[1]苏珮茹,罗香雅,余丽娜,等.紫菀酮对体外破骨细胞分化和活性的影响及作用机制研究[J].中医正骨,2023,35(06):24-35.
　SU Peiru,LUO Xiangya,YU Lina,et al.Effects of shionone on differentiation and activity of osteoclasts in vitro and its mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2023,35(06):24-35.
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紫菀酮对体外破骨细胞分化和活性的影响及作用机制研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第35卷
期数:
2023年06期
页码:
24-35
栏目:
基础研究
出版日期:
2023-06-20

文章信息/Info

Title:
Effects of shionone on differentiation and activity of osteoclasts in vitro and its mechanism of action
作者:
苏珮茹1; 罗香雅1; 余丽娜2; 曾春平1; 周琳1
(1.广州医科大学附属第五医院,广东 广州 510700; 2.华北理工大学公共卫生学院,河北 唐山 063210)
Author(s):
SU Peiru1; LUO Xiangya1; YU Lina2; ZENG Chunping1; ZHOU Lin1
1.The Fifth Affiliated Hospital of Guangzhou Medical University,Guangzhou 510700,Guangdong,China 2.School of Public Health,North China University of Science and Technology,Tangshan 063210,Hebei,China
关键词:
骨质疏松;  破骨细胞;  细胞分化;  紫菀酮;  体外研究技术;  信号传导;  NF-κB;  组织蛋白酶K;  腺苷三磷酸酶;  抗酒石酸酸性磷酸酶;  NFATC转录因子
Keywords:
osteoporosis;  osteoclasts;  cell differentiation;  shionone;  in vitro techniques;  signal transduction;  NF-kappa B;  Cathepsin K;  adenosine triphosphatases;  tartrate-resistant acid phosphatase;  NFATC transcription factors
摘要:
目的:探讨紫菀酮对体外破骨细胞分化和活性的影响及作用机制。方法:①分析紫菀酮对核因子-κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)诱导Raw 264.7细胞向破骨细胞分化的影响。将Raw 264.7细胞分为空白对照组、阳性对照组、紫菀酮1 μmol·L-1干预组、紫菀酮2.5 μmol·L-1干预组、紫菀酮5 μmol·L-1干预组和紫菀酮10 μmol·L-1干预组,空白对照组加入完全培养基,阳性对照组加入含25 ng·mL-1RANKL的完全培养基,其余各组分别加入含有相应浓度紫菀酮及25 ng·mL-1RANKL的完全培养基,培养5 d后进行抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色,统计各组破骨细胞数; ②分析紫菀酮对Raw 264.7细胞活力的影响。将Raw 264.7细胞分为空白对照组、紫菀酮1 μmol·L-1干预组、紫菀酮2.5 μmol·L-1干预组、紫菀酮5 μmol·L-1干预组和紫菀酮10 μmol·L-1干预组,空白对照组加入完全培养基,其余各组分别加入含有相应浓度紫菀酮的完全培养基,分别培养48 h、120 h后测定各组细胞活力; ③分析紫菀酮对破骨细胞纤维状肌动蛋白环形成的影响。将Raw 264.7细胞分为空白对照组、阳性对照组、紫菀酮5 μmol·L-1干预组和紫菀酮10 μmol·L-1干预组,空白对照组加入完全培养基,阳性对照组加入含25 ng·mL-1RANKL的完全培养基,紫菀酮5 μmol·L-1干预组和紫菀酮10 μmol·L-1干预组分别加入含有相应浓度紫菀酮及25 ng·mL-1RANKL的完全培养基,培养5 d后采用鬼笔环肽和4',6-二脒基-2-苯基吲哚染色,观察各组破骨细胞纤维状肌动蛋白环形成情况。④分析紫菀酮对核因子-κB(nuclear factor-κB,NF-κB)转录活性的影响。将稳定转染pGL4.32[luc2P/NF-κB-RE/Hygro]载体的RAW 264.7细胞分为空白对照组、阳性对照组和紫菀酮 10 μmol·L-1干预组,空白对照组及阳性对照组加入完全培养基,紫菀酮10 μmol·L-1干预组加入终浓度为10 μmol·L-1紫菀酮的完全培养基; 培养2 h后,阳性对照组及紫菀酮10 μmol·L-1干预组分别加入RANKL溶液,使培养基中RANKL的终浓度为25 ng·mL-1; 继续培养6 h后,检测各组细胞荧光素酶的荧光强度。⑤分析紫菀酮对核因子-κB抑制蛋白激酶α亚基(inhibitor of nuclear factor kappa-B kinase subunit α,IκBα)蛋白表达的影响。将Raw 264.7细胞分为阳性对照组和紫菀酮10 μmol·L-1干预组,每组6孔,分别编号1～6。待细胞贴壁后,阳性对照组加入完全培养基,紫菀酮10 μmol·L-1干预组加入终浓度为10 μmol·L-1紫菀酮的完全培养基,继续培养2 h后,在2组的1号孔加入RANKL溶液使RANKL终浓度为25 ng·mL-1,在1号孔加入RANKL溶液后30 min、40 min、50 min、55 min,分别在2号、3号、4号、5号孔加入RANKL溶液至RANKL终浓度为25 ng·mL-1。在1号孔加入RANKL后60 min,收集各孔细胞,采用蛋白印迹法检测IκBα蛋白的表达量。⑥分析紫菀酮对破骨细胞特异性基因转录的影响。将Raw 264.7细胞分为阳性对照组、紫菀酮5 μmol·L-1干预组和紫菀酮10 μmol·L-1干预组。阳性对照组加入含 25 ng·mL-1RANKL的完全培养基,紫菀酮5 μmol·L-1干预组和紫菀酮10 μmol·L-1干预组分别加入含有相应浓度紫菀酮和25 ng·mL-1RANKL的完全培养基,培养5 d后,收集各组细胞,采用实时定量PCR检测组织蛋白酶K、空泡型ATP酶d2亚基(vacuolar ATPase subsnit D2,V-ATPase d2)、TRAP的mRNA表达量。⑦分析紫菀酮对活化T-细胞核因子c1(nuclear factor of activated T cells c1,NFATc1)蛋白表达的影响。将Raw 264.7细胞分为阳性对照组和紫菀酮10 μmol·L-1干预组,每组4孔,分别编号1～4。待细胞贴壁后,阳性对照组加入完全培养基,紫菀酮10 μmol·L-1干预组加入终浓度为10 μmol·L-1紫菀酮的完全培养基。继续培养2 h后,在2组的1号孔加入RANKL溶液使RANKL终浓度为25 ng·mL-1,在1号孔加入RANKL溶液后2 d、4 d,分别在2号、3号孔加入RANKL溶液使RANKL终浓度为25 ng·mL-1。在1号孔加入RNAKL后5 d,收集各孔细胞。采用蛋白印迹法检测NFATc1蛋白的表达量。结果:①紫菀酮对RANKL诱导Raw 264.7细胞向破骨细胞分化影响的分析结果。空白对照组之外的5组破骨细胞数比较,差异有统计学意义[(187.667±14.503)个,(180.000±14.422)个,(174.333±11.060)个,(152.667±5.033)个,(130.667±11.015)个,F=11.767,P=0.001]。紫菀酮10 μmol·L-1干预组、紫菀酮5 μmol·L-1干预组破骨细胞数均少于阳性对照组、紫菀酮1 μmol·L-1干预组和紫菀酮2.5 μmol·L-1干预组(P=0.004,P=0.000; P=0.017,P=0.000; P=0.047,P=0.001); 紫菀酮10 μmol·L-1干预组破骨细胞数少于紫菀酮5 μmol·L-1干预组(P=0.044)。②紫菀酮对Raw 264.7细胞活力影响的分析结果。紫菀酮干预48 h、120 h时,5组Raw 264.7细胞活力比较,组间差异均无统计学意义(48 h:0.960±0.101,0.938±0.051,0.916±0.072,0.915±0.079,1.009±0.105,F=0.647,P=0.641; 120 h:1.347±0.161,1.388±0.047,1.423±0.473,1.398±0.067,1.357±0.034,F=0.400,P=0.805)。③紫菀酮对破骨细胞纤维状肌动蛋白环形成影响的分析结果。与空白对照组比较,阳性对照组、紫菀酮5 μmol·L-1干预组和紫菀酮10 μmol·L-1干预组均可见破骨细胞和纤维状肌动蛋白环; 与阳性对照组比较,紫菀酮5 μmol·L-1干预组和紫菀酮10 μmol·L-1干预组破骨细胞纤维状肌动蛋白环形成均受到抑制,且其受到抑制的程度随紫菀酮干预浓度增加而增强。④紫菀酮对破骨细胞NF-κB转录活性影响的分析结果。3组荧光素酶相对荧光强度比较,组间差异有统计学意义(0.058±0.003,1.000±0.044,0.753±0.040,F=613.132,P=0.000)。阳性对照组和紫菀酮10 μmol·L-1干预组的荧光素酶相对荧光强度高于空白对照组(P=0.000,P=0.000),紫菀酮10 μmol·L-1干预组的荧光素酶荧光强度低于阳性对照组(P=0.000)。⑤紫菀酮对IκBα蛋白表达影响的分析结果。2组破骨细胞IκBα蛋白相对表达量随诱导时间延长均呈先下降后上升趋势,但2组的趋势不完全一致(1.000±0.000,0.414±0.171,0.285±0.104,0.132±0.021,0.157±0.038,0.802±0.066,F=49.839,P=0.000; 0.980±0.130,0.632±0.102,0.347±0.037,0.302±0.070,0.546±0.142,1.502±0.345,F=21.435,P=0.000); 诱导20 min、30 min、60 min,紫菀酮10 μmol·L-1干预组破骨细胞IκBα蛋白相对表达量高于阳性对照组(t=-4.018,P=0.016; t=-4.586,P=0.010; t=-3.444,P=0.026)。⑥紫菀酮对破骨细胞特异性基因转录影响的分析结果。3组破骨细胞V-ATPase-d2、组织蛋白酶K、TRAP的mRNA表达量比较,组间差异均有统计学意义(1.000±0.089,0.879±0.100,0.530±0.054,F=25.553,P=0.001; 1.000±0.030,0.725±0.153,0.719±0.111,F=6.293,P=0.034; 1.000±0.326,0.834±0.030,0.656±0.327,F=88.003,P=0.000)。紫菀酮5 μmol·L-1干预组破骨细胞组织蛋白酶K、TRAP的mRNA表达量均低于阳性对照组(P=0.023,P=0.001); 紫菀酮10 μmol·L-1干预组破骨细胞V-ATPase-d2、TRAP的mRNA表达量均低于紫菀酮5 μmol·L-1干预组和阳性对照组(P=0.000,P=0.002; P=0.000,P=0.000); 紫菀酮10 μmol·L-1干预组破骨细胞组织蛋白酶K的mRNA表达量低于阳性对照组(P=0.021)。⑦紫菀酮对NFATc1蛋白表达影响的分析结果。2组破骨细胞NFATc1蛋白相对表达量随诱导时间延长均呈上升趋势,但2组的上升趋势不完全一致(1.000±0.000,1.175±0.007,4.700±0.742,19.430±1.763,F=250.352,P=0.000; 1.042±0.035,1.334±0.290,3.531±0.583,15.690±0.823,F=525.669,P=0.000); 诱导后5 d,紫菀酮10 μmol·L-1干预组破骨细胞NFATc1蛋白相对表达量低于阳性对照组(t=3.329,P=0.029)。结论:紫菀酮能够抑制体外破骨细胞的分化和活性,其作用机制与抑制NF-κB和NFATc1信号通路有关。
Abstract:
Objective:To explore the effects of shionone on differentiation and activity of osteoclasts in vitro and its mechanism of action.Methods:①To analyze the effects of shionone on the differentiation of Raw 264.7 cells into osteoclasts induced by receptor activator of nuclear factor-κB ligand(RANKL).The Raw 264.7 cells were divided into blank control group,positive control group,1 μmol/L shionone intervention group,2.5 μmol/L shionone intervention group,5 μmol/L shionone intervention group and 10 μmol/L shionone intervention group.The Raw 264.7 cells in blank control group were cultured in complete culture medium,the ones in positive control group were cultured in complete culture medium supplemented with 25 ng/mL RANKL,and the ones in the rest groups were cultured in complete culture medium supplemented with 25 ng/mL RANKL and shionone at concentration of 1,2.5,5 and 10 μmol/L respectively.After 5-day culture,tartrate-resistant acid phosphatase(TRAP)staining was performed,and the number of osteoclasts in each group was counted.②To analyze the effects of shionone on the viability of Raw 264.7 cells.The Raw 264.7 cells were divided into blank control group,1 μmol/L shionone intervention group,2.5 μmol/L shionone intervention group,5 μmol/L shionone intervention group and 10 μmol/L shionone intervention group.The Raw 264.7 cells in blank control group were cultured in complete culture medium,and the ones in the other groups were cultured in complete culture medium supplemented with shionone at concentration of 1,2.5,5 and 10 μmol/L respectively.After 48- and 120-hour culture,the viability of Raw 264.7 cells were detected in each group.③To analyze the effects of shionone on the formation of fibrous actin rings(F-actin rings)in osteoclasts.The Raw 264.7 cells were divided into blank control group,positive control group,5 μmol/L shionone intervention group and 10 μmol/L shionone intervention group.The Raw 264.7 cells in blank control group were cultured in complete culture medium,the ones in positive control group were cultured in complete culture medium supplemented with 25 ng/mL RANKL,and the ones in the remained 2 groups were cultured in complete culture medium supplemented with 25 ng/mL RANKL and shionone at concentration of 5 and 10 μmol/L respectively.After 5-day culture,the staining was performed with phalloidin and 4',6-diamidino-2-phenylindole(DAPI)to observe the formation of F-actin rings in osteoclasts in each group.④To analyze the effects of shionone on the transcriptional activity of nuclear factor-κB(NF-κB).The RAW 264.7 cells stably transfected with pGL4.32[luc2P/NF-κB-RE/Hygro] vector were divided into blank control group,positive control group and 10 μmol/L shionone intervention group.The transfected cells in blank control group and positive control group were cultured in complete culture medium,and the ones in 10 μmol/L shionone intervention group were cultured in complete culture medium supplemented with shionone at final concentration of 10 μmol/L.After 2-hour culture,the cells in positive control group and 10 μmol/L shionone intervention group were added with RANKL solution respectively till its final concentration was 25 ng/mL in the medium.After an additional 6-hour culture,the fluorescence intensity of luciferase in each group was detected.⑤To analyze the effects of shionone on the protein expression of inhibitor of nuclear factor kappa-B kinase subunit α(IκBα).The Raw 264.7 cells were divided into positive control group and 10 μmol/L shionone intervention group,with 6 wells in each group numbered from 1 to 6 respectively.After cell adhesion,the cells in positive control group were cultured in complete culture medium,and the ones in 10 μmol/L shionone intervention group were cultured in complete culture medium supplemented with shionone at final concentration of 10μmol/L.After an additional 2-hour culture,the No.1 wells in the 2 groups were added with RANKL solution respectively till its final concentration was 25 ng/mL in the medium.At 30,40,50,and 55 minutes after adding RANKL solution to the No.1 wells,the No.2,3,4 and 5 wells in the 2 groups were added with RANKL solution respectively till its final concentration was 25 ng/mL in the medium.At 60 minutes after adding RANKL to the No.1 wells,the cells were collected from each well,and the protein expression level of IκBα was detected by using Western blot.⑥To analyze the effects of shionone on the transcription of osteoclast-specific genes.The Raw 264.7 cells were divided into positive control group,5 μmol/L shionone intervention group and 10 μmol/L shionone intervention group.The cells in positive control group were cultured in complete culture medium supplemented with 25 ng/mL RANKL,and the ones in the other two groups were cultured in complete culture medium supplemented with 25 ng/mL RANKL and shionone at concentration of 5 and 10 μmol/L respectively.After 5-day culture,the cells were collected from each group,and the mRNA expression levels of cathepsin K,vacuolar ATPase subunit D2(V-ATPase d2)and TRAP were detected by using real-time quantitative PCR(RT-qPCR).⑦To analyze the effects of shionone on the protein expression of nuclear factor of activated T cells c1(NFATc1).The Raw 264.7 cells were divided into positive control group and 10 μmol/L shionone intervention group,with 4 wells in each group numbered from 1 to 4 respectively.After cell adhesion,the cells in positive control group were cultured in complete culture medium,and the ones in 10 μmol/L shionone intervention group were cultured in complete culture medium supplemented with shionone at final concentration of 10 μmol/L.After an additional 2-hour culture,the No.1 wells in the 2 groups were added with RANKL solution respectively till its final concentration was 25 ng/mL in the medium.At 2 and 4 days after adding RANKL solution to the No.1 wells,the No.2 and 3 wells in the 2 groups were added with RANKL solution respectively till its final concentration was 25 ng/mL in the medium.At 5 days after adding RANKL solution to the No.1 wells,the cells were collected from each well,and the protein expression level of NFATc1 was detected by using Western blot.Results:①The results of analysis on the effects of shionone on RANKL-induced differentiation of Raw 264.7 cells into osteoclasts.There was statistical difference in the number of osteoclasts among the 5 groups except for the blank control group(187.667±14.503,180.000±14.422,174.333±11.060,152.667±5.033,130.667±11.015,F=11.767,P=0.001).The number of osteoclasts was less in 10 μmol/L shionone intervention group and 5 μmol/L shionone intervention group compared to positive control group,1 μmol/L shionone intervention group and 2.5 μmol/L shionone intervention group(P=0.004,P=0.000; P=0.017,P=0.000; P=0.047,P=0.001),and it was less in 10 μmol/L shionone intervention group compared to 5 μmol/L shionone intervention group(P=0.044).②The results of analysis on the effects of shionone on the viability of Raw 264.7 cells.There was no statistical difference in the viability of Raw 264.7 cells among the 5 groups after 48- and 120-hour intervention(48-hour:0.960±0.101,0.938±0.051,0.916±0.072,0.915±0.079,1.009±0.105,F=0.647,P=0.641; 120-hour:1.347±0.161,1.388±0.047,1.423±0.473,1.398±0.067,1.357±0.034,F=0.400,P=0.805).③The results of analysis on the effects of shionone on the formation of F-actin rings in osteoclasts.Compared with the blank control group,the osteoclasts and F-actin rings were observed in positive control group,5 μmol/L shionone intervention group and 10 μmol/L shionone intervention group.Compared with the positive control group,the formation of F-actin rings in osteoclasts was inhibited in 5 μmol/L shionone intervention group and 10 μmol/L shionone intervention group,and the degree of inhibition enhanced with the increasement of intervention concentration of shionone.④The results of analysis on the effects of shionone on the NF-κB transcriptional activity in osteoclasts.There was statistical difference in the relative fluorescence intensity of luciferase among the 3 groups(0.058±0.003,1.000±0.044,0.753±0.040,F=613.132,P=0.000).The relative fluorescence intensity of luciferase was higher in positive control group and 10 μmol/L shionone intervention group compared to blank control group(P=0.000,P=0.000),and it was higher in positive control group compared to 10 μmol/L shionone intervention group(P=0.000).⑤The results of analysis on the effects of shionone on the expression of IκBα protein.The relative expression level of IκBα protein in osteoclasts displayed a trend of going downward firstly and upward subsequently with the induction-time in the 2 groups,while the 2 groups were inconsistent with each other in the variation tendency(1.000±0.000,0.414±0.171,0.285±0.104,0.132±0.021,0.157±0.038,0.802±0.066,F=49.839,P=0.000; 0.980±0.130,0.632±0.102,0.347±0.037,0.302±0.070,0.546±0.142,1.502±0.345,F=21.435,P=0.000).The relative expression level of IκBα protein in osteoclasts was higher in 10 μmol/L shionone intervention group compared to positive control group when the cells were induced for 20,30 and 60 minutes respectively(t=-4.018,P=0.016; t=-4.586,P=0.010; t=-3.444,P=0.026).⑥The results of analysis on the effects of shionone on the transcription of osteoclast-specific genes.There was statistical difference in the mRNA expression levels of V-ATPase-d2,cathepsin K and TRAP in osteoclasts among the 3 groups(1.000±0.089,0.879±0.100,0.530±0.054,F=25.553,P=0.001; 1.000±0.030,0.725±0.153,0.719±0.111,F=6.293,P=0.034; 1.000±0.326,0.834±0.030,0.656±0.327,F=88.003,P=0.000).The mRNA expression levels of cathepsin K and TRAP in osteoclasts were lower in 5 μmol/L shionone intervention group compared to positive control group(P=0.023,P=0.001).The mRNA expression levels of V-ATPase-d2 and TRAP in osteoclasts were lower in 10 μmol/L shionone intervention group compared to 5 μmol/L shionone intervention group and positive control group(P=0.000,P=0.002; P=0.000,P=0.000).The mRNA expression level of cathepsin K in osteoclasts was lower in 10 μmol/L shionone intervention group compared to positive control group(P=0.021).⑦The results of analysis on the effects of shionone on the expression of NFATc1 protein.The relative expression level of NFATc1 protein in osteoclasts displayed an upward trend with the induction-time in the 2 groups,while the 2 groups were inconsistent with each other in the variation tendency(1.000±0.000,1.175±0.007,4.700±0.742,19.430±1.763,F=250.352,P=0.000; 1.042±0.035,1.334±0.290,3.531±0.583,15.690±0.823,F=525.669,P=0.000).After 5-day induction,the relative expression level of NFATc1 protein in osteoclasts was lower in 10 μmol/L shionone intervention group compared to positive control group(t=3.329,P=0.029).Conclusion:Shionone can inhibit the differentiation and activity of osteoclasts in vitro,and its mechanism may be that it can inhibit NF-κB and NFATc1 signaling pathways.

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备注/Memo

备注/Memo:
基金项目:广东省基础与应用基础研究基金项目(2019A1515110723) 通讯作者:周琳 E-mail:912395010@qq.com
更新日期/Last Update: 1900-01-01