Objective To observe the effect of caffeine of different concentrations on the metabolic activity and DNA methylation levels of rat osteoblasts.Method Osteoblasts were isolated from the skull of neonatal SD rats with pancreatien-collagenase digestion.Alkaline phosphatase(ALP)staining and Alizarin red staining were used to identify rat osteoblasts.Osteoblasts were treated with 0.00,0.25,0.50,1.00,and 2.00 mmol/L of caffeine for 48 h.Osteoblast metabolic viability was detected with CCK-8.ALP activity in osteoblasts was detected by microenzyme tagging.Bone Gla-protein(BGP),5-methylcytosine(5-mC)and 5-hydroxymethylcytosine(5-hmC)contents were measured by ELISA.DNA methyltransferase 1(DNMT1)and methylcytosine dioxygenase 2(TET2)protein expression were detected with Western blot.Results Compared with the 0.00 mmol/L group,the metabolic viability of rat osteoblasts increased in the 0.25 mmol/L caffeine group,while the metabolic viability of osteoblasts decreased in the 2.00 mmol/L caffeine group,and the differences were statistically significant(P＜0.05);Compared with the 0.00 mmol/L group,ALP activity and BGP content in rat osteoblasts increased in the 0.50 mmol/L caffeine group,while ALP activity and BGP content of osteoblasts decreased in the 2.00 mmol/L caffeine group,and the differences were statistically significant(P＜0.05);Compared with the 0.00 mmol/L group,DNMTl protein expression was reduced in the 0.50 mmol/L caffeine group and TET2 protein expression increase in rat osteoblasts,and the differences were statistically significant(P＜0.05).After the treatment with 2.00 mmol/L caffeine was performed,there was a significant increase in DNMT1 protein expression and a significant decrease in TET2 protein expression in osteoblasts,with statistical significance(P＜0.05);Compared with the 0.00 mmol/L group,the 5-mC content decreased and the 5-hmC content increased in rat osteoblast DNA in the 0.25 mmol/L,and 0.50 mmol/Lgroups.Conversely,and the differences were statistically significant(P＜0.05).The 5-mC content increased and the 5-hmC content decreased in osteoblast DNA in the 1.00 mmol/L,and 2.00 mmol/L groups,with statistical significance(P＜0.05).Conclusion Low-dose caffeine can enhance rat osteoblast metabolic activity and promotes DNA hydroxymethylation,and reduce global DNA methylation levels,while high-dose caffeine is able to suppress rat osteoblast metabolic activity and inhibit DNA hydroxymethylation,leading to reducing global DNA methylation levels.
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