A Study On The Role Of Caspases In The Stretch-Induced PCD In HPDLCs

Purpose Force is closely related to periodontal health.Appropriate mechanical stimulation is indispensable in maintaining periodontal homeostasis and in regulating periodontal remodeling,while aberrant mechanical stimulation can be associated with destruction and resorption of periodontal tissue.Probing into the internal mechanism of periodontal remodeling and destruction in order to prevent the progress of periodontal diseases and to explore a new therapeutic target is an important topic in stomatology contemporarily.In this study,the human periodontal ligament cells(HDPLCs)were cultured in vitro.Then,the cells were subjected to stretching load and the morphological changes were observed before and after the stretching force.The programmed cell death(PCD)rates were detected,and the protein expression level and the activity of caspase-3,-5,-7,-8 and-9 were analyzed.Afterwards,the function and the regulatory mechanism of caspases in the stretch-induced PCD in HPDLCs were detected.The study could play an important role in revealing the mechanism of the mechanical force in periodontium.Methods 1.Healthy premolars were extracted for orthodontic reason and the periodontal tissue was scraped from the root surface.The HPDLCs were cultured in vitro by using tissue-cultured method.The immunohistochemistry was applied to identify the origin of the cells and the alkaline phosphatase staining was used to identify the cultured cells from gingival fibroblasts.The in vitro cultured HPDLCs were subject to 20% cyclic stretch for 6h or 24 h,and the morphological changes were observed under an inverted phase-contrast light microscope.The PCD rates were detected by Annexin V/PI double staining method and flow cytometry.2.The in vitro cultured HPDLCs were subject to 20% cyclic stretch for 6h or 24 h.The activation of caspase-3,-5,-7,-8 and-9 was analyzed by Western Blot,and the activity of caspase-3,-5,-8 and-9 was measured by colorimetric assay.3.The in vitro cultured HPDLCs were subject to 20% cyclic stretch for 6h or 24 h.The influence of caspase-8 and caspase-9 inhibition on the stretch-induced PCD and the protein expression level of caspase-3 was evaluated;the influence of caspase-3 and caspase-5 inhibition on the stretch-induced PCD was measured;the influence of caspase-3 inhibition on the activity and the protein expression level of caspase-5 was detected;the influence of caspase-5 inhibition on the activity and the protein expression level of caspase-3 was analyzed.Whether caspase-3 was immunoprecipitated with caspase-5 or not in response to cyclic stretch was determined by co-immunoprecipitation.Results 1.HPDLCs were successfully cultured in vitro by using tissue-cultured method.After 20% cyclic stretch for 6h and 24 h,the cells were elongated and prone to be paralleled to each other,with their long axis aligned perpendicularly to the stretch force vector.Cyclic stretch induced PCD in HPDLCs.After 6h stretch loading,the PCD rates increased mainly due to the up-regulation of the early PCD rates,while cyclic stretch for 24 h raised the PCD rates with more cells entering the late stage of PCD.2.20% cyclic stretch induced activation of caspase-3,-5,-7,-8 and-9 in HPDLCs.The protein expression level and the activity of caspase-3(P<0.05)and the protein expression level of caspase-7 were up-regulated by 24 h stretch(P<0.01).The protein expression level and the activity of caspase-5(P<0.01),-8(P<0.05),-9(P<0.01)were up-regulated by 6h and 24 h stretches.3.The stretch-induced PCD(P<0.01)and the protein expression level of the activated caspase-3(P<0.01)were inhibited after inhibiting both caspase-8 and caspase-9 in both 6h and 24 h stretched cells and after inhibiting caspase-9 alone in 24 h stretched cells.In contrast to the up-regulation of PCD rates after stretch loading,the addition of the caspase-3(P<0.01)or caspase-5(P<0.01)inhibitor significantly inhibited the PCD rates in HPDLCs.Meanwhile,the inhibition of caspase-5 inhibited the protein expression level(P<0.05)and activity(P<0.01)of caspase-3,while the inhibition of caspase-3 inhibited the protein expression level(P<0.01)and activity(P<0.05)of caspase-5.The result of co-immunoprecipitation revealed that the expression of caspase-3 was immunoprecipitated with caspase-5.Conclusion After subjected to 20% cyclic stretch,the in vitro cultured HPDLCs were changed in the shape and the alignment.PCD was induced in response to cyclic stretch,but the cells entered different PCD stages after 6h and 24 h stretches.The stretch-induced PCD was caspase-3,-5,-7,-8 and-9 dependent.Caspase-8 and caspase-9 functioned differently in different PCD stages in response to different stretching durations.Caspase-3 and caspase-5 could interact with each other after mechanical stretch loading,but the precise mechanism remains unknown.