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Specnuezhenide alleviates senile osteoporosis by activating of TGR5/FXR signaling in bone marrow mesenchymal stem cells and RANKL-induced osteoclasts
Specnuezhenide 通过骨髓间充质干细胞和 RANKL 诱导的破骨细胞中 TGR5/FXR 信号传导缓解罗河骨质疏松症

Xuehui Deng 2,*, Bingfeng Lin 1,*, Wenlong Xiao 3, Fang Wang 2, Pingcui Xu 1, Nani Wang 1, 2, 3
雪辉 2,* 秉峰 1,*肖文龙 3王芳 2徐平翠1王纳尼 12, 3

1 Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, Zhejiang, People’s Republic of China;
1 浙江省中医科学院医学系,中华人民共和国浙江省杭州市;

2 School of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China;
2 浙江中医大学药学院中华人民共和国浙江省杭州市;

3 School of Pharmacy, Hangzhou Medical College, Hangzhou, Zhejiang, People’s Republic of China
3 杭州医学院药学院,中华人民共和国浙江省杭州

* These authors contributed equally to this work
*这些作者对这项工作的贡献相同

Correspondence: Nani Wang, Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, 132 Tianmushan Road, Hangzhou, Zhejiang 310007, People’s Republic of China, Tel/Fax +86-571-88849089, Email wnn8511@163.com
通讯地址:Nani Wang浙江省中医研究院医学部,浙江省310007市杭州天目山路132号,中华人民共和国,电话/传真:+86-571-88849089,邮箱wnn8511@163.com

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Background: Specnuezhenide (SPN) is an iridoid glycoside isolated from Fructus Ligustri Lucidi, a prescribed herb for senile osteoporosis treatment. However, the direct role of SPN on bone metabolism remains unclear. In this study, the effects of SPN on D-galactose (D-gal)-induced mice, bone marrow mesenchymal stem cells (BMSCs) and nuclear factor-κB ligand-induced osteoclasts (OC) were examined.
背景Specnuezhenide (SPN) 是从 FructusLigustriLucidi 中分离的环烯醚萜,Fructus Ligustri Lucidi 是一种治疗老年性骨质疏松症的处方草药。然而,SPN 对骨代谢的直接作用仍不清楚。在本研究中,研究了 SPN 对 D-半乳糖 (D-gal) 诱导的小鼠骨髓间充质干细胞 (BMSC) 和透明因子-κB配体诱导的破骨细胞 (OC) 的影响

Methods: In this study, micro-CT was used to observe the bone microstructure. The osteogenesis was examined using western blot and alkaline phosphatase staining. The osteoclastogenesis was examined using western blot and F-actin ring staining. The senescence-associated β-galactosidase was used to detect cell senescence. Moreover, the expression of Takeda G protein-coupled receptor 5 (TGR5) / farnesoid X receptor (FXR) signaling pathway-related genes and proteins was determined through quantitative real-time PCR and immunofluorescence.
方法: 本研究采用 micro-CT 观察骨微观结构。 使用western blot 和碱性磷酸酶染色检查 ost发生 使用 western blot 和F-肌动蛋白环染色检查破骨细胞生成采用衰老相关的 β-半乳糖苷酶检测细胞衰老。此外, 通过实时定量 PCR 和免疫荧光测定 Takeda G 蛋白偶联受体 5 (TGR5) /法尼醇 X 受体 (FXR) 信号通路相关基因和蛋白的表达。

Results: Oral administration of SPN improved the bone microstructure in D-gal-induced mice, and increased bone mineral density, bone volume, trabecular thickness, and trabecular number. SPN also upregulated the osteogenesis markers osteocalcin, bone morphogenetic protein 2, and runt-related transcription factor 2 and downregulated the osteoclasis markers tartrate-resistant acid phosphatase, nuclear factor-κB, nuclear factor of activated T-cells in the D-gal-induced bone. Furthermore, SPN increased the alkaline phosphatase staining, inhibited F-actin ring formation, and reduced the senescence-associated β-galactosidase in vitro. Mechanistically, SPN activated TGR5/FXR pathway in D-gal-induced BMSCs and OC. The protective effects of SPN were abolished after addition of the TGR5 inhibitor SBI-115 or FXR inhibitor DY268. Moreover, SPN could elevated protein and mRNA levels of TGR5, FXR, and the downstream small heterodimer partner in D-gal-induced bone.
结果: Oral 给予 SPN 改善了 D-gal 诱导小鼠 骨微观结构增加了骨密度、骨体积、小梁厚度小梁数量。SPN 还上调了成骨标志物骨钙素、骨形态发生蛋白 2 和 runt 相关转录因子 2下调了骨质形成标志抗酒石酸盐酸性磷酸酶核因子-κB活化 T 细胞的核因子在 D-gal 诱导的骨骼中。此外,SPN 在体外增加了碱性磷酸酶染色抑制了 F-肌动蛋白环的形成降低了衰老相关的 β-半乳糖苷酶实际上,SPN 激活了 D-gal 诱导的 BMSCs 和 OC 中的 TGR5/FXR 通路。加入 TGR5 抑制剂 SBI-115 或 FXR 抑制剂 DY268 后,SPN 的保护作用被消除。此外,SPN 可以提高 D-gal 诱导的骨骼中 TGR5 、 FXR 和下游小异二聚体伴侣的蛋白质和 mRNA 水平。

Conclusion: SPN alleviated senile osteoporosis and cell senescence via activating TGR5/FXR pathway.
结论: SPN 通过诱人的 TGR5/FXR 通路减轻老年性骨质疏松症和细胞衰老

Keywords: Specnuezhenide, Takeda G protein-coupled receptor 5, Farnesoid X receptor, Osteoporosis.
关键词SpecnuezhenideTakeda G 蛋白偶联受体 5法尼醇 X 受体O骨质细胞病。

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Introduction

Senile osteoporosis is characterized by deteriorated bone microstructure and reduced bone strength, resulting in high fracture risk.1 This disease has been considered a public health challenge worldwide owing to the dramatic increase in the aging population.2 The causes of bone loss during aging are closely related with the senescence of bone marrow mesenchymal stem cells (BMSCs).3 In addition, senescent-cell conditioned medium impaired enhanced osteoclast-progenitor survival, leading to increased osteoclastogenesis.4 Bile acid receptors Takeda G protein-coupled receptor 5 (TGR5) and farnesoid X receptor (FXR) were downregulated in the aging bone. TGR5 knockdown induces osteoclast differentiation to decrease bone mass in aged mice.5 Activation of FXR promotes the differentiation of BMSC into osteoblasts.6 FXR agonists also suppressed osteoclast differentiation from bone marrow macrophages.7 Increasing evidence have proven that TGR5 and FXR are important regulators in the development of senile osteoporosis. Thus, the TGR5/FXR signaling pathway is a promising target for mitigating senile osteoporosis.
Senile 骨质疏松症的特点是骨微结构恶化和骨强度降低,导致骨折风险高1 由于人口老龄化的急剧增加,这种疾病一直被认为是全球范围内的公共卫生挑战。2 agin g 期间骨质流失的原因骨髓间充质干细胞 (BMSC)衰老密切相关3此外,S烯细胞条件培养基损害了破骨细胞祖细胞存活率的增强,导致破骨细胞生成增加4胆汁酸受体武田 G 蛋白偶联受体 5 (TGR5) 和法尼醇 X 受体 (FXR) 在衰老骨骼中 TGR5 敲除诱导破骨细胞分化,从而降低老年小鼠的骨量。5FXR 的激活促进 BMSC 分化为成骨细胞。6 FXR 激动剂还抑制骨髓巨噬细胞的破骨细胞分化。7 越来越多的证据表明,TGR5 和 FXR 是老年性骨质疏松症发展的重要调节因子。因此,TGR5/FXR 信号通路是缓解老年性骨质疏松症的一个有前途的靶点。

Specnuezhenide (SPN) is an iridoid glycoside isolated from Fructus Ligustri Lucidi,8 which is clinically used in the treatment of senile osteoporosis.9,10 The extract of Fructus Ligustri Lucidi could restore bone mass in D-galactose (D-gal)-induced mice.11 SPN showed anti-osteoporosis effects in streptozotocin-induced rats.12 However, the effect of SPN on the senile osteoporosis have not been elucidated, especially the role of SPN on the BMSCs and OC senescence remains unclear. In the present study, we examined the effects of SPN on the bone mass of D-gal-induced mice and osteogenic activity of D-gal-induced BMSCs and OC. Our results demonstrated that SPN improved bone microarchitecture, promoted bone formation and suppressed bone resorption in vivo. SPN could inhibit the senescence of cells, increase the osteogenesis activity and decrease osteoclasis activity in vitro. Mechanistically, SPN exerted osteoprotective effects, at least partly, through activating the TGR5/FXR pathway. Our study provided evidence to support SPN as a potential therapeutic agent to preventing and treating senile osteoporosis.
Specnuezhenide (SPN) 是从 FructusLigustri Lucidi 8 中分离的环烯醚萜糖苷,临床上用于治疗老年性骨质疏松症。9,10 FructusLigustriLucidi 的提取物 可以恢复 D-半乳糖 (D-gal) 诱导的小鼠的骨量。11SPN链脲佐菌素诱导的大鼠中显示出抗骨质疏松作用12然而,SPN 对老年性骨质疏松症的影响尚未阐明,尤其是 SPN 对 BMSC 和 OC 衰老的作用仍不清楚。在本研究中,我们检查了 SPN 对 D-gal 诱导的小鼠骨量和 D-gal 诱导的 BMSCs 和 OC 的成骨活性的影响。我们的结果表明,SPN 改善了骨微结构促进了骨形成抑制了体内吸收。SPN 在体外可抑制细胞衰老,增加成骨活性降低骨质形成活性。从机制上讲,SPN 至少 部分通过激活 TGR5/FXR 通路发挥了骨保护作用。我们的研究提供了证据,支持 SPN 作为预防和治疗老年性骨质疏松症的潜在治疗剂。

Materials and methods
M文献和方法

Mice and treatment
小鼠和治疗

The male ICR mice (8 weeks), weighing 20 - 22 g, were obtained from Hangzhou Medical College (Hangzhou, Zhejiang, China). All animal experiments were carried out in an accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23; revised 1978) and performed under the Guidelines for Animal Experiments of the Zhejiang Academy of Traditional Chinese Medicine (Approval No. 2021-001). The protocol was approved by the Institutional Animal Ethics Committee. SPN (#102728, purity ≥ 98%, Yongjian Pharmaceutical, Jiangsu, China, Figure 1A) was dissolved in sterile saline. The mice were randomly divided into the following several groups: normal control group (CON, sterile saline daily gastric irrigation (ig) + subcutaneous injection (sq) of sterile saline daily, n=10), model group (MOD, sterile saline daily ig + 150 mg/kg/d D-gal daily sq, n=10), SPN with low dosage (SPN-L, 5 mg/kg/d SPN daily ig + 150 mg/kg/d D-gal daily sq, n=10), and SPN with high dosage (SPN-H, 10 mg/kg/d SPN daily ig + 150 mg/kg/d D-gal daily sq, n=10) for three months. The experimental diagram was shown in the text (Figure 1B). The number of animals per group was determined by previous reports.13 The dose and duration of SPN were used according to the previous reports.14,15
雄性 ICR 小鼠 (8 周),体重 20 - 22 g,购自杭州医学院 (中国浙江省杭州市)。所有动物实验均按照 NIH 实验动物护理和使用指南(NIH 出版物第 80-23 号;1978 年修订)进行,并根据浙江中医科学院动物实验指南(批准号 2021-001)进行。该协议得到了机构动物伦理委员会的批准。 SPN(#102728,纯度≥ 98%,中国江苏永制药,图 1A)溶于无菌盐水中 将小鼠随机分为 以下组:正常对照组(CON,无菌生理盐水每日g次冲洗ig)+皮下注射sq)每日一次,n=10),模型发育组(MOD,无菌生理盐水每日ig + 150 mg/kg/d D-gal每日一次q,n=10),低剂量 SPN(SPN-L,5 mg/kg/d SPN每天 ig+ 150 mg/kg/d D-gal 每天s q,n=10)和高剂量 SPN(SPN-H,10 mg/kg/d SPN每天 i g + 150 mg/kg/d D-gal 每天s q,n=10),持续三个月。 实验图如图 1B 所示每组动物的数量 以前的报告确定13 根据之前的报告使用 SPN 的剂量和持续时间1415

Microcomputed tomography bone analysis
显微计算机断层扫描骨分析

Femurs were removed and kept in 4% formalin. Fixed femurs were scanned using a micro-computed tomography (micro-CT) scanner (Skyscan 1172, Bruker, Belgium). Values for bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) were given.
去除股骨并保存在 4% 福尔马林中。 使用显微计算机断层扫描 (micro-CT) 扫描仪 (Skyscan 1172, Bruker, Belgium) 扫描固定股骨。给出了骨矿物质密度(BMD) 、骨体积/总体积 (BV/TV) 、小梁厚度 (Tb.Th) 和小梁数 (Tb.N) 的值。

Dynamic histomorphometry measurement of bone formation
骨形成的动态组织形态测量法测量

The dynamic histomorphometry parameters measured included mineral apposition rate (MAR).16 The mice were injected with calcein (35 mg/kg, ip, Sangon Biorech, Shanghai, China) and tetracycline (20 mg/kg, ip, Aladdin, Shanghai, China) dissolved in sterile saline at 10 and 2 days prior to harvest. Femurs were embedded in methyl methacrylate (#M813511, Macklin, Shanghai, China), blocks, and sectioned into 10-μm slices. The sections were viewed under a fluorescent Pannoramic MIDI (3DHISTECH).
测量的动态组织形态学参数包括矿物质附着率 (MAR)。16 小鼠收获前 10 天和 2 天用钙黄绿素(35 mg/kg,ip,Sangon Biorech,中国上海)和四环素(20 mg/kg,ip阿拉丁,中国上海)溶于无菌盐水中。将股骨包埋在甲基丙烯酸甲酯 (#M813511, Macklin, Shanghai, China) 中,块状,并切成 10 μm 切片。这些切片是在荧光 Pannoramic MIDI (3DHISTECH) 下观察的。

Western blotting analysis
Western 印迹分析

The tibia was homogenized and extracted in RIPA lysis buffer (Beyotime, Shanghai, China). The protein content was measured by a BCA kit (Keygentec, Nanjing, China). The primary antibodies included anti-FXR (#DF12402, Affinity, Jiangsu, China), anti-TGR5 (#DF14087, Affinity), anti-small heterodimer partner (SHP, #AF6244, Affinity), anti-alkaline phosphatase (ALP, #BM4284, Boster, Hubei, China), anti-runt-related transcription factor 2 (Runx2, #DF0171, Boster), anti-bone morphogenetic protein 2 (BMP2, #AF5163, Affinity), anti-nuclear factor-κB (RANK, #DF12532, Affinity), anti-nuclear factor of activated T-cells (NFATc1, #D0522, Santa cruz biotechnology). The membrane was incubated with goat anti rabbit IgG. Protein band densities were analyzed using ImageJ software and normalized to GAPDH.
在 RIPA 裂解缓冲液(Beyotime,上海,中国)中均质化和提取胫骨 w。蛋白质含量由 BCA 试剂盒 (Keygentec, Nanjing, China) 测量。一抗包括抗 FXR (#DF12402Affinity, Jiangsu, China)、抗 TGR5 (#DF14087, Affinity)、抗小异二聚体伴侣 (SHP, #AF6244Affinity)、抗碱性磷酸酶 (ALP, #BM4284, Boster, Hubei, China)、抗 runt 相关转录因子 2 (Runx2, #DF01 71,Boster)、抗骨形态发生蛋白 2(BMP2,#AF5163,亲和力),抗核因子-κ B (RANK,#DF12532,亲和力),活化 T 细胞的抗核因子(NFATc1,#D0522,圣克鲁斯生物技术)。将膜与 g燕麦抗兔 IgG 一起孵育。使用 ImageJ 软件分析蛋白质条带密度 并归一化为 GAPDH。

Immunohistochemistry
免疫组化

Femurs were collected, fixed with 4% formalin for 48 hours, decalcified in 10% ethylenediamine tetraacetic acid (EDTA), paraffin-embedded and used for tartrate-resistant acid phosphatase (TRAP) and Masson’s trichrome staining. 5 μm sections were incubated with primary antibodies against osteocalcin (OCN, #DF12303, Boster), TGR5, FXR, P16 (#AF0228, Affinity), P21 (#DF6423, Affinity), and P53 (#AF0879, Affinity). The images were analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, USA).
收集股骨,用 4% 福尔马林固定 48 小时,在 10% 乙绔呤diaminetetraaceticacidEDTA) 中脱钙,对ffin 包埋并用于抗酒石酸盐酸性磷酸酶 (TRAP) 和 Masson 三色染色。将 5 μm 切片与初级切片一起孵育抗骨钙素 (OCN, #DF12303, Boster), TGR5 FXR, P16 (#AF0228,亲和力), P21 (#DF6423,亲和力) 和 P53 (#AF0879,亲和力) 的抗体。通过 Image-Pro Plus 6.0 软件 (Media Cybernetics, Silver Spring, USA) 对图像进行分析。

Cell culture and treatment
Cell 培养和处理

BMSCs and RAW 264.7 cells were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). Cells were cultured in Dulbecco’s modified essential medium supplemented with 10% FBS and 1% antibiotics, and cultured until confluent at 37℃ in a humidified atmosphere of 5% CO2. To generate osteoclasts, RAW 264.7 cells (3 × 103 cells/well) were cultured in the presence of 50 ng/mL of nuclear factor-κB ligand (RANKL) and various additives. BMSCs and OC were respectively divided into several groups: blank control group, D-gal group (D-gal, 50 mM), D-gal + SPN group (D-gal, 50 mM; SPN, 10-1 μM), D-gal + SPN + SBI-115 group (D-gal, 50 mM; SPN, 10-1 μM; SBI-115, 1 μM), D-gal + SPN + DY268 group (D-gal, 50 mM; SPN, 10-1 μM; DY268, 1 μM), D-gal + SPN + SBI-115 + DY268 group (D-gal, 50 mM; SPN, 10-1 μM; SBI-115, 1 μM; DY268, 1 μM). After treating for 48h, the cells were collected for senescence-associated β-galactosidase (SA-β-gal) staining.
BMSCs RAW 264.7 细胞 购自国家认证细胞培养物保藏中心(中国上海)。将细胞在补充有 10% FBS 和 1% 抗生素的 Dulbecco 改良必需培养基中培养,并在 37°C 和 5% CO2 的潮湿气氛中培养至汇合。为了生成破骨细胞,在 50 ng/mL nuclear factor-κB 配体 (RANKL) 和各种添加剂存在下培养 RAW 264.7 细胞 (3 ×103 个细胞/孔)。 将 BMSCs 和 OC 分别分为几组:空白对照组、D-gal 组 (D-gal, 50 mM)、D-gal + SPN 组 (D-gal, 50 mM; SPN,10-1 μM),D-gal + SPN + SBI-115(D-gal,50 mM;SPN,10-1 μM; SBI-115,1 μM),D-gal + SPN + DY268 组(D-gal,50 mM;SPN,10-1 μM;DY268,1 μM),D-gal + SPN + SBI-115 + DY268 组(D-gal,50 mM;SPN,10-1 μM;SBI-115,1 μM;DY268,1 μM)。 处理 48 小时后,收集细胞进行衰老相关 β-半乳糖苷酶 (SA-β-gal) 染色。

SA-β-gal staining
SA-β-gal 染色

SA-β-gal staining was performed according to the manufacturer’s instructions (#C0602, Beyotime). Briefly, cells were fixed in 4% paraformaldehyde for 15 min and incubated with SA-β-gal staining solution at 37 for 12 h. Cells were washed with PBS, and photographed under a microscope (TI-DH, Nikon, Tokyo, Japan). The percentage of SA-β-gal positive cells was expressed as the ratio of blue-stained cells to the total cells.
根据制造商的说明 (#C0602, Beyotime) 进行 S A-β-gal 染色。简而言之,将细胞在 4% 多聚甲醛中固定 15 分钟,并与 SA-β-gal 染色溶液在 37°C 下孵育 12小时。 用 PBS 洗涤细胞,并在显微镜下拍照 (TI-DH, Nikon, Tokyo, Japan)。SA-β-gal 阳性细胞的百分比表示为蓝色染色细胞与总细胞的比率。

ALP staining
ALP 染色

After the BMSCs reached 80% confluence, the medium was replaced with an osteogenic induction medium (100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid). D-gal, SPN, SBI-115, and DY268 were added to the osteogenic induction medium according to the experiment design. After culturing for 7 days, ALP staining measurements were conducted. For ALP staining, cells were washed with PBS three times and stained using an ALP staining kit (#C3206, Beyotime). Cells were washed with water, and photographed under a microscope.
在 BMSCs 达到 80% 汇合后,用成骨诱导培养基(100 nM 地塞米松、10 mM β-甘油磷酸和 50 μM 抗坏血酸)替换培养基。根据实验设计,将 D-gal 、 SPN 、 SBI-115 和 DY268 添加到成骨诱导培养基中。培养 7 天后,进行 ALP 染色测定。 对于 ALP 染色,用 PBS 洗涤细胞 3 次,并使用 ALP 染色试剂盒 (#C3206, Beyotime 染色。用水洗涤细胞,并在显微镜下拍照。

F-actin ring staining
F 肌动蛋白环染色

After 5 d of OC induction, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 5 min and then stained with fluorescein isothiocyanate (FITC)-phalloidin solution (#C1033, Beyotime) in darkness for 1 h. After washing with PBS, the cells were incubated with a DAPI solution for 10 min to stain the nuclei. The images of F-actin rings were visualized under a confocal microscope (LSM800, Zeiss, Oberkochen, Germany).
OC 诱导 5 天后,用 4% 多聚甲醛固定细胞, 用 PBS 中的 0.1% Triton X-100 透化 5 分钟,然后用硫氰酸荧光素 (FITC)-鬼笔环肽溶液 (#C1033,Beyotime) 在黑暗中染色 1 h。用 PBS 洗涤后,将细胞与 DAPI 溶液孵育 10 分钟细胞核进行染色。在共聚焦显微镜 (LSM800, Zeiss, Oberkochen, Germany) 下观察 F 肌动蛋白环的图像

RNA sequencing
RNA 测序

RNA was isolated using TRIzol reagent. Sequencing libraries were generated using the ALFA-SEQ Directional RNaLib Prep Kit (mChip, Guangzhou, China). The mRNA was purified by the poly-T oligo-attached magnetic beads. First strand cDNA was prepared using random hexamer primers and M-MuLV reverse transcriptase (RNase H). Second strand cDNA was synthesized using DNA polymerase I and RNase H. Library quality was assessed on a Qsep400 high-throughput nucleic acid protein analysis system (Houze Biotechnology Co., Ltd., Hangzhou, China). Genes were considered differentially expressed when the fold changes were ≥ 2 and the corrected P value was < 0.05.
使用 TRIzol 试剂分离 RNA 。使用 ALFA-SEQ 定向 RNaLib Prep Kit (mChip, Guangzhou, China) 生成测序文库。mRNA 由 poly-T 寡核苷酸连接的磁珠纯化。 使用随机六聚体引物和 M-MuLV 逆转录酶 (RNase H) 制备第一链 cDNA 使用 DNA 聚合酶 I 和 RNase H 合成第二链 cDNA,在 Qsep400 高通量核酸蛋白分析系统(厚泽生物科技有限公司,中国杭州)上评估文库质量。当倍数变化≥ 2 且校正后的 P 值为 < 0.05 时,认为基因差异表达。

Immunofluorescence staining
免疫荧光染色

Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Trion X-100 (omit this step for TGR5). Cells were respectively incubated with anti-TGR5 (dilution 1:500) and anti-FXR (dilution 1:500) antibodies at 4℃ for 12 h. Cells were incubated with Goat pAb to Rb lgG (Alexa Fluor® 488, #ab150077, Abcam) at room temperature for 1 h. The slides were mounted with DAPI and photographed under a confocal microscope.
用 4% 多聚甲醛固定细胞 15 分钟,并 用 0.1% Trion X-100 透化(TGR5 省略此步骤)。细胞分别在4°C下与抗TGR5(稀释度1:500)和抗FXR(稀释度1:500)抗体孵育d12小时。 CellsGoat pAbRblgG (Alexa Fluor® 488#ab150077, Abcam 在室温下孵育 1 小时。用 DAPI 封片并在共聚焦显微镜下拍摄载玻片

RNA isolation and real-time reverse transcription polymerase chain reaction
RNA 分离和实时逆转录聚合酶链反应

Total RNA was extracted from tibias using TRIzol (Invitrogen, Carlsbad, CA, US). The RNA sample was reverse transcribed using a TOBOBlue qRT Premix with a gDNA Eraser 2.0 kit (#RTQ202, Toroivd Technology, Shanghai, China). The mRNA expression of SHP, TGR5, FXR were measured in a 7500 quantitative real-time reverse transcription polymerase chain reaction system (RT-PCR, Applied Biosystems, Waltham, MA, US) using SYBR Green reagents (#QPS201, Toyobo, Osaka, Japan). The primer sequences were listed in Table S1. Relative mRNA expression was quantified by comparing cycle threshold values. GAPDH was used as a housekeeping gene, and data were shown as the fold change relative to controls.
使用 TRIzol (Invitrogen, Carlsbad, CA, US) 从胫骨中提取总 RNA。使用带有 gDNA Eraser 2.0 试剂盒(#RTQ202,Toroivd Technology,上海,中国)的 TOBOBlueqRT Premix 对 RNA 样品进行逆转录 使用 SYBR Green 试剂 (#QPS201, Toyobo, Osaka, Japan) 在 7500 定量实时逆转录聚合酶链反应系统 (RT-PCR, Applied Biosystems, Waltham, MA, US) 中测量 SHP TGR5 、 FXR mRNA 表达。引物序列于表 S1 中。 通过比较循环阈值来定量相对 mRNA 表达。GAPDH 用作管家基因,数据显示为相对于对照的倍数变化。

Statistical analysis
统计分析

Statistical analysis was conducted using GraphPad Prism 9.0 (GraphPad, CA, US). Data are presented as the means ± SD. Statistical comparisons were performed using one-way or two-way analysis of variance followed by Dunnett corrections to compare multiple groups. P < 0.05 was considered statistically significant.
使用 GraphPad Prism 9.0 (GraphPad, CA, US) 进行统计分析。数据作为 SD ±均值表示。使用单向或双向方差分析进行统计比较,然后进行 Dunnett 校正以比较多组。 P < 0.05 被认为具有统计学意义。

Results

SPN promotes osteoporosis in D-gal-induced mice
SPN 促进 D-gal 诱导小鼠的骨质疏松症

To determine the effect of SPN on bone microstructure, micro-CT analysis was performed on mouse femurs. The SPN-L and SPN-H groups showed an increase in BMD (Figure 1C, P < 0.01), BV/TV (P < 0.01), Tb.N ( P < 0.01), and Tb.Th (P < 0.01), in comparison to the MOD group. These findings suggested that SPN improved osteoporosis in D-gal-induced mice.
为了确定 SPN 对骨微结构的影响,对小鼠股骨进行了显微 CT 分析。与 MOD 组相比,SPN-L 和 SPN-H 组显示 BMD (图1 C,P < 0.01)、BV/TV (P < 0.01)、Tb.NP< 0.01) 和 Tb.ThP < 0.01) 增加。这些发现表明 SPN 改善了 D-gal 诱导小鼠的骨质疏松症。

SPN increases bone formation in D-gal-induced mice
SPN 增加 D-gal 诱导小鼠的 b 1 形成

OCN is specifically secreted by osteoblasts and considered as an essential biomarker of bone formation.17 The protein expression of OCN was downregulated in the MOD group compared to that in the CON group (Figure 2A, P < 0.01), while SPN restored this change (P < 0.01). Besides, SPN promoted the expression of ALP (Figure 2B, P < 0.05 for SPN-L and P < 0.01 for SPN-H), BMP2 (P < 0.01 for SPN-H), and Runx2 (P < 0.01) in D-gal-induced tibias compared to those in the MOD group. MAR were significantly reduced in MOD mice compared to CON mice (Figure S1, P < 0.01), whereas SPN reduced this suppression (P < 0.01). Masson’s trichrome staining showed that SPN increased the osteoblast numbers in the D-gal-induced mice (P < 0.01). These results showed that SPN could promote bone formation in the D-gal-induced mice.
OCN 由成骨细胞特异性分泌,被认为是骨形成的重要生物标志物17 与 CON 组相比,MOD 组 OCN的蛋白表达下调 (图 2A,P < 0.01),而 SPN 恢复了这种变化 (P < 0.01)。此外,SPN 促进 ALP 的表达 [2 B,SPN-L P < 0.05 SPN-H 的 P < 0.01BMP2 (SPN-H 的 P < 0.01) 和 Runx2 (P < 0.01) 的表达与MOD 组中的 THE THE GROUP 中的用户。与 CON 小鼠相比,MOD 小鼠的 MAR 显著降低 (图 S1,P < 0.01),而 SPN 降低这种抑制 (P < 0.01)。Masson 三色染色显示 SPN 增加了 D-gal 诱导小鼠的成骨细胞数量 (P < 0.01)。这些结果表明,SPN 可以促进 D-gal 诱导小鼠的骨形成。

SPN decreases bone resorption in D-gal-induced mice
SPN 降低 D-gal 诱导小鼠的 b1 再吸收

TRAP-staining was applied to examine whether SPN alleviated D-gal-induced bone resorption. D-gal increased osteoclasts in the femur compared to that in the CON group (Figure 3A, P < 0.01), but the osteoclast number was reduced by SPN (P < 0.01). These results indicated that SPN suppressed bone resorption in D-gal-induced mice. The protein expression of RANK and NFATc1 was upregulated in the MOD group compared to that in the CON group (Figure 3C, P < 0.01), while SPN restored this change (P < 0.01).
应用 TRAP 染色检测 SPN 是否减轻 D-gal 诱导的吸收CON相比,D-gal 股骨破骨细胞增加 (图 3A,P < 0.01),但破骨细胞数量因 SPN 而减少 (P < 0.01)。这些结果表明,SPN 抑制 D-gal 诱导小鼠的骨吸收。 与 CON 组相比,MOD 组 RANK 和 NFATc1 蛋白表达上调 (图 3C,P < 0.01),而 SPN 恢复了这种变化 (P < 0.01)。

SPN attenuates bone senescence in D-gal-induced mice.
SPN 减轻 D-gal 诱导小鼠的骨衰老

Cellular senescence is a master issue in the development of age-related osteoporosis, so we measured the effect of SPN in the senescence markers in mouse femurs. The expressions of senescence-related proteins P16, P21, and P53 were significantly increased in model mice (Figure 4, P < 0.01) compared with those of the control group. SPN downregulated the expressions of these proteins as compared to that in the model group in a dose-dependent manner (P < 0.01).
Cellular 衰老是年龄相关性骨质疏松症发展的主要问题,因此我们测量了 SPN 对小鼠股骨衰老标志物的影响。与对照组相比,模型小鼠衰老相关蛋白 P16 、 P21 和 P53 的表达显著增加 [图 4,P < 0.01]。与 模型组相比,SPN 以剂量依赖性方式下调这些蛋白的表达 (P < 0.01)。

SPN attenuates cell senescence by the TGR5/FXR pathway.
SPN 通过 TGR5/FXR 通路减弱细胞衰老

The SA-β-gal-stained positive cells were detected to evaluate the anti-senescent effect of SPN. As expect, D-gal treatment increased the SA-β-gal-stained positive cells (Figure 5, P < 0.01). However, the ratio of SA-β-gal-stained cell in SPN group was lower than those in the MOD group (P < 0.01). Additionally, the combination of SBI-115 and DY268 increased the senescent cells compared with the D-gal + SPN + SBI-115 group (P < 0.01) or D-gal + SPN + DY268 group (P < 0.01).
检测 SA-β-gal 染色的阳性细胞,评价SPN 的 nti-senescent 效应。 正如预期的那样,D-gal 处理增加了 SA-β-gal 染色的阳性细胞 5,P< 0.01)。SPN 组 SA-β-gal 染色细胞比例低于 MOD 组 (P< 0.01)。 此外,与 D-gal + SPN + SBI-115 组 (P< 0.01) 或 D-gal + SPN + DY268 组 P< 0.01) 相比,SBI-115 和 DY268 的组合增加了衰老细胞

SPN promoted osteogenic differentiation of BMSCs and blocked osteoclast differentiation of RAW 264.7 cells by the TGR5/FXR pathway.
SPN 促进 BMSCs 的成骨分化,并通过 TGR5/FXR 通路阻断 RAW 264.7 细胞的破骨细胞分化。

ALP is an early-stage osteogenic differentiation marker.18 The osteogenic induction medium induced osteogenic differentiation of BMSCs along with the upregulation of ALP expression. SPN also increased expression of ALP (Figure 6A). To confirm whether activating TGR5 and FXR can induce ALP generation in BMSCs, we detected the level of ALP in BMSCs after stimulation with the TGR5 antagonist SBI-115 or FXR antagonist DY268. SBI-115 and DY268 suppressed ALP expression. As a result, the osteogenic differentiation effect of SPN was blocked in the presence of the SBI-115 or DY268.
ALP 是一种早期成骨分化标志物18成骨诱导培养基诱导 BMSCs 成骨分化以及 ALP 表达上。SPN 还增加了 ALP 的表达 (图 6A)。 为了确认激活 TGR5 FXR 是否诱导 BMSC 中 ALP 的产生,我们在用 TGR5 刺激后检测了 BMSC 中 ALP 的水平 同轴剂 SBI-115 或 FXR 拮抗剂 DY268。SBI-115 和 DY268 抑制 ALP 表达。结果在 SBI-115 或 DY268 存在下,SPN 的成骨分化作用被阻断。

Formation of F-actin rich adhesive structures by osteoclasts is an essential step in bone resorption.19 RANKL stimulation increased well-defined F-actin sealing rings with a higher intensity ring height. The F-actin ring in the MOD group displayed a huge typical ring structure (Figure 6B), while the that in the SPN group was obviously smaller. RANKL-induced RAW 264.7 cells were also abrogated by TGR5 antagonist SBI-115 or FXR antagonist DY268. The result suggested that SPN inhibited osteoclast differentiation.
破骨细胞对富含 F-肌动蛋白的粘附结构进行 F 或交配是骨吸收的重要步骤。19 RANKL 刺激增加了具有更高强度环高度的清晰定义的 F-肌动蛋白密封环。MOD 组中的 F-肌动蛋白环表现出巨大的典型环状结构 (图 6B),而 SPN 组中的 F-肌动蛋白环明显较小。RANKL 诱导的 RAW 264.7 细胞也被 TGR5 拮抗剂 SBI-115 或 FXR 拮抗剂 DY268 消除。结果表明 SPN 抑制破骨细胞分化。

SPN upregulates TGR5 and FXR expression in D-gal-induced BMSCs and OC
SPN 上调 D-gal 诱导的 BMSCs 和 OC 中 TGR5 和 FXR 的表达

Next, we performed BMSCs RNA-sequence analysis to explore the molecular mechanism of SPN. Compared with the model group, FXR was predominately high among all the up-regulated genes (Figure 7A). Gene set enrichment analysis (GSEA) also revealed that SPN could regulated bile acid biosynthetic process-related genes (Figure 7B and 7C). To confirm whether SPN functions through TGR5-FXR activation, we detected TGR5 and FXR expression in vitro (Figure 7D and 7E). D-gal exposure decreased the levels of TGR5 (P < 0.01) and FXR (P < 0.01), but SPN significantly elevated TGR5 (P < 0.01) and FXR (P < 0.01) in D-gal-induced BMSCs and OC.
接下来,我们进行 BMSCs RNA 序列分析,探讨 SPN 的分子机制。与模型组相比, FXR 在所有上调的基因中主要较高(图7A)。基因集富集分析 (GS, E A) 还显示 SPN 可以调控胆汁酸生物合成过程相关基因 (图7B 和 7C)。为了确认 SPN 是否通过 TGR5-FXR 激活发挥作用,我们在体外检测到 TGR5 和 FXR 表达 (图7D 和 7E)。D-gal 暴露降低了 D-gal 诱导的 BMSCs 和 OCs 中 TGR5 (P< 0.01) 和 FXR (P< 0.01) 水平,但 SPN 显著升高了 TGR5 (P< 0.01) 和 FXR (P< 0.01)。

SPN activates TGR5 and FXR in D-gal-induced mice
SPN 在 D-gal 诱导的小鼠中激活 TGR5 和 FXR

We next examined the effect of SPN on TGR5 and FXR in bone tissues. The protein levels of TGR5 (Figure 8A, P < 0.01) and FXR (P < 0.01) were downregulated in the MOD group as compared to those in the CON group, while SPN increased TGR5 and FXR expression (P < 0.01). Western blot results showed that SPN significantly upregulated the protein expression of TGR5 (Figure 8B, P < 0.01), FXR (P < 0.01), and its downstream SHP (P < 0.01) levels in mouse tibias compared to those in the MOD group.
We 接下来检查 SPN 对骨组织中 TGR5 和 FXR 的影响。与 CON 组相比,MOD 组 TG R5 [图 8A, P < 0.01] 和 FXR (P < 0.01) 蛋白水平下调,而 SPN 增加 TGR5 和 FXR 表达 (P < 0.01)。Western blot 结果显示,与 MOD 组相比,SPN 显著上调小鼠胫骨 TGR5 蛋白表达 (图 8B,P < 0.01)、FXR (P < 0.01) 及其下游 SHP (P < 0.01) 水平。

SPN treatment also upregulated the mRNA expression of TGR5 (Figure 8C, P < 0.01), FXR (P < 0.01), and SHP (P < 0.01) in D-gal tibias. These data demonstrated that SPN treatment stimulated the TGR5-FXR pathway in D-gal-induced bone.
SPN 处理还上调了 D-gal 胫骨中 TGR5 的 mRNA 表达 [图 8C,P < 0.01] 、FXR (P < 0.01 和 SHP (P < 0.01)。这些数据表明,SPN 治疗刺激了 D-gal-in 诱导骨中的 TGR5-FXR 通路

Discussion
D震荡

Currently, a variety of active ingredients have found in Fructus Ligustri Lucidi. Previous investigations by our group and other researchers have shown that the aqueous extract and some compounds of Fructus Ligustri Lucid are beneficial on bone metabolism.20 However, the direct osteoprotective effect of SPN, the major active component of Fructus Ligustri Lucidi, in the aging models remains to be elucidated. The present work demonstrated for the first time that the administration of SPN increased bone mass of D-gal-induced mice and attenuated cell senescence. The mechanism underlying this anti-osteoporosis effect may be linked to the improvement of bone formation and inhibition of bone resorption through promoting osteoblastic differentiation of BMSCs and suppressing osteoclastic differentiation of RAW 264.7 cells via activating the TGR5/FXR pathway.
目前,在 FructusLigustriLucidi 中发现了多种活性成分。我们小组和其他研究人员之前的研究表明,FructusLigustri Lucid 的水提取物和一些化合物对骨骼代谢有益。20 然而, SPN 的直接骨保护作用,FructusLigustriLucidi 的主要活性成分,在衰老模型中的直接骨保护作用仍有待阐明。目前的工作首次证明,SPN 的给药增加了 D-gal 诱导的小鼠的骨量并减轻了细胞衰老。这种抗骨质疏松作用的机制可能与通过激活 TGR5/FXR 通路促进 BMSC 成骨细胞分化和抑制 RAW 264.7 细胞的骨形成和抑制骨吸收有关。

D-gal was applied to establish senile osteoporosis models.21 Exposure of mice to D-gal triggers the acceleration of natural senescence, which results in the induction of senile osteoporosis.22 Cell senescence and impaired osteogenic differentiation also could be found in D-gal-induced BMSCs.23 In this study, D-gal injection significantly decreased bone mass in mice. We also observed the senescent cells ratio was increased in D-gal-exposed BMSCs and OC. Importantly, SPN effectively improved bone microarchitecture and increased the osteogenesis markers, such as ALP, OCN, BMP2, and Runx2. Furthermore, the activities of SPN regulates osteoclast differentiation by down-regulating the expression of TRAP, RANK, and NFATc1 and inhibiting the F-actin formation. Altogether, these results suggest that SPN can stimulate the osteoblastic differentiation of BMSCs, suppress osteoclast differentiation of RAW 264.7 cells and attenuate senile osteoporosis.
应用 D-gal 建立老年性骨质疏松症模型。21 小鼠暴露于 D-gal 会触发自然衰老的加速,从而导致老年性骨质疏松症的诱导。22 在 D-gal 诱导的 BMSCs.23 中也可以发现细胞衰老和成骨分化受损。这项研究,D-gal 注射显着降低了小鼠的骨量。我们还观察到 D-gal 暴露的 BMSCs 和 OC 中的衰老细胞比例增加。重要的是,SPN 有效地改善了骨微结构并增加了成骨 标志物,如 ALP 、 OCN 、 BMP2 和 Runx2。此外,SPN 的活性通过下调 TRAP 、 RANK 和 NFATc1 的表达并抑制 F-肌动蛋白的形成来调节破骨细胞分化。总之,这些结果表明 SPN 可以刺激 BMSC 的成骨细胞分化,抑制 RAW 264.7 细胞的破骨细胞分化 并减轻老年性骨质疏松症。

TGR5 and FXR are bile acid receptors, which are essential regulators in the development of senile osteoporosis.24,25 It has been reported that TGR5 deletion markedly decreased bone mass in aged and ovariectomized mice.6 Upregulation of TGR5 could promote osteoblast mineralization and increase osteoblast differentiation marker genes.21 TGR5 regulates osteoclast differentiation to reduce bone loss.26 FXR also suppressed osteoclast differentiation and promoted osteoblast differentiation.6 FXR typically heterodimerizes with the retinoid X receptor in FXR response elements associated with the promoters of target gene Runx2, which is a bone-specific transcription factor. Deletion of FXR in vivo leads to a significant bone loss.27 In this study, we observed SPN increased levels of TGR5 and FXR, the bone formation biomarkers Runx2, OCN, BMP2, and ALP in D-gal-induced mice. In addition, SPN suppressed the bone resorption biomarkers TRAP, RANK, NFATc1, and F-actin. SPN might play bone protective roles by activating TGR5/FXR pathway in D-gal-induced bone. These results agreed with previous studies that demonstrated the promotion effect of TGR5 and FXR on the bone protection. The present study also confirmed the role of the TGR5/FXR pathway in the effect of SPN on D-gal-induced BMSCs and OC by using TGR5 antagonist (SBI-115) and FXR antagonist (DY268). According to the present findings, SPN effectively promoted osteoblastic differentiation of BMSCs, inhibited osteoclastic differentiation of RAW 264.7 cells, and alleviate cell senescence via activating the TGR5/FXR pathway.
TGR5 和 FXR 是胆汁酸受体,是老年性骨质疏松症发展的重要调节因子24,25 据报道,TGR5 缺失显着降低了老年和卵巢切除小鼠的骨量6TGR5 的上调可以促进成骨细胞矿化并增加成骨细胞分化标志基因。21TGR5 调节破骨细胞分化以减少骨质流失。26FXR 还抑制破骨细胞分化并促进成骨细胞分化。6FXR 通常与靶基因 Runx2(一种骨特异性转录因子)启动子相关的 FXR 反应元件中的类视黄醇 X 受体异二聚化。体内 FXR 的缺失 会导致严重的骨质流失。27在这项研究中,我们观察到 SPN 在 D-gal 诱导的小鼠中增加了 TGR5 和 FXR、骨形成生物标志物 Runx2 、 OCN 、 BMP2 和 ALP 的水平。此外,SPN 抑制骨吸附生物标志物 TRAP 、 RANK 、 NFATc1 和 F-肌动蛋白。SPN 可能通过激活 D-gal 诱导的骨骼中的 TGR5/FXR 通路来发挥骨骼保护作用。这些结果与先前的研究一致,这些研究证明了 TGR5 和 FXR 对骨骼保护的促进作用。 本研究还通过使用 TGR 5 拮抗剂 (SBI-115) 和 FXR 拮抗剂 (DY268) 证实了 TGR5/FXR 通路在 SPN 对 D-gal 诱导的 BMSCs 和 OC 的影响中的作用。根据目前的研究结果,SPN 通过激活 TGR5/FX R 通路有效促进 BMSCs 的成骨细胞分化,抑制 RAW 264.7 细胞破骨细胞分化,并缓解细胞衰老

Conclusions
结论

In summary, we are the first to demonstrate that SPN alleviates senile osteoporosis. This effect was associated with activating the TGR5/FXR pathway, stimulating osteoblastic differentiation of BMSCs, inhibiting osteoclastic differentiation of RANKL-induced RAW 264.7 cells and attenuating cell senescence. Future work might identify the toxicity of this compound and the anti-osteoporosis effect on female. This work provides a theoretical basis that SPN might be a functional factor supplement in preventing and treating osteoporosis.
总之,我们是第一个证明 SPN 缓解老年性骨质疏松症的人。这种作用与激活 TGR5/FXR 通路、刺激 BM SCs 的成骨细胞分化、抑制 RANKL 诱导的 RAW 264.7 细胞的破骨细胞分化和减弱细胞衰老有关。 未来的工作可能会确定这种化合物的毒性和对女性的抗骨质疏松症作用。这项工作为 SPN 可能是预防和治疗骨质疏松症的功能因子补充剂提供了理论基础。

Abbreviations
缩写

alkaline phosphatase (ALP), bone mesenchymal stem cells (BMSCs), bone mineral density (BMD), bone morphogenetic protein 2 (BMP2), bone volume/total volume (BV/TV), D-galactose (D-gal), ethylenediamine tetraacetic acid (EDTA), farnesoid X receptor (FXR), gene set enrichment analysis (GSEA), gastric irrigation (ig), immunohistochemistry (IHC), micro-computed tomography (micro-CT), osteoclast (OC), osteocalcin (OCN), nuclear factor-κB ligand (RANKL), nuclear factor-κB (RANK), nuclear factor of activated T-cells (NFATc1), real-time reverse transcription polymerase chain reaction (RT-PCR), runt-related transcription factor 2 (Runx2), subcutaneous injection (sq), senescence-associated β-galactosidase (SA-β-gal), small heterodimer partner (SHP), specnuezhenide (SPN), Takeda G protein receptor 5 (TGR5), trabecular number (Tb.N), trabecular thickness (Tb.Th), Tartrate-resistant acid phosphatase (TRAP)
碱性磷酸酶 (ALP),间充质干细胞 (BMSC),骨密度 (BMD),骨形态发生蛋白 2 (BMP2),骨体积/总体积 (BV/TV),D-半乳糖 (D-gal),dt乙酸 acidEDTA)、法尼醇 X 受体 (FXR)、g烯集富集分析 (GSEA)、g胃腔冲洗 (ig)、免疫组织化学 (IHC)、显微计算机断层扫描 (micro-CT)破骨细胞 (OC)、骨钙素 (OCN)、nuclear 因子-κB配体 (RANKL)、核因子-κB (RANK)、活化 T 细胞核因子 (NFATc1)、实时逆转录聚合酶链反应RT-PCR)、Runt 相关转录因子 2 (Runx2)皮下注射sq)、衰老相关 β-半乳糖苷酶 (SA-β-gal)、小异二聚体伴侣 (SHP)、特殊异二聚 (SPN)、田 G 蛋白受体 5 (TGR5)、小梁数 (Tb.N)、小梁厚度 (Tb.Th)、抗酒石酸盐酸性磷酸酶 (TRAP)

Funding
资金

This work was supported by Zhejiang Provincial Natural Science Foundation of China (LY21H280001, 2021C03047), National Natural Science Foundation of China (81973447), China Postdoctoral Science Foundation (2020M681364), Zhejiang Provincial Medicine Foundation (2021ZQ018, 2022ZX002, 2021ZYY28, GZY-ZJ-KJ-23006, 2024ZR001), and Zhejiang Province Chai Xiujuan Famous old TCM expert inheritance studio construction project.
这项工作得到了浙江省级自然科学基金(LY21H280001,2021C03047)、国家自然科学基金(81973447)、中国博士后科学基金(2020M681364)、浙江省医药基金(2021ZQ018、2022ZX002、2021ZYY28、GZY-ZJ-KJ-23006、2024ZR001)和浙江省柴秀娟的支持。著名中医老专家传承工作室建设项目。

Disclosure
披露

The authors report no conflicts of interest in this work.
作者报告称,这项工作没有利益冲突。

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References
R引用

1. Qadir A, Liang S, Wu Z, et al. Senile osteoporosis: the involvement of differentiation and senescence of bone marrow stromal cells [J]. Int J Mol Sci, 2020, 21: 349.
1.QadirA、Liang S、Wu Z 人。老年o骨质细胞增多症: the involvement of differentiation and senescence of bone marrow stromal cells [J]。 国际分子科学杂志202021: 349.

2. Guo Y, Jia X, Cui Y, et al. Sirt3-mediated mitophagy regulates AGEs-induced BMSCs senescence and senile osteoporosis [J]. Redox Biol, 2021, 41: 101915.
2.Guo Y, JiaX, Cui Yet al.Sirt3 介导的线粒体自噬调控 AGEs 诱导的 BMSCs 衰老和老年性骨质疏松症 [J]. 氧化还原生物学2021, 41: 101915.

3. Hu M, Xing L, Zhang L, et al. NAP1L2 drives mesenchymal stem cell senescence and suppresses osteogenic differentiation [J]. Aging cell, 2022, 21: e13551.
3. 胡 M, 邢 L, 张 Let al.NAP1L2 驱动间充质干细胞衰老并抑制成骨分化 [J]。 老化细胞2022,21:e13551

4. Farr JN, Xu M, Weivoda MM, et al. Targeting cellular senescence prevents age-related bone loss in mice [J]. Nat Med, 2017, 23: 1072-1079.
4.Farr JN、XuMWeivodaMM 人。 靶向细胞衰老可防止小鼠与年龄相关的骨质流失 [J]。 自然医学201723: 1072-1079.

5. Li L, Chen B, Zhu R, et al. Fructus Ligustri Lucidi preserves bone quality through the regulation of gut microbiota diversity, oxidative stress, TMAO and Sirt6 levels in aging mice [J]. Aging, 2019, 11: 9348-9368.
5. Li L, ChenB, ZhuRet al. FructusLigustriLucidi 通过调节衰老小鼠肠道菌群多样性、氧化应激、TMAO 和 Sirt6 水平来保持骨骼质量 [J]。 老龄化2019, 11: 9348-9368.

6. Boufker HI, Lagneaux L, Fayyad-Kazan H, et al. Role of farnesoid X receptor (FXR) in the process of differentiation of bone marrow stromal cells into osteoblasts [J]. Bone, 2011, 49: 1219-1231.
6.Boufker HI、LagneauxL、Fayyad-KazanH 人。法尼醇 X 受体 (FXR) 在骨髓基质细胞分化为成骨细胞过程中的作用 [J]. 骨骼2011, 49: 1219-1231.

7. Cho SW, An JH, Park H, et al. Positive regulation of osteogenesis by bile acid through FXR [J]. J Bone Miner Res, 2013, 28: 2109-2121.
7. Cho SW, AnJH, Park H,et al. 通过 FXR 胆汁酸正向调节成骨 [J]。 骨矿工研究杂志2013, 28: 2109-2121.

8. Wu J, Ke X, Fu W, et al. Inhibition of hypoxia-induced retinal angiogenesis by specnuezhenide, an effective constituent of Ligustrum Lucidum Ait., through suppression of the HIF-1α/VEGF signaling pathway [J]. Molecules, 2016, 21: 1756.
8. Wu JKeX, FuWet al.通过抑制 HIF-1α/VEGF p athway 诱导 r etinal angiogenesis Specnuezhenide一种 effective constituent of LigustrumLucidumAit.)诱导的 hypoxia-i 的发生 [J]。分子,2016, 21:1756.

9. Chen B, Wang L, Li L, et al. Fructus Ligustri Lucidi in osteoporosis: a review of its pharmacology, phytochemistry, pharmacokinetics and safety [J]. Molecules, 2017, 22: 1469.
9. Chen B, Wang L, LiLet al. 毛孔菌LigustriLucidi骨质疏松症中: pharmacology、phytochemistrypharmacokinetics 和 safety 的视图 [J] 分子,2017, 22: 1469.

10. Hu D, Huang S, Ding Y, et al. Specnuezhenide reduces carbon tetrachloride-induced liver injury in mice through inhibition of oxidative stress and hepatocyte apoptosis [J]. J Pharm Pharmacol, 2022, 74: 191-199.
10. 胡 D, 黄S, 丁Y.Specnuezhenide 通过抑制氧化应激和肝细胞凋亡来减少四氯化碳诱导的小鼠肝损伤 [J]。 理学杂志2022, 74: 191-199.

11. Li Z, Huang J, Wang F, et al. Dual targeting of bile acid receptor-1 (TGR5) and farnesoid X receptor (FXR) prevents estrogen-dependent bone loss in mice [J]. J Bone Miner Res, 2019, 34:765-776.
11. Li Z, HuangJ, WangFet al. bile acid receptor-1 (TGR5) 和 farnesoid X receptor (FXR) prevents estrogen-d ependent bone loss in mice双重t排列 [J]。 骨矿工研究杂志2019, 34:765-776.

12. Ye X, Jiang J, Yang J, et al. Specnuezhenide suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis [J]. Acta Biochim Biophys Sin, 2022, 54: 1080-1089.
12. 叶 X, 江J, 杨J.Specnuezhenide 通过抑制 RANKL 诱导的破骨细胞生成来抑制糖尿病诱导的骨质流失 [J]。 生物物理学杂志,2022, 54: 1080-1089.

13. Dell RB, Holleran S, Ramakrishnan R. Sample size determination [J]. ILAR J, 2002, 43: 207-213.
13. 戴尔 RB、霍勒兰S、拉马克里希南R.样本量测定 [J]。 ILAR J,2002,43:207-213

14. Ma C, Zhou X, Xu K, et al. Specnuezhenide decreases interleukin-1β-induced inflammation in rat chondrocytes and reduces joint destruction in osteoarthritic rats [J]. Front Pharmacol, 2018, 9: 700.
14. 马 C, 周XKet al.Specnuezhenide decreases interleukin-1β-i 诱导 inflammation in rat chondrocytes 和 reduces joint destruction in osteoarthritic rats [J]. 药理学,2018, 9: 700.

15. Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human [J]. J Basic Clin Pharm, 2016, 7: 27-31.
15. Nair ABJacobS. 动物和人类剂量转换的简单实践指南 [J]。 基本 Clin 药学杂志,2016, 7: 27-31.

16. Chin KY, Abdul-Majeed S., Mohamed N et al. The effects of tocotrienol and lovastatin co-supplementation on bone dynamic histomorphometry and bone morphogenetic protein-2 expression in rats with estrogen deficiency [J]. Nutrients, 2017, 9: 143.
16. Chin KY、Abdul-Majeed S.、MohamedN 等人 tocotrienollovastatin co-s 增强对 bone dynamic h异形测量法bone m产卵蛋白-2 expression 在 rats 中的影响estrogen defficiency [J]. 营养物质,2017, 9: 143.

17. Rifai OA, Chow J, Lacombe J, et al. Proprotein convertase furin regulates osteocalcin and bone endocrine function [J]. J Clin Invest, 2017, 127: 4104-4117.
17.Rifai OA、ChowJ、LacombeJ 等人前蛋白转化酶furin 调节骨钙素和骨内分泌功能 [J]. 临床 投资杂志,2017, 127: 4104-4117.

18. Zhang J, Zhang W, Dai J, et al. Overexpression of Dlx2 enhances osteogenic differentiation of BMSCs and MC3T3-E1 cells via direct upregulation of osteocalcin and ALP [J]. Int J Oral Sci, 2019, 11: 12.
18. Zhang J, ZhangW, DaiJet al.Dlx2 过表达通过直接上调 osteocalcinALP 增强 BMSCs 和 MC3T3-E1 细胞的成骨分化 [J]。国际口腔科学杂志2019, 11: 12.

19. Jeong JW, Ji SY, Lee H, et al. Fermented sea tangle (Laminaria japonica Aresch) suppresses RANKL-induced osteoclastogenesis by scavenging ROS in RAW 264.7 cells [J]. Foods, 2019, 8: 290.
19.Jeong JW、JiSY、LeeH 等人。发酵的 sea tangle (Laminaria japonicaAreschs 通过在 RAW 264.7 cells 中去除 ROS 来抑制 RANKL-i 诱导的 o脂肪碎屑生成 [J]食品2019, 8: 290.

20. Wang C, Gao H, Cai E, et al. Protective effects of Acanthopanax senticosus - Ligustrum lucidum combination on bone marrow suppression induced by chemotherapy in mice [J]. Biomed Pharmacother, 2019, 109: 2062-2069.
20. Wang C, GaoHCaiE等.刺五加-灵贞组合对小鼠化疗诱导的骨髓抑制保护作用 [J]. 生物医学药剂学2019, 109: 2062-2069.

21. Xu P, Lin B, Deng X, et al. VDR activation attenuates osteoblastic ferroptosis and senescence by stimulating the Nrf2/GPX4 pathway in age-related osteoporosis [J]. Free Radic Biol Med, 2022, 193: 720-735.
21.平, 林B, 邓Xet al.VDR 激活 通过刺激年龄相关性骨质疏松症中的 Nrf2/GPX4 通路来减轻成骨细胞铁死亡和衰老 [J]。 Free RadicBiol Med,2022, 193: 720-735.

22. El-Baz FK, Saleh DO, Abdel Jaleel GA, et al. Heamatococcus pluvialis ameliorates bone loss in experimentally-induced osteoporosis in rats via the regulation of OPG/RANKL pathway [J]. Biomed Pharmacother, 2019, 116: 109017.
22. El-Baz FK、SalehDO、Abdel JaleelGA 等人雨生血球菌通过调节 OPG/RANKL 通路改善实验诱导的大鼠骨质疏松症中的骨质流失 [J]。 生物医学药剂学2019, 116: 109017.

23. Yang Q, Zou Y, Wei X, et al. PTP1B knockdown alleviates BMSCs senescence via activating AMPK-mediated mitophagy and promotes osteogenesis in senile osteoporosis [J]. Biochim Biophys Acta Mol Basis Dis, 2023, 1869: 166795.
23. Yang Q, ZouY, WeiXet al. PTP1B 敲低通过激活 AMPK 介导的线粒体自噬减轻 BMSCs 衰老,促进 老年性骨质疏松症的成骨 [J]。 生物化学生物物理学杂志Mol Basis Dis,2023, 1869: 166795.

24. Wang Q, Wang G, Wang B, et al. Activation of TGR5 promotes osteoblastic cell differentiation and mineralization [J]. Biomed Pharmacother, 2018, 108: 1797-1803.
24. Wang Q, WangG, WangB, et al.TGR5 的激活促进成骨细胞分化和矿化 [J]。 生物医学药剂学2018, 108: 1797-1803.

25. Fujimori K, Iguchi Y, Yamashita Y, et al. Synthesis of novel farnesoid X receptor agonists and validation of their efficacy in activating differentiation of mouse bone marrow-derived mesenchymal stem cells into osteoblasts [J]. Molecules, 2019, 24: 4155.
25. Fujimori K、IguchiY、YamashitaY 等人。n ovel farnesoid X receptor agonists 和 validation of their efficacy in activating differentiation of mouse bone marrow-d erived mesenchymalStem cells into osteoblasts [J]. 分子,2019, 24: 4155.

26. Zhang Y, Wei J, Feng X, et al. Folic acid supplementation prevents high body fat-induced bone loss through TGR5 signaling pathways [J]. Food Funct, 2024, 15: 4193-4206.
26. 张 Y, 魏J, 冯X .补充叶酸通过 TGR5 信号通路防止高体脂肪诱导的骨流失 [J]。 食品功能2024, 15: 4193-4206.

27. Liu M, Jin F, Zhang S, et al. Activation of farnesoid X receptor signaling by geniposidic acid promotes osteogenesis [J]. Phytomedicine, 2022, 103: 154258.
27. Liu M, JinF, Zhang S,et al.geniposidic acid 激活法尼醇 X 受体信号传导促进成骨 [J]。 植物医学2022, 103: 154258.

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Figure Captions
Figure 字幕

Figure 1 SPN improved bone microarchitecture in D-gal-induced mice. (A) SPN structure. (B) Experimental diagram. (C) Micro-CT images of trabecular bone at the femur and trabecular microarchitecture parameters (n=5). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 compared with the MOD group.
图 1SPN 改善了 D-gal 诱导小鼠的骨骼微结构。 (A) SPN 结构 (B) E实验图。 (C) 股骨小梁和小梁微结构参数 (n=5Micro-CT 图像。数据表示为 SD ±平均值。*P < 0.05,**P < 0.01MOD 组相比。

Figure 2 SPN improved bone formation in D-gal-induced mice. (A) IHC analysis of OCN in the femur (black arrows, n=5). (B) Western blotting analysis of ALP, BMP2 and Runx2 in tibias (n=3). Data are expressed as the mean ± SD. **P < 0.01 compared with the MOD group.
2 SPN 改善了 D gal 诱导的小鼠的形成(A)I 股骨 OCNHC 分析 (黑色箭头n=5)。(B) 胫骨 (n=3)ALP 、 BMP2 和 Runx2 Western blotting 分析数据表示为 SD ±平均值。**P < 0.01 与 MOD 组相比。

Figure 3 SPN suppressed bone resorption in D-gal-induced mice. (A) Representative TRAP-stained histological sections of femurs from D-gal-induced mice. (B) The number of osteoclasts/The area of tissue (N.OC/T.Area) was analyzed. (C) Western blotting analysis of RANK and NFATc1 in tibias (n=3). Data are expressed as the mean ± SD. **P < 0.01 compared with the MOD group.
Figure 3SPN 抑制 D-gal 诱导小鼠的骨吸收。(A) 来自 D-gal 诱导的小鼠股骨的代表性 TRAP 染色组织学切片。(B) 分析破骨细胞数量/组织面积 (N.OC/T.Area)。(C)胫骨 RANK 和 NFATc1 的蛋白质印迹分析 (n=3)。 数据表示为 SD ±平均值。**P < 0.01 与 MOD 组相比。

Figure 4 SPN attenuated senescence in D-gal-induced mice. Immunohistochemistry of P16, P21, and P53, scale bar = 50 μm (n = 5). Data are expressed as the mean ± SD. **P < 0.01 compared with the MOD group.
Figure 4 SPN 减弱了 D-gal 诱导小鼠的衰老。P16、P21 和 P53 的免疫组织化学,比例尺 = 50 μm (n = 5)。数据表示为 SD ±平均值。**P < 0.01 与 MOD 组相比。

Figure 5 SPN inhibited the SA-β-gal-stained positive cells. (A) SA-β-gal staining of BMSCs. (B) SA-β-gal staining of OC. **P < 0.01 compared with D-gal group. ## P < 0.01 compared with D-gal + SPN group. $$ P < 0.01 compared with D-gal + SPN + SBI-115 + DY268 group.
Figure 5 SPN 抑制 SA-β-gal 染色的阳性细胞。(一)BMSC 的 SA-β-gal 染色。 (B) OC 的 SA-β-gal 染色。**P < 0.01 与 D-gal 组相比。## P < 0.01 与 D-gal + SPN 组相比。$$ P < 0.01 与 D-gal + SPN + SBI-115 + DY268 组相比。

Figure 6 SPN promoted of ALP expression and inhibited of F-actin formation. (A) ALP staining. (B) F-actin staining.
图 6 SPN 促进 ALP 表达并抑制 F-肌动蛋白形成。(A) ALP 染色。(B) F-肌动蛋白染色。

Figure 7 SPN increased TGR5 and FXR expression in D-gal-induced BMSCs and OC. (A) Changed genes in D-gal + SPN group compared with D-gal group displayed in volcano plot assay. (B) GSEA showing the bile acid biosynthetic process in D-gal + SPN group compared with D-gal group. (C) Changed bile acid biosynthetic process-related genes in D-gal + SPN group compared with D-gal group displayed in heatmap assay. Immunofluorescence analysis of TGR5 and FXR in (D) BMSCs and (E) OC (n=6). Scale bar, 10 μm. Data are expressed as the mean ± SD. **P < 0.01 compared with D-gal group.