| Objective:Pyroptosis is a recently discovered inflammatory programmed cell death,closely related to oral infectious diseases.However,whether it is involved in the periodontal tissue lesions caused by occlusal traumatic diseases still needs to be further explored.Methods:Male SD rats,aged 7-week-old,weighing 250 ±20 g,were randomly divided into five groups:control group(group C),primary occlusal trauma group(group T),experimental periodontitis group(group L),secondary occlusal trauma group(group T+L)and inhibitor group(group T+L+I).The rat model of group T was established by using raised 1 mm CAD/CAM Co-Cr metal crown bonding on the right maxillary first molar.The rat model of e group L was established by periodontal wire ligation combined with LPS gingival sulcus injection.The rat model of group T+L was established by subgingival margin 1.5 mm and raised 1 mm Co-Cr metal crown bonding combined with LPS gingival sulcus injection.The rat model of group T+L+I was established by intraperitoneal injection of glyburide at the same time of the establishment of the model of group T+L.The rats were sacrificed at 1,2,3 and 4 weeks after modeling.QRT-PCR analyzed the expression of pyroptosis-related,inflammatory and osteoclast-related gene.Micro-CT analyzed the changes of bone morphological parameters.Morphological changes of periodontal tissue were observed by H&E staining.TRAP staining was used to observe the expression of osteoclasts.IHC staining was used to detect the expression and distribution of NLRP3,caspase-1,GSDMD and IL-1β.Caspase-1/Tunel immunofluorescence staining was used to analyze the location of cell death and caspase-1 positive cells.Results:1.The results of qRT-PCRThe expression of NLRP3,ASC,caspase-1 and GSDMD in group T were significantly higher in group C(P<0.05),and the expression of RANKL peaked in the 2nd week(P<0.05),was still higher in group C in the 4th week.The expression of NLRP3,ASC and caspase-1 in group L gradually increased,and reached the highest level in the 3rd week(P<0.05),and the expression of GSDMD was significantly higher in group C in the 2nd week(P<0.05).The expression of NLRP3,caspase-1,GSDMD in group T+L were significantly increased in the 1st week(P<0.05),and still had a higher expression in the 3rd and 4th week(P<0.05).Compared with group T and L,the expression of pyroptosis-related factors,RANKL and IL-1β in group T+L was significantly increased(P<0.05),while the expression of OPG was significantly decreased(P<0.05).The expression of pyroptosis-related factors,IL-1β,and RANKL in group T+L+I was significantly decreased(P<0.05),and the expression of OPG was increased(P<0.05),compared with group T+L.2.The results of IHC stainingThe HSCORE of NLRP3 and caspase-1 in group T reached the peak in the 3rd and 2nd week respectively,and the HSCORE of GSDMD increased with time.The HSCORE of NLRP3,GSDMD and caspase-1 in group L was the highest in the 3rd and 2nd week respectively.The HSCORE of NLRP3,caspase-1 and IL-1β in group T+L was the highest in the 2nd and 3rd week,and continued to be expressed in the 3rd and 4th week respectively.The HSCORE of GSDMD in group T+L increased with time.Compared with group T and L,the HSCORE of NLRP3,caspase-1,GSDMD and IL-1β were higher in group T+L.3.The results of H&E and caspase-1/TUNEL fluorescence stainingH&E staining showed that with the passage of time,the arrangement of periodontal ligament fibers was disordered and the infiltration of inflammatory cells increased.The surface of alveolar bone in the root bifurcation area showed Howship lacunar.The results of caspase-1/TUNEL fluorescence staining showed that the expression of pyroptosis-like cells in group T+L was the strongest in the 2nd week(P<0.05).4.The results of TRAP stanningIn the root bifurcation area of the right maxillary first molar,TRAP positive cells were continuously expressed in group T from the 1st to the 4th week.The expression of TRAP positive cells was the highest in group L in the 3rd week.Compared with group T and L,a large number of TRAP positive cells were found in group T+L in the 2nd week,and a small number of TRAP positive cells were still found in the 4th week.5.The results of Micro-CTThe indexes of BMD,BV/TV and Tb.Th in group T+L were significantly lower than those in group C,T,and L in the 4th week(P<0.05).The indexes of BMD,BV/TV,and Tb.Th in group T+L+I were significantly higher than those in group T+L(P<0.05).Conclusion:1.Primary occlusal trauma can induce increased expression of pyroptosis-associated factors in the periodontal tissue of rats in the early stage.2.LPS can induce the rapid expression of pyroptosis-associated factors in the periodontal tissue of rats in the middle stage.3.Secondary occlusal trauma can still induce the high expression of pyroptosis-associated factors in the periodontal tissue of rats in the middle and late-stage4.The secondary occlusal trauma can promote the obvious production of pro-inflammatory cytokines in the periodontal tissues leading to severe bone resorption5.Glyburide,the NLRP3 inhibitor,can attenuate pyroptosis and reverse the bone resorption caused by secondary occlusal trauma. |
