ... -  《-》

Ketamine inhibits LPS-induced HGMB1 release in vitro and in vivo.

High mobility group box 1 (HMGB1) has been identified to be a critical mediator of severe sepsis. Ketamine has been shown to reduce sepsis-induced pathological complications. These effects are because of the reduced expression and release of several inflammatory mediators. However, whether ketamine affects the expression and release of HMGB1 is not known. We investigated the effect of ketamine on HMGB1 release in lipopolysaccharide (LPS)-induced macrophages in vitro and in cecal ligation and puncture (CLP)-induced septic rats in vivo, and determined its molecular mechanism of action. RAW264.7 cells or primary macrophages were incubated with or without LPS (500 ng/mL) in the presence or absence of ketamine, a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580), a nuclear factor-kappa B (NF-κB) inhibitor (pyrimidine dithiocarbamate), or small interfering RNA. The protein and expression levels of inflammatory mediators, such as HMGB1, tumor necrosis factor-α, and interleukin-1β were measured using enzyme-linked immunosorbent assays and real-time polymerase chain reaction. The effect of ketamine on NF-κB and p38 MAPK activation was evaluated using enzyme-linked immunosorbent assays, Western blot analysis, and electrophoretic mobility shift assay. Western blotting was used to observe changes in translocation of HMGB1 from the nucleus to cytoplasm. In addition, CLP-induced septic rats were treated with ketamine (0.5, 5, 10 mg/kg) or saline (10 mL/kg) 3h after sepsis, and the levels of HMGB1 and functional parameters of multiple organs were determined using several detection kits. Seven-day survival was also assessed. Ketamine inhibited HMGB1 release in LPS-activated RAW264.7 cells and CLP-induced septic rats. Translocation of HMGB1 from the nucleus to cytosol and expression of HMGB1 mRNA were inhibited significantly by ketamine. Ketamine inhibited the translocation of NF-κB from the cytoplasm to the nucleus and phosphorylation of p38 MAPK in LPS-activated RAW264.7 cells. Rats treated with ketamine improved survival in rats and significantly reduced CLP-induced dysfunction/injury of organs. Ketamine suppresses LPS-induced HMGB1 release in LPS-activated RAW264.7 cells and a CLP-induced model of sepsis in rats by partially inhibiting NF-κB/p38 MAPK pathways. Ketamine increased survival time induced by CLP and reduced organ dysfunction in septic peritonitis.

Zhang Z ，Zhang L ，Zhou C ，Wu H ... -  《-》

Penehyclidine hydrochloride inhibits the release of high-mobility group box 1 in lipopolysaccharide-activated RAW264.7 cells and cecal ligation and puncture-induced septic mice.

High-mobility group box 1 (HMGB1) is a critical mediator in the pathogenesis of many inflammatory diseases. Penehyclidine hydrochloride (PHC) has been proven to reduce sepsis-related mortality and sepsis-induced pathological complications. These effects are because of the reduced expression and release of many inflammatory mediators, although it is not clear whether PHC affects the expression and release of HMGB1. In this study, we explored the effect of PHC on the release of HMGB1 in lipopolysaccharide (LPS)-activated RAW264.7 cells and cecal ligation and puncture (CLP)-induced septic mice. RAW264.7 cells were incubated with LPS in the presence or absence of various concentrations of PHC. The expression levels of HMGB1 in the culture supernatant were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Western blotting was used to observe changes in the translocation of HMGB1 from the nucleus to the cytoplasm, and the nuclear factor (NF)-κB activity in the nuclear extract was detected by the NF-κB p50/p65 Transcription Factor Assay Kit. In addition, 48 CLP-induced septic BALB/c were treated with different concentrations of PHC 1 h before performing the CLP, and the level of serum HMGB1 and the functional parameters of multiple organs were determined using several detection kits. We found that PHC inhibited the release of HMGB1 in LPS-activated RAW264.7 cells and CLP-induced septic mice. PHC inhibited the translocation of HMGB1 from the nucleus to the cytoplasm and also suppressed the expression of HMGB1 messenger RNA. Furthermore, PHC inhibited the translocation of NF-κB from the cytoplasm to the nucleus in LPS-activated RAW264.7 cells in a dose-dependent manner. Compared with the CLP alone group, the levels of alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine, and creatine kinase were significantly decreased in mice treated with 0.45 mg/kg of PHC (P < 0.01). Our study demonstrates that PHC inhibits the translocation of HMGB1 from the nucleus to the cytoplasm and the expression of HMGB1 messenger RNA in a dose-dependent manner. The mechanism responsible for these effects involves the NF-κB signaling pathway. Moreover, PHC can significantly protect important organs, such as the liver, kidney, and heart in mice with sepsis.

Yang Q ，Liu X ，Yao Z ，Mao S ，Wei Q ，Chang Y ... -  《-》

Magnesium sulfate inhibits the secretion of high mobility group box 1 from lipopolysaccharide-activated RAW264.7 macrophages in vitro.

High mobility group box 1 (HMGB1) is an important inflammatory factor that is closely related to mortality in patients with sepsis. High magnesium therapy has been proved to reduce sepsis-related mortality and sepsis-induced pathologic complications. These effects result from reduced expression and release of many inflammatory cytokines, although it is not clear whether high magnesium affects the expression and release of HMGB1. In the present study, we explored the effect of magnesium sulfate on the expression and release of HMGB1 in lipopolysaccharide (LPS)-activated macrophages. RAW264.7 cells were incubated with LPS in the presence or absence of various concentrations of magnesium sulfate. An enzyme-linked immunosorbent assay was used to detect the levels of HMGB1 in the culture supernatant. Real-time polymerase chain reaction was used to assess the expression of HMGB1 mRNA. The nuclear/cytoplasm extraction kit was used to extract the nuclear and cytoplasmic proteins. Western blotting was used to observe the changes in the translocation of HMGB1 from the nucleus to the cytoplasm. The nuclear factor (NF)-κB p50/p65 Transcription Factor Assay Kit was used to analyze NF-κB activity in the nuclear extract. We found that magnesium sulfate inhibited translocation of HMGB1 from the nucleus to the cytoplasm and its extracellular release in LPS-activated macrophages and also suppressed the expression of HMGB1 mRNA. Furthermore, magnesium sulfate inhibited the translocation of NF-κB from the cytoplasm to the nucleus in LPS-activated macrophages in a dose-dependent manner. Our study has demonstrated that magnesium sulfate inhibits the translocation of HMGB1 from the nucleus to the cytoplasm and the expression of HMGB1 mRNA in a dose-dependent manner. The mechanism responsible for these effects involves the NF-κB signaling pathway.

Liu Z ，Zhang J ，Huang X ，Huang L ，Li S ，Wang Z ... -  《-》

Magnesium deficiency promotes secretion of high-mobility group box 1 protein from lipopolysaccharide-activated macrophages in vitro.

High-mobility group box 1 (HMGB1) is a critical mediator of sepsis that is closely related to sepsis lethality. Magnesium deficiency predisposes to worse outcomes from endotoxin challenge by promoting the production of cytokines. However, whether magnesium deficiency affects the expression and release of HMGB1 is not currently known. In the present study, we explored the effect of magnesium deficiency on the expression and secretion of HMGB1 in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. RAW264.7 cells were incubated with LPS in normal magnesium (1 mmol/L magnesium sulfate) or low magnesium (0.1 mmol/L magnesium sulfate) in Roswell Park Memorial Institute 1640 medium. An enzyme-linked immunosorbent assay was used to detect HMGB1 levels in the culture supernatant. Real-time polymerase chain reaction was used to assess the HMGB1 mRNA levels. A nuclear/cytoplasm extraction kit was used to extract the nuclear and cytoplasmic proteins. Western blotting was used to observe the changes in translocation of HMGB1 from the nucleus to the cytoplasm. A nuclear factor κ-light chain enhancer of activated B cells (NF-κB) p50/p65 transcription factor assay kit was used to analyze the NF-κB activity in nuclear extracts. Magnesium deficiency promoted translocation of HMGB1 from the nucleus to the cytoplasm and its extracellular secretion in LPS-activated macrophages, while enhancing the expression of HMGB1 mRNA. Furthermore, magnesium deficiency promoted the translocation of NF-κB from the cytoplasm to the nucleus in LPS-activated macrophages. Magnesium deficiency promotes the translocation of HMGB1 from the nucleus to the cytoplasm and the expression of HMGB1 mRNA. Magnesium deficiency also activates the NF-κB signaling pathway.

Liu Z ，Chang Y ，Zhang J ，Huang X ，Jiang J ，Li S ，Wang Z ... -  《-》