Roles And Regulation Of Low-dose YC-1 And NAC On Energy Metabolism In Hypoxiainducible Goat Temporomandibular Joint Disc Cells

Objective:The normal temporomandibular joint（TMJ）disc is located in an ischemia and hypoxia environment,and the continuous loading would lead to the nutritional and metabolic impairment of the TMJ disc cells.The roles and regulatory mechanisms of hypoxia-inducible factor-1αspecific inhibitor（Lificiguat,YC-1）and antioxidants on the energy metabolism of TMJ disc cells are currently unclear.Therefore,in this study,we used the chemical hypoxia inducer cobalt chloride（Co Cl₂）,2%O₂ physical hypoxia and chemical-physical dual hypoxia to simulate the physiological hypoxic environment of TMJ disc cells,and explored the roles and regulation of low-dose of YC-1 and N-acetylcysteine（NAC）on the energy metabolism in hypoxia-inducible goat TMJ disc cells.Methods:Isolation and culture of goat TMJ disc cells to p₂ generation.1.Establishment of a cobalt chloride-mediated in vitro goat TMJ disc cells chemical hypoxia model.The experimental groups were divided into 0,50,100,200,300,400,500,600,700μM Co Cl₂ groups.Cell proliferation by CCK-8 assay,the relative m RNA of hypoxia-inducible factor-1α（HIF-1α）by RT-q PCR and intracellular reactive oxygen species（ROS）generation by DCFH-DA probe were used to determine the Co Cl₂concentration for subsequent experiments.2.The effects of low-dose YC-1 on energy metabolism in hypoxia-inducible goat TMJ disc cells.The experimental groups were divided into the 21%O₂ group（control group,YC-1 group,Co Cl₂ group,Co Cl₂+YC-1 group）and the 2%O₂ group（control group,YC-1 group,Co Cl₂ group,Co Cl₂+YC-1 group）.The cell proliferation,glucose consumption and lactate production in culture supernatant,intracellular adenosine triphosphate（ATP）production,ROS expression and the relative m RNA level of HIF-1α,glucose transporter 1（GLUT1）,lactate dehydrogenation A（LDHA）,pyruvate kinase M2（PKM2）and phosphoglycerate kinase1（PGK1）were detected respectively.3.The effects of NAC on energy metabolism in goat TMJ disc cells exposed to hypoxia.The experimental groups included control group,NAC group,Co Cl₂ group and Co Cl₂+NAC group,which were exposed to 21%O₂ and 2%O₂respectively.The study aimed to detect the cell proliferation,glucose consumption,lactic acid production and intracellular ATP production,as well as ROS generation and the relative m RNA level of HIF-1α,GLUT1,LDHA,PKM2 and PGK1.Results:1.The proliferation of goat TMJ disc cells were inhibited by Co Cl₂.When the concentration of Co Cl₂≤200μM,it had no obvious effects on cell proliferation at24 h and 48 h（P>0.05）,and when the concentration of Co Cl₂≥300μM,the proliferation of goat temporomandibular joint disc cells was inhibited（P<0.05）,meanwhile the inhibition was time-dependent and dose-dependent.Co Cl₂ would reduce the m RNA expression of HIF-1α,and the inhibitory effect increased with the increase of the concentration of Co Cl₂.Co Cl₂ significantly increased the expression of ROS,which was dose-dependent.2.Low-dose YC-1 inhibited the cell proliferation of goat TMJ disc cells,inhibited glucose consumption and lactate production in the culture supernatant of goat TMJ disc cells（P<0.05）,and inhibited both ROS regeneration and intracellular ATP production（P<0.05）,meanwhile inhibited the m RNA expression of HIF-1α,GLUT1,LDHA,PKM2 and PGK1（P<0.05）.3.5 m M NAC could promote the proliferation of goat TMJ disc cells exposed to hypoxia,the Co Cl₂ group was promoted significantly（P<0.05）;and promoted glucose consumption in the culture supernatant under hypoxia（P>0.05）;while significantly reduced the production of lactate in the culture supernatant（P<0.01）;and effectively eliminated the expression of intracellular ROS（P<0.001）.However,NAC promoted the intracellular ATP production（P<0.05）;but it inhibited the relative m RNA level of HIF-1α,GLUT1,LDHA,PKM2 and PGK1（P<0.05）,with the most significant inhibition of PGK1.Conclusion:1.300μM Co Cl₂ inhibited the proliferation of goat TMJ disc cells and suppressed HIF-1αm RNA expression,while promoted intracellular ROS expression.It was determined that 300μM Co Cl₂ could be used for the study of Co Cl₂-induced oxidative stress damage in goat TMJ disc cells under hypoxic environment.2.Low-dose YC-1 inhibited the glycolysis of goat TMJ disc cells through the inhibition of HIF-1α.3.NAC reduced the lactate production and inhibited the expression of key enzymes of glycolysis in hypoxia-inducible goat TMJ disc cells.However,it promoted glucose consumption and intracellular ATP production.NAC would transfer energy metabolism toward oxidative phosphorylation under hypoxia,and would become a potential clinical therapeutic target for temporomandibular joint disc diseases and temporomandibular joint disorders.