柚皮苷通过调控RAW264.7细胞功能影响MC-3T3-E1细胞的成骨分化

Naringin regulates the function of RAW264.7 macrophages to affect the osteogenic differentiation of MC-3T3-E1 cells

背景:柚皮苷作为一种中药单体,具有抗炎、促进成骨和抗癌等作用;RAW264.7巨噬细胞在骨破坏过程中发挥重要作用。有研究发现降低炎症水平可以促进MC-3T3-E1细胞的成骨分化,所以通过抑制炎症相关通路,可能对成骨细胞分化具有促进作用。目的:观察柚皮苷通过调控RAW264.7细胞功能是否会影响MC-3T3-E1细胞的成骨分化。方法:采用CCK-8法检测不同浓度柚皮苷对脂多糖诱导的RAW264.7细胞的毒性。将RAW264.7细胞分成7组:即对照组(DMEM培养基)、炎症模型组(1 mg/L脂多糖)和柚皮苷组(1 mg/L脂多糖+50,100,150,200,250μmol/L柚皮苷),通过RT-PCR检测炎症因子m RNA水平变化,筛选出抗炎浓度较好的3组(150,200,250μmol/L柚皮苷组)数据用于后续实验。将RAW264.7细胞分成4组:炎症模型组、150,200,250μmol/L柚皮苷组分别取各组的上清液制备炎症上清液。最后将上述制取的炎症上清液与成骨诱导培养基1∶1比例混合共同培养MC-3T3-E1细胞并分为5组:对照组、炎症模型组、150,200,250μmol/L柚皮苷组,培养7,14 d进行成骨相关基因的检测,并进行碱性磷酸酶染色、碱性磷酸酶活性实验。结果与结论:①1 mg/L的脂多糖对巨噬细胞无毒性作用,200μmol/L柚皮苷对脂多糖诱导的巨噬细胞有细胞毒性作用(P<0.05);②筛选药物抗炎浓度过程中,与对照组相比柚皮苷浓度为150,200,250μmol/L时抗炎效果较好(P<0.05),此3组浓度用于后续实验;③碱性磷酸酶染色结果显示,与对照组相比柚皮苷浓度200μmol/L时染色效果最好,且14 d比7 d染色效果更佳;④与对照组相比,200μmol/L柚皮苷组的成骨基因mRNA表达水平最接近对照组,成骨效果最差的为炎症模型(P<0.05);⑤碱性磷酸酶活性实验中,7 d时与对照组相比,柚皮苷浓度为200μmol/L时碱性磷酸酶活性最强(P<0.05);与炎症模型组相比,柚皮苷浓度为200μmol/L时最佳(P<0.05);14 d时,与对照组相比,200μmol/L组无显著差异;与炎症模型组相比,200μmol/L组活性最强;⑥以上结果证明,柚皮苷通过调节RAW264.7巨噬细胞功能可改善炎症状态下MC-3T3-E1细胞的成骨分化。

BACKGROUND:Naringin,as a monomer of traditional Chinese medicine,has the anti-inflammatory and anti-cancer effects and can promote osteogenesis.RAW264.7 cells play an important role in bone destruction.Some studies have found that reducing inflammation can promote the osteogenic differentiation of MC-3T3-E1 cells.So,it may promote osteogenic differentiation by inhibiting inflammation-related pathways.OBJECTIVE:To observe whether naringin can regulate the function of RAW264.7 cells to affect the osteogenic differentiation of MC-3T3-E1 cells.METHODS:The cytotoxicity of different concentrations of naringin to RAW264.7 cells induced by lipopolysaccharide was tested by cell counting kit-8method.RAW264.7 cells were divided into seven groups:control(DMEM culture),model group(1 mg/L lipopolysaccharide),and naringin groups(1 mg/L lipopolysaccharide+50,100,150,200,250μmol/L naringin).In different concentration naringin groups,the mRNA levels of inflammatory factors were detected by RT-PCR.Data of three naringin groups with better anti-inflammatory concentrations(150,200,250μmol/L)were selected for follow-up experiments.RAW264.7 cells were divided into four groups:inflammation model,150,200,250μmol/L naringin groups.The above four groups were used as inflammatory supernatant.The inflammatory supernatant prepared above was mixed with osteogenic induction medium in a ratio of 1:1 to co-culture MC-3T3-E1 cells.Culture cells were divided into five groups:control group,inflammation model group,150,200,250μmol/L naringin groups.Osteogenesis-related gene detection,alkaline phosphatase staining,and alkaline phosphatase activity experiment were carried out on 7 and 14 days of culture.RESULTS AND CONCLUSION:(1)1 mg/L lipopolysaccharide has no toxic effect on macrophages,but 200μmol/L naringin showed a cytotoxic effect on macrophages induced by lipopolysaccharide(P<0.05).(2)In the process of screening the anti-inflammatory concentration of naringin,the concentration of naringin at 150,200,and 250μmol/L compared with the control group had good anti-inflammatory effects(P<0.05).Therefore,150,200,and 250μmol/L naringin were used for subsequent experiments.(3)Alkaline phosphatase staining results showed that compared with the control group,200μmol/L naringin had the best dyeing result and the dyeing effect was better on 14 days than 7 days.(4)The mRNA expression level of osteogenesis-related genes in the 200μmol/L naringin was the closest to that in the control group and the inflammation model group had the worst osteogenic effect(P<0.05).(5)In the alkaline phosphatase activity experiment,compared with the control group and inflammation model group,200μmol/L naringin showed the strongest alkaline phosphatase activity on 7 days of culture(P<0.05).Compared with the control group and inflammation model group,200μmol/L naringin showed no statistical significance in the alkaline phosphatase activity on 14 days of culture.Whilst compared with the inflammation group,the strongest alkaline phosphatase activity was found in the 200μmol/L naringin group.(6)The above results show that naringin can improve the osteogenic differentiation of MC-3T3-E1 cells in the inflammatory state by regulating the function of RAW264.7 macrophages.