The Study On The Role Of ROS In The Stretch-induced Inflammatory Reaction In HPDLCs

PurposeMechanical force plays a very important role in maintaining periodontal tissue health.Excessive mechanical force stimulation can cause aseptic inflammation of periodontal tissue.Periodontal ligament(PDL)comprises the primary target of mechanical forces.It has been reported that cyclic stretch could induce inflammatory reaction such as pyroptosis and the release of IL-1β in human periodontal ligament cells(HPDLCs),through activating inflammasomes and related caspases.Though reactive oxygen species(ROS)has been reported to be involved in the pathogen-induced periodontal inflammatory reaction,the role of ROS in the mechanical force-related periodontal diseases has not been well clarified.Recent studies have shown that reactive oxygen species(ROS)play an important role in the inflammatory reaction induced by mechanical forces,and there was a hypothesis that ROS is the upstream factor in the activation of inflammasomes.Therefore,this study aimed to explore the role of ROS in the mechanical forces induced inflammatory reaction in HPDLCs in order to further reveal the mechanism of mechanical stimulation induced inflammatory reaction in HPDLCs.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 3 h,6 h,12 h and 24 h.Using 2’,7’-dichlorofluorescein diacetate(DCFDA)as fluorescence probe,the Level of ROS in h PDLCs was detected by flow cytometry.3.HPDLCs were subjected to 20% mechanical stretch for 3 h,6 h,12 h and 24 h,and the secretion of IL-1βand IL-18 in the cell-culture medium of HPDLCs were measured by Enzyme linked immunosorbent assay(ELISA).HPDLCs were subjected to 20% mechanical stretch for 6 h,and stretch-induced pyroptosis were labelled with Annexin V-FITC/PI and analyzed by flow cytometry.4.With the inhibition of ROS by apocynin,HPDLCs were exposed to 20% stretch strain for 6h.Stretch-induced 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),IL-18,NLRP3 and caspase-1 were detected by western blot;the secretion of IL-1β and IL-18 in the cell-culture medium of HPDLCs were measured by ELISA;caspase-1 enzyme activity test kit was used to detect the level of activated caspase-1.The role of ROS in stretch induced expression and release of IL-1β and IL-18,and activation of NLRP3 and caspase-1 were identified.5.With the inhibition of caspase-1,HPDLCs were exposed to 20% stretch strain for 6h.Stretch-induced 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),IL-18,NLRP3 and caspase-1 were detected by western blot;the secretion of IL-1β and IL-18 in the cell-culture medium of HPDLCs were measured by ELISA.To clarify the role of caspase-1 in the process of stretch induced inflammation of HPDLCs,and the relationship between caspase-1 and ROS.Results1.HPDLCs were successfully cultured and passaged by using tissue-cultured method.2.Result of cellular ROS detection showed that the expression of ROS in HPDLCs increased in response to 3 h and 6 h cyclic stretch(P < 0.01 vs control for 3 h,P <0.001 vs control for 6 h)3.The results of ELISA revealed that the appearance of IL-1β and IL-18 in the cell culture medium of HPDLCs varied in response to 3,6,12,and 24 h cyclic stretches.IL-1β level increased in response 6 h cyclic stretch(P < 0.001 vs non-stretched control).The appearance of IL-18 in the cell culture medium of HPDLCs increased in response to 6 h and 12 h cyclic stretch(P < 0.05 vs control for 6 h,P < 0.01 vs control for 12 h).Flow cytometric analysis demonstrated that the pyroptotic rate of HPDLCs in response to 6 h cyclic stretch increased significantly,compared with non-stretched control(P < 0.001)4.The increased pyroptotic rate of HPDLCs in response to 6 h cyclic stretch was significantly inhibited with the addition of apocynin,the ROS inhibitor,in comparison with the non-inhibited 6 h stretched cells(P < 0.001).ELISA results showed that the increased appearance of IL-1β in response to 6 h stretch was significantly inhibited with the addition of apocynin,in comparison with the non-inhibited 6 h stretched cells(P < 0.05),and the increased appearance of IL-18 in response to 6 and 12 h stretches was significantly inhibited with the addition of apocynin,in comparison with the non-inhibited 6 h and 12 h stretched cells(P < 0.05 for 6 h,P < 0.01 for 12 h).Western blot results showed that 6 h cyclic stretch increased the expression of the IL-1β and IL-18 moderately,while the protein level of IL-1β and IL-18 then declined sharply with the addition of apocynin in response to6 h cyclic stretch,in comparison with the non-inhibited stretched cells.Western blot results showed the increased expression of NLRP3 inflammasome and caspase-1 in response to 6 h cyclic stretch,whereas the increased expression of NLRP3 and caspase-1 was significantly inhibited with the addition of apocynin.Caspase-1activity assay showed increased caspase-1 enzyme level in response to 6 h cyclic stretch(P<0.001),whereas the increased caspase-1 enzyme level was significantly inhibited with the addition of apocynin,in comparison with the non-inhibited 6 h stretched cells.5.Flow cytometric analysis demonstrated that the increased pyroptotic rate in response to 6 h cyclic stretch was significantly inhibited with the addition of caspase-1inhibitor,in comparison with the non-inhibited 6 h stretched cells(P < 0.05).Western blot results showed that the stretch-induced expression of IL-1β and IL-18 were inhibited with the addition of caspase-1 inhibitor,in comparison with the non-inhibited 6 h stretched cells(Figure 5(e),Figure 5(f)).Results of ELISA also revealed that the appearance of IL-1β and IL-18 in the cell culture medium of HPDLCs were significantly inhibited with the addition of caspase-1 inhibitor,in comparison with the non-inhibited 6 h stretched cells(P<0.05 as for IL-1β,P<0.001 as for IL-18).ConclusionROS was involved in the inflammatory reaction of HPDLCs induced by machenical forces.20% cyclic stretch increased the expression of ROS in HPDLCs,and ROS was involved in the stretch induced pyroptosis of HPDLCs and the expression and release of inflammatory factors such as IL-1 β and IL-18 via a caspase-1 dependent way.This study provides experimental data for further study of stretch induced inflammatory reaction in HPDLCs and periodontal tissue,and provides a reference for antioxidant as a new strategy for the prevention and treatment of machenical forces related periodontal inflammatory diseases in the future.