The Role And Mechanism Of RIP3/Caspase-8 In Human Periodontal Ligament Stem Cells Mediated Periodontal Tissue Regeneration In Inflammatory Environment

Background:Periodontitis is one of the most common oral diseases in adults,is the leading cause of adult tooth deficiency,incidence rate is as high as 50%above,serious,can increase the risk of heart disease,diabetes,arthritis,and the risk of pregnancy complications.The pathogenesis of chronic periodontitis is due to the bacteria or their products gathering on the surface of dental or periodontal tissue,which invade periodontal tissue and cause the body to chronic inflammation reaction,the disease which is associated with immune can lead to the destruction of the periodontal tissue and the absorption of alveolar bone,resulting in loose tooth loss.Therefore,periodontitis treatment is aimed at the regeneration of periodontal tissue and the recovery of structure and function of periodontal tissue in the inflammatory environment.But the current treatment methods,such as periodontal scraping and guided periodontal tissue regeneration which are affected by cure periodontitis pathogenesis complex,bacteria can't completely clear,and the body's immune response to disease development,cannot obtain the functional periodontal tissue regeneration.One of the important cell groups of periodontal regeneration is human periodontal ligament stem cells(hPDLSCs)in periodontal ligament.Due to its multi-directional differentiation function,immune regulation,high proliferation and self-renewal.hPDLSCs can form periodontal ligament cells and cementum cells in vitro differentiation,and form cementum/periodontal membrane structure in vivo.Its discovery and research provide new ideas and new methods for periodontal tissue regeneration,which is of great significance.Recent studies have shown that RIP3 and Caspase-8 regulate bacteria induced autoimmune and programmed necrosis,also known as necroptosis.However,in the inflammatory state,hPDLSCs differentiation and periodontal tissue regeneration decreased,and the immune response increased,and its specific molecular mechanism was not clear.Therefore,it is of great clinical significance and application value to solve the problem of how to use hPDLSCs to regulate inflammatory response and promote periodontal regeneration.Gram-negative bacteria are the main pathogenic bacteria in periodontal pathogens,and the endotoxin is a pathogenic substance which is gram-negative bacteria sole own.The main ingredient lipopolysaccharide(LPS)is a component in the cell wall of gram-negative bacteria,among which the gingival porphyrins(Pg)is the main pathogenic bacteria causing chronic periodontitis.We simulated periodontitis with LPS which is Pg-derived.A recent study found that receptors interacting protein 3(RIP3)which is from the silk/threonine protein kinase family induced a kind of programmed necrosis(necroptosis)under LPS stimulation,and it regulate the immune response which IL-1(3 participate in through the NLRP3(inflammatory corpuscle).Objectives:In vitro and vivo Study in this paper,plan to investigate necroptosis happened in hPDLSCs in inflammatory conditions and its effect on periodontal tissue regeneration in RIP3 and Caspase-8 signaling pathways,and it will provide a new strategy for the treatment and theoretical basis in the future.Material and Methods:1.hPDLSCs' isolation,culture and identification:this experiment adopts the limited dilution method to cultivate hPDLSCs.MTT assay cell proliferation;the cell cycle and mesenchymal stem cell markers(STRO-1,CD146,CD34 and CD45)were detected by flow cytometry.Immunofluorescence staining was used to detect vimentin monoclonal antibody and keratin polyclonal antibody.multidirectional differentiation detection:toluidine blue staining detect clone formation rate calculation,oil red O staining into fat,alizarin red staining mineralized nodules,PCR detection of hPDLSCs osteogenesis after induction of osteogenesis related gene protein(COL1,RUNX-2,BSP,OCN).2.In this experiment,it is necessary to determine that in the inflammatory environment,there exists necroptosis,inhibiting RIP3/Caspase-8 protect the biological characteristics of hPDLSCs.The experiment was divided into five groups:control group:hPDLSCs were cultured in the alpha-mem culture medium;LPS group:hPDLSCs were cultured in medium containing LPS(10ug/ml);Caspase-8 suppression group(LPS+Z-VAD-FMK):hPDLSCs were cultured in the medium containing LPS(10ug/ml)+ Z-VAD-FMK(20?M);RIP3 inhibited group(LPS+GSK'872):hPDLSCs were cultured in the medium containing LPS(10 ug/ml)+ GSK'872(3?M);RIP3/Caspase-8 suppression group(LPS+ Z-VAD-FMK +GSK'872):hPDLSCs were cultured in the medium containing LPS(10ug/ml)+Z-VAD-FMK(20?M)+GSK'872(3?M).The necroptosis cells was observed under transmission electron microscope.The cell apoptosis and necrosis level were quantitatively evaluated by Annexin V-PI double-labeled flow cytometry.The immunofluorescence technique was used to detect the RIP1/RIP3 complex,further determine whether there is necroptosis;WB detect RIP1,RIP3,MLKL;RT-PCR,alizarin red staining,ALP staining and quantitative evaluate the osteoclast capacity of the cells.3.In this part of the experiment,we need to clarify.RIP3/Caspase-8 regulates the immune response of hPDLSCs in the inflammatory environment,which is achieved through necroptosis.Furthermore,inhibition of RIP3/Caspase-8 reduced the inflammatory response and immune characteristics of hPDLSCs.In the same way,immunofluorescence was used to detect NLRP3,caspase-1 and the co-location of NLRP3,caspase-1 and both in the downstream of N1RP3-IL-1? signaling pathway and RT-PCR and ELISA were used to detect the expression of inflammatory cytokines TNF-alpha,I1-1?and IL-18.4.Inhibiting RIP3/Caspase-8 can effectively promote the regeneration of periodontal tissue of hPDLSCs in inflammatory state.The potential of hPDLSCs' forming cementum has been further verified by experiments in animal models.Subcutaneous heterotopic transplantation of nude mice:HE and Masson staining were used to observe the formation of periodontal tissue,and we analyzed the bone mass formed by Image pro-plus analysis using computer Image analysis software;Immunofluorescence staining was used to detect periodontal related osteogenic proteins(COL1,OCN,BSP,RUNX-2).Experimental study on the cell aggregate graft and bone graft of SD rat periodontal defect:Micro-CT was used to observe the periodontal tissue of the living body in situ.Bone absorption was observed in TRAP staining,while HE and Masson staining were used to observe the formation of periodontal tissue,using computer Image analysis software Image pro-plus to analyze the bone mass.Results:1.after osteoinduction,the hPDLSCs alizarin red staining was positive.After lipid induction,oil red O staining positive;vimentin monoclonal antibody dyed positive,and keratin polyclonal antibody negative;Flow cytology tested positive expression of STRO-1 and CD 146,while negative expression of CD34 and CD45;RT-PCR gene expression in osteogenesis,all of these showed hPDLSCs,which were cultured with a limited diluent method,were consistent with the characteristics of mesenchymal stem cells,and which is also the stem cells required for this experiment.2.In Inflammatory condition,TEM,Annexin V-PI double marking streaming and immunofluorescence can confirm hPDLSCs exist necroptosis.WB results confirm RIP3/Caspase-8 regulate hPDLSCs'signal;RT-PCR and alizarin red staining,ALP staining showed that RIP3/Caspase-8 inhibition have certain protective effect on hPDLSCs' biological characteristics.3.Immunofluorescence detection showed that RIP3/Caspase-8 regulates the immune response of hPDLSCs,indicating that the downstream signaling pathway exists,which is achieved through necroptosis.RT-PCR and ELISA detection further show that inhibiting RIP3/Caspase-8 lower the inflammatory response and immune characteristics of hPDLSCs.4.Micro-CT,HE and TRAP staining showed that injection LPS can be successfully used as a model of inflammatory periodontal defect in rats.The results of CT and histological results in vivo experiments showed that hPDLSCs had the ability of forming cementum and alveolar bone,and inhibition of RIP3/Caspase-8 was particularly effective in promoting the regeneration of periodontal tissue.Conclusion:It is concluded that inhibiting RIP3/Caspase-8 can effectively promote periodontal tissue regeneration in the inflammatory microenvironment.