TolCV1 Has Multifaceted Roles During Vibrio vulnificus Infection.

RtxA1 is a major cytotoxin of Vibrio vulnificus (V. vulnificus) causing fatal septicemia and necrotic wound infections. Our previous work has shown that RpoS regulates the expression and secretion of V. vulnificus RtxA1 toxin. This study was conducted to further investigate the potential mechanisms of RpoS on RtxA1 secretion. First, V. vulnificus TolCV1 and TolCV2 proteins, two Escherichia coli TolC homologs, were measured at various time points by Western blotting. The expression of TolCV1 was increased time-dependently, whereas that of TolCV2 was decreased. Expression of both TolCV1 and TolCV2 was significantly downregulated in an rpoS deletion mutation. Subsequently, we explored the roles of TolCV1 and TolCV2 in V. vulnificus pathogenesis. Western blot analysis showed that RtxA1 toxin was exported by TolCV1, not TolCV2, which was consistent with the cytotoxicity results. Furthermore, the expression of TolCV1 and TolCV2 was increased after treatment of the host signal bile salt and the growth of tolCV1 mutant was totally abolished in the presence of bile salt. A tolCV1 mutation resulted in significant reduction of V. vulnificus induced-virulence in mice. Taken together, TolCV1 plays key roles in RtxA1 secretion, bile salt resistance, and mice lethality of V. vulnificus, suggesting that TolCV1 could be an attractive target for the design of new medicines to treat V. vulnificus infections.

Anti-allergic Inflammatory Components from the Leaves of Piper crocatum Ruiz & Pav.

Piper crocatum Ruiz & Pav. (P. crocatum), a traditional medicinal plant, has been shown to possess various pharmacological activities, including anticancer activity, antioxidant activity, antibacterial activity, anti-hyperglycemic activity, anti-allergic inflammatory activity and others. To identify the potential anti-allergic inflammatory effective constituents of P. crocatum, 13 single compounds were isolated from the methanol extract of P. crocatum leaves, and their structures were identified by contrasting their NMR spectroscopic data and previously published papers. First, the anti-allergic inflammatory activities of these single compounds were examined by accessing immune function related biomarkers such as nitric oxide (NO) and ß-hexosaminidase. We found that the methanol extract and catechaldehyde (compound 1) potently suppressed NO production. Additionally, Western blot analysis showed that P. crocatum methanol extract and compound 1 suppressed the production of NO by reducing inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Consistent with these observations, P. crocatum methanol extract and compound 1 remarkably decreased ß-hexosaminidase release from RBL-2H3 cells stimulated with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA)-specific immunoglobulin E (IgE) antibodies. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay indicated that P. crocatum methanol extract and compound 1 exhibited no cytotoxicity to RAW264.7 and RBL-2H3 cells. Based on these findings, compound 1 is suggested as an active anti-allergic inflammatory component of P. crocatum.

DIDS inhibits Vibrio vulnificus cytotoxicity by interfering with TolC-mediated RtxA1 toxin secretion.

Vibrio vulnificus (V. vulnificus) infection, frequently resulting in fatal septicemia, has become a growing health concern worldwide. The present study aimed to explore the potential agents that could protect against V. vulnificus cytotoxicity, and to analyze the possible underlying mechanisms. First, we observed that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) significantly suppressed V. vulnificus cytotoxicity to host cells by using a lactate dehydrogenase (LDH) assay. DIDS did not exhibit any effect on host cell viability, bacterial growth, microbial adhesion and swarming motility. DIDS effectively lowered V. vulnificus RtxA1 toxin-induced calcium influx into host mitochondria and RtxA1 binding to host cells. To further elucidate the underlying mechanism, the synthesis and secretion of RtxA1 toxin were investigated by Western blotting. Intriguingly, DIDS selectively inhibited the secretion of RtxA1 toxin, but did not influence its synthesis. Consequently, the outer membrane portal TolC, a key conduit for RtxA1 export coupled with tripartite efflux pumps, was examined by RT-PCR and Western blotting. We found that DIDS significantly reduced the expression of TolCV1 protein at the transcriptional level. Taken together, these results suggest that DIDS is a promising new paradigm as an antimicrobial drug that targets TolC-mediated toxin.

Anti-Melanogenic Effect of Dendropanax morbiferus and Its Active Components via Protein Kinase A/Cyclic Adenosine Monophosphate-Responsive Binding Protein- and p38 Mitogen-Activated Protein Kinase-Mediated Microphthalmia-Associated Transcription Factor Downregulation.

Dendropanax morbiferus H. Lév has been reported to have some pharmacologic activities and also interested in functional cosmetics. We found that the water extract of D. morbiferus leaves significantly inhibited tyrosinase activity and melanin formation in α-melanocyte stimulating hormone (MSH)-induced B16-F10 cells. D. morbiferus reduced melanogenesis-related protein levels, such as microphthalmia-associated transcription factor (MITF), TRP-1, and TRP-2, without any cytotoxicity. Two active ingredients of D. morbiferus, (10E)-9,16-dihydroxyoctadeca-10,17-dien-12,14-diynoate (DMW-1) and (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol (DMW-2) were identified by testing the anti-melanogenic effects and then by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis. DMW-1 and DMW-2 significantly inhibited melanogenesis by the suppression of protein kinase A (PKA)/cyclic AMP (cAMP)-responsive binding protein (CREB) and p38 MAPK phosphorylation. DMW-1 showed a better inhibitory effect than DMW-2 in α-MSH-induced B16-F10 cells. D. morbiferus and its active component DMW-1 inhibited melanogenesis through the downregulation of cAMP, p-PKA/CREB, p-p38, MITF, TRP-1, TRP-2, and tyrosinase. These results indicate that D. morbiferus and DMW-1 may be useful ingredients for cosmetics and therapeutic agents for skin hyperpigmentation disorders.

Inhibitory effects of ChondroT and its constituent herbs on RANKL-induced osteoclastogenesis.

BACKGROUND: ChondroT is a complex herbal medicine consisting of water extracts of Ostericum koreanum (Maxim.) Kitag., Lonicera japonica Thunb., Angelica gigas Nakai, Clematis manshurica Rupr., and Phellodendron amurense Rupr. (6:4:4:4:3). Previous studies have reported that ChondroT possesses chondroprotective and anti-inflammatory, anti-osteoarthritic, and anti-hyperuricemic activities. The study is aim to demonstrate the effects of ChondroT and its five constituent herbs on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and the underlying mechanisms. METHODS: Osteoclastogenesis was identified in bone marrow-derived macrophages (BMDMs) by tartrate-resistant acid phosphatase (TRAP) staining assay, actin ring formation assay and the bone resorption assay. For the molecular mechanisms, activation of RANKL-induced NF-κB and MAPK signaling pathways and the expression levels of osteoclast-specific proteins were investigated by Western blotting. Cell viability was assessed by MTT assay. Actin ring formation and NF-κB translocation were evaluated by immunostaining. RESULTS: ChondroT and each of its constituent herbs significantly suppressed osteoclast differentiation dose dependently, and decreased actin ring formation as well as bone-resorbing capacity. Mechanistically, ChondroT and its constituent herbs downregulated the expressional levels of osteoclast-specific proteins such as NFATc1, c-Fos, Cathepsin K, and matrix metalloproteinase 9 (MMP9) by suppressing NF-κB translocation to nucleus and MAPKs phosphorylation at different levels. Compared to its five constituent herbs, ChondroT exhibited the best inhibitory efficiency against osteoclastogenesis. CONCLUSIONS: Taken together, ChondroT has anti-osteoclastogenesis properties by inhibiting NF-κB and MAPKs pathways. It could be considered as a potential therapeutic candidate for the treatment of osteoclast-related bone diseases.

Anti-Inflammatory Effects of Canavalia gladiata in Macrophage Cells and DSS-Induced Colitis Mouse Model.

Canavalia gladiata, known as sword bean, has been used as a Chinese traditional medicine for anti-inflammatory effects. However, the action mechanisms of sword bean have not yet been clearly defined. In the present study, the whole parts of a ripened sword bean (RSB) and the green sword bean (GSB) containing bean pod were extracted with ethanol by reflux extraction. The two crude extracts (RSBE and GSBE) from RSB and GSB were validated by a liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis of gallic acid as a reference chemical. The anti-inflammatory effects of two sword bean extracts were extensively investigated using LPS-stimulated macrophage cells. First, RSBE and GSBE significantly inhibited the production of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandinE2 (PGE2), and nitric oxide (NO) in LPS-induced RAW264.7 cells. RSBE and GSBE showed no cytotoxicity to RAW264.7 cells and mouse peritoneal macrophage cells. In addition, the overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) induced by LPS in RAW264.7 cells was significantly decreased by RSBE and GSBE. Western blotting and immunostaining analysis showed that RSBE and GSBE inhibited the nuclear translocation of NF-κB subunits, which correlated with the inhibitory effects on inhibitor kappa B (IκB) degradation. In dextran sulfated sodium (DSS)-induced colitis mice model, RSBE restored body weight, colon length, and the levels of pro-inflammatory cytokines, such as TNF-α, IL-6, interleukin-1ß (IL-1ß), and interferon-γ (IFN-γ). In addition, RSBE significantly suppressed the expression of COX-2, iNOS, and NF-κB.

Vibrio vulnificus RtxA1 cytotoxin targets filamin A to regulate PAK1- and MAPK-dependent cytoskeleton reorganization and cell death.

Cytoskeletal rearrangement and acute cytotoxicity occur in Vibrio vulnificus-infected host cells. RtxA1 toxin, a multifunctional autoprocessing repeats-in-toxin (MARTX), is essential for the pathogenesis of V. vulnificus and the programmed necrotic cell death. In this study, HeLa cells expressing RtxA1 amino acids 1491-1971 fused to GFP were observed to be rounded. Through yeast two-hybrid screening and subsequent immunoprecipitation validation assays, we confirmed the specific binding of a RtxA11491-1971 fragment with host-cell filamin A, an actin cross-linking scaffold protein. Downregulation of filamin A expression decreased the cytotoxicity of RtxA1 toward host cells. Furthermore, the phosphorylation of JNK and p38 MAPKs was induced by the RtxA1-filamin A interaction during the toxin-mediated cell death. However, the phosphorylation of these MAPKs was not observed during the RtxA1 intoxication of filamin A-deficient M2 cells. In addition, the depletion of pak1, which appeared to be activated by the RtxA1-filamin A interaction, inhibited RtxA1-induced phosphorylation of JNK and p38, and the cells treated with a pak1 inhibitor exhibited decreased RtxA1-mediated cytoskeletal rearrangement and cytotoxicity. Thus, the binding of filamin A by the RtxA11491-1971 domain appears to be a requisite to pak1-mediated MAPK activation, which contributes to the cytoskeletal reorganization and host cell death.

Anti-bacterial effects of components from Sanguisorba officinalis L. on Vibrio vulnificus and their soluble epoxide hydrolase inhibitory activity.

Sanguisorba officinalis L. is a traditional herbal medicine, which is prevailingly applied to cure hemorrhoids, wounds and ulcers in Eastern Asian countries. The purpose of this study was to investigate the antibacterial and soluble epoxide hydrolase (sEH) inhibitory effects of the extracts and components from S. officinalis. The methanol extract was divided into ethyl acetate (EtOAc), n-butanol (n-BuOH), and water layers. In our screening procedure, the EtOAc and n-BuOH extracts and compounds (1-2) remarkably suppressed the growth of V. vulnificus in a dose-dependent manner. In addition, the EtOAc extract and compound 1 exhibited significant inhibitory effect on the V. vulnificus induced cytotoxicity on HeLa cells. Furthermore, compound 4 displayed an inhibition against sEH with an IC50 value of 7.0 ± 0.5 µM. A kinetic analysis demonstrated that the inhibitory effect of compound 4 was a mixed type, with an inhibitory constant (Ki) 0.22 ± 0.0 µM.

Anti-allergic inflammatory components from Sanguisorba officinalis L.

Sanguisorba officinalis L. was well known as a traditional herbal medicine to treat inflammation and allergic skin diseases. The aim of this research was to indentify compounds with anti-allergic inflammatory property. Twenty-five compounds (1-25) were isolated from S. officinalis including two new compounds (1 and 8), and their chemical structures were identified by NMR and ESIMS analysis. Consequently, the anti-allergic inflammatory activities of these isolates were investigated by inhibiting ß-hexosaminidase and IL-4 production in PMA/A23187-stimulated RBL-2H3 cells. Compounds 6, 8, 13, 17-18 and 25 significantly inhibited ß-hexosaminidase release and IL-4 production. Additionally, compounds 8, 17 and 25 effectively suppressed the activation of NF-κB and NF-κB p65 translocation into the nucleus. Anti-inflammatory effects of isolated compounds were evaluated in LPS-stimulated RAW264.7 macrophages, and they showed dramatic inhibition on LPS-induced overproduction of nitric oxide (NO) and TNF-α. Consistently, the protein levels of iNOS and COX-2 were remarkably decreased by the single compounds 8, 13 and 25. These results showed that compounds 8, 13 and 25 from S. officinalis may have a therapeutic potential for allergic inflammatory diseases.

Vibrio vulnificus RtxA1 Toxin Expression Upon Contact With Host Cells Is RpoS-Dependent.

The expression of virulence genes in bacteria is known to be regulated by various environmental and host factors. Vibrio vulnificus, an estuarine bacterium, experiences a dramatic environmental change during its infection process. We reported that V. vulnificus RtxA1 toxin caused acute cell death only when close contact to host cells was allowed. A sigma factor RpoS is a very important regulator for the maximal survival of pathogens under stress conditions. Here, we studied the role of RpoS in V. vulnificus cytotoxicity and mouse lethality. The growth of rpoS mutant strain was comparable to that of wild-type in heart infusion (HI) media and DMEM with HeLa cell lysate. An rpoS mutation resulted in decreased cytotoxicity, which was restored by in trans complementation. Interestingly, host contact increased the expression and secretion of V. vulnificus RtxA1 toxin, which was decreased and delayed by the rpoS mutation. Transcription of the cytotoxic gene rtxA1 and its transporter rtxB1 was significantly increased after host factor contact, whereas the activity was decreased by the rpoS mutation. In contrast, the rpoS mutation showed no effect on the transcriptional activity of a cytolytic heamolysin gene (vvhA). Additionally, the LD50 of the rpoS mutant was 15-fold higher than that of the wild-type in specific pathogen-free CD-1 female mice. Taken together, these results show that RpoS regulates the expression of V. vulnificus RtxA1 toxin and its transporter upon host contact.

Anti-allergic Inflammatory Effects of the Essential Oil From Fruits of Zanthoxylum coreanum Nakai.

Zanthoxylum coreanum Nakai is a rare shrub which grows in Korea and China. Pericarp of Z. coreanum has been used as a crude medicine, but there are few researches about the pharmacologic activities. The present study was designed to investigate the anti-allergic inflammatory activities of the essential oil from fruits of Zanthoxylum coreanum Nakai (ZCO). Our findings showed that ZCO inhibited both the IgE-antigen complex or PMA/A23187-induced ß-hexosaminidase release and IL-4 production dose-dependently in RBL-2H3 mast cells, and confirmed that ZCO at the tested concentrations did not show cytotoxicity to RBL-2H3 cells by MTS assay. Additionally, we found that ZCO showed the significant inhibition on LPS-induced overproduction of TNF-α, IL-6 and NO. Consistently, the protein levels of iNOS and COX-2 were also remarkably decreased by ZCO treatment. Herein, Our mechanistic studies revealed that ZCO significantly suppressed the activation of transcription factor NF-κB in PMA-activated 293T cells, and further inhibited NF-κB p65 translocation into the nucleus in LPS-stimulated RAW264.7 cells. Further investigation identified that ZCO down-regulated LPS-induced phosphorylation of MAPK (JNK, ERK, and p38) signal pathway. For incremental research, we established an DNCB-induced atopic dermatitis model in BALB/c mice, and found that ZCO remarkably inhibited DNCB-induced ear swelling and AD-like symptoms. Based on these findings, ZCO is suggested to have a therapeutic potential for the allergic inflammatory diseases.