Hypoxia And Lipopolysaccharide Synergistically Induce NLRP3 Inflammasome Activation In Human Gingival Fibroblasts

Objective:Periodontitis has various etiologies and exhibits a high rate of clinical incidence;nevertheless,there is currently no specific treatment method.The development and progression of periodontitis are dependent on the local microenvironment,and hypoxia and inflammation are two critical factors that shape the periodontitis microenvironment.Recent studies have confirmed that the activation of NLRP3inflammasome plays a key role in the pathogenesis of periodontitis.The establishment of a complete model of inflammation has attracted extensive attention in the clinical treatment of periodontal disease.Therefore,the purpose of our study was to investigate the effects of hypoxia and Porphyromonas gingivalis-lipopolysaccharide（P.gingivalis-LPS）on activation of the NACHT leucine-rich repeat protein 3（NLRP3）inflammasome in human gingival fibroblasts（HGFs）,and to provide a preliminary experimental basis for better clinical prevention and treatment of periodontitis.Methods:1.Primary culture and identification of human gingival fibroblasts and experimental grouping processing:the primary human gingival fibroblasts were cultured by tissue block culture,and cell morphology and specific protein markers were identified by immunofluorescence method.The cultured cells were divided into 6 groups:control group,LPS stimulation group,hypoxia 3 hours stimulation group,hypoxia 3 hours+LPS stimulation group,hypoxia 6 hours stimulation group and hypoxia 6 hours+LPS stimulation group.Cellular experiments involving the normoxic group were performed using a standard incubator（37°C,5%CO₂,21%O₂,balanced N₂）.For the hypoxic group,oxygen concentrations in the incubator were reduced to 1%by changing the nitrogen concentrations while maintaining CO₂ at 5%.Briefly,the cells were seeded on 6-well culture plates at density of 3×10⁵ cells per well.After 24 h,the cells adhered and the media in the HGF cultures were changed to serum-free MEM-α.The LPS group was stimulated with LPS for 12 h and the concentration of LPS used in this experiment was 1μg/ml.The hypoxia group was cultured in incubators at 1%O₂ for 3 h and 6 h,respectively.The difference was that the hypoxia+LPS group was prestimulated with LPS for 6 hours before being placed in the incubator.2.Detection of related indicators of NLRP3 inflammasome activation:The expression levels of genes and proteins of hypoxia-inducible factor-1α（HIF-1α）,interleukin-1β（IL--1β）,gasdermin D（GSDMD）and the NLRP3 inflammasome,including NLRP3,apoptosis-associated speck-like protein containing CARD（ASC）,caspase-1 and its activated forms,were measured using quantitative real-time polymerase chain reaction（q RT-PCR）and western blot.Enzyme-linked immunosorbent assay（ELISA）was used to detect and determine levels of the inflammatory factor interleukin-1βin cell supernatants.Lactate dehydrogenase（LDH）release assay,caspase-1 activity assay and Hoechst33342/Propidium Iodide（PI）staining were performed to further verify the presence of pyroptosis.Results:1.Primary culture and identification of human gingival fibroblasts:immunofluorescence results showed that the primary cells were human gingival fibroblasts.2.The results of real-time fluorescence quantitative polymerase chain reaction and western blot showed that the expressions of m RNA and protein of NLRP3 inflammasome（NLRP3,ASC,Caspase-1）were up-regulated after P.gingivalis-LPS and hypoxia stimulation,and it was not affected by individual stimulation using P.gingivalis-LPS or hypoxia.The combination of both hypoxia and P.gingivalis-LPS stimulation significantly enhanced inflammasome activation and promoted the expression of interleukin-1β,gasdermin D and HIF-1αat gene and protein levels.At the same time,the expression of IL-1βin the supernatant of hypoxia+LPS group was the highest by Enzyme-linked immunosorbent assay.3.Cellular membrane integrality was detected by Hoechst 33342 and PI staining.HGFs were doubly stained with Hoechst 33324/PI,the nuclei of cells was stained by Hoechst 33324,while PI could penetrate into the dying cells with the loss of cell membrane integrity.We observed the ratio of membrane-damaged cells（PI permeable,red）to the total cells（Hoechst33342,blue）that was induced by hypoxia and P.gingivalis-LPS treatment was observably increased compared to normoxia group,hypoxia group and LPS group.Similarly,the cell contents LDH release was remarkable increased in hypoxia and P.gingivalis-LPS group compared with other groups.Hypoxia and P.gingivalis-LPS significantly increased the level of activated caspase-1 in HGFs,which was consistent with the results of cell staining and LDH release.Taken together,these data suggest that hypoxia and P.gingivalis-LPS induced cell pyroptotic death via the activation of caspase-1 in HGFs.Conclusion:Hypoxia and P.gingivalis-LPS synergistically induced NLRP3inflammasome activation in HGFs,and subsequently high levels of IL-1βand GSDMD-mediated pyroptosis can cause an HGF inflammatory response,which plays an important role in the pathogenesis of periodontitis.