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Vibrio alginolyticus is a food-borne marine Vibrio that causes gastroenteritis, otitis media, otitis externa, and septicemia in humans. The pathogenic mechanisms of V. alginolyticus have previously been studied in aquaculture animals; however, the underlying mechanisms in mammals remain unknown. In this study, an in vitro model of mouse peritoneal macrophages infected with V. alginolyticus was established. qPCR results revealed that V. alginolyticus induced the transcription levels of various cytokines, including IL-1β, IL-12, IL-18, TNF-α, IL-17, IL-6, IFN-γ, and IL-10, and the secretion level of IL-1β is the most significant. Inhibition assays with Ac-YVAD-CHO (a caspase-1 inhibitor) and Z-VAD-FMK (a pan-caspase inhibitor) were conducted to determine whether caspase-1 or caspase-11 is involved in V. alginolyticus-triggered IL-1β secretion. Results showed that IL-1β secretion was partly inhibited by Ac-YVAD-CHO and absolutely blocked by Z-VAD-FMK. To explore the sensed pattern recognition receptors, several NLR family members and the AIM2 receptor were detected and many receptors were upregulated especially NLRP3. Moreover, the NLRP3 protein displayed a puncta-like surrounding cell nucleus, which signified that the NLRP3 inflammasome was activated in response to V. alginolyticus infection. Inhibition assays with glyburide and CA-074 methyl ester (K+ outflow inhibitor and cathepsin B inhibitor) blocked IL-1β secretion, which demonstrated the essential role of the NLRP3 inflammasome in inflammatory response. To better understand how V. alginolyticus affects IL-1β release, the NLRP3 inflammasome was detected with doses ranging from 0.1 to 10 MOIs and time periods ranging from 3 to 12 h. Results showed that V. alginolyticus-mediated NLRP3 inflammasome activation was in a time- and dose-dependent manner and IL-1β release peaked at MOI of 1 for 12 h. Most importantly, blocking the NLRP3 inflammasome with inhibitors and the use of NLRP3-/- and caspase-1/11-/- mice could attenuate pro-inflammatory cytokine secretion, such as IL-1β, IL-6, IL-12, and TNF-α. Taken together, our study first found that the NLRP3 inflammasome plays vital roles in V. alginolyticus triggered inflammatory response in mouse peritoneal macrophages. This may provide reference information for the development of potential anti-inflammatory treatments against V. alginolyticus infection.

Background Astragaloside IV (AS‐IV), a natural herbal compound from Astragalus membranaceus, has inhibitory effects on receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis, and RANKL signal helps to regulate odontoblast differentiation. However, whether and how AS‐IV affects odontoblastic differentiation remains unclear. Methods Lipopolysaccharide (LPS)‐stimulated MDPC‐23 cells and rat pulpitis were treated with AS‐IV, cell viability, and LDH leakage was analyzed by CCK‐8 assay and LDH Leakage assay. The production of TNF‐α and IL‐6 was determined by ELISA and qRT‐PCR assay. The expression of alkaline phosphatase (ALP) was detected using an ALP assay kit, and the expression of dentin sialophos‐phoprotein (DSPP), dentin matrix protein‐1 (DMP1), basic fibroblast growth factor (FGF2), and phosphorylated extracellular signal‐regulated kinase (p‐ERK) was determined by western blot. Results AS‐IV dose dependently increased in cell viability and decreased the overproduction of TNF‐α and IL‐6 in LPS‐stimulated MDPC‐23 cells. AS‐IV also counteracted LPS‐induced downregulation of ALP, DSPP, and DMP1 in MDPC‐23 cells. Furthermore, AS‐IV significantly decreased the expression of FGF2 and p‐ERK in LPS‐stimulated MDPC‐23 cells. More important, the addition of FGF2 partly neutralized AS‐IV‐mediated inhibition of FGF2/ERK signaling, abolished AS‐IV‐induced reduction of TNF‐α and IL‐6, and counteracted AS‐IV‐induced upregulation of DSPP and DMP‐1 in these cells. Meanwhile, AS‐IV inhibited the excessive production of TNF‐α and IL‐6, suppressed the downregulation of DSPP and DMP1, and disturbed the up‐regulation of FGF2 and p‐ERK in the pulp tissues of rat pulpitis model. Conclusions AS‐IV exerted anti‐inflammatory and pro‐differentiation effects in LPS‐stimulated MDPC‐23 cells and rat pulpitis via inhibiting the FGF2/ERK signaling pathway.

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