A Study On The Role Of Gasdermin-d Played In The Cyclic Stretch-induced Inflammatory Reaction In Human Periodontal Ligament Cells

PurposeMechanical stimulation is closely related to the health of periodontal tissues.It has been shown that cyclic stretch could induce programmed cell death(PCD)and the release of IL-1βin human periodontal ligament cells(HPDLCs),through activating inflammasome and related caspases.However,the exact signaling regulating mechanism remains unclear.The latest research showed that Gasdermin-d(GSDMD)is a crucial executioner in inflammatory reaction.Therefore,in this study,we aim to explore the role of GSDMD plays in inducing inflammatory reaction of HPDLCs by mechanical stimulation,and further elucidate the exact mechanism of it.Methods1.Human periodontal ligament(PDL)tissues were acquired from extracted premolars of teenagers,pieces of PDL tissues were scraped from the middle of the root surface with a sterile scalpel.The HPDLCs were cultured and passaged in vitro by using tissue-cultured method.2.HPDLCs were subjected to 20% mechanical stretch by Flexcell FX-5000 tension system for 6h and 24 h,and the morphological changes were observed under an inverted phase-contrast light microscope.Stretch-induced PCD and pyroptosis were labelled with Annexin V-FITC/PI and analyzed by flow cytometry.The protein expression of IL-1β(pro-IL-1β,mature-IL-1β)and IL-18 were detected by western blot,and the secretion of IL-1βand IL-18 in the cell-culture medium of HPDLCs were measured by Enzyme linked immunosorbent assay(ELISA).3.HPDLCs were exposed to 20% cyclic stretch for 6h and 24 h,the mRNA and protein expression levels of GSDMD were dected by real-time quantitative PCR and western blot respectively.After being blocked with GSDMD siRNA,The pyroptosis rate were analyzed by Annexin V-FITC/PI and flow cytometry;The expression and secretion of IL-1βand IL-18 in HPDLCs were evaluated by western blot and ELISA in response to 6h and 24 h cyclic stretch;To identify the effect of GSDMD blocking on the expression and secretion of IL-1βand IL-18 as well as pyroptosis.4.With the inhibition of caspase-1,HPDLCs were exposed to 20% stretch strain for6 h and 24 h,the protein expression of activated GSDMD was detected by western blot.Results1.HPDLCs were successfully cultured and passaged by using tissue-cultured method.2.After exposing to 20% cyclic stretch for 6h,the PCD and pyroptosis rates of HPDLCs up-regulated in comparison with control group(P<0.05).The protein expression levels of IL-1βand IL-18 increased compared with control group in response to 6h cyclic stretch(P<0.05).6h and 24 h cyclic stretch up-regulated the concentration of IL-1βand IL-18 in the cell culture medium,when compared with control group(P<0.05).3.After exposing to 20% cyclic stretch for 6h and 24 h,the mRNA and protein expression of GSDMD in HPDLCs both increased(P<0.05).With the addition of GSDMD siRNA,the pyroptosis rates decreased in response to 6h stretch,when compared with non-inhibited 6h stretched group(P<0.05);Similarly,The expression and secretion of IL-1βand IL-18 in HPDLCs were both dropped compared with non-inhibited 6h stretched group(P<0.05).4.The expression of the activated GSDMD decreased with the addition of caspase-1inhibitor after 6h cyclic stretch,compared with the non-inhibited 6h stretched cells(P<0.05).ConclusionThe present study demonstrated that GSDMD was involved in the inflammatory reaction in HPDLCs subjected to mechanical stretch,for 20% cyclic stretch could up-regulated the mRNA and protein expression of GSDMD.In addition,GSDMD may be an important regulating effector in the secretion of inflammatory cytokines IL-1 β and IL-18 during the process of stretch-induced pyroptosis and may be regulated by caspase-1.These findings indicated a more precise signaling of stretch-induced inflammatory response in HPDLCs.