Experimental research into the potential therapeutic effect of GYY4137 on Ovariectomy-induced osteoporosis.

BACKGROUND: Evidence has shown that endogenous H2S plays an important role in the physiological and pathophysiological processes of many organs. The study aimed to explore whether exogenous H2S has a potential therapeutic effect on a rat ovariectomy-induced model of osteoporosis. METHODS: The OVX osteoporosis model was established in female Sprague-Dawley rats by full bilateral ovariectomy. The rats were randomly divided into four groups, with the two experimental groups receiving an intraperitoneal injection of GYY4137 or sodium alendronate. The level of H2S in the plasma was determined and common laboratory indicators to diagnose osteoporosis, such as alkaline phosphatase (ALP) activity and the levels of osteocalcin (OCN), calcitonin, parathyroid hormone and leptin were measured. The bone mineral density (BMD) of the 4th and 5th lumbar vertebrae was measured using dual-energy X-ray absorptiometry. The maximum stress of femoral fracture was obtained through a three-point bending test of the femur. RESULTS: The OVX osteoporosis model was successfully established. GYY4137 was injected to increase the level of H2S in the plasma in one group, designated OVX-GYY during the observation period (p < 0.05). At 12 weeks, the BMD value of the fourth lumbar vertebra in the OVX-GYY group had increased (p < 0.05). The BMD femur value in the OVX-vehicle group had decreased (p < 0.05). Bilateral ovariectomy leads to biochemical disorders related to bone metabolism and hormone levels in rat plasma (all p < 0.05). Ovariectomy also reduced blood calcium, blood phosphate and calcitonin, and increased parathyroid hormone and leptin. The opposite results were obtained for the groups with alendronate sodium or GYY4137 treatment (all p < 0.05). CONCLUSIONS: Through the slow release of H2S, GYY4137 did an excellent job of simulating endogenous neuroendocrine gaseous signaling molecules. Exogenous H2S had a regulatory effect on osteoporosis in ovariectomized rats, showing potential value for the treatment of human postmenopausal osteoporosis.

GYY4137 stimulates osteoblastic cell proliferation and differentiation via an ERK1/2-dependent anti-oxidant mechanism.

OBJECTIVE: Oxidative stress plays a critical role in the development of osteoporosis. Hydrogen sulfide (H2S), produces anti-oxidant effect in various biological systems. The present study found that GYY4137, a slow H2S releasing compound, stimulated both mRNA level and activity of alkaline phosphatase, the marker of osteoblast differentiation. This research aims to explore the mechanism on how GYY4137 stimulates osteoblastic cell proliferation and differentiation via an ERK1/2-dependent anti-oxidant approach. METHODS: The MC3T3-E1 osteoblast-like cell line was cultured in plate. After pretreatment with GYY4137 (100 µM) for 30 min, the cells were washed twice with PBS solution and then incubated in freshly prepared low serum medium containing 400 µM H2O2 for 4 h. Cells viability was evaluated with the MTT. Cell apoptosis was evaluated by the Hoechst 33342. Then, ALP activity, NO and the superoxide dismutase (SOD) activity is determined by assay kit accordingly, ALP mRNA is identified by RT-PCR. ERK1/2 was analyzed by Western blot. The ROS production was measured with a fluorescence reader. All data was analyzed by SPSS 16.0. RESULTS: We found in the present study that GYY4137, a slow H2S releasing compound, stimulated both mRNA level and activity of alkaline phosphatase, the marker of osteoblast differentiation. RT-PCR shows that GYY4137 stimulated the transcriptional levels of Runx2, a key transcription factor associated with osteoblast differentiation. These data suggest that GYY4137 may stimulate osteoblastic cell proliferation and differentiation. Moreover, GYY4137, which alone at 1-1000 µM had no significant effect, protected MC3T3-E1 osteoblastic cells against hydrogen peroxide (H2O2)-induced cell death and apoptosis. This was mediated by its anti-oxidant effect, as GYY4137 reversed the reduced superoxide dismutase activity and the elevated productions of reactive oxygen species and nitric oxide in the osteoblastic cells treated with H2O2. Western blotting analysis showed that the protective effects of GYY4137 were mediated by suppression of ERK1/2. CONCLUSIONS: GYY4137 stimulates osteoblastic cell proliferation and bone differentiation via an ERK1/2-dependent anti-oxidant mechanism. Our findings suggest that GYY4137 may have a potentially therapeutic value for osteoporosis.

Hydrogen sulfide protects MC3T3-E1 osteoblastic cells against H2O2-induced oxidative damage-implications for the treatment of osteoporosis.

Osteoporosis is a bone disease that leads to an increased risk of fracture. Oxidative damage is an important contributor to the morphological and functional changes in the development of osteoporosis. We found in this study that hydrogen sulfide (H2S), a novel endogenous gaseous mediator, protected MC3T3-E1 osteoblastic cells against hydrogen peroxide (H2O2)-induced oxidative injury. NaHS, an H2S donor, increased cell viability and reduced cell apoptosis caused by H2O2. NaHS also stimulated osteoblast proliferation by enhancing both transcription and activity of alkaline phosphatase in MC3T3-E1 osteoblastic cells. Moreover, treatment with NaHS stimulated the transcriptional level of osteocalcin, the main bone matrix protein, and the protein expression of collagen, a major constituent of bone tissue. The above effects were mediated by the antioxidant effect of H2S. NaHS reversed the reduced superoxide dismutase activity, decreased reactive oxygen species production, and suppressed NADPH oxidase activity in H2O2-treated osteoblasts. In addition, NaHS treatment also produced anti-inflammatory effects via inhibition of the production of nitric oxide and TNF-α, suggesting an anti-inflammatory effect of H2S. Cell viability and Western blotting analysis demonstrated that the protective effects of H2S were mediated by p38 and ERK1/2 MAPKs. In conclusion, H2S protects osteoblastic cells against oxidative stress-induced cell injury and suppression of proliferation and differentiation via a MAPK (p38 and ERK1/2)-dependent mechanism. Our findings suggest that H2S may have a potentially therapeutic value for osteoporosis.

[Biocompatibility of polylactic-co-glycolic acid for culturing bFGF gene-transfected bone marrow stromal cells and application of the cell complex for repairing rabbit cartilage defect].

OBJECTIVE: To evaluate the biocompatibility of polylactic-co-glycolic acid (PLGA) for culturing bFGF gene-transfected bone marrow stromal cells (BMSCs) and assess the feasibility of this cell complex for repairing cartilage defect in rabbits using tissue engineering method. METHODS: BMSCs transfected by bFGF gene were cultured on PLGA matrix to assess the biocompatibility of PLGA. The cell complex was then implanted into the cartilage defect in rabbits, and its effect in cartilage defect repair was evaluated by histological observation and immunohistochemical staining. RESULTS: BMSCs transfected by bFGF gene grew normally on PLGA matrix. After implantation, the complex showed good effect for cartilage defect repair in rabbits. CONCLUSION: PLGA has good biocompatibility with the transfected BMSCs, and the cell complex can be used for repairing rabbit cartilage defect and may potentially serve as a substitute of cartilage autograft.

[Construction of eukaryotic expression vectors of basic fibroblast growth factor and transfection in rabbit bone marrow stromal cells].

OBJECTIVE: To investigate approach and possibility of transferring basic fibroblast growth factor (bFGF) gene into rabbit bone marrow stromal cells (BMSCs). METHODS: The eukaryotic expression vectors harboring bFGF cDNA were constructed and transfected into rabbit BMSCs mediated by liposome. The transcription and expression of bFGF gene in the transfected BMSCs were detected by means of morphological observation, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and RT-PCR. The changes in the biological characteristics of the transfected MSCs were also observed. RESULTS: Stable overexpression of bFGF protein was detected in the transfected BMSCs, which showed differentiation towards chondrocyte lineage. CONCLUSION: Stable expression of bFGF gene in transfected BMSCs can induce cell differentiation into chondrocyte lineage.