六味地黄丸通过CLCF1/NFATc1调控破骨细胞膜融合治疗小鼠绝经后骨质疏松症

doi:10.12122/j.issn.1673-4254.2026.08.03

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (8): 1743-1753.doi: 10.12122/j.issn.1673-4254.2026.08.03

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六味地黄丸通过CLCF1/NFATc1调控破骨细胞膜融合治疗小鼠绝经后骨质疏松症

郑若曦¹^,²(), 谢丽华², 黄小彬², 葛继荣²()

^1.福建中医药大学中医骨伤及运动康复教育部重点实验室，福建福州 350122
^2.福建省中西医结合防治骨质疏松重点实验室（福建省中医药科学院、福建中医药大学附属康复医院），福建福州 350001

收稿日期:2025-12-31 出版日期:2026-08-20 发布日期:2026-08-01
通讯作者: 葛继荣 E-mail:296986072@qq.com;gjrrjgcy@163.com
作者简介:郑若曦，硕士，助理实验师，E-mail: 296986072@qq.com
基金资助:
国家自然科学基金(82374483);国家自然科学基金(82274563);国家中医药管理局高水平中医药重点学科建设项目(zyyzdxk-2023106);福建中医药大学中医骨伤科学学科开放课题(XGS2023011);福建省省属公益类科研院所基本科研专项项目(2024R1003008)

Liuwei Dihuang Pill regulate osteoclast membrane fusion to improve postmenopausal osteoporosis in mice: the CLCF1/NFATc1 signaling axis as a therapeutic target

Ruoxi ZHENG¹^,²(), Lihua XIE², Xiaobin HUANG², Jirong GE²()

^1.Ministry of Education Key Laboratory of Orthopedics and Traumatology of Traditional Chinese Medicine and Rehabilitation, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
^2.Fujian Key Laboratory of Integrated Traditional Chinese and Western Medicine for Osteoporosis Prevention and Treatment (Fujian Academy of Chinese Medical Sciences, Rehabilitation Hospital Affiliated to Fujian University of Traditional Chinese Medicine), Fuzhou 350001, China

Received:2025-12-31 Online:2026-08-20 Published:2026-08-01
Contact: Jirong GE E-mail:296986072@qq.com;gjrrjgcy@163.com
Supported by:
National Natural Science Foundation of China(82374483)

摘要/Abstract

摘要：

目的探讨六味地黄丸（LWDHW）通过心肌营养蛋白样细胞因子1（CLCF1）/活化T细胞核因子1（NFATc1）调控破骨细胞膜融合治疗绝经后骨质疏松症（PMOP）的作用机制。方法将C57小鼠分为假手术组、模型组、治疗组，10只/组，去势模拟绝经后骨质疏松症并灌胃六味地黄丸治疗。将RAW264.7细胞分为对照组（空白血清）、模型组（核因子κB受体活化因子配体（RANKL）+空白血清）、治疗组（RANKL+六味地黄丸含药血清），用RANKL诱导为破骨细胞并用六味地黄丸含药血清干预。si-CLCF1慢病毒转染敲低细胞中CLCF1表达，将细胞分为空载体（si-NC）组、si-NC+LWDHW组、si-CLCF1组、si-CLCF1+LWDHW组。骨密度分析系统检测各组小鼠骨密度；抗酒石酸酸性磷酸酶（Trap）染色观察各组破骨细胞和胫骨组织中破骨细胞成熟情况；鬼笔环肽免疫荧光观察计算各组细胞膜融合效率及纤丝状肌动蛋白（F-actin）环形成；实时聚合酶链反应和蛋白质免疫印迹试验检测各组胫骨组织和细胞中CLCF1、NFATc1、树突状细胞特异性跨膜蛋白（DC-STAMP）、破骨细胞刺激跨膜蛋白（OC-STAMP）和空泡型ATP酶V0结构域2（ATP6V0D2）基因和蛋白表达。结果六味地黄丸治疗可增加PMOP小鼠骨密度，减少破骨细胞成熟，降低破骨细胞融合效率（P<0.05），减少F-actin环形成。与模型组相比，六味地黄丸干预可升高胫骨组织和细胞中CLCF1基因和蛋白表达（P<0.05），降低NFATc1、DC-STAMP、OC-STAMP和ATP6V0D2基因和蛋白表达（P<0.05）。与si-CLCF1组相比，si-CLCF1+LWDHW组六味地黄丸含药血清干预后破骨细胞融合效率下降（P<0.05），F-actin环形成减少，NFATc1、DC-STAMP、OC-STAMP和ATP6V0D2基因和蛋白表达降低（P<0.05）。结论六味地黄丸可通过上调CLCF1表达，下调NFATc1、DC-STAMP、OC-STAMP和ATP6V0D2表达，调节破骨细胞膜融合防治绝经后骨质疏松症。

关键词: 六味地黄丸, 绝经后骨质疏松症, 破骨细胞, 膜融合

Abstract:

Objective To explore the mechanism of Liuwei Dihuang Pill (LWDHW) for improving postmenopausal osteoporosis (PMOP). Methods Thirty female C57BL/6 mice were randomized equally into sham-operated group, PMOP model group, and LWDHW treatment group. The mice in the latter two groups underwent ovariectomy to simulate PMOP, followed by gavage of normal saline and LWDHW (twice daily) for 60 days, respectively. Bone mineral density (BMD) of the mice was detected, and TRAP staining was used to observe osteoclast and osteoclast maturation in the tibia tissue. In RANKL-induced RAW264.7 cells with or without lentivirus-mediated CLCF1 knockdown, the effects of incubation with normal rat serum or LWDHW-medicated rat serum on cell membrane fusion rate and F-actin loop formation were observed using immunofluorescence staining with phalloidin peptide; the mRNA and protein expressions of CLCF1, NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2 were detected using RT-qPCR and Western blotting. Results In PMOP mice, LWDHW treatment significantly increased BMD, reduced osteoclast maturation, osteoclast membrane fusion rate, and F-actin ring formation, upregulated the mRNA and protein expressions of CLCF1 in tibial tissues, and lowered the expressions of NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2. In RANKL-induced RAW264.7 cells with CLCF1 knockdown, treatment with LWDHW-medicated rat serum significantly decreased osteoclast membrane fusion rate, F-actin ring formation, and the expressions of NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2. Conclusion LWDHW improves PMOP in mice by regulating osteoclast membrane fusion through upregulating CLCF1 expression and downregulating NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2 expressions.

Key words: Liuwei Dihuang Pill, postmenopausal osteoporosis, osteoclasts, membrane fusion

郑若曦, 谢丽华, 黄小彬, 葛继荣. 六味地黄丸通过CLCF1/NFATc1调控破骨细胞膜融合治疗小鼠绝经后骨质疏松症[J]. 南方医科大学学报, 2026, 46(8): 1743-1753.

Ruoxi ZHENG, Lihua XIE, Xiaobin HUANG, Jirong GE. Liuwei Dihuang Pill regulate osteoclast membrane fusion to improve postmenopausal osteoporosis in mice: the CLCF1/NFATc1 signaling axis as a therapeutic target[J]. Journal of Southern Medical University, 2026, 46(8): 1743-1753.

图/表 16

表1 基因引物序列

Tab.1 Primer sequences for RT-qPCR

Gene name	Sequence (5'→3')
GAPDH	Forward: TGGAAAGCTGTGGCGTGATG
GAPDH	Reverse: TACTTGGCAGGTTTCTCCAGG
CLCF1	Forward: AACGAGCCTGACTTCAATCC
CLCF1	Reverse: GACTGTACGCCTCATAGTTCTG
NFATc1	Forward: CCGTCACATTCTGGTCCATAC
NFATc1	Reverse: TTCATTCTCCAAGTAACCGTGTAG
DC-STAMP	Forward: GTAGGAACGCTTTGATTGCGG
DC-STAMP	Reverse: TAGATTCAGCGGAGTGGCAAG
OC-STAMP	Forward: CATCCGCTCCCTATTTGTGC
OC-STAMP	Reverse: CACGCACATTGCCTAAGACG
ATP6V0D2	Forward: AGTCTTACCTTGAGGCATTCTACA
ATP6V0D2	Reverse: TCTCCCTGTCTTCTTTGCTTAGT

图1 各组小鼠胫骨TRAP染色情况比较

Fig.1 Tibial TRAP staining in different groups. LWDHW： Liuwei Dihuang Pill group.

表2 各组小鼠骨密度比较

Tab.2 Comparison of bone mineral density among the 3 groups(Mean±SD, n=10, g/cm2)

Group	BMD
Sham	0.065±0.02
Model	0.043±0.02*
LWDHW	0.054±0.02^#

图2 各组小鼠胫骨中膜融合相关基因表达水平比较

Fig.2 Comparison of mRNA expression levels of CLCF1 (A), NFATc1 (B), DC-STAMP (C), OC-STAMP (D), and ATP6V0D2 (E) related to tibial medial membrane fusion in each group of mice (n=3). *P<0.05 vs Sham group; #P<0.05 vs Model group.

图3 各组小鼠胫骨中膜融合相关蛋白表达水平比较

Fig.3 Expression levels of membrane fusion-related proteins in the 3 groups of mice. A: Western blotting for detecting expressions of CLCF1, NFATc1, DC-STAMP, OC-STAMP, and ATP6V0D2 in mouse tibia. B-F: Expression levels of CLCF1, NFATc1, DC-STAMP, OC-STAMP, and ATP6V0D2 (n=3). *P<0.05 vs Sham group; #P<0.05 vs Model group.

图4 各组细胞的破骨分化成熟情况

Fig.4 Osteoclast differentiation and maturation in each group (Original magnification: ×200).

图5 各组细胞融合发生率比较

Fig.5 Cell membrane fusion rates in each group. *P<0.05 vs Control group; #P<0.05 vs Model group.

图6 各组细胞中膜融合相关基因表达水平比较

Fig.6 Transcriptional levels of the membrane fusion-related genes in the 3 groups. A: CLCF1. B: NFATc1. C: DC-STAMP. D: OC-STAMP. E: ATP6V0D2. *P<0.05 vs Control group; #P<0.05 vs Model group.

图7 各组细胞中膜融合相关蛋白表达水平比较

Fig.7 Expression levels of membrane fusion-related proteins in each group. A: Western blotting for detecting expressions of CLCF1, NFATc1, DC-STAMP, OC-STAMP, and ATP6V0D2 in the osteoclasts. B-F: Expression levels of CLCF1, NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2. *P<0.05 vs Control group; #P<0.05 vs Model group.

图8 CLCF1慢病毒不同条件下EGFP荧光强度

Fig.8 EGFP fluorescence intensity in RAW264.7 cells under different conditions of CLCF1 lentivirus infection (×100). A-C: MOI=100. D-F: MOI=50. G-I: MOI=10. A, D, G: Complete medium. B, E, H: HitransG A. C, F, I. HitransG P.

图9 不同慢病毒对CLCF1蛋白表达的影响

Fig.9 Effects of different lentiviruses on expression of CLCF1 protein. A: Western blotting for detecting expressions of CLCF1 in the osteoclasts. B: CLCF1. *P<0.05 vs si-NC group; #P<0.05 vs si-CLCF1-2 group.

图10 CLCF1敲低表达后各组破骨细胞TRAP染色情况

Fig.10 TRAP staining of RAW264.7 cells in each groups after CLCF1 knockdown (×200).

图11 CLCF1敲低表达后各组破骨细胞膜融合情况

Fig.11 Osteoclast membrane fusion in each group after CLCF1 knockdown (×200).

图12 CLCF1敲低表达后各组细胞融合发生率比较

Fig.12 Cell membrane fusion in each group after CLCF1 knockdown. *P<0.05 vs si-NC; #P<0.05 vs si-NC+LWDHW; ▲P<0.05 vs si-CLCF1.

图13 CLCF1敲低表达对细胞膜融合相关基因表达水平比较

Fig.13 Transcriptional levels of genes related to cell membrane fusion after CLCF1 knockdown. A: NFATc1. B: DC-STAMP. C: OC-STAMP. D: ATP6V0D2. *P<0.05 vs si-NC, #P<0.05 vs si-NC+LWDHW, ▲P<0.05 vs si-CLCF1.

图14 CLCF1敲低表达对细胞膜融合相关蛋白表达水平比较

Fig.14 Effect of CLCF1 knockdown on expression levels of cell membrane fusion-related proteins. A: Western blotting for detecting expressions of NFATc1, DC-STAMP, OC-STAMP, and ATP6V0D2 in osteoclast. B: NFATc1. C: DC-STAMP. D: OC-STAMP. E: ATP6V0D2. *P<0.05 vs si-NC; #P<0.05 vs si-NC+LWDHW; ▲P<0.05 vs si-CLCF1.

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六味地黄丸通过CLCF1/NFATc1调控破骨细胞膜融合治疗小鼠绝经后骨质疏松症

Liuwei Dihuang Pill regulate osteoclast membrane fusion to improve postmenopausal osteoporosis in mice: the CLCF1/NFATc1 signaling axis as a therapeutic target

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