| Objective: The effects of folic acid on bone health and osteogenic/adipogenic differentiation of hBMSCs in high-fat environment were studied through in vivo and in vitro experiments,and the role and mechanism of Nrf2 signaling pathway in this biological process were explored,so as to provide new ideas and experimental basis for the prevention and treatment of osteoporosis.Methods: The 8-week-old male C57/BL6 J mice were divided into normal diet group(Control group),high fat diet group(HFD group)and high fat diet supplemented with folic acid group(HFD+FA group).After 16 weeks of feeding,the mice were euthanized and the femurs and tibias were removed.Micro-CT and H&E staining were used to observe the effects of folic acid on bone microstructure and bone morphology in mice.The m RNA and protein levels of Runx2,PPAR-γ,CEBP-α,Nrf2,HO-1,NQO1 and GCLM were detected by q RT-PCR and Western Blot respectively.The intervention dose of palmitic acid and folic acid was determined by CCK-8 assay,which was used to detect the effects of them on the viability of hBMSCs.The osteogenic/adipogenic differentiation of hBMSCs was induced for 16 days.Alizarin red staining was used to observe the formation of calcium nodules in hBMSCs after 16 days of osteogenic differentiation.Oil red O staining was used to observe the formation of lipid droplets in hBMSCs after 16 days of adipogenic differentiation.The protein levels of Runx2,PPAR-γ,CEBP-α,Nrf2,HO-1,NQO1 and GCLM in hBMSCs after 16 days of osteogenic/adipogenic differentiation were detected by Western Blot.Results:(1)Compared with the HFD group,the body fat percentage of the mice in the HFD+FA group decreased significantly,but there was no significant change in body weight.Compared with the Control group,the trabecular sparse bone in the HFD group was destroyed,the bone mass was reduced,the BV/TV,BS/TV and Th.N was significantly decreased,the SMI was significantly increased,and folic acid intervention ameliorated these changes(P<0.05).Compared with the HFD group,the expressions of Runx2 was significantly increased in HFD+FA group,while the expression of PPAR-γ and CEBP-α were inhibited,and the expressions of Nrf2,HO-1,NQO1 and GCLM were significantly increased(P<0.05).(2)After 16 days of osteogenic induction of hBMSCs,compared with the high-fat control group,obvious calcium nodules appeared after folic acid intervention,but there was no significant difference in staining results between the intervention groups.Compared with the blank control group,the protein level of Runx2 was decreased in hBMSCs in the high-fat control group after 16 days of osteogenic induction(P<0.05).After FA at the dose of 50μmol/L treatment,the protein level of Runx2 was significantly increased(P<0.05).And the protein levels of Nrf2,HO-1,NQO1 and GCLM were significantly decreased after 16 days of osteogenic induction in high-fat environment(P<0.05).After adding folic acid,the protein levels of Nrf2,HO-1,NQO1 and GCLM were significantly increased(P<0.05).(3)After 16 days of adipogenesis induction,compared with the high-fat control group,the number and morphology of lipid droplets of hBMSCs were decreased after the intervention with FA.Compared with the blank control group,the protein expression levels of PPAR-and CEBP-α in hBMSCs of the high-fat control group were significantly increased after 16 days of adipogenic induction,and the expressions were significantly decreased after folic acid intervention(P<0.05).And after50μmol/L folic acid treatment,the protein levels of Nrf2,HO-1,NQO1 and GCLM were significantly increased compared with the high-fat control group(P<0.05).Conclusion:(1)Folic acid at the dose of 20μg/m L can alleviate high-fat diet-induced osteoporosis and improve bone microstructure in mice.(2)The improving effect of folic acid on osteoporosis in mice and the pro-osteogenic effect on hBMSCs may be through the activation of Nrf2 signaling pathway.(3)In terms of protein level,folic acid can activate the expression of Runx2 to promote osteogenic differentiation of hBMSCs,and reduce the expression of PPAR-γ and CEBP-α to inhibit the adipogenic differentiation of hBMSCs. |
PDF Full Text Request