title: GYY4137 stimulates osteoblastic cell proliferation and differentiationviaan ERK1/2-dependent anti-oxidant mechanism.
abstract: 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 H2O2for 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.
authors.affiliation: Department of Orthopaedics, 2nd Clinical Medical College of Jinan University (Shenzhen People's Hospital) Shenzhen 518020, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Lv
authors.affiliation: Department of Orthopaedics, 2nd Clinical Medical College of Jinan University (Shenzhen People's Hospital) Shenzhen 518020, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Orthopaedics, 2nd Clinical Medical College of Jinan University (Shenzhen People's Hospital) Shenzhen 518020, China.
authors.forename: Ting-Hui
authors.initials: TH
authors.lastname: Xiao
authors.affiliation: Department of Orthopaedics, 2nd Clinical Medical College of Jinan University (Shenzhen People's Hospital) Shenzhen 518020, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Orthopaedics, 2nd Clinical Medical College of Jinan University (Shenzhen People's Hospital) Shenzhen 518020, China.
authors.forename: Bo-Wen
authors.initials: BW
authors.lastname: Lin
authors.affiliation: Department of Orthopaedics, 2nd Clinical Medical College of Jinan University (Shenzhen People's Hospital) Shenzhen 518020, China.
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Zhang
authors.affiliation: Department of Orthopaedics, 2nd Clinical Medical College of Jinan University (Shenzhen People's Hospital) Shenzhen 518020, China.
authors.forename: Yi-Miao
authors.initials: YM
authors.lastname: Lin
authors.affiliation: Department of Orthopaedics, 2nd Clinical Medical College of Jinan University (Shenzhen People's Hospital) Shenzhen 518020, China.
authors.forename: Zhong-Shi
authors.initials: ZS
authors.lastname: Xu
keywords: ERK1/2; Oxidative stress; bone formation; hydrogen sulfide; osteoporosis; reactive oxygen species