Effects Of Porphyromonas Gingivalis Lipopolysaccharide On The Pyroptosis Of Gingival Fibroblasts

Purpose: Chronic periodontitis(CP)is a common infectious and immune disease in oral cavity,which is one of the main reasons for tooth loss.Porphyromonas gingivalis(P.gingivalis)is one of the main pathogenic bacterium and it can destroy periodontium by releasing multiple secretions including lipopolysaccharide(LPS).Gingivitis is a precursor of periodontitis and the continuity and extension of inflammation is an important factor in processing from gingivitis to periodontitis.However,gingival inflammation shows no difference between gingivitis and periodontitis.Thus,gingival fibroblast status plays a vital role in the development of periodontal diseases.Pyroptosis is an inflammatory cell death form and exists in many inflammatory diseases.Considering the importance of gingival fibroblasts,the current study is to investigate whether P.gingivalis-LPS could activate the pyroptosis in gingival fibroblast and cause the destruction of gingival fibroblasts,which leads to development of gingivitis.Methods:1.Clinical inflammatory gingival samples from chronic periodontitis patients were collected to examine the protein level of NLRP3,caspase-1,caspase-4,caspase-5,IL-18 and IL-1β by immunohistochemistry.2.Primary cultured HGF and the effects of P.gingivalis-LPS on the cell growth and caspase-1 secretion were detected.3.Flow cytometry and LDH release were conducted to investigated the change of membrane permeability after P.gingivalis-LPS or capase-1 inhibitor Z-YVAD-FMK(YVAD)stimulation.4.The mRNA levels of NLRP3,caspase-4,caspase-5,IL-18,IL-1β and GSDMD were examined by qPCR after the addition of P.gingivalis-LPS or YVAD.5.Rat gingivitis model was established using P.gingivalis-LPS,and clinical indexes were observed.NLRP3,caspase-1,caspase-11,IL-18 and IL-1β expressions were detected by immunohistochemistry.Results: 1.The expression levels of NLRP3,caspase-1,caspase-4,caspase-5,IL-18 and IL-1β were higher in inflammatory gingival tissue samples from CP patients than those in healthy subjects.2.HGF growth was significantly inhibited when the concentration of P.gingivalis-LPS ≥50 μg/m L,and caspase-1 secretion was increased after 1,10,50 μg/m L P.gingivalis-LPS stimulation.3.The proportion of double positive cell number by PI and Annexin Ⅴ-FITC staining and LDH release were increased after P.gingivalis-LPS infection,but the results were reversed with the addition of YVAD.4.qPCR showed NLRP3,caspase-4,caspase-5,IL-18,IL-1β and GSDMD mRNA levels were upregulated to certain degrees.5.After injection of P.gingivalis-LPS in rat gingival sulcus,the bleeding on probing(BOP)(+)index and probing depth(PD)were increased.The expression levels of NLRP3,caspase-1,caspase-11,IL-18 and IL-1β were higher in gingivitis group.Conclusion: P.gingivalis-LPS could activate the reaction of pyroptosis in HGF and induce inflammation,which leads to gingivitis.