蛇床子素对RAW264.7细胞向破骨细胞分化的影响及其机制

Influence of osthole on RAW264.7 cells differentiating to osteoclasts and mechanism study

目的探讨蛇床子素(OST)对小鼠单核细胞RAW264. 7细胞系向破骨细胞分化的影响及相关机制。方法通过使用RAW264. 7细胞系体外诱导培养破骨细胞;将RAW264. 7细胞系分为阴性组、对照组及OST组。以核因子κB受体活化因子配基(RANKL)诱导细胞分化,OST组以高、中、低浓度(100、10、1μmol/L)根据实验要求干预细胞系相应时间;采用CCK-8法筛选无细胞毒性的药物浓度组;通过抗酒石酸酸性磷酸酶(TRAP)染色法测定阳性细胞数和融合指数;通过测定骨吸收板上溶解坑面积和培养液中的荧光强度评估OST对骨吸收功能的影响;使用荧光素酶报告基因法观察OST对RAW264. 7细胞系中活化T细胞核因子(NFAT)和核因子κB(NF-κB)表达的影响;应用q-PCR法检测OST对RAW264. 7细胞系中相关破骨基因活化T细胞核因子1(NFATc1)、组织蛋白酶K(CTSK)、基质金属蛋白酶9(MMP-9)、TRAP、干扰素β3(INTE-BETAβ3)、酪氨酸激酶(c-Src)表达的影响;应用Western Blot技术检测OST对RAW264. 7细胞系中NF-κB信号通路上相关蛋白膜p65、IκB、p-IκB、核p65表达的影响。结果通过CCK-8法筛选出1μmol/L和10μmol/L 2组药物浓度; OST组中的TRAP阳性细胞数和融合指数较对照组明显减少(P <0. 05),且中浓度组的抑制效果更明显(P <0. 05); OST组的骨吸收相对面积和荧光强度较对照组均受到明显抑制(P <0. 05),且中浓度组的抑制效果更明显(P <0. 05); OST可以明显抑制转录因子NFAT和NF-κB的启动(P <0. 05); OST可以抑制NFATc1等破骨相关基因的表达,除了Integrin-BETA3、c-Src外,2个浓度组组间比较差异均有统计学意义(P <0. 05);与对照组相比,OST可明显抑制细胞膜中p-IκB蛋白的表达(P <0. 05),促进IκB、p65的表达(P <0. 05),同时也抑制了细胞核中p65的蛋白表达(P <0. 05)。结论 OST可通过抑制NF-κB信号通路的表达而引起NFATc1等相关转录因子的下调来抑制破骨细胞的分化。

Objective To investigate the influence of osthole( OST) on RAW264. 7 cell lines of mouse monocytes differentiating to osteoclasts and study the related mechanism. Methods Osteoclasts were induced and cultured in vitro by using RAW264. 7 cell lines,and then RAW264. 7 cell line was divided into negative group,control group and OST group. RAW264. 7 cell lines were induced to differentiation by using RANKL,and OST group was intervened with high-dose,mid-dose and low-dose OST( 100μmol/L,10μmol/L,1 μmol/L,high-dose,mid-dose and low-dose OST groups) for corresponding time. CCK-8 method was used to screen non-cytotoxic medicinal concentration group,and anti-tartaric acid phosphatase( TRAP) staining method was used to determine number of TRAP positive cells and fusion index. The influence of OST on bone absorptive function was reviewed through detecting area of dissolving pit and fluorescence intensity in bone absorption board. The influence of OST on expressions of nuclear factor of activated T cell( NFAT) and nuclear factor-κB( NF-κB) in RAW264. 7 cell lines were observed by using luciferase reporter gene method. The influence of OST on expressions of nuclear factor of activated T cell-1( NFATc1),CTSK,MMP-9,TRAP,INTE-BETAβ3 and c-Src was detected by using q-PCR. The influence of OST on expressions of p65,IκB,p-IκB and p65 in NF-κB signaling pathway was detected by using Western blotting assay. Results There were 2 medicinal concentration groups( 1 μmol/L and 10 μmol/L,low-dose group and mid-dose group) screened by using CCK-8 method. TRAP positive cells and fusion index were significantly lower in all OST groups than those in control group( P < 0. 05),which was more significant in mid-dose OST group( P < 0. 05). The related area of bone absorption and fluorescence intensity were significantly inhibited in OST group( P < 0. 05),which was more significant in mid-dose OST group( P < 0. 05). OST inhibited significantly the initiations of NFAT and NF-κB( P < 0. 05) and expressions of osteoclast related genes including NFATc1,and except of Integrin-BETA3 and c-Src,other related genes had statistical difference between low-dose group and mid-dose group( P < 0. 05). Compared with control group,the protein expression of p-IκB was inhibited significantly( P < 0. 05),expressions of IκB and p65 were improved( P < 0. 05),and protein expression of p65 was inhibited( P < 0. 05) in all OST groups. Conclusion OST can induce downregulations of NFATc1 and related transcription factors to control differentiation of osteoclasts through inhibiting expression of NF-κB signaling pathway.