The Mechanism Of Tocopherol In Maintaining Cell Stemness By Inhibiting Ferroptosis Of BMSCs Under Oxidative Stress

Background:Osteoporosis is a chronic bone metabolic disease characterized by the reduction of bone mass and destruction of osteocyte structure.About 200 million people in the global population suffer from osteoporosis,and the incidence is higher than other common diseases in the world.In China,about 88 million people have osteoporosis,and the incidence of osteoporosis among people over 50 years old is 19.2%,among which the proportion of women is 32.1%and men is 6.0%.The incidence of osteoporosis in those over 65 years reached 32.0%,with 51.6%female and 10.7%male.Studies have shown that in the occurrence of osteoporosis,the patients’alveolar bone is first to be destroyed,prone to alveolar bone resorption,which leads to the jaw bone defect.Implant can maximize the chewing efficiency of patients with missing teeth and is the preferred method to repaire missing teeth.However,patients with osteoporosis often face the clinical problem of insufficient bone mass when they are undergoing dental implants.Bone metabolism is abnormal in patients with osteoporosis,which seriously affects the bone fusion of bone implants and their own bone tissue,increases the risk of failed repair of jaw defects.Therefore,improving the level of bone metabolism in patients with osteoporosis is important for the treatment of osteoporosis jaw defects.The formation of new bone not only depends on the bone resorption function of osteoclasts and osteoblasts,but also on the differences in the survival and apoptosis rates of these two cells.The decline of the number and function in BMSCs is considered to be the key factor to the bone mass loss and the repair of jaw defects.Studies have shown that the level of bone metabolism in osteoporosis patients is closely related to oxidative stress.ROS level is high in osteoporosis patients,which induces apoptosis of osteoblasts and osteocytes,inhibits cell mineralization and osteogenesis.Ferroptosis is a new type of cell death characterized by elevated reactive oxygen species and lipid peroxidation.Ferroptosis occurs in osteoblasts under oxidative stress can accelerate the development of osteoporosis.Therefore,reducing the level of oxidative stress and ferroptosis could be crucial for the regulation of osteoblast function.At present,the main drugs for the treatment of osteoporosis include raloxifene,calcitonin,bisphosphonates and so on,but long-term use will lead to mandible necrosis,digestive tract and other adverse reactions.Tocopherol is a strong antioxidant,and previous studies have also shown that tocopherol plays an important role in inhibiting inflammation,regulating immunity,inhibiting proliferation and differentiation of osteoclast.In this study,hydrogen peroxide was used to construct an oxidative stress model in vitro,to explore the effect of tocopherol on hydrogen peroxide-stimulated BMSCs and provide a new idea for improving the function of BMSCs under oxidative stress.Aims:1.Exploring the effects of tocopherol on the cell viability,oxidative stress and osteogenic differentiation in hydrogen peroxide-stimulated BMSCs.2.Exploring whether tocopherol suppresses ferropotosis in hydrogen peroxide-stimulated BMSCs via the PI3K/AKT/mTOR signaling pathway to maintain the cell stemness.Methods:1.Exploring the role of tocopherol on the cell viability,intracellular reactive oxygen species level and osteogenic differentiation in hydrogen peroxide-stimulated BMSCs.1)To determine the appropriate concentration of H₂O₂,an oxidative stress model in vitro was constructed with different concentrations of hydrogen peroxide.2)CCK8 examined the effect of tocopherol on cell viability under oxidative stress.3)The ROS fluorescence probe and flow cytometric analysis detected the effect of tocopherol on changing levels of intracellular reactive oxygen species under oxidative stress.Western Blot and qPCR determined the effect of tocopherol on antioxidant related proteins（NRF2,KEAP1）and mRNA（Nrf2,Cat,Sod-1,Sod-2）of BMSCs under oxidative stress.4)After replacing the osteogenic induction medium and co-cultivating with tocopherol,alkaline phosphatase（ALP）staining and alizarin red staining（ARS）observed the effect of tocopherol on osteogenic differentiation of BMSCs under oxidative stress;Western Blot and qPCR tested the effect of tocopherol on protein levels of osteogenic differentiation（ALP,OPN）and mRNA（Alp,Opn,Runx2）of BMSCs under oxidative stress.2.Exploring whether tocopherol promotes osteogenic differentiation of hydrogen peroxide-stimulated BMSCs by inhibiting ferropotosis.1)Western Blot and qPCR detected the effect of tocopherol on the change levels of ferroptosis-related proteins（ACSL4,xCT,GPX4）and mRNA（Gpx4,Ptgs2,Ncoa4）under oxidative stress.2)Confocal laser scanning microscope examined the effect of tocopherol on ferroptosis under oxidative stress.3)After the addition of ferroptosis agonist（Erastin）,Western Blot and qPCR further tested the effects of tocopherol on the change levels of cellular ferroptosis-related proteins（ACSL4,xCT,GPX4）and mRNA（Nrf2,Gpx4,Ptgs2,Ncoa4）under oxidative stress.4)After the addition of ferroptosis agonist（Erastin）,Western Blot and qPCR further tested the effects of tocopherol on the change levels of cellular osteogenesis-related proteins（ALP,RUNX 2）and mRNA（Alp,Ocn,Opn,Runx 2）under oxidative stress.5)After the addition of ferroptosis agonist（Erastin）,alkaline phosphatase（ALP）staining and Alizarin red staining observed the effect of tocopherol on osteogenic differentiation of BMSCs under oxidative stress.3.Exploring whether tocopherol promotes osteogenic differentiation of hydrogen peroxide-stimulated BMSCs by inhibiting ferroptosis via the PI3K/AKT/mTOR signaling pathway.1)Western Blot tested the effect of tocopherol on the change levels of oxidative stress-related signaling pathway proteins（P-PI3K,PI3K,P-AKT,AKT,P-mTOR,mTOR,P-AMPK,AMPK,P-ERK,ERK,P-P38,P38）.2)After the addition of ferroptosis inhibitor（Ferrostatin-1,Fer-1）,Western Blot tested whether the effect of tocopherol on the oxidative stress-related signaling pathway proteins（P-PI3K,PI3K,P-AKT,P-mTOR,mTOR,P-AMPK,AMPK,P-ERK,ERK,P-P38,P38）were consistent with the Fer-1.3)After the addition of PI3K agonist（740-YP）,Western Blot and qPCR examined the effects of tocopherol on the levels of ferroptosis-related proteins（ACSL4,xCT,GPX4）and mRNA（Gpx4,Nrf2,Ptgs2,Ncoa4）under oxidative stress.4)After the addition of PI3K agonist（740-YP）,alkaline phosphatase（ALP）staining and Alizarin red staining（ARS）were used to observe the effect of tocopherol on osteogenic differentiation of BMSCs under oxidative stress.Results:Part 1:1.The concentration of hydrogen peroxide at 200μM was able to significantly inhibit cell viability.2.Tocopherol was able to maintain the cell viability in hydrogen peroxide-stimulated BMSCs and upregulated the expression levels of intracellular antioxidative stress-related proteins and genes.3.Tcopherol was able to promote osteogenic activity of hydrogen peroxide-stimulated BMSCs and upregulated the expression levels of intracellular osteogenesis-related proteins and genes.Part 2:1.Tocopherol was able to inhibit the expression of ferropotosis-related proteins and genes in BMSCs stimulated by hydrogen peroxide,and reduce the level of ferrous iron level in cell mitochondria.2.Tocopherol upregulated the expression of osteogenesis-related markers in hydrogen peroxide-stimulated BMSCs by inhibiting ferropotosis.Part 3:1.Tocopherol suppressed ferropotosis levels in hydrogen peroxide-stimulated BMSCs through the PI3K/AKT/mTOR signaling pathway.2.Tocopherol promoted osteogenic differentiation of hydrogen peroxide-stimulated BMSCs by inhibiting ferropotosis through the PI3K/AKT/mTOR signaling pathway.Conclusion:Tocopherol maintains the cell stemness of BMSCs under oxidative stress by inhibiting ferropotosis through the PI3K/AKT/mTOR signaling pathway.