[Andrographolide inhibits extracellular signal-regulated kinase 1/2 signaling pathway in activated macrophages].

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.

Production of Anti-P16 Sera by Genetic Immunization and Protein Immunization.

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.

An essential role for RIG-I in toll-like receptor-stimulated phagocytosis.

Retinoic acid-inducible gene-I (RIG-I) plays an important role in antiviral response by recognizing double-stranded RNA. Here we demonstrate an unanticipated role of RIG-I in Toll-like receptor (TLR)-stimulated phagocytosis. Stimulation with lipopolysaccharide (LPS), a ligand of TLR4, induced the expression of RIG-I in macrophages. Depletion of RIG-I by RNAi or gene targeting inhibited the LPS-induced phagocytosis of bacteria. Cellular processes involved in phagocytosis, such as small GTPase Cdc42/Rac1 activation, actin polymerization, and actin-regulator Arp2/3 recruitment, were also impaired in RIG-I-deficient macrophages activated by LPS. Moreover, RIG-I(-/-) mice were found to be more susceptible to infection with Escherichia coli as compared to wild-type mice. Thus, the regulatory functions of RIG-I are strikingly broad, including a role not only in antiviral responses but in antibacterial responses as well.

Andrographolide inhibits the production of TNF-alpha and interleukin-12 in lipopolysaccharide-stimulated macrophages: role of mitogen-activated protein kinases.

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.

A new phenanthrene with a spirolactone from Dendrobium chrysanthum and its anti-inflammatory activities.

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.

Beta-arrestin 2 functions as a G-protein-coupled receptor-activated regulator of oncoprotein Mdm2.

Oncoprotein Mdm2 is a master negative regulator of the tumor suppressor p53 and has been recently shown to regulate the ubiquitination of beta-arrestin 2, an important adapter and scaffold in signaling of G-protein-coupled receptors (GPCRs). However, whether beta-arrestin 2 has any effect on the function of Mdm2 is still unclear. Our current results demonstrated that the binding of Mdm2 to beta-arrestin 2 was significantly enhanced by stimulation of GPCRs. Activation of GPCRs led to formation of a ternary complex of Mdm2, beta-arrestin 2, and GPCRs and thus recruited Mdm2 to GPCRs at plasma membrane. Moreover, the binding of beta-arrestin 2 to Mdm2 suppressed the self-ubiquitination of Mdm2 and consequently reduced the Mdm2-mediated p53 degradation and ubiquitination. Further experiments revealed that overexpression of beta-arrestin 2 enhanced the p53-mediated apoptosis while suppression of endogenous beta-arrestin 2 expression by RNA interference technology considerably attenuated the p53-mediated apoptosis. Our study thus suggests that beta-arrestin 2 may serve as a cross-talk linker between GPCR and p53 signaling pathways.