Inhibition Of Glutamate Uptake In Senescent Chondrocytes Increases Their Susceptibility To Ferroptosis

Purpose: This study aimed to discover the possible mechanism of the long-term survival of Sen Chos by comparing the metabolic differences between senescent chondrocytes(Sen Chos)and non-senescent chondrocytes(Non-Sen Chos)in order to achieve the purpose of selectively clearing Sen Chos.Materials and methods: Human primary chondrocytes was induced senescence by radiation.Untargeted metabolomics was performed on five pairs of Non-Sen Chos and Sen Chos.According to metabolomic analysis,western blotting,q PCR and assay kits were used to observe the activation of ferroptosis metabolic system in Sen Chos.The responses of Non-Sen Chos and Sen Chos to the typical ferroptosis inducer RSL3 were detected from the aspects of cell viability,cell dead staining and lipid peroxidation.Responses to RSL3-induced ferroptosis in Sen Chos were observed by glutamine deprivation(Gln-)and glutamate supply during glutamine deprivation(Gln-Glu+).Excitatory amino acid transporter 1(EAAT1)was identified by comparing the differences in the expression of glutamate metabolism-related genes between Non-Sen Chos and Sen Chos by q PCR.EAAT1 specific inhibitor UCPH-101 and EAAT1 specific si RNA was used to observe the response of Sen Chos to RSL3-induced ferroptosis.Results: Compared with Non-Sen Chos,ferroptosis metabolic system was more active in Sen Chos.RSL3 treatment induced Non-Sen Chos ferroptosis,whereas Sen Chos had higher survival rate and less cell death,despite higher levels of lipid peroxidation and MDA in Sen Chos.Compared with normal culture,with RSL3 treatment Sen Chos were responsive to ferroptosis in Gln-,and less reactive to ferroptosis in Glu+.EAAT1 inhibition or knockdown of Sen Chos sensitized to RSL3-induced ferroptosis.Conclusions: Sen Chos has an active ferroptosis metabolism system compared with Non-Sen Chos and resist RSL3-induced ferroptosis.Resistance to RSL3-induced ferroptosis of Sen Chos is dependent on cellular glutamate uptake,which is mainly regulated by increased expression of EAAT1 in SenChos.