ABSTRACT
OBJECTIVE: To investigate the effects of andrographolide on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and tumor necrosis factor-α (TNF-α) expression in lipopolysaccharide (LPS)-activated macrophages. METHODS: LPS-activated mouse peritoneal macrophages were cultured in media with different concentrations of andrographolide. Cytotoxicity of andrographolide was detected by cell counting kit-8. The macrophages were lysed, and then expressions of phosphorylated ERK1/2, JNK and p38 and nuclear factor-κB inhibitor (IκBα) protein were detected by Western blotting and TNF-α mRNA expression was detected by reverse transcription-polymerase chain reaction. Supernatants of the macrophages were used to detect content of TNF-α protein by enzyme-linked immunosorbent assay. RESULTS: Andrographolide at 1-100 µg/mL showed no cytotoxicity on LPS-activated mouse peritoneal macrophages. Andrographolide inhibited ERK1/2 phosphorylation in LPS-activated murine peritoneal macrophages, which was concentration-dependent (P<0.01). Andrographolide at 1-25 µg/mL had no effects on phosphorylation levels of JNK and p38 and IκBα degradation in LPS-stimulated mouse peritoneal macrophages. In activated macrophages, TNF-α expression was inhibited by 12 µg/mL andrographolide and 20 µmol/L PD98059 (inhibitor of ERK1/2 signaling pathway) at both mRNA expression and protein secretion levels. CONCLUSION: In LPS-activated macrophages, andrographolide may inhibit the expression of TNF-α by inhibiting ERK1/2 signaling pathway.
Subject(s)
Diterpenes/pharmacology , MAP Kinase Signaling System , Macrophages/drug effects , Macrophages/metabolism , Animals , Cells, Cultured , Female , Macrophage Activation , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Andrographolide has been reported to possess a variety of pharmacological activities. In this study, we have investigated the effect of andrographolide on the production of TNF-alpha and IL-12 (Interleukin-12) in murine peritoneal macrophages. Andrographolide decreased TNF-alpha, IL-12a and IL-12b at mRNA level, and reduced the production of TNF-alpha and IL-12p70 proteins in a concentration-dependent manner. Furthermore, we have found that addition of andrographolide inhibited the activation of ERK1/2 MAP kinase, but not that of JNK, p38 or NF-kappaB. These results suggested that andrographolide inhibit LPS-induced production of TNF-alpha via suppression of the ERK1/2 signaling pathway.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cytokines/antagonists & inhibitors , Diterpenes/pharmacology , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/drug effects , Mitogen-Activated Protein Kinases/metabolism , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Interleukin-12/antagonists & inhibitors , Macrophage Activation/drug effects , Macrophages, Peritoneal/enzymology , Macrophages, Peritoneal/immunology , Male , Mice , Mice, Inbred C57BL , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
Investigation of phenolic patterns from the stems of Dendrobium chrysanthum by HPLC-PDA-MS has led to the isolation of a new phenanthrene derivative with a spirolactone ring, dendrochrysanene (1), that proved to suppress the mRNA level of TNF-alpha, IL8, IL10, and iNOS in murine peritoneal macrophages. The structure of 1 was characterized on the basis of various NMR (1H, 13C, 1H-1H COSY, HMQC, and HMBC), mass spectrometry, and X-ray crystal diffraction data.
Subject(s)
Dendrobium/chemistry , Phenanthrenes/chemistry , Phenanthrenes/pharmacology , Spiro Compounds/chemistry , Spiro Compounds/pharmacology , Spironolactone/chemistry , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Cells, Cultured , Crystallography, X-Ray , Drugs, Chinese Herbal/chemistry , Interleukins/metabolism , Macrophages, Peritoneal/drug effects , Male , Mice , Mice, Inbred C57BL , Phenanthrenes/metabolism , Spiro Compounds/metabolism , Tumor Necrosis Factors/metabolismABSTRACT
Genetic immmunization is a new method of producing antibody, which is different from protein immunization. In order to produce anti-P16 sera and to find out the difference between genetic immunization and protein immunization, fusion protein GST-P16 and eukaryotic expression vector pCMV-p16 were injected in rabbit respectively. Western blot analysis showed that the titer of sera from protein immunization was 1:625, which was much higher than the titer from genetic immunization sera. Our results indicate that much has to be done on the characteristics of the genetic immunization and methods for enhancing its immunization effect, before we can use it widely to produce antibody.
