丁酸钠对RAW264.7细胞活性及破骨细胞分化的影响

Effects of sodium butyrate on activity of RAW264.7 cells and osteoclast differentiation

目的探讨丁酸钠对RAW264.7细胞活性及破骨细胞分化的影响。方法应用0、0.25、0.50、1.00、2.00、3.00、4.00、5.00mmol/L丁酸钠处理RAW264.7细胞,每种浓度设置3个复孔,细胞计数试剂盒-8检测其对RAW264.7细胞的毒性。活细胞染色液检测0、0.25、0.50、1.00mmol/L丁酸钠对RAW264.7细胞凋亡的影响。应用破骨细胞分化因子将RAW264.7细胞诱导成破骨细胞,实验分为两组(n=3):诱导成熟后实验组加入1.00mmol/L丁酸钠处理,对照组培养基中加入等体积溶剂,观察并比较两组破骨细胞的数量和骨再吸收区域面积。蛋白质免疫印迹法检测0、0.25、0.50、1.00mmol/L丁酸钠对RAW264.7细胞中核因子活化B细胞κ-轻链增强子(NF-κB)相关信号通路的作用。结果与0mmol/L丁酸钠比较,1.00、2.00、3.00、4.00、5.00mmol/L丁酸钠均可显著降低RAW264.7细胞的活性,差异均有统计学意义(P<0.05)。1.00mmol/L丁酸钠处理RAW264.7细胞24h后可以诱导细胞凋亡。对照组与实验组破骨细胞数量分别为9.33±2.08、4.67±1.16,差异有统计学意义(t=3.395,P=0.027);破骨细胞骨再吸收区域面积百分比分别为52.43%±5.38%、14.28%±2.72%,差异有统计学意义(t=10.970,P<0.001)。与其他浓度丁酸钠相比,1.00mmol/L丁酸钠可使NF-κB激酶的α/β亚基磷酸化水平降低,细胞质中亚基p65含量增多,B淋巴细胞瘤-2相关X蛋白、被剪切的半胱氨酸天冬氨酸蛋白酶3表达量及组蛋白H3的乙酰化均升高。结论随着丁酸钠浓度的升高,NF-κB的活性随之受到抑制,RAW264.7细胞凋亡增多。1.00mmol/L丁酸钠可减少破骨细胞的生成和骨再吸收区域面积。

Objective To investigate the effects of sodium butyrate on the activity of RAW264.7 cells and the osteoclast differentiation. Methods The RAW264.7 cells were treated by sodium butyrate at concentrations of 0, 0.25, 0.50, 1.00, 2.00, 3.00, 4.00 and 5.00 mmol/L, with 3 double pores for each concentration. The cytotoxicity of sodium butyrate on RAW264.7 cells was detected by a CCK-8 kit. The effects of sodium butyrate (0, 0.25, 0.50 and 1.00 mmol/L) on apoptosis of RAW264.7 cells were detected by Hoechst33342 staining. RAW264.7 cells were induced into osteoclasts by osteoclast differentiation factors. The experiment was carried out in 2 groups (n=3). After induced maturation, the experimental group was treated with 1.00 mmol/L sodium butyrate and the control medium was added only with the same volume of solvent. The number of osteoclasts and the area of bone resorption were observed and compared. The differentiation of RAW264.7 cells was detected by tartrate-resistant acid phosphatase (TRAP) staining. Western blotting was used to detect the effects of sodium butyrate (0, 0.25, 0.50 and 1.00 mmol/L) on NF-κB-related signaling pathway in RAW264.7 cells. Results Compared with the group of 0 mmol/L sodium butyrate, the activity of cells treated with 1.00, 2.00, 3.00, 4.00 and 5.00 mmol/L sodium butyrate for 24 h was significantly decreased (P<0.05). Treatment with 1.00 mmol/L sodium butyrate for 24 h induced apoptosis. The number of osteoclasts in the control group and the experimental group were 9.33±2.08 and 4.67±1.16, respectively, showing a significant difference between the 2 groups (t=3.395, P=0.027). The percentages of bone resorption area in the control group and the experimental group were 52.43%±5.38% and 14.28%±2.72%, respectively, also showing a significant difference between the 2 groups (t=10.970, P<0.001). Western blot results showed that, compared with other concentrations of sodium butyrate, treatment with 1 mmol/L sodium butyrate on RAW264.7 cells for 24 h led to an increase in the expression levels of cytoplasmic p65, B lymphoma-2 associated X protein and cleaved-caspase 3 and the acetylation of Histone H3 but a decrease in the phosphorylation level of α/β subunit of NF-κB kinase. Conclusions With the increased concentration of sodium butyratecan, the activity of NF-κB may be suppressed and the number of apoptotic cells may increase. 1.00 mmol/L sodium butyrate can reduce osteoclast formation and bone resorption area.