Influence of methylene blue on the proliferation and apoptosis of intervertebral disc annulus fibrosus cells

English Keywords:Methylene blue  Annulus fibrosus cells  Caspase-3  TGF-β1  TIMP-1  Bcl-xl/bax  Proliferation  Apoptosis

English Abstract:

  【Abstract】 Objectives: To investigate the effect of methylene blue at different concentrations on the proliferation and apoptosis of intervertebral disc annulus fibrosus cells(AFCs) in vitro. Methods: AFCs were isolated and cultured with collagenase and sequential trypsin digestion from the caudal intervertebral disc annulus fibrosus of 3-month-old clean-grade Sprague-Dawley rats. Cellular morphology was observed, and cellular proliferation was detected by CCK-8 assay. The third generation AFCs were incubated with control group and various concentrations of methylene blue groups, as low group(2μg/ml), medium group(20μg/ml) and high group(200μg/ml) for 2h, 4h, 6h, 12h, 24h and 72h. The activity of caspase-3 was detected by caspase-3 kit. The mRNA and protein expressions of collagen type I, basic transforming growth factor β1(TGF-β1), fibroblast growth factor(bFGF), tissue inhibitor of metalloproteinase-1(TIMP-1), caspase-3, bcl-xl and bax were detected by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and Western blot. Results: Primary AFCs could form cell colonies and exhibit clonal growth slowly. The passaged cells showed faster growth rate and spindle-shaped with pseudo-foot out. Methylene blue showed negative effect on the proliferation of AFCs and could induce apoptosis of AFCs, the apoptosis rate of medium and high group was 74.95% and 90.09% respectively, with a statistically significant differences compared with control group. There were also significant differences among the low group, medium group and high group(P<0.05). Methylene blue also had a negative effect on the mRNA and protein expressions of collagen type I, TGF-β1, bFGF, TIMP-1 and bcl-xl, but it could increase the expressions of caspase-3 and bax. There were significant differences between the control group and various methylene blue concentration groups(P<0.05). Conclusions: Methylene blue can inhibit proliferation and promote apoptosis of AFCs in a dose and time dependent manner, and the bcl-xl/bax mediated pathway may be involved in the apoptosis mechanism. Methylene blue can also inhibit the expressions of extracellular matrix of AFCs, and the TIMP-1 mediated pathway may be involved in the mechanism.