A Study On The Role Of NLRP Inflammasomes In Stretch-induced Programmed Cell Death And Release Of IL-1β In Human Periodontal Ligament Cells

Purpose Mechanical stimulation is closely related to the health of periodontal tissue.In the present study,we explored the stretch-induced release of inflammatory mediator IL-1β,programmed cell death and expressions of nucleotide-binding oligomerization domain-like receptor family members containing pyrin domain(NLRP)inflammasome related factors in human periodontal ligament cells(HPDLCs).We aimed to explicit the contribution of NLRP inflammasomes to the stretch-induced programmed cell death and release of inflammatory mediator,and to reveal the mechanism of stretch-induced periodontal inflammation.Methods 1.HPDLCs were cultured in vitro,and immunohistochemistry was used to determine the source of cells cultured.HPDLCs were subjected to mechanical stretch by Flexcell FX-5000 tension system with a proper elongation magnitude and proper time of strain,and the morphological change was observed by immunofluorescence to verify the effectiveness of stretch loading.Stretch-induced programmed cell death and pyroptosis were labelled with Annexin V-FITC/PI and analyzed by flow cytometry.The levels of IL-1β in the cell-culture medium of HPDLCs in response to stretch were detected by ELISA.2.HPDLCs were exposed to 20% stretch strain for 6h or 24 h,and the m RNA and protein expression levels of caspase-1,caspase-5,ASC,NLRP1,NLRP3 and IL-1β were detected by real-time quantitative PCR and western blot.Caspases’ activities were measured using the corresponding caspase colorimetric assay kits.The interactions between caspase-1 or caspase-5 and ASC were detected respectively by co-immunoprecipitation.3.With the inhibition of caspase-1,stretch-induced programmed cell death,pyroptosis and change of IL-1β were detected.With the inhibition of caspase-5,stretch-induced programmed cell death,pyroptosis and release of IL-1β in the cell-culture medium of HPDLCs were detected.Results 1.HPDLCs were successfully cultured,and the application of stretch altered the morphology.It inclined parallel to each other and aligned their long axis perpendicular to the stretching force vector.Compared with control group,the programmed cell death rates of HPDLCs increased significantly in response to 20% strain for 6h and 24h(P<0.05).Compared with control group,the content of IL-1β in the cell-culture medium of HPDLCs increased significantly in response to 20% stretch for 4h and 6h(P<0.05).2.The m RNA and protein expression levels of NLRP3,caspase-1,caspase-5 and IL-1β were up-regulated with 20% stretch for 6h(P<0.05).The protein expression levels of NLRP1 and ASC were up-regulated with 20% stretch for 6h(P<0.05),but the m RNA expression levels did not change significantly.The protein expression level of NLRP3 was up-regulated with 20% stretch for 24h(P<0.05).The results of co-immunoprecipitation revealed that the protein expressions of caspase-1 or caspase-5 were immunoprecipitated with ASC.3.The addition of the caspase-1 inhibitor significantly inhibited programmed cell death,pyroptosis,the protein expression and release of IL-1β,compared with the non-inhibited 6h stretched cells(P<0.05).The addition of the caspase-5 inhibitor significantly inhibited programmed cell death,pyroptosis and release of IL-1β,compared with the non-inhibited 6h stretched cells(P<0.05).Conclusion Stretch induced programmed cell death,including pyroptosis,and the release of IL-1β in HPDLCs.NLRP1 and NLRP3 inflammasomes contributed to the stretch-induced programmed cell death and the release of IL-1β in HPDLCs.Caspase-1 and caspase-5 were indispensable for the stretch-induced programmed cell death and release of IL-1β in HPDLCs.This study provides experimental data for elucidating the mechanism of stretch-induced programmed cell death and inflammation in HPDLCs,and has important implications on the mechanism of stretch-induced periodontal inflammation.