Effect Of Simulated Microgravity On Ferroptosis In Osteosarcoma

In the space environment,the most common problems faced by astronauts are a series of diseases caused by microgravity,such as sensory nervous system disorders,fluid head-on transfer,cardiovascular system dysfunction,muscle atrophy,bone loss and so on.The results showed that simulated microgravity could induce iron overload and oxidative stress response of osteoblasts,resulting in a significant increase in the level of oxidative stress,including a significant decrease in oxidized glutathione and antioxidant enzymes,an increase in the levels of reactive oxygen species(ROS),malondialdehyde(MDA)and superoxide dismutase(SOD)m RNA;The main characteristics of ferroptosis are the increase of iron content and oxidative stress response,but it is not clear whether ferroptosis occurs in simulated microgravity environment.In this paper,a cell model simulating microgravity effect was constructed by using a rotating cell culture system(RCCs).Human osteosarcoma MG63 cells were divided into six groups and cultured for three days.They were ground group,ground plus ferroptosis inducer rsl3 group,ground plus ferroptosis inhibitor ferrostatin-1 group,simulated microgravity group,simulated microgravity plus ferroptosis inducer rsl3 group,and simulated microgravity plus ferroptosis inhibitor ferrostatin-1 group;MTT assay was performed on MG63 cells to screen the appropriate concentration of ferroptosis related drugs to establish an ferroptosis cell model;After repeated simulated microgravity experiments and sample acquisition,the cell samples under simulated microgravity were detected in three aspects,and the cell phenotypes were observed by MTT,cell count and morphology;QRT PCR and Western blot were performed at the molecular level to detect the m RNA and protein expression of ferroptosis marker genes GPx4 and SLC7A11;At the metabolic level,the ferroptosis related indicators GSH,Fe,ROS,MDA and SOD were detected with the kit;The molecular mechanism of its occurrence was preliminarily explored.The m RNA expression of p53,ACSl4 and Nrf2 in the ferroptosis related signal pathway was detected by QRT PCR,and the protein expression of p53 was detected by Western blot.The results showed that the survival rate and growth state of MG63 cells decreased significantly under simulated microgravity;The m RNA and protein levels of GPx4 and SLC7A11 were significantly down regulated;Iron content increased significantly;The cells in each group under simulated microgravity had oxidative stress response,which showed that GSH level was down regulated,ROS level was up regulated,MDA level was up regulated and SOD level was up regulated.The m RNA levels and p53 protein levels of p53,ACSl4 and Nrf2 in the ferroptosis related signal pathway are inconsistent with the characteristics of ferroptosis.In conclusion,MG63 cells have ferroptosis under simulated microgravity conditions,and promote rsl3 induced ferroptosis.Its occurrence may not be related to p53,ACSl4,Nrf2 mediated ferroptosis signaling pathway.