NAC Protects Periodontal Ligament Cells From Pyroptosis And Osteogenic Differentiation Damage Through The SIRT1/NF-κB/Caspase-1 Signaling Pathway

Objective: Periodontitis is a chronic inflammatory disease,the onset and progression of periodontitis is characterized by the production of large amounts of reactive oxygen species(ROS).Pyroptosis caused by excessive ROS is a new type of programmed cell death,which is accompanied by cell membrane rupture and release of a large number of inflammatory factors,increasing inflammatory response and bone resorption.N-acetylcysteine(NAC)is an antioxidant that can scavenge ROS and inhibit cell apoptosis,but its role and specific mechanism in pyroptosis and osteogenic differentiation needs further study.When cells are stimulated,the transcription of nuclear factor-κB(NF-κB)into the nucleus can stimulate the NOD-like receptor pyrin domain-containing protein 3(NLRP3)inflammasome,and then activate Caspase-1,which is the classical pathway of pyroptosis.The silent information regulator 2homolog 1(SIRT1)is a deacetylase,which can play an anti-inflammatory and antioxidant role by deacetylating key proteins,while the activity of SIRT1 can be inhibited by overproduction of ROS.Therefore,the purpose of this study was to investigate whether NAC could restore SIRT1 expression by scavenging excess ROS,and then regulate the expression of NF-κB/Caspase-1 signaling pathway,so as to reduce pyroptosis and osteogenic differentiation dysfunction of h PDLCs.Methods: Part I: To observe the effect of NAC on pyroptosis and osteogenic differentiation of h PDLCs stimulated by lipopolysaccharides(LPS)/ adenosine triphosphate(ATP).HPDLCs were isolated from human periodontium and identified.The cells were divided into three groups:control group,LPS/ATP group and LPS/ATP+NAC group.First,Cell Counting Kit-8 assay was employed to determine the appropriate concentration of NAC for the follow-up experiments.The staining of ROS was observed by fluorescence microscope.The level of ROS and the incidence of pyroptosis were detected by flow cytometry.The osteogenic differentiation of h PDLCs was observed using alkaline phosphatase(ALP)and alizarin red staining.Part Ⅱ: To investigate the mechanism of NAC in alleviating pyroptosis and osteogenic differentiation dysfunction of h PDLCs.The cells were divided into seven groups: Control group,LPS/ATP group,LPS/ATP+NAC group,LPS/ATP+NAC+EX-527(SIRT1 inhibitor)group,LPS/ATP+SRT1720(SIRT1 activator)group,LPS/ATP+BAY-11-7082(NF-κB inhibitor)group and LPS/ATP+VX-765(Caspase-1 inhibitor)group.The incidence of pyroptosis was detected by flow cytometry.The osteogenic differentiation of cells was observed by ALP and alizarin red staining.Pathway-related genes(SIRT1,NF-κB p65,ASC,NLRP3 and Caspase-1)and osteogenic genes(ALP,OCN and RUNX-2)were detected by q RT-PCR.Pathway-related proteins(SIRT1,NF-κB p65,acetylated-NF-κB p65,ASC,NLRP3,Caspase-1 and GSDMD-N)and osteogenic proteins were detected by Western Blot.The expression levels of interleukin-18(IL-18),interleukin-1β(IL-1β)and lactate dehydrogenase(LDH)were determined by ELISA.Results: Part I: The optimal concentration of NAC was selected as10 m M.Compared with the control group,LPS/ATP stimulation significantly increased the level of ROS and the incidence of pyroptosis in h PDLCs.Meanwhile,ALP and alizarin red staining showed that the osteogenic differentiation ability of h PDLCs was significantly decreased.In the LPS/ATP+NAC group,the level of ROS and the incidence of pyroptosis in h PDLCs were significantly decreased compared with LPS/ATP group,and osteogenic differentiation ability was improved.Part Ⅱ: Compared with the control group,LPS/ATP stimulation increased the incidence of pyroptosis and decreased the osteogenic differentiation ability.q RT-PCR and Western Blot results showed that LPS/ATP stimulation decreased the expression of SIRT1,ALP,OCN and RUNX-2.The expression of NF-κB p65,acetylated NF-κB p65,ASC,NLRP3,Caspase-1 and GSDMD-N were increased.ELISA results showed that the expression of IL-18,IL-1β and LDH were increased.Compared with the LPS/ATP group,the incidence of pyroptosis in the LPS/ATP+NAC and LPS/ATP+SRT1720 groups was decreased,the osteogenic differentiation ability was improved,and the expression of SIRT1,ALP,OCN and RUNX-2 were increased.The expression of NF-κB p65,acetylated-NF-κB p65,ASC,NLRP3,Caspase-1 and GSDMD-N were decreased,and the expressions of IL-18,IL-1β and LDH were decreased.Compared with the LPS/ATP+NAC group,the incidence of pyroptosis in the LPS/ATP+NAC+EX-527 group was increased,the osteogenic differentiation ability was decreased,and the expression of SIRT1,ALP,OCN and RUNX-2 were decreased.The expression of NF-κB p65,acetylated-NF-κB p65,ASC,NLRP3,Caspase-1 and GSDMD-N were increased and the expression of IL-18,IL-1β and LDH were increased.Compared with the LPS/ATP group,the incidence of pyroptosis in the LPS/ATP+BAY-11-7082 group was decreased,the osteogenic differentiation ability was improved,the expression of ALP,OCN and RUNX-2 were increased,while the expression of SIRT1 was not significantly changed.The expression of NF-κB p65,acetylated-NF-κB p65,ASC,NLRP3,Caspase-1 and GSDMD-N were significantly decreased,and the expression of IL-18,IL-1β and LDH were decreased.Compared with the LPS/ATP group,the incidence of pyroptosis in the LPS/ATP+VX-765 group was decreased,the osteogenic differentiation ability was improved,and the expression of ALP,OCN and RUNX-2 were increased.The expression of SIRT1,NF-κB p65,acetylated-NF-κB p65,ASC and NLRP3 were not significantly changed,but the expression of Caspase-1 and GSDMD-N were significantly decreased,and the expression of IL-18,IL-1β and LDH were decreased.Conclusion: NAC could inhibit pyroptosis and osteogenic differentiation dysfunction of h PDLCs by regulating the SIRT1/NF-κB/Caspase-1 signaling axis,which provides a theoretical basis for the application of NAC in periodontitis.