Anti-inflammatory And Osteogenic Effects Of Human ?-defensin 3 Gene Transfection Along With Gold Nanoparticle In Vitro And In Vivo

[Objective]To observed the effect of human ?-defensin 3(hBD3)gene transfection on cell viability,anti-inflammatory and osteogenesis of human periodontal ligament cells(hPDLCs)in inflammatory microenvironment in vitro.To investigate the osteogenic changes of hPDLCs after adding gold nanoparticles(AuNPs)and its potential mechanism.In vivo,rat periodontal ligament cells(rPDLCs)treated with hBD3 gene and AuNPs were transplanted into SD rats in chronic periodontitis progress to explore the periodontitis changes and its potential mechanism.[Method]In vitro,hPDLCs were treated with recombinant adenovirus vector containing hBD3 gene(Ad-hBD3)and AuNPs.Cell viability was observed by CCK 8 method.After adding E.coli-LPS,the levels of cytokines of hPDLCs was detected by enzyme-linked immunosorbent assay(ELISA)to observed inflammatory reactions.Effects of hBD3 gene transfection alone or along with AuNPs on the expression of osteogenic related proteins and osteogenic differentiation of hPDLCs were discussed further.After transfection and then treated with AuNPs for 12h,the p38 and p-p38 protein levels were detected by western blot.After adding p38 MAPK pathway inhibitor SB203580(10?mol/L)into cells,experiments mentioned above were repeated to investigate whether the osteogenic differentiation process was regulated by p38/MAPK pathway.In vivo,rat periodontal ligament cells(hPDLCs)were cultured and identified.Then choose the p3-p5 cells for hBD3 gene transfection and AuNPs treatment.Five-week-old female Sprague-Dawley rats were randomly divided into different groups.The bilateral maxillary second molars were ligated by silk thread soaked ahead for 2h in Porphyromonas gingivalis bacterium solution to create the experimental periodontitis model.Cell suspension was injected into mesio,middle and distal palatal gingival of ligated molars.After execution,local and systemic changes in SD rats among the groups were observed by ELISA,Micro-CT and immunohistochemistry.[Results]hPDLCs transfected by hBD3 gene could successfully express both at mRNA and protein levels.There was no significant negative effect on cell viability after transfection and addition of AuNPs.ELISA results showed the inflammatory cytokines levels were significantly decreased in cells after transfection with or without AuNPs.qPCR and western blot results displayed that the bone-related mRNA and protein levels were increased significantly in experimental groups compared with the control.ALP staining,ALP activity assay and ARS staining results showed that the osteogenic differentiation ability of experimental groups was extremely higher than that of the control group.After transfection and then AuNPs treated for 12h,p-p38 protein levels was significantly higher in experimental groups.After adding SB203580(10?mol/L),the inhibitor of p38 MAPK pathway,the bone-related mRNA and protein levels,ALP and ARS staining results of those groups were significantly decreased.In vivo experiment,ELISA results showed-that the inflammatory cytokines levels was lower compared with control group.Micro-CT results displayed the less alveolar bone absorption and higher bone mineral density index in experimental groups.Immunohistochemical results also revealed the less gingival and bone destruction in experimental group,and the p-p38 expression levels in experimental group was higher than that in control group.[Conclusion]Under the stimulation of E.coli-LPS,the inflammatory response of hPDLCs transfected with hBD3 gene was significantly inhibited,however AuNPs seemed not to have significant effects on this results.In the inflammatory microenvironment,hBD3 gene transfection significantly increased the bone-related genes and protein expression of hPDLCs,while adding AuNPs could further promote this process.hBD3 gene transfection and the addition of AuNPs were also beneficial to the osteogenic differentiation of hPDLCs.These were potentially related to the p38 MAPK pathway.During the experimental periodontitis process in SD rats,local injection of rPDLCs treated with hBD3 gene transfection and AuNPs reduced the inflammation reaction and destruction of alveolar bone.p38 MAPK pathway may also be involved in this process.