蛇床子素/壳聚糖衍生物胶束对破骨细胞分化及其特异性分子表达的影响

Effects of osthol/chitosan derivative micelles on osteoclast differentiation and specific molecular expression

目的观察蛇床子素(osthale,OST)/壳聚糖衍生物胶束(osthole-loaded N-octyl-O-sulfonyl chitosan micelles,NSC-OST)对破骨细胞分化及其特异性分子表达的影响,初步探讨其抗骨质疏松的分子机制。方法通过使用大鼠骨髓单核细胞体外建立破骨细胞诱导模型;将细胞分为空白组、对照组、OST组、NSC-OST组,以巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)和核因子κB受体活化因子配基(receptor activator of nuclear factor kappa-Βligand,RANKL)诱导细胞分化,药物组以相同的有效浓度干预细胞;采用CCK-8法观察NSC-OST对细胞活性的影响;通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法测定阳性细胞数;制备骨磨片并将其与细胞共培养,通过甲苯胺蓝染色测定各组骨吸收陷窝面积;通过扫描电镜观察骨吸收陷窝的微观结构;使用荧光素酶报告基因法观察NSC-OST对细胞中活化T细胞核因子(nuclear factor of activated T cells,NFAT)转录表达的影响;应用Western blot法检测NSC-OST对细胞中NFATc1等相关破骨特异分子蛋白表达的影响。结果CCK-8法发现OST和NSC-OST对骨髓前体细胞均没有细胞毒性;对照组、OST组、NSC-OST组TRAP染色阳性细胞数分别为148.8±12.5、107.6±9.7、75.0±7.4(个),OST和NSC-OST均可抑制破骨细胞的生成(P<0.05),但NSC-OST的抑制效果更明显(P<0.05);该3组诱导的破骨细胞生成的骨陷窝吸收面积分别为:1344942±164150、824080±37484、597716±14659(μm^2/片),两药物组的骨吸收活性均受到明显抑制(P<0.05),且NSC-OST组的抑制效果更明显(P<0.05);对照组、CsA组、OST组和NSC-OST组的荧光素酶报告基因表达量分别为29174±1540、9073±824、21661±1430、13434±879,两药物组均可明显抑制NFAT的转录表达,且NSC-OST组的抑制效果更好;与对照组相比,药物组均可抑制破骨特异分子NFATc1、Fos蛋白(cellular oncogene fos,c-Fos)、组织蛋白酶K(cathepsin K,CTSK)、抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)的蛋白表达(P<0.05),且NSC-OST组的效果更显著(P<0.05)。结论NSC-OST可以抑制破骨细胞的分化以及NFATc1等相关破骨特异分子的表达,且其效果优于OST。

Objective To observe the effects of osthol-loaded N-octyl-O-sulfonyl chitosan micelles(NSC-OST)on osteoclasts differentiation and specific molecular expression,and to explore the molecular mechanism of its anti-osteoporosis.Methods Osteoclast induction model was established using rat bone marrow mononuclear cells in vitro.The cells were divided into blank group,control group,osthole(OST)group,and NSC-OST group.Cell differentiation was induced by macrophage colony stimulating factor(M-CSF)and nuclear factor kappa B receptor activating factor ligand(RANKL).The drug group was intervened with the same effective concentration.Cells,CCK-8 method was used to observe the effects of NSC-OST on cell activity.Tartrate-resistant acid phosphatase(TRAP)staining was used to determine the number of positive cells.Bone abrasive tablets were prepared and co-cultured with cells,and the area of bone resorption lacunae in each group was measured by toluidine blue staining.The microscopic nodules of bone resorption lacunae were observed by scanning electron microscopy.Luciferase reporter gene method was used to observe the effects of NSC-OST on the transcription and expression of activated T cell nuclear factor(NFAT).Western blot was used to detect the effects of NSC-OST on the expression of osteoclast-specific molecules such as NFATc1 in cells.Results CCK-8 assay showed that OST and NSC-OST had no cytotoxicity to bone marrow precursor cells.The numbers of TRAP staining positive cells in the control group,OST group,and NSC-OST group were 148.8±12.5,107.6±9.7,and 75.0±7.4,respectively,suggesting that both OST and NSC-OST can inhibit the production of osteoclasts(P<0.05),but the inhibitory effect of NSC-OST is more obvious(P<0.05);the absorption areas of osteoclasts induced by these three groups were 1344942±164150,824080±37484,597716±14659(μm^2/piece),suggesting that the bone resorption activity of both drug groups was significantly inhibited(P<0.05),and the inhibitory effect of the NSC-OST group was more significant(P<0.05);the control group,CsA group,OST group and NSC-OST group The luciferase reporter gene expression levels were 29174±1540,9073±824,21661±1430,13434±879,both drug groups can significantly inhibit the transcriptional expression of NFAT,and the NSC-OST group has better inhibition effect.Compared with the control group,the drug group could inhibit the osteoclast-specific molecules NFATc1,Fos protein(c-Fos),cathepsin.K(CTSK),tartrate-resistant acid phosphatase(TRAP)protein expression(P<0.05),and the effect of NSC-OST group was more significant(P<0.05).Conclusion NSC-OST can inhibit the differentiation of osteoclasts and the expression of osteoclast-specific molecules such as NFATc1,and its effect is better than OST.