摘要
目的 观察不同浓度咖啡因对大鼠成骨细胞活性及DNA甲基化水平的影响。方法 胰酶-胶原酶消化法提取新生SD大鼠颅骨成骨细胞,碱性磷酸酶(ALP)染色和茜素红染色鉴定大鼠成骨细胞;分别以0.00、0.25、0.50、1.00、2.00 mmol/L的咖啡因处理成骨细胞48 h后,采用CCK-8法检测成骨细胞代谢活力,微量酶标法检测成骨细胞培养液中ALP活性,ELISA检测骨钙素(BGP)、5-甲基胞嘧啶(5-mC)和5-羟甲基胞嘧啶(5-hmC)的含量,Western blot检测DNA甲基化转移酶1 (DNMT1)和甲基胞嘧啶双加氧酶2 (TET2)蛋白表达的情况。结果 与0.00 mmol/L咖啡因组比较,0.25 mmol/L咖啡因组大鼠成骨细胞代谢活力增加,2.00 mmol/L咖啡因组成骨细胞代谢活力降低,差异均有统计学意义(P<0.05);与0.00 mmol/L咖啡因组比较,0.50 mmol/L咖啡因组大鼠成骨细胞中ALP活性和BGP含量升高,2.00 mmol/L咖啡因组成骨细胞中ALP活性和BGP含量降低,差异均有统计学意义(P<0.05);与0.00 mmol/L咖啡因组比较,0.50 mmol/L的咖啡因组大鼠成骨细胞中DNMT1蛋白表达降低、TET2蛋白表达增加、差异均有统计学意义(P<0.05),2.00 mmol/L的咖啡因组成骨细胞中DNMT1蛋白表达增加、TET2蛋白的表达降低、差异均具有统计学意义(P<0.05);与0.00 mmol/L咖啡因组比较,0.25 mmol/L和0.50 mmol/L组大鼠成骨细胞DNA中5-mC含量降低、5-hmC含量增加、差异均有统计学意义(P<0.05),1.00 mmol/L和2.00 mmol/L组的5-mC含量增加、5-hmC含量降低、差异均有统计学意义(P<0.05)。结论 低剂量咖啡因促进大鼠成骨细胞代谢活力,促进DNA羟甲基化降低整体DNA甲基化水平;高剂量咖啡因抑制大鼠成骨细胞代谢活力,抑制DNA羟甲基化,增加整体DNA甲基化水平。
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,DNMT1 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/L groups.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.
作者
黄徐
陈永福
杨晶晶
尹从玉
潘雪莉
HUANG Xu;CHEN Yongfu;YANG Jingjing;YIN Congyu;PAN Xueli(School of Public Health,Guizhou Medical University&Key Laboratory of Enviromental Pollution Monitoring and Disease Control of Ministry of Education,Guiyang 550025,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2024年第2期185-190,197,共7页
Journal of Guizhou Medical University
基金
2021年贵州省大学生创新训练计划项目(S202110660051)。
