| Objective: To construct an overloaded cell model,verify the pro-apoptotic effect of high tensile stress on goat temporomandibular joint disc（TMJ disc）cells.Then we explored whether resveratrol（RSV）could reverse the apoptosis of TMJ disc cells under high-intensity stress,finally achieve the anti-apoptotic effect.Experimental design: Fresh 3-month-old goat heads were selected.TMJ disc cells were isolated,cultured in vitro,and passed to the third generation.Subsequently,the TMJ disc cells were seeded in the silica membrane chamber,and the cells were subjected to uniaxial periodic tensile stress using an in vitro cell augmentation system（B-Bridge cell stretcher）.The cells were divided into blank control groups（0% tensile strain rate）and 2%,4%,and 8% tension groups（parameters: 0.5Hz,1h/d,3d）.The changes of cytoskeleton were observed by immunofluorescence staining.The cell proliferation level and apoptosis rate were detected by CCK-8 method and flow cytometry,respectively.It was preliminarily verified that 8% tension could be applied as high-intensity stress to promote cell apoptosis.Then,different concentrations of RSV（0,2.5,5,7.5,10,20 μM）were added to TMJ disc cells,and the optimal concentration of RSV intervention was screened out by CCK-8.Then the experimental groups were divided into blank group,RSV group,2% tension group,2%+RSV group,8% tension group and 8%+RSV group.The cells were also subjected to stress for 3 days,1 hour a day,using CCK-8,Hoechst 33259 staining and flow cytometry to detect cell proliferation and apoptosis,RT-q PCR detection of Bcl-2,Bax and Caspase-3 gene expression.Data were analyzed using paired t-test and one-way ANOVA.Main results:1.Effects of different tensile stress on cell morphology,proliferation,and apoptosis: Immunofluorescence staining showed that the fibrous skeleton in the 2%tension group was elongated and a complete fibrous network structure was observed.The cytoskeleton of the 4% tension group appeared dissociated and deformed to a certain extent.The fiber skeleton of the 8% tension group was extremely elongated,and fiber fracture even occurred.The results of CCK-8 showed that 2% tensile force could promote cell proliferation（P<0.01）,while 8% tension significantly inhibited cell proliferation（P<0.01）.The results of flow cytometry showed that the apoptosis rate increased most obviously under 8% tension（P<0.01）.In summary,the 8% tensile stress exceeded the normal physiological range and can be used to construct a highstrength stress model.2.The optimal concentration of RSV intervention: CCK-8 results showed that the concentration of RSV in the range of 2.5-7.5 μM promoted cell proliferation.When it was ≥10 μM,it inhibited cell proliferation.Under 8% tension,7.5 μM of RSV had the highest ability to promote cell proliferation.Therefore,7.5 μM of RSV was used for the following experiments.3.Effects of RSV on cell proliferation and apoptosis under 8% tensile force:CCK-8 results showed that compared with the 8% tension group,the cell proliferation in the 8%+RSV group was significantly increased（P<0.05）.The results of Hoechst33259 staining showed that the cells of the blank control group and 2% tension group had a small amount of apoptosis,while obvious apoptosis was observed in the 8%tension group.Compared with the 8% tension group,the apoptotic cells in the8%+RSV group were significantly reduced（P<0.05）.This was basically consistent with the apoptosis rate detected by flow cytometry.4.PCR detection of Bcl-2,Bax,Caspase-3 gene expression: The expression of Bcl-2 in the 8% tension group was decreased（P<0.05）,Bax expression was increased（P<0.01）,the expression of Caspase-3 was increased（P<0.05）.Compared with the8% tension group,the expression of Bcl-2 in the 8%+RSV group was increased（P>0.05）,the expression of Bax was decreased（P<0.05）,and the expression of Caspase-3 was decreased（P<0.05）.Conclusion:1.8% tensile stress as a high-intensity stress model significantly promoted the apoptosis of goat TMJ disc cells.2.RSV could effectively reverse the apoptosis of goat TMJ disc cells under high tensile stress through the mitochondrial pathway. |
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