[1]侯成志,赵勇,夏迪,等.补肾活骨汤含药血清对小鼠RAW264.7细胞向破骨细胞分化的影响及其作用机制[J].中医正骨,2026,38(03):1-7,29.
　HOU Chengzhi,ZHAO Yong,XIA Di,et al.Effects and mechanism of Bushen Huogu Tang(补肾活骨汤)medicated serum on the differentiation of mouse RAW264.7 cells into osteoclasts[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2026,38(03):1-7,29.
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补肾活骨汤含药血清对小鼠RAW264.7细胞向破骨细胞分化的影响及其作用机制()

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第38卷
期数:
2026年03期
页码:
1-7,29
栏目:
基础研究
出版日期:
2026-03-20

文章信息/Info

Title:
Effects and mechanism of Bushen Huogu Tang(补肾活骨汤)medicated serum on the differentiation of mouse RAW264.7 cells into osteoclasts
作者:
侯成志; 赵勇; 夏迪; 俞张镜泽; 秦伟凯; 董永丽; 魏光成; 顾金光
(中国中医科学院望京医院,北京 100102)
Author(s):
HOU Chengzhi; ZHAO Yong; XIA Di; YU Zhangjingze; QIN Weikai; DONG Yongli; WEI Guangcheng; GU Jinguang
Wangjing Hospital of CACMS,Beijing 100102,China
关键词:
骨关节炎;  补肾活骨汤;  破骨细胞;  分化;  RAW264.7细胞
Keywords:
osteoarthritis;  Bushen Huogu Tang;  osteoclasts;  differentiation;  RAW 264.7 cells
摘要:
目的:探究补肾活骨汤含药血清对小鼠RAW264.7细胞向破骨细胞分化的影响及其作用机制。方法:①补肾活骨汤含药血清和空白血清制备方法。将24只SD大鼠随机分为补肾活骨汤含药血清制备组和空白血清制备组,每组12只,分别给予补肾活骨汤药液和蒸馏水灌胃,连续灌胃7 d后,采集腹主动脉血,制备补肾活骨汤含药血清和空白血清。②补肾活骨汤含药血清干预浓度的筛选方法。取第3代小鼠RAW264.7细胞,分为空白对照组及10%、15%、20%、25%补肾活骨汤含药血清组。空白对照组加入10%的空白血清,10%、15%、20%、25%补肾活骨汤含药血清组分别加入10%、15%、20%、25%的补肾活骨汤含药血清。培养24 h后,采用MTT法检测各组小鼠RAW264.7细胞的活力,确定干预浓度。③补肾活骨汤含药血清对小鼠RAW264.7细胞向破骨细胞分化影响的分析方法。取第3代小鼠RAW264.7细胞,分为空白对照组、诱导分化组及10%、15%、20%补肾活骨汤含药血清组。除空白对照组外,其余各组均加入终浓度为100 ng·mL-1的巨噬细胞集落刺激因子(macrophage colony-stimula-ting factor,M-CSF)和50 ng·mL-1的核因子κB受体激活蛋白配体(receptor activator of nuclear factor-κB ligand,RANKL); 同时,空白对照组和诱导分化组加入10%的空白血清,10%、15%、20%补肾活骨汤含药血清组分别加入10%、15%、20%的补肾活骨汤含药血清。培养5 d后,进行抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色,并统计各组小鼠RAW264.7细胞分化的破骨细胞数量。④补肾活骨汤含药血清影响小鼠RAW264.7细胞向破骨细胞分化作用机制的分析方法。取第3代小鼠RAW264.7细胞,分为空白对照组、诱导分化组、20%补肾活骨汤含药血清组。除空白对照组外,其余各组均加入终浓度为100 ng·mL-1的M-CSF和50 ng·mL-1的RANKL; 空白对照组和诱导分化组加入10%的空白血清,20%补肾活骨汤含药血清组加入20%的补肾活骨汤含药血清。培养5 d后,采用实时定量PCR检测TRAP和活化T细胞核因子c1(nuclear factor of activated T cells c1,NFATc1)的mRNA表达水平,采用Western Blot法检测NFATc1、组织蛋白酶K、多囊蛋白1、PDZ结合基序转录共激活因子(transcriptional coactivator with PDZ-binding motif,TAZ)的蛋白表达水平。结果:①补肾活骨汤含药血清干预浓度的筛选结果。空白对照组及10%、15%、20%补肾活骨汤含药血清组小鼠RAW264.7细胞活力组间两两比较,差异均无统计学意义(P=0.745,P=0.922,P=0.981,P=0.958,P=0.999,P=0.993),25%补肾活骨汤含药血清组小鼠RAW264.7细胞活力低于空白对照组及10%、15%、20%补肾活骨汤含药血清组(P=0.000,P=0.000,P=0.000,P=0.000)。确定补肾活骨汤含药血清干预浓度为10%、15%、20%。②补肾活骨汤含药血清对小鼠RAW264.7细胞向破骨细胞分化影响的分析结果。空白对照组无破骨细胞。10%补肾活骨汤含药血清组小鼠RAW264.7细胞分化的破骨细胞数量与诱导分化组的差异无统计学意义(P=0.209),15%、20%补肾活骨汤含药血清组小鼠RAW264.7细胞分化的破骨细胞数量均少于诱导分化组和10%补肾活骨汤含药血清组(P=0.000,P=0.001; P=0.016,P=0.002); 20%补肾活骨汤含药血清组小鼠RAW264.7细胞分化的破骨细胞数量少于15%补肾活骨汤含药血清组(P=0.046)。③补肾活骨汤含药血清影响小鼠RAW264.7细胞向破骨细胞分化作用机制的分析结果。诱导分化组、20%补肾活骨汤含药血清组小鼠RAW264.7细胞TRAP、NFATc1的mRNA相对表达量和NFATc1、组织蛋白酶K、多囊蛋白1、TAZ的蛋白相对表达量均高于空白对照组(P=0.000,P=0.000; P=0.002,P=0.011; P=0.000,P=0.000; P=0.000,P=0.000; P=0.001,P=0.002; P=0.000,P=0.001),20%补肾活骨汤含药血清组小鼠RAW264.7细胞TRAP、NFATc1的mRNA相对表达量和NFATc1、组织蛋白酶K、多囊蛋白1、TAZ的蛋白相对表达量均低于诱导分化组(P=0.000,P=0.000,P=0.008,P=0.005,P=0.006,P=0.022)。结论:补肾活骨汤含药血清能够抑制小鼠RAW264.7细胞向破骨细胞分化,其作用机制可能通过下调多囊蛋白1、TAZ的表达进而抑制NFATc1、组织蛋白酶K等破骨细胞分化相关基因的表达。
Abstract:
Objective:To investigate the effects of Bushen Huogu Tang(补肾活骨汤,BSHGT)medicated serum on the differentiation of mouse RAW264.7 cells into osteoclasts,and explore its underlying mechanisms.Methods:①Preparation of BSHGT medicated serum and blank serum.Twenty-four SD rats were randomized into a BSHGT medicated serum preparation group and a blank serum preparation group,with 12 ones in each group.The rats in the 2 groups were intervened by intragastric administration with BSHGT solution and distilled water,respectively,for consecutive 7 days.One hour after the end of the last intervention,the blood was drawn from the abdominal aorta of rats in each group for making BSHGT medicated serum and blank serum.②Screening method for the intervention concentration of BSHGT medicated serum.The third generation of mouse RAW264.7 cells were collected and divided into blank control group,and 10%,15%,20%,and 25% BSHGT medicated serum groups.The mouse RAW264.7 cells in blank control group were intervened by 10% blank serum,while the ones in treatment groups by 10%,15%,20%,and 25% BSHGT medicated serum,respectively,for 24 hours.After the end of the intervention,the viability of the mouse RAW264.7 cells in each group was assessed by MTT assay to determine the intervention concentration.③Analysis method for the effect of BSHGT medicated serum on the differentiation of mouse RAW264.7 cells into osteoclasts.The third generation of mouse RAW264.7 cells were fetched and divided into a blank control group,an induced differentiation group,and 10%,15%,and 20% BSHGT medicated serum groups.All groups but the blank control group were intervened by macrophage colony-stimulating factor(M-CSF)at a final concentration of 100 ng/mL and receptor activator of nuclear factor-κB ligand(RANKL)at a final concentration of 50 ng/mL.Meanwhile,the blank control group and the induced differentiation group were intervened by 10% blank serum,and the 10%,15%,and 20% BSHGT medicated serum groups by 10%,15%,and 20% BSHGT medicated serum,respectively.After 5-day culture,the number of osteoclasts differentiated from mouse RAW264.7 cells in each group was counted by tartrate-resistant acid phosphatase(TRAP)staining.④Analysis method for the mechanism of BSHGT medicated serum affecting the differentiation of mouse RAW264.7 cells into osteoclasts.The third generation of mouse RAW264.7 cells were selected and divided into a blank control group,an induced differenti-ation group,and a 20% BSHGT medicated serum group.All groups but the blank control group were intervened by M-CSF at a final concentration of 100 ng/mL and RANKL at a final concentration of 50 ng/mL.Meanwhile,the blank control group and the induced differentiation group were intervened by 10% blank serum,while the 20% BSHGT medicated serum group by 20% BSHGT medicated serum.After 5-day culture,the mRNA expression levels of TRAP and nuclear factor of activated T cells c1(NFATc1)were detected by real-time quantitative PCR,and the protein expression levels of NFATc1,cathepsin K,polycystin-1,and transcriptional coactivator with PDZ-binding motif(TAZ)were detected by Western Blot.Results:①Screening results of intervention concentrations of BSHGT medicated serum.Pairwise comparisons of the viability of mouse RAW264.7 cells among the blank control group,and 10%,15%,and 20% BSHGT medicated serum groups showed no statistically significant differences(P=0.745,P=0.922,P=0.981,P=0.958,P=0.999,P=0.993).However,the 25% BSHGT medicated serum group showed lower cell viability compared to the blank control group and the 10%,15%,and 20% BSHGT medicated serum groups(P=0.000,P=0.000,P=0.000,P=0.000).Therefore,the intervention concentrations of BSHGT medicated serum were determined to be 10%,15%,and 20% for subsequent experiments.②Analysis results of the effect of BSHGT medicated serum on the differentiation of mouse RAW264.7 cells into osteoclasts.No osteoclasts were observed in the blank control group.There was no statistically significant difference in the number of osteoclasts differentiated from mouse RAW264.7 cells between the 10% BSHGT medicated se-rum group and the induced differentiation group(P=0.209).However,the number of osteoclasts differentiated from mouse RAW264.7 cells was less in both 15% and 20% BSHGT medicated serum groups compared to the induced differentiation group and the 10% BSHGT medicated serum group(P=0.000,P=0.001; P=0.016,P=0.002),and was less in the 20% BSHGT medicated serum group compared to the 15% BSHGT medicated serum group(P=0.046).③Analysis results of the mechanism of BSHGT medicated serum affecting the differentiation of mouse RAW264.7 cells into osteoclasts.The relative mRNA expression levels of TRAP and NFATc1,as well as the relative protein expression levels of NFATc1,cathepsin K,polycystin-1,and TAZ in mouse RAW264.7 cells were all higher in the induced differenti-ation group and the 20% BSHGT medicated serum group compared to the blank control group(P=0.000,P=0.000; P=0.002,P=0.011; P=0.000,P=0.000; P=0.000,P=0.000; P=0.001,P=0.002; P=0.000,P=0.001).However,they were all lower in the 20% BSHGT medicated serum group compared to the induced differentiation group(P=0.000,P=0.000,P=0.008,P=0.005,P=0.006,P=0.022).Conclusion:BSHGT medicated serum can inhibit the differentiation of mouse RAW264.7 cells into osteoclasts.It may work by inhibiting the expression of osteoclast differentiation-related genes such as NFATc1 and cathepsin K through downregulating the expression of polycystin-1 and TAZ.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(82505627); 中国中医科学院望京医院高水平中医医院建设项目(WJZJ-202316); 中国中医科学院望京医院自主选题项目(WJYY-ZZXT-2022-03)
通信作者:赵勇 E-mail:zhaoyong423@163.com
(收稿日期:2025-09-02 本文编辑:吕宁)
更新日期/Last Update: 2026-03-20