The Mechanism Of Mechanical Stress Regulating Inflammatory Response Of Chondrocytes Through Affecting Mitochondrial Function

Objective1.To explore the effects of mechanical stress stimulation with different intensities on the proliferation and apoptosis of rat inflammatory chondrocytes.2.To investigate the regulation of mitochondrial function in rat inflammatory chondrocytes by mechanical stress stimulation with different intensities.Methods1.The knee articular chondrocytes were isolated from newborn Sprague Dawley rats and cultured in vitro,intervening with 5ng/m L IL-1? to construct chondrocyte inflammatory response model.2.The inflammatory chondrocytes were given 2000?strain and 5000?strain mechanical stress stimulation by the Four Point Bending system at a frequency of 1Hz for 2h.EDU cell proliferation fluorescent staining was used to detect the proliferation ability of the cells.Western Blot assay was used to detect the proteins Caspase3 and Bax expressions.Annexin V-FITC flow cytometry was used to observe the proportion of apoptotic cells.3.The inflammatory chondrocytes were given 2000?strain and 5000?strain mechanical stress stimulation by the Four Point Bending system at a frequency of 1Hz for 2h.The changes of mitochondrial membrane potential and ROS level were detected by fluorescence staining and flow cytometry.The expressions of mitochondrial dynamics-associated proteins MFN1,MFN2,OPA1,DRP1 and mitochondrial autophagy-related proteins ULK1,Parkin,PINK1,LC3 were detected by Western Blot assays.Results1.Inflammatory chondrocytes were subjected to low-intensity stress stimulation(2000?strain)group showed higher proportion of EDU-positive cells and lower ratio of apoptotic cells compared with control group and high-intensity stress stimulation(5000?strain)group.Meanwhile,2000?strain group could down-regulate the expressions of proteins Caspase3 and Bax,while control group and 5000?strain group were opposite.2.The low-intensity stress stimulation group(2000?strain)promoted the recovery of mitochondrial membrane potential and inhibited the production of ROS compared with the control group and the high-intensity stress stimulation group(5000?strain);In contrast to control group,2000?strain stress stimulation could promote the expressions of proteins MFN1,MFN2,OPA1,DRP1 simultaneously up-regulate the expressions of proteins ULK1,Parkin,PINK1,LC3,while 5000?strain stress stimulation inhibited proteins MFN1,MFN2,OPA1 expressions and up-regulated proteins DRP1,ULK1,Parkin,PINK1,LC3 expressions.Conclusion1.Low-intensity mechanical stress stimulation could promote the proliferation of inflammatory chondrocytes and inhibit the apoptosis of inflammatory chondrocytes.2.Mechanical stress stimulation could affect the apoptosis level of inflammatory chondrocytes by regulating the function of mitochondria.Low-intensity mechanical stress stimulation could effectively accelerate mitochondrial circulation,maintain mitochondrial homeostasis and reduce the apoptosis level of chondrocytes,while the effect of high intensity mechanical stress stimulation was reversed,which resulting in the apoptosis of chondrocytes by breaking the balance of mitochondrial fusion and fission.