Effect And Mechanism Of Histone Demethylase PHF8 In Weightlessness Osteoporosis

Background: Skeletal system dysfunction caused by weightlessness has become one of the major problems to be solved by astronauts in manned space flight.Its main performance is weightlessness osteoporosis,which progresses continuously and the protective effect is limited with the extension of time,seriously endangering the health and safety of astronauts.Therefore,it is important to investigate the mechanism of weightlessness osteoporosis and find effective protective measures.Objective: This project aims to reveal the characteristics of weightlessness osteoporosis and the role of bone marrow mesenchymal stem cells in it,to study the expression characteristics of PHF8 molecules in bone marrow mesenchymal stem cells under simulated weightlessness,and to reveal the role and regulatory mechanism of PHF8 in the abnormal function of BMSCs induced by simulated weightlessness.It provides novel ideas and clues for screening new drug targets to prevent or slow down weightless osteoporosis and disuse osteoporosis.Methods: 1.The bone tissue samples of lumbar 4 vertebra were collected from the normal group and disuse osteoporosis group.The expression characteristics of the osteogenesis-related transcription factor PHF8 and the bone-formation-related molecule OPN were detected by q RT-PCR.2.BMSCs were cultured in vitro and divided into Ground group(Ground)and Simulated microgravity group(SMG).BMP-2 was used to induce osteogenic differentiation and the SMG group was placed in a 2D-rotator to simulate weightlessness.The protein and m RNA levels of osteogenic-related proteins OPN and OCN,apoptotic factors Bax and Bcl-2,cell proliferation factor PCNA,cell differentiation factor ALP and osteoblast-associated transcription factors PHF8,Hoxa2 and Runx2 were measured in both groups of BMSCs using Western blot and q RT-PCR assays after 48 h of simulated weightlessness;Annexin V-FITC/PI flow cytometry was used to detect the apoptotic rate of BMSCs in the two groups of samples;In addition,the fluorescence intensity of PHF8,Hoxa2 and Runx2 in the two groups of samples was performed by immunofluorescence assay.3.Plasmid that overexpress and silence PHF8 were constructed,and after infecting BMSCs with plasmids,cells that overexpress PHF8 and silence PHF8 were obtained,respectively.The transfected cells were induced into osteogenic differentiation by BMP-2 and simulated weightlessness for 48 h.The expressions of PHF8,Hoxa2 and Runx2 were detected by Western blot.Results: 1.Compared with the ground group,the disuse osteoporosis group showed reduced expression of the bone-related transcription factor PHF8 and OPN,which is related to bone formation.2.After rotating the simulated weight loss for 48 h,the results of Western blot and q RT-PCR showed that: Compared with the ground group,the expressions of OPN and OCN,Bax,Bcl-2,PCNA and ALP,PHF8 and Runx2 were decreased,and Hoxa2 were increased.Flow cytometry results indicated that the total apoptosis rate of BMSCs in the simulated microgravity group increased by 1.54 times compared with the ground group.Immunofluorescence results showed that the fluorescence intensity of PHF8 and Runx2 decreased and that of Hoxa2 increased in simulated weightlessness compared with the ground group.3.After osteogenic induction and differentiation of cells with overexpression and silence of PHF8 and 48 h of simulated weightlessness,Western blot test results showed that compared with the simulated weightlessness,the expression of PHF8 with silence of PHF8 showed a decreasing trend,but there was no statistical significance.After overexpression of PHF8,the expression of simulated weightlessness PHF8 was significantly increased,which was statistically significant.In addition,the expressions of PHF8,Runx2 and Hoxa2 in the ground group and simulated microgravity group after overexpression of PHF8 were similar.Conclusions: 1.The expression of PHF8 in bone tissue samples of disuse osteoporosis patients was decreased.2.Simulated weightlessness can lead to changes in BMSCs function and the expression of PHF8 in BMSCs was decreased under simulated weightlessness.3.Overexpression of PHF8 gene can partially alleviate osteoporosis caused by simulated weightlessness.