Study Of Activation Effect Of Enterococcus Faecalis And LTA On NLRP3 Inflammasome And Drug Intervention

Periapical periodontitis was an immune inflammatory response which was primarily inflected by bacteria.Enterococcus faecalis(E.faecalis)is one of the common pathogenic bacteria with prevalence ranging from 29%to 77%in the endodontic periradicular lesions.E.faecalis has multiple virulence factors,including,protein adhesion,gelatinase,lipoteichoic acid(LTA)and so on.LTA,one of the main pathogenic factors,has a strong immunogenicity and is the main virulence factor.LTA can tolerate antibiotics and fungicides,triggering a series of inflammatory cytokine release and osteoblast apoptosis.The study found that E.faecalis LTA can interact with TLR2 of murine macrophage cell RAW264.7,then activated the NF-?B signaling pathway to promote the secretion of inflammatory cytokines TNF-a and NO,which may be one of the reasons causing periapical inflammation and bone destruction.As the first line of defense,innate immunity recognizes foreign pathogens by three kinds of pattern recognization receptors(PRR).Now three kinds of PRRs found mainly include the RIG-I like receptor(RLR),toll-like receptor(TLR),and NOD-like receptor(NLR).RLR mainly identify the virus,on the study of immune response of periapical disease caused by bacteria,although more evidence show that Toll-like receptor pathway and endodontic immune inflammatory disease has a closely related,but when Toll-like receptor pathway is inhibited,there is still the presence of inflammation,suggesting that another pathway may plays a role in periapical bone resorption,while NLR signaling pathways would be the new breakthrough.The researches on NLR signaling pathways focus on IL-1?-generation-related NLRP3 inflammasome which has high expression in stratified epithelium cells and myeloid cells,indicating that it may play a key role in the body's first line of defense against pathogenic microorganisms.In response to external stimuli,by oligomerization,NLRP3 gathers a large number of Pro-Caspase-1,forms NLRP3 inflammasome and activates Caspase-1,and then promotes the Pro-IL-1?expression,which are prerequisites for IL-1? secretion,IL-1?is a sign of activation of NLRP3 inflammasome in the innate immune defense,then causes the inflammation.Compared with reversible pulpitis,in irreversible pulpitis,NLRP3/Caspase 1 has significantly high erexpression.When used the E.faecalis act on macrophages THP-1,and found that E.faecalis can induce Caspase-1 activation and Pro-IL-1?expression,then lead to overproduction of IL-1?and cause exaggerated pulpitis.So we speculate that NLRP3 inflammasome signaling pathways will be the future new direction to research the pathogenesis of periapical periodontitis.Dioscin is a natural product present in the rootstocks of dioscorea nipponica makino,commonly known as diosgenin,is an important basic raw material for the production of steroid hormone drug.Many researches have shown that dioscin has lots of medicinal effects,such as hepatoprotective properties,treatment of osteoporosis,anti-inflammatory,lipid-lowering,anti-tumor.Studies have demonstrated that Dioscin suppressed the expression of Reactive oxygen species(ROS),IL-1 and IL-6 by LPS-induced were through downregulated of CK2,JNK,NF-?B and AP-1 activation.However,the effects of dioscin on NLRP3 inflammasome is yet unknown.The present study divided into the in vivo and in vitro experiments.In the vivo experiments part,we first examined the expression of NLRP3 inflammasome in human chronic periapical inflammatory lesions,the results show that NLRP3 inflammasome expression was significantly higher than in normal tissues,indicating NLRP3 inflammasome involved in the progression of human chronic periapical inflammatory,which provided the basis for the further research.Enterococcus faecalis is the main pathogens in the refractory apical periodontitis,so we enclosed the E.faecalis sealed into the root canal to build the rat periapical periodontitis model,and to explore whether E.faecalis can activate the NLRP3 inflammasome and whether the inflammasome involved in the body in the progress of periapical inflammation and periapical bone resorption in rats.In the vitro experiments aim to detect the activation mechanism of the virulence factor of Enterococcus faecalis LTA on NLRP3 inflammasome,and explore the role of traditional Chinese medicine Dioscin in this process,as well as the drugs'bone formation function in the presence of LTA,and provide the laboratory basis for the clinical refractory periapical treatment.Specific studies were as follows:Part 1:Expression and role of NLRP3 inflammasome in periapical periodontitis Experiment 1:Expression of NLRP3 inflammasome in clinical chronic periapical periodontitisObjective:To investigate the expression of NLRP3 inflammasome in clinical chronic periapical periodontitis,and provided the clinical application basis for next research.Methods:We collected the apical lesions of chronic periapical periodontitis in patients who need surgery or tooth extraction,we used the method of Real-time qPCR and immunochemistry to detecte the expression of NLRP3 inflammasome.Results:NLRP3,Caspase-1 and IL-1?in human periapical periodontitis expression was significantly higher than the control group(p<0.05).Correlation analysis showed that,NLRP3 and Caspase-1 expression are associated with a modest,IL-1?and the expression of Caspase-1 and NLRP3 had low correlation.Conclusion:NLRP3 inflammasomes involved in human chronic periapical inflammation.Experiment 2:Establish the E.faecalis infected rat periapical periodontitis model and investigate the expression and role of NLRP3 inflammasomesObjective:Establish the E.faecalis infected rat periapical periodontitis model,observed and analyzed the inflammation and bone resorption during different pathological periapical lesions.Detected the expression of NLRP3,Caspase-1 and IL-1?in rat periapical lesions,and analyze the possible role and the relationship between periapical bone resorption and NLRP3 inflammasomes.Methods:E.faecalis is a major pathogen for the refractory periapical,so we enclosed the E.faecalis sealed into the root canal to build the rat periapical periodontitis model,and enclosed PBS cotton as experimental controls.Micro-CT and HE staining was confirmed the progression of inflammation,immunohistochemical to detect the protein localization and expression of NLRP3,and through Real-time qPCR to detect the mRNA of NLRP3,Caspase-1 and the IL-1?,while using an enzyme staining method to detect the osteoclasts expression,analysis the relationship of osteoclast cells with IL-1?,suggesting the possible role of NLRP3 inflammasome in the bone resorption of periapical periodontitis.Results:(1)Micro-CT and HE staining showed that we successfully constructed E.faecalis induced rat periapical animal model,periapical inflammation in 2 weeks was the acute phase,3 weeks was the beginning of chronic inflammation.The areas of periapical bone resorption from 1 week,and reached the maximum at 3 weeks(p<0.05),at 4 weeks the area with the 3 weeks.(2)NLRP3,Caspase-1 and IL-1? protein and mRNA were expressed in every group,but the expression of NLRP3 inflammasome sealing bacteria group was significantly higher than enclosed PBS group,the expression of NLRP3 inflammasomes was different as inflammatory processes,the significant changes during the acute phase of 2 weeks,the expression of all of the factors are to the highest value(p<0.05).(3)Correlation analysis showed that,NLRP3 and Caspase-1 was associated with a modest,the expression of NLRP3 and IL-1?,Caspase-1 and IL-1?has highly correlated in E.faecalis+group,and in E.faecalis-group the relationship between the three groups was moderate correlation.(4)Osteoclast expression also reached the peak at 2 weeks,sealing bacteria group was significantly higher than enclosed PBS group,the expression of osteoclast-positive cells with IL-1?showed a moderate correlation.Conclusion:In the periapical periodontitis caused by E.faecalis,the expression of NLRP3,Caspase-1 and IL-1?significantly increased,suggested that the E.faecalis can activate of the NLRP3 inflammasome,the high expression of IL-1? and the expression of osteoclasts has a strong correlation.Part 2.The activation mechanism of NLRP3 inflammasome by E.faecalis LTAObjective:Detected the E.faecalis LTA whether the virulence factor for E.faecalis activate the NLRP3 inflammasome,and to explore the activation mechanism of NLRP3 inflammasome.Methods:1.We chose the major virulence factor of E.faecalis LTA as the only stimulus actingon murine macrophage cells RAW264.7,using Real-time qPCR,Western blot and ELISA methods to detect the expression of NLRP3-associated inflammatory cytokine mRNA and protein.2.We used the NF-?B inhibitor BAY 11-7082 inhibiting NF-?B signaling pathway to detect NLRP3 inflammasome activation mechanism by LTA whether by means of NF-?B signaling pathway.In order to confirm the inhibitors suppression efficiency,we used immunofluorescence staining to detect the expression of NF-?B.3.We used the nicotinamide adenine dinue leotide phosphate(NADPH)oxidase inhibitor DPI inhibited the expression of ROS to verify LTA for NLRP3 activation mechanism.In order to confirm the inhibitors suppression efficiency,we used staining and flow cytometry to detect ROS.Results:1.LTA can directly activate NLRP3 inflammasome without the need of ATP.LTA stimulate the murine macrophage two hours,it can promote the high expression of protein of NLRP3,Caspase-1 and IL-1?,protein expression had no significant difference in 2 hours after 24 hours.NLRP3 mRNA expression at day 1 was the minimum(p<0.05),3 days and 5 days was not statistically significant(p>0.05).The mRNA expression of Caspase-1 had no significant difference between 1,3,5 days,while the mRNA expression of IL-1?at the peak in 3 days(p<0.05).2.NF-?B inhibitors can effectively inhibit NF-?B P65 nuclear transfer,and once NF-?B signaling pathway is inhibited,the mRNA and protein expression of NLRP3,Caspase-1 and IL-1?were significantly reduced.3.DPI can effectively inhibit the production of ROS,once the expression of ROS is suppressed,the mRNA and protein expression of NLRP3,Caspase-1 and IL-1?were also decreased significantly.Conclusion:LTA was the virulence factor for E.faecalis activate the NLRP3 inflammasome,E.faecalis LTA does not depend on the exogenous ATP,can directly activate the protein and mRNA of NLRP3 inflammasome expression in mouse macrophage cell line RAW264.7,mainly through the activation of NF-?B signal pathway and ROS production.Part 3.Experimental Study of Dioscin on the activation of NLRP3 inflammasome stimulated by LTA and the effected on bone formation Objective:We explored whether the medicine Dioscin can inhibit the activation of NLRP3 inflammasome by LTA,as well as the drugs' bone formation function in the presence of LTAm and provide the laboratory basis for the clinical refractory periapical treatment.Methods:1.We used the traditional Chinese medicine Dioscin as the experiment drug to acted on the RAW264.7,Real-time qPCR method to detect the expression of NLRP3 inflammasome mRNA,Western blot and ELISA methods to detect the expression of NLRP3 inflammasome protein.By immunofluorescence staining to detect Dioscin on NF-?B expression,staining and flow cytometry for expression of ROS.2.Dioscin acted on the mouse embryonic osteoblast precursor cells MC3T3-E1,Real-time qPCR method to detect the expression of osteogenic factor ALP?Runx2 and OCN mRNA,and detected the formation of mineralized nodules by alizarin red staining.Results:1.Results of Real-time qPCR and Western blot showed that even with the presence of LTA,traditional chinese medicine Dioscin,can significantly reduce the expression of NLRP3,Caspase-1 and IL-1?mRNA and protein.Test results immunofluorescence staining and flow cytometry confirmed that the inhibition mechanism of Dioscin mainly through the inhibiting activation of NF-?B and ROS released.2.Chinese medicine dioscin has a strong role to promote the bone formation,when the drug acts on osteoblast precursor cells in the third days,the expression of early osteogenic factor Runx2 and ALP is higher than the normal control group(p<0.05).With the extension of time,the expression of Runx2 and ALP has gradually increased(p<0.05).Bone formation markers OCN in the 7th day was higher than the normal control group(p<0.05),the highest expression at 14 days(p<0.05),while inducing extended to 21 days,the formation of a greater amount of mineralization knotsection,indicating bone formation has occurred.Conclusion:(1)Dioscin NLRP3 can significantly inhibit the expression of NLRP3 inflammasome,and the mechanism is by inhibiting the activation of NF-?B signaling pathway and the release of ROS.(2)Dioscin has a strong role to promote bone formation.(3)Dioscin can be used as a drug candidates for the clinical refractory endodontic disease treatment.