| Objective: To investigate the effects of homocysteine(Hcy) on the osteogenic and adipogenic differentiation capability of rat bone marrow mesenchymal stromal cells(BMSCs), and the intervention of resveratrol and puerarin on the differentiation capability of rat BMSCs. To study the effects and mechanisms of homocysteine, resveratrol and puerarin on the osteoporosis.Method: 1. Rat BMSCs were isolated by whole bone marrow adherenence method, and identified surface markers of CD44, CD90, CD34 and CD45 by flow cytometry. 2. The effects of different concentration homocysteine, resveratrol and puerarin on cell proliferation were examined by MTT assay. 3. The P3 of BMSCs divided into: control group, induced differentiation(osteogenic or adipogenic differentiation, OM/AM group), R or P group(10μmol/L Resveratrol or Puerarin), H group(100μmol/L Hcy), R+H or P+H group(10μmol/L Resveratrol or Puerarin +100μmol/L Hcy), R+H+L/P+H+L group(10μmol/L Resveratrol or Puerarin+100μmol/L Hcy+10μmol/L LY294002). Cells osteogenic and adipogenic differentiation capability were detected by alizarin red staining and oil red O staining. The level of Runx2, BMP2, Collagen ?, OPG, PPAR-γ, Gadd45 and β-catenin m RNA were detected by q RT-PCR assays. The level of ALP activity were detected by kit. 3. The level ROS was detected by flow cytometry, and the level of cell MDA, GSH-PX and SOD were detected by kit. 4. AKT, p-AKT, Fox O1 proteins expression were detected by western blot assays.Results: 1. Isolate, culture and identification of rat BMSCs: BMSCs were isolated from SD rat’s bone marrow of femur and tibia. BMSCs phenotypes were analyzed by FACS, expanded BMSCs expressed CD 44 and CD 90 at(99.86±5.934)% and(95.10±2.013)%, and expressed CD34, CD45 at(0.68±0.612)% and(0.31±0.117)%. 2. MTT assay results showed: 1~1000μmol/L Hcy concentration-dependently decreased proliferation of BMSCs. 0.1~100μmol/L resveratrol increased proliferation of BMSCs, especially at the concentration of 10μmol/L(p<0.05). Puerarin(10~100μmol/L)increased proliferation of BMSCs, especially at the concentration of 10μmol/L(p<0.05). Resveratrol and puerarin decreased this inhibitory effects of Hcy on cell viability(p<0.05). 3. Alizarin red staining, oil red O staining and Real-time PCR results: Calcium nodules after alizarin red staining decreased and percentage of positive cells after oil O staining increased in H group when compared with control group. Hcy had inhibitory effects on the expression of Runx2, BMP2, Collagen ?, OPG and Gadd45 m RNA, and promoted the expression of PPAR-γ and β-catenin m RNA(p<0.05). Resveratrol and puerarin reduced this effect of Hcy, but this effect can partly blocked by LY294002(p<0.05). Cells treated with Hcy showed lower ALP activity compared with OM group(p<0.05), resveratrol and puerarin reduced this effect of Hcy, but this effect can partly blocked by LY294002(p<0.05). 4. ROS, MDA, SOD and GSH-PX: Hcy group showed higher ROS and MDA level, lower SOD and GSH-PX level(p<0.05). Resveratrol and puerarin reduced this effect of Hcy on cell ROS, MDA, SOD and GSH-PX level(p<0.05), but this reduce effect can partly blocked by LY294002. Cells treated with adipogenic medium showed higher ROS and MDA level, and lower SOD and GSH-PX level than groups of control and OM(p<0.05). 5. The result of WB: Cells treated with osteogenic medium showed higher expression of p-AKT/AKT(p<0.05), the cells treated with Hcy showed lower expression of p-AKT/AKT and Fox O1 than OM group(p<0.05), resveratrol and puerarin reduced this effect of Hcy, but this reverse effect can partly blocked by LY294002(p<0.05).Conclusion: Homocysteine leads to the development of osteoporosis by promoting the osteogenesis and inhibiting adipogenic of BMSCs via inducing oxidative stress. Resveratrol and puerarin had the effects of antiosteoporosis. The mechanism maybe was activation PI3K/AKT/Fox O1 pathway to inhibiting oxidative stress, thus protecting the osteogenesis and inhibiting the adipogenic of BMSCs. |
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