Effects Of Glucose Concentration And Oxygen Partial Pressure On Respiratory Metabolism Of TMJ Disc Cells In Goats

Objective:To explore the pathway of metabolism of goat temporomandibular joint disc cells,simulate the cell growth environment with glucose concentration and oxygen concentration as observation indexes,and observe the effects of changes in nutritional environment on cell proliferation,matrix synthesis and metabolism.Experiment design:primary goat TMJ disc cells were isolated from fresh goat heads which in March to June month,and then passaged cells to the third generation.According to different glucose concentration,the experimental groups were divided into glocose free group（0 m M）,micro glocose group（0.5 m M）,low glocose group（3m M）,normal glocose group（5.5 m M）and high glocose control group（22.5 m M）.According to different oxygen partial pressure,they were divided into normoxia group（21%O₂）and hypoxia group（2%O₂）.They were cultured in different nutritional environment and tested the indicators.Cell proliferation was detected by CCK-8assay.XFe Seahorse glycolysis kit was used to detect the extracellular acidification rate（ECAR）and cellular oxygen consumption rate（OCAR）;enzyme linked immunosorbent assay（ELISA）kit was used to detect the concentration of respiratory metabolism related factors:lactate dehydrogenase（LDH）,reactive oxygen species（ROS）,superoxide dismutase（SOD）;RT-q PCR was used to detect the expression of some metabolism related genes:glucose transporter 1（GLUT1）and Adenosine 5’-monophosphate（AMP）-activated protein（AMPKα1）and some extracellular matrix related genes:collagen type I（COL-Ⅰ）,collagen type II（COL-Ⅱ）and aggrecan.Mitotracker-green mitochondrion fluorescence probe staining,flow cytometry（FCM）analysis and laser confocal microscopy（CLSM）observation and image collection.The data were analyzed by one-way ANOVA and two-way ANOVA.Results:1.Cell proliferation rate:Cultured in 21%O₂for a short time（9 days）,the glucose concentration had no significant effect on cell proliferation;under 2%O₂,the cell proliferation rate of high glucose group was 60%of that of normoxia group,while that of low glucose group was arrested.2.PCR results:no matter what the oxygen partial pressure was,the expression of COL-I and COL-II genes was significantly up-regulated with the increase of glucose concentration（p<0.0001）.The expression of COL-I was significantly down-regulated in hypoxia（p<0.0001）.3.Results of Seahorse energy analysis:in normoxic condition,the group with lower sugar concentration had higher ocar and higher glycolysis ability.Ecar（p<0.0001）and ocar（p=0.0004）of TMJ disc cells in hypoxia were significantly lower than those in normoxia.In hypoxia,the energy metabolism of goat TMJ disc cells was inhibited.4.Enzyme linked immunosorbent assay（ELISA）kit results:in normal oxygen,the concentration of LDH decreased with culture time,but the concentration of ROS and SOD increased,and there was no significant difference between different glucose groups（p>0.05）.In hypoxia,the concentration of LDH increased with culture time,and the concentration of ROS and SOD increased more obviously.5.Mitotracker staining:with the increase of glucose concentration,the number of mitochondria increased.Compared with normoxia group,the number of mitochondria in hypoxia group increased significantly.The morphology of mitochondria in high glucose group was normal,while mitochondria in non-glucose group appeared swelling and deformation.Conclusion:glycolysis may be the main pathway of energy metabolism of TMJ disc cells,and its metabolic pathway will be regulated with the change of nutritional environment.Glucose is an essential nutrient to ensure the survival of TMJ articular disc cells,the synthesis of extracellular matrix and the maintenance of mitochondrial function.