The Effect And Mechanism Of Caspase-11 On Promoting NLRP3 Inflammasome Activation Via The Cleavage Of Pannexin1 In Periodontitis

Purpose: Periodontitis,which was regarded as one of the main cause of tooth loss,is a chronic inflammation caused by bacterial dysbiosis.The clinical manifestations of periodontitis include gingival inflammation and alveolar bone resorption.The occurrence and development of periodontitis is regulated by multiple inflammatory factors.NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome is a multiprotein complex located in the cytoplasm.It participates in the innate immune response of the body by activating proinflammatory cytokines such as interleukin-1β(IL-1β)and interleukin-18(IL-18),and mediates pyroptosis.It plays an important role in maintaining immune balance and regulating the inflammatory process.However,the specific mechanism of NLRP3 inflammasome activation in periodontitis is still unclear.In this experiment,we inhibited the expression of Caspase-11 and Pannexin1,and detected the activation of NLRP3 inflammasome to explore the mechanism of how Caspase-11 activates the NLRP3 inflammasome in periodontitis.Methods: 1.Raw264.7 cells were pretreated with P.gingivalis-LPS,and western blot was used to detect the protein expression of Caspase-11,Pannexin1,NLRP3,Caspase-1 and IL-1β.2.Caspase-11 expression in Raw264.7 cells was knocked down using si-Casp-11 transfection method,and western blot was used to detect the protein expression of Caspase-11 to evaluate transfection efficiency.Using P.gingivalis-LPS pre-treated Raw264.7,western blot was used to detect the protein expression of Caspase-11,Pannexin1,NLRP3,Caspase-1 and IL-1β.3.Pannexin1 expression in Raw264.7 cells was knocked down using si-Panx1 transfection method,and western blot was used to detect the protein expression of Pannexin1 to evaluate transfection efficiency.Using P.gingivalis-LPS pre-treated Raw264.7,western blot was used to detect the protein expression of Caspase-11,Pannexin1,NLRP3,Caspase-1 and IL-1β.4.CBX and AZD9056 were used to inhibit the function of Pannexin1 and P2X7 receptor.Using P.gingivalis-LPS pre-treated Raw264.7,western blot was used to detect the protein expression of Caspase-1 and IL-1β.5.The rat model of periodontitis was established by orthodontic wire ligation.Micro-CT was used to detect alveolar bone resorption and analyze bone parameters.The rats in the experimental group were treated with local injection of CBX or AZD9056.Western blot and histological staining were used to detect the changes of Caspase-1 and IL-1β expression in rats gingival tissueResults:1.Western blot results showed that the protein expression of Caspase-11,Pannexin1,NLRP3,Caspase-1 and IL-1β in the LPS group were significantly upregulated compared to the control group.2.Western blot results showed that the protein expression of Caspase-11 in the si-Casp-11 group decreased by about 60% compared to the negative control group,and the cleavage of Pannexin1 and the activation of NLRP3 inflammasome induced by P.gingivalis-LPS were significantly inhibited.3.Western blot results showed that the protein expression of Pannexin1 in the si-Panx1 group decreased by about 60% compared to the negative control group,and the activation of NLRP3 inflammasome induced by P.gingivalis-LPS were significantly inhibited.4.Western blot results showed that the activation of NLRP3 inflammasome induced by P.gingivalis-LPS was significantly inhibited in the CBX group and AZD9056 group.5.Micro-CT analysis showed that the distance of CEJ-ABC was 3.0 times greater in rats with maxillary first molars with significant alveolar bone resorption than in control rats.Western blot and histological staining results showed that the expression of Caspase-1 and IL-1β in the gingival tissues of the inhibitor group rats was reduced compared with that of the periodontitis group.Conclusion:1.Caspase-11 promotes NLRP3 inflammasome activation via the cleavage of Pannexin1 in periodontitis.2.The cleavage of Pannexin1 leads to the activation of NLRP3 inflammasome through the release of extracellular ATP.