INTS7/ABCD3 Interaction Stimulates The Proliferation And Osteoblastic Differentiation Of MouseBone Marrow Mesenchymal Stem Cells By Suppressing Oxidative Stress

Background:Abnormal proliferation of bone marrow mesenchymal stem cells(BMMSCs),impaired osteogenic differentiation ability and enhanced lipid formation ability are the main pathogenic factors of osteoporosis.The molecular malregulation of BMMSCs is the main cause of abnormal differentiation and proliferation.The discovery of new molecules and related mechanisms for the regulation of BMMSCs differentiation will help to understand the balance of osteogenic and adipogenic differentiation,and provide new ideas for the prevention and treatment of osteoporosis.Aims:We aimed to uncover the effects of the Ints7-Abcd3/Hdlbp interaction on BMMSCs biological behaviors and the potential molecular mechanism.Methods:(1)Immunofluorescent staining was performed to detect the subcellular localization of Ints7 protein,Ints7 was knocked down,CCK-8 assays and EdU-based flow cytometric analysis were used to determine the proliferation capacity,flow cytometric analysis was used to determine the cell-cycle distribution of BMMSCs,TUNEL assays were performed to determine BMMSC apoptosis,Alizarin Red S dye solution was used to stain differentiated osteoblasts,Oil Red O dye solution was used to stain differentiated adipocytes and qRT-PCR detection of osteogenic or adipogenic-related key genes.(2)Ints7 protein binding was found by immunoprecipitation assays.The bound proteins were knocked down,CCK-8 assays and EdU-based flow cytometric analysis were used to determine the proliferation capacity,respectively.TUNEL assays were performed to determine BMSC apoptosis,Alizarin Red S dye solution was used to stain differentiated osteoblasts,Oil Red O dye solution was used to stain differentiated adipocytes and qRT-PCR detection of osteogenic or adipogenic-related key genes.(3)After knocked down of Ints7 and Abcd3,the changes of ROS and DNA damage were detected by immunofluorescent staining.The mRNA level of antioxidants was detected by qRT-PCR,and the effect of antioxidants rescued by the knocked down of Ints7 and Abcd3 on the proliferation of BMMSCs was detected by CCK-8 assays.Results:(1)Immunofluorescent staining showed that Ints7 was mainly localized in the cytoplasm rather than the nucleus.Knockdown of Ints7 in BMMSCs cells showed decreased cell proliferation,increased apoptosis and cell cycle arrest in G0/G1 phase;Alizarin Red S and Oil Red O staining showed that osteogenic differentiation was inhibited and adipogenic differentiation was enhanced.(2)It was found by immunoprecipitation assays that Abcd3 and Hdlbp could bind to Ints7 protein.Subsequently,by knockdown of Abcd3 and Hdlbp,it was found that Abcd3 protein,but not Hdlbp protein,was involved in the proliferation and osteogenic differentiation of BMMSCs.After Abcd3 knockdown,the phenotype is similar to that of Ints7 knockdown e,that is,the proliferation of BMMSCs is decreased,the apoptosis is increased,the expression of osteogenic differentiation and its related marker genes Runx2,Alp,Osx and Ocn is decreased,and the adipogenesis and its related marker gene Ppay,C/ebpα and Adipsin expression was enhanced.(3)We further found that the knockdown of Ints7 and Abcd3 can increase the amount of ROS and lead to increased DNA damage due to excess ROS,while the expression of antioxidant-related genes such as Sod1,Gpx1,Gpx4,Txnrdl and Cat decreased.Additionally,antioxidants rescued the effects of Ints7 and Abcd3 interference on BMMSCs cell proliferation.Conclusion:Ints7-Abcd3 interaction stimulates proliferation and osteoblast differentiation of mouse bone marrow mesenchymal stem cells by suppression of oxidative stress.