Study On The Regulation Of NLRP3 Inflammasome Pathway In Human Gingival Fibroblast By Drug-loaded Nanoparticles

Objective: A polyelectrolyte complex nanoparticle comprising chitosan(CS)and carboxymethyl chitosan(CMCS)was prepared(CS/CMCS-NPs)by ionic gelation,which was then used as a doxycycline(Dox)carrier(Dox:CS/CMCS-NPs).To study its physical and chemical properties,cell compatibility,bacteriostatic and antiinflammatory effects in the NLRP3 inflammasome model in human gingival fibroblasts(HGFs)induced by Porphyromonas gingivalis lipopolysaccharide(P.gingivalis-LPS)and adenosine triphosphate(ATP).Methods: 1.Primary culture and identification of human gingival fibroblasts and construction and suppression of NLRP3 inflammasome model: primary gingival fibroblasts were cultured by tissue block culture and identified by immunofluorescence method.Porphyromonas gingivalis lipopolysaccharide combined with adenosine triphosphate induced human gingival fibroblasts to construct NLRP3 inflammasome model,and the expressions of related mRNA and proteins were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR),enzyme-linked immunosorbent assay(ELISA)and Western blotting assay.2.Preparation and property detection of Dox:CS/CMCS-NPs and CS/CMCS-NPs: preparation of CS/CMCS-NPs and CS/CMCS-NPs coated with Dox(Dox:CS/CMCSNPs)by ionic gelation method.The morphology was observed by SEM and TEM and the particle size and dispersion were determined by laser particle analyzer.Encapsulation efficiency and loading capacity of Dox:CS/CMCS-NPs were detected by spectrophotometer.3.Detection of cell compatibility and antimicrobial activity of CS/CMCS-NPs and Dox:CS/CMCS-NPs composite nanoparticle system: CCK-8 was used to determine the cell activity of CS/CMCS-NPs and Dox:CS/CMCS-NPs on human gingival fibroblasts for 24,48 and 72 hours at concentrations of 0,62.5,125,250,500 and 1000 ?g/ml respectively.The antimicrobial activity of CS/CMCS-NPs and Dox:CS/CMCS-NPs against Porphyromonas gingivalis was determined by colony counting method.4.Dox:CS/CMCS-NPs on human gingival fibroblasts in the role of the NLRP3 inflammasome: using doxycycline,Dox:CS/CMCS-NPs and CS/CMCS-NPs stimulated NLRP3 inflammasome model in human gingival fibroblasts,and the mRNA and protein expression were detected by the real-time fluorescence quantitative polymerase chain reaction,enzyme-linked immunosorbent and Western blotting method.Results: 1.Primary culture and identification of human gingival fibroblasts and construction and suppression of NLRP3 inflammasome model: immunofluorescence results showed that the primary cells were human gingival fibroblasts.The results of realtime fluorescence quantitative polymerase chain reaction,ELISA and western blot showed that the expressions of mRNA and protein of NLRP3 inflammasome(NLRP3,ASC,Caspase-1,IL-1?)were up-regulated after LPS+ATP stimulation,and down-regulated after doxycycline stimulation,showing statistical differences compared with the blank group.2.Preparation and property detection of Dox:CS/CMCS-NPs and CS/CMCS-NPs:(2)Both CS/CMCS-NPs and Dox:CS/CMCS-NPs had regular morphology and stable structure.(2)The loading capacity of Dox:CS/CMCS-NPs was 28±4.01,and the encapsulation rate was(75±7.21)%.3.Detection of cell compatibility and antimicrobial activity of CS/CMCS-NPs and Dox:CS/CMCS-NPs composite nanoparticle system: CCK-8 results showed that the cell activity of CS/CMCS-NPs and Dox:CS/CMCS-NPs on human gingival fibroblasts for 24,48,72 hours was not statistically different from that of the blank group at 0,62.5,125,250,500 and 1000 ?g/ml concentrations.Colony count results showed that Dox:CS/CMCS-NPs could inhibit the growth of Porphyromonas gingivalis more effectively than CS/CMCS-NPs and blank group.4.Dox:CS/CMCS-NPs on human gingival fibroblasts in the role of the NLRP3 inflammasome: real-time fluorescence quantitative polymerase chain reaction,ELISA and Western blot results showed that Dox:CS/CMCS-NPs could down-regulate the expression in mRNA and protein levels of NLRP3 inflammasome(NLRP3,ASC,Caspase-1,IL-1?),compared with the blank group and CS/CMCS-NPs stimulation.The results were statistically significant.Conclusion: 1.Dox:CS/CMCS-NPs had stable properties,good cell compatibility and bacteriostasis,and was a good drug-carrying nanoparticle.2.Porphyromonas gingivalis lipopolysaccharide combined with adenosine triphosphate can activate NLRP3 inflammasome pathway in human gingival fibroblasts,and doxycycline and Dox:CS/CMCS-NPs could down-regulate their expression.