ABSTRACT
Objective:To explore the effect of prostaglandin E2 (PGE2) on the expression of high mobility group box-1 protein (HMGB1) in peritoneal macrophages of septic mice and its possible mechanisms.Methods:Ihe mouse peritoneal macrophages were isolated and cultured by conventional methods.The model of inflammation was established by using lipopolysaccharide (LPS) to incubate with mouse peritoneal macrophages.The PGE2,prostaglandin E receptor (EP) 4 agonist,EP4 RNAi,and DN.CREB inhibitory plasmid were used to interfere with the LPS-treated mouse peritoneal macrophage.The levels of HMGB 1 was determined by Western blot.Results:Compared with LPS alone treatment,the expression of HMGB 1 in peritoneal macrophages was increased obviously after 24 h by treatment with PGE2 and LPS,and it was also increased after the combined treatment of EP4 receptor agonist with LPS for 24 h (both P0.05);compared with LPS alone treatment,the combined treatment of EP4 receptor agonist with LPS for 24 h could up-regulate the phosphorylation of epidermal growth factor receptor (EGFR) and protein kinase B (Akt) thr308 (P<0.05),which were blocked by EGFR inhibitor.Once Akt specific inhibitor was used before EP4 and LPS treatment,the expression of HMGB1 was declined (P<0.05).Conclusion:PGE2 can up-regulate the expression of HMGB1 in sepsis of peritoneal macrophages through EP4 receptor,which may be related to the activation of EGFR/PI3K/Akt signaling pathway.
ABSTRACT
Lipopolysaccharides (LPS) contamination in herbal crude polysaccharides is inevitable. The present study was performed to explore the effect of polymyxin B on abolishing the influence of LPS contamination in mononuclear cells. LPS was pretreated with polymyxin B sulfate (PB) at different concentrations for 1, 5 or 24 h, and then used to stimulate RAW264.7 and mouse peritoneal macrophages (MPMs). The nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in cell culture supernatant, as the indications of cell response, were assayed. Bupleurum chinensis polysaccharides (BCPs) with trace amount contamination of LPS was treated with PB. 30 μg·mL of PB, treating LPS (10 and 1 000 ng·mL in stimulating RAW264.7 and MPMs respectively) at 37 °C for 24 h, successfully abolished the stimulating effect of LPS on the cells. When the cells were stimulated with LPS, BCPs further promoted NO production. However, pretreated with PB, BCPs showed a suppression of NO production in MPMs and no change in RAW264.7. In the in vitro experiments, LPS contamination in polysaccharide might bring a great interference in assessing the activity of drug. Pretreatment with PB (30 μg·mL) at 37 °C for 24 h was sufficient to abolish the effects of LPS contamination (10 and 1 000 ng·mL).
Subject(s)
Animals , Mice , Bupleurum , Chemistry , Drug Contamination , Drugs, Chinese Herbal , Pharmacology , Lipopolysaccharides , Macrophages , Metabolism , Nitric Oxide , Metabolism , Polymyxin B , Pharmacology , Polysaccharides , Pharmacology , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
Endothelin-1 (ET-1) has been characterized as a potent vasoconstrictor secreted by the endothelium, and play a major role in the regulation of vascular tone. It has been also known to participate in inflammatory reactions. The production of ET-1 by macrophages during infection and inflammation is related to tissue perfusion and leukocyte extravasation. The aim of this study is to investigate the role of IL-8/CXCL8, as a major inflammatory chemokine, for ET-1 expression in macrophges. Expression of ET-1 mRNA in mouse peritoneal macrophages (PeM phi) was weaker than that in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). However, expression of IL-8/CXCL8-induced ET-1 mRNA in PeM phi was much more stronger than that in SHR and WKY VSMCs. Maximum expression of ET-1 mRNA was observed at 50 ng/ml dose of IL-8/CXCL8 and occurred at 2 h after addition of IL-8/CXCL8. Expression of ET-1 by IL-8/CXCL8 was dependent on NF-kappaB activation and ERK1/2 phosphorylation. Baicalein, a 12-lipoxygenase (LO) inhibitor, inhibited the expression of IL-8/CXCL8-induced ET-1 mRNA. This inhibitory action of baicalein was mediated via ERK1/2 inactivation. Induction of 12-LO mRNA by IL-8/CXCL8 and expression of ET-1 mRNA by 12-LO metabolite, 12(S)-HETE were also detected. The expression of IL-8/CXCL8-induced ET-1 mRNA was not detected in PeM phi transfected with 12-LO siRNA. These results suggest that IL-8/CXCL8 can act as one of main inducers of ET-1 in vascular inflammatory reactions, and ET-1 expression by IL-8/CXCL8 is related to 12-LO pathway in PeM phi.
Subject(s)
Animals , Mice , Rats , Arachidonate 12-Lipoxygenase , Endothelin-1 , Endothelium , Flavanones , Inflammation , Leukocytes , Macrophages , Macrophages, Peritoneal , Muscle, Smooth, Vascular , NF-kappa B , Perfusion , Phosphorylation , Rats, Inbred SHR , RNA, Messenger , RNA, Small InterferingABSTRACT
AIM To establish radioligand binding assay of PAF (platelet activating factor) receptors in mouse peritoneal macrophages and observe the characteristics of PAF receptors. METHODS PAF receptor radioligand binding was studied in intact adherent mouse peritoneal macrophages by -PAF. The radioactivity was counted with an LS6500 scintillation system. RESULTS The PAF receptor binding was shown to be saturable with an equilibrium K D of 3.2 nmol?L -1 and a B max of 100.2 fmol?1?10 6 cells -1. The competitive analysis showed that such specific binding could be inhibited by BN52021. CONCLUSION Utilizing the adherent character of macrophages, the binding ligands could be separated from non-binding ligands without negative pressure filtration, then cells could reserve fine activity, and PAF receptors could be near to physiological properties for screening of PAF antagonist.