Praeruptorin B inhibits osteoclastogenesis by targeting GSTP1 and impacting on the S-glutathionylation of IKK
Praeruptorin B 通过靶向 GSTP1 和影响 IKK 的 S-谷胱甘肽化来抑制破骨细胞生成 .

Praeruptorin B 普拉鲁托林 B

GSTP1

Nuclear factor  核因子

Receptor activator of nuclear factor ligand
核因子 配体的受体激活剂

Osteoclastogenesis 破骨细胞生成

Osteoporosis 骨质疏松症

In a normal body, bone undergoes continuous remodeling maintained by a balance between the functions of osteoclasts (OCs) and osteoblasts. Enhanced OC differentiation, together with the functional limitation of osteoblasts, can cause a myriad of osteolytic bone diseases, including osteoporosis [1], rheumatoid arthritis[2], osteoarthritis [3] and periodontal disease [4]. In recent years, with the aging of the population and the extension of life expectancy, the incidence of osteoporosis has continually increased [5]. Osteoporotic fracture, a common complication of osteoporosis, produces significant morbidity and mortality [6]. Moreover, long-term bed rest can lead to further loss of bone mass, thereby forming a vicious cycle [7]. This accounts for a substantial disease burden and costs for society. Although many medications to treat osteoporosis are available, none of these existing drugs are free of side effects when used over the long term. Therefore, it is necessary to develop new drugs with a low cost; low risk; and limited, minor, adverse effects to treat osteoporosis.
在正常人体中，骨骼在破骨细胞（OC）和成骨细胞功能的平衡下不断重塑。破骨细胞分化增强，加上成骨细胞功能受限，可导致多种溶骨性骨病，包括骨质疏松症[1]、类风湿性关节炎[2]、骨关节炎[3]和牙周病[4]。近年来，随着人口老龄化和预期寿命的延长，骨质疏松症的发病率持续上升[5]。骨质疏松性骨折是骨质疏松症的常见并发症，会导致严重的发病率和死亡率[6]。此外，长期卧床会导致骨量进一步流失，从而形成恶性循环 [7]。这造成了巨大的疾病负担和社会成本。虽然目前有许多治疗骨质疏松症的药物，但长期使用这些药物都不会产生副作用。因此，有必要开发成本低、风险小、不良反应有限且轻微的新药来治疗骨质疏松症。

OCs are derived from monocyte-macrophage hematopoietic lineage cells in response to stimulation with RANKL and M-CSF. Activation of RANK by RANKL induces downstream signaling pathways, including the pathway, which also plays a vital role in osteoclastic differentiation . During NF- signaling, RANKL recruits its adaptor, TRAF6, and regulates TRAF6 polyubiquitination, subsequently promoting the phosphorylation of IKK, inducing the phosphorylation and degradation of IкB, and finally activating NF-кB to translocate into the nucleus, where it initiates OC-related gene transcription. Previous studies have shown that NF- is modified through S-glutathionylation, which prevents the degradation of IкB and subsequent DNA binding of RelA/p65 dimers . Thus, the regulation of S-glutathionylation is tightly linked with NF-kB signaling.
OC是单核-巨噬细胞造血系细胞在RANKL和M-CSF刺激下产生的。RANKL激活RANK诱导下游信号通路，包括 通路，该通路在破骨细胞分化 中也起着至关重要的作用。在NF- 信号传导过程中，RANKL会招募其适配体TRAF6，并调节TRAF6的多泛素化，随后促进IKK的磷酸化，诱导IкB的磷酸化和降解，最后激活NF-кB转位到细胞核中，启动OC相关基因的转录。以前的研究表明，NF- 通过 S-谷胱甘肽化被修饰，从而阻止 IкB 的降解和随后 RelA/p65 二聚体 的 DNA 结合。因此，S-谷氨酰化的调节与 NF-kB 信号转导密切相关。

Glutathione S-transferase Pi 1 (GSTP1), a ubiquitously expressed
谷胱甘肽 S 转移酶 Pi 1 (GSTP1)是一种普遍表达的

Received 22 April 2022; Received in revised form 26 July 2022; Accepted 8 August 2022
2022 年 4 月 22 日收到；2022 年 7 月 26 日收到修订稿；2022 年 8 月 8 日接受

Available online 26 August 2022
2022 年 8 月 26 日在线提供

0753-3322/C 2022 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
0753-3322/C 2022 作者。由 Elsevier Masson SAS 出版。本文为 CC BY-NC-ND 许可下的开放存取文章 ( http://creativecommons.org/licenses/by-nc-nd/4.0/)。
protein, is an accepted catalyst of protein S-glutathionylation reactions following oxidative stress [12]. GSTP1 responds to various stimuli, such as hypoxia, and can negatively regulate downstream signaling pathways [13]. GSTP1 has been shown to play an important role in modulating that involves the S-glutathionylation of IKK proteins and interaction with NF-kB family members. The loss of GSTP1 induces oxidative stress in cell and affects NF-кB signal transduction. Additionally, GSTP1 associates with TRAF2, which is a regulator of NF-кB. Therefore, GSTP1 is a crucial regulatory factor of NF-кB [14-16]. Additionally, several studies have demonstrated that GSTP1/NF-кB signaling is correlated with proinflammatory cytokine production, which indicates the effect of GSTP1 and NF-kB on inflammatory pathways [17,18]. Although S-glutathionylation is key to the regulation of , the role of the interaction of GSTP1 with NF-кB in OCs remains to be further studied. Here, we emphasize the roles of GSTP1 and S-glutathionylation in RANKL-induced osteoclastogenesis.
蛋白，是氧化应激后蛋白质 S-谷胱甘肽化反应的公认催化剂 [12]。GSTP1 可对缺氧等各种刺激做出反应，并可对下游信号通路进行负向调节 [13]。研究表明，GSTP1 在调节 中发挥着重要作用，其中涉及 IKK 蛋白的 S-谷氨酰化以及与 NF-kB 家族成员的相互作用。GSTP1 的缺失会诱发细胞 中的氧化应激，并影响 NF-кB 信号转导。此外，GSTP1 与 TRAF2 相关联，而 TRAF2 是 NF-кB 的调节因子。因此，GSTP1 是 NF-кB 的一个重要调节因子 [14-16]。此外，多项研究表明，GSTP1/NF-кB 信号传导与促炎细胞因子的产生相关，这表明 GSTP1 和 NF-kB 对炎症通路有影响 [17,18]。虽然S-谷胱甘肽化是调控 的关键，但GSTP1与NF-кB在OCs中的相互作用仍有待进一步研究。在这里，我们强调了GSTP1和S-谷氨酰化在RANKL诱导的破骨细胞生成中的作用。

Natural products and their derivatives are indispensable to modern medicine and serve as important sources for anti-inflammatory and antimalarial drug discovery and development [19,20]. Praeruptorin B (Pra-B), a seselin-type coumarin, is one of the main components found in the roots of Peucedanum praeruptorum Dunn and exhibits obvious biological activity, acting mainly as an anti-inflammatory and antitumor agent. Previous studies have demonstrated that Pra-B is also linked to signaling as an inhibitor of the nuclear transportation of NF-кB [21]. However, the role of Pra-B in OC formation and function remains unclear. Hence, we aimed to explore the effects of Pra-B on RANKL-induced osteoclastogenesis and dissect the underlying molecular mechanisms.
天然产物及其衍生物是现代医学中不可或缺的物质，也是抗炎和抗疟药物发现和开发的重要来源[19,20]。Praeruptorin B（Pra-B）是一种芝麻甙类香豆素，是Peucedanum praeruptorum Dunn根部的主要成分之一，具有明显的生物活性，主要用作抗炎剂和抗肿瘤剂。先前的研究表明，Pra-B 作为 NF-кB 核运输的抑制剂，也与 信号转导有关 [21]。然而，Pra-B 在 OC 形成和功能中的作用仍不清楚。因此，我们旨在探索Pra-B对RANKL诱导的破骨细胞生成的影响，并剖析其潜在的分子机制。

Alpha modification of Eagle's minimal essential medium ( -MEM), penicillin, and FBS were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA). Pra-B was obtained from Chengdu Herb Purify Co., Ltd. in Chengdu, and a stock solution of Pra-B at in dimethyl sulfoxide (DMSO) was prepared. Then, the Pra-B solution was diluted in -MEM for cell culture [22]. The M-CSF and GST-rRANKL applied in the experiments were obtained from R&D Systems (Minneapolis, MN, USA). Cell Counting Kit-8 (CCK-8) was purchased from Engreen Biosystems (Beijing, China). Oligo-dT primers were acquired from Imgenex (Littleton, CO, USA). TRIzol and rhodamine-conjugated phalloidin were purchased from Thermo Fisher Scientific in San Jose. A TRAcP staining kit was acquired from Sigma-Aldrich in Sydney. Small interfering RNA (siRNA) for RNA interference was obtained from GenePharma in Shanghai. Specific antibodies against CTSK (sc-48353, 1:500), histone H3 (sc-517576, 1:1000), NFATc1 (sc-7294, 1:100) and -actin (sc-47778, 1:2000) were acquired from Santa Cruz Biotechnology in San Jose. Specific antibodies against integrin (#4702, 1:1000), IKK (#2370, 1:1000), GSTP1 (#3369, 1:1000), p65 (#8242, 1:1000), p-p65 (#3033, 1:1000), and IкB (#4814, 1:1000) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-GSH antibody (#101-A, 1:1000) was purchased from (Virogen, Watertown, MA).
阿尔法改良老鹰最低限度基本培养基（ -MEM）、青霉素和 FBS 均来自 Thermo Fisher Scientific (Carlsbad, CA, USA)。Pra-B 从成都草本净化有限公司获得，并在二甲基亚砜（DMSO）中制备了 的Pra-B储备溶液。然后将Pra-B溶液稀释在 -MEM中进行细胞培养[22]。实验中使用的 M-CSF 和 GST-rRANKL 均来自 R&D Systems 公司（Minneapolis, MN, USA）。细胞计数试剂盒-8（CCK-8）购自英格林生物系统公司（中国北京）。Oligo-dT 引物购自 Imgenex 公司（美国科罗拉多州利特尔顿）。TRIzol 和罗丹明连接的类磷脂酰蛋白购自圣何塞的 Thermo Fisher Scientific 公司。TRAcP 染色试剂盒购自悉尼的 Sigma-Aldrich。用于干扰 RNA 的小干扰 RNA（siRNA）购自上海的 GenePharma 公司。针对CTSK（sc-48353，1:500）、组蛋白H3（sc-517576，1:1000）、NFATc1（sc-7294，1:100）和 -actin（sc-47778，1:2000）的特异性抗体购自圣何塞的圣克鲁斯生物技术公司。针对整合素 (#4702, 1:1000), IKK (#2370, 1:1000), GSTP1 (#3369, 1:1000), p65 (#8242, 1:1000), p-p65 (#3033, 1:1000) 和 IкB (#4814, 1:1000) 的特异性抗体购自 Cell Signaling Technology (Beverly, MA, USA)。抗 GSH 抗体 (#101-A, 1:1000) 购自 (Virogen, Watertown, MA)。

Bone marrow-derived macrophages (BMMs) were derived from the femur and tibia of 6-week-old C57BL/6 mice. The bone marrow cells in the medullary cavity of the femurs and tibia were isolated, filtered and centrifuged. Then, the cells were transferred to complete -MEM medium containing and M-CSF, and cultured in a humidified incubator with at . The solution was changed every other day. Cell passage was carried out after the cell contact rate reached . The cells of the 1 st to 3rd generation were used for the experiments. All procedures were approved by Wenzhou
骨髓衍生巨噬细胞（BMMs）取自 6 周大的 C57BL/6 小鼠的股骨和胫骨。分离、过滤和离心股骨和胫骨髓腔中的骨髓细胞。然后，将细胞转移到含有 和 M-CSF的完全 -MEM培养基中，在 的 加湿培养箱中培养。培养液每隔一天更换一次。细胞接触率达到 后进行细胞传代。实验使用第 1 代至第 3 代细胞。所有实验程序均经温州
Medical University. Then, the BMMs were cultured with -MEM containing and .
医科大学。然后，用含有 和 的 -MEM培养BMM。

To test the cytotoxicity of Pra-B, BMMs were first seeded in 96-well plates at a concentration of cells/well. Next, the BMMs were incubated in complete -MEM containing M-CSF ( ) for . The cells were incubated with various concentrations of Pra-B for another or , followed by incubating with of CCK-8 solution for . Finally, OD450 was detected using a microplate reader (Multiskan Spectrum; Thermo LabSystems, Chantilly, VA, USA) [23].
为了测试 Pra-B 的细胞毒性，首先在 96 孔板中以 细胞/孔的浓度接种 BMM。然后，将 BMM 在含有 M-CSF 的完全 -MEM （ ）中培养 。细胞再与不同浓度的 Pra-B 培养 或 ，然后与 的 CCK-8 溶液培养 。最后，使用微孔板阅读器（Multiskan Spectrum; Thermo LabSystems, Chantilly, VA, USA）检测 OD450 [23]。

Tartrate-resistant acid phosphatase (TRAcP), an enzyme whose biological function still remains unknown, is mainly expressed in activated macrophages and OCs with bone resorption functions. Mice lacking TRACP developed mild osteopetrosis, while those with over-expressed TRACP developed osteoporosis, suggesting that the enzyme plays a crucial part in bone resorption [24,25]. BMMs were inoculated into 96-well plates at cells/well overnight and then incubated with GST-rRANKL (50 ng/mL) under Pra-B stimulation. Complete medium containing fresh GST-rRANKL and Pra-B was exchanged every 2 days until day 6 . Then, the cells were rinsed with phosphate-buffered saline (PBS). Then, for TRAcP staining, glutaraldehyde was used to fix with the cells for . To count down the cells, all TRAcP-positive multinucleated cells ( nuclei) were considered OCs.
抗酒石酸磷酸酶（TRAcP）是一种生物功能尚不清楚的酶，主要表达于活化的巨噬细胞和具有骨吸收功能的 OC。缺乏 TRACP 的小鼠会出现轻度骨质软化症，而过度表达 TRACP 的小鼠则会出现骨质疏松症，这表明该酶在骨吸收中起着至关重要的作用 [24,25]。以 细胞/孔的数量将 BMMs 接种到 96 孔板中过夜，然后在 Pra-B 刺激下与 GST-rRANKL（50 ng/mL）孵育。每两天更换一次含有新鲜 GST-rRANKL 和 Pra-B 的完全培养基，直到第 6 天。然后，用磷酸盐缓冲盐水（PBS）冲洗细胞。然后，用 戊二醛固定细胞 ，进行TRAcP染色。计数细胞时，将所有 TRAcP 阳性的多核细胞（ 核）视为 OC。

The demineralization function of OCs was determined by the hydroxyapatite absorption method. Osteoclastic BMMs were inoculated in collagen-coated 6-well plates at a density of cells/well, with an exchange of culture medium containing fresh GST-rRANKL and M-CSF ( ) every 2 days. Then, at the given time points, the cells were extracted and transferred to a hydroxyapatite-coated 96-well plate or bone slice (CLS3989, Corning, NY, USA) at a density of cells per well. Then, complete -MEM containing fresh M-CSF ( ) and GST-rRANKL ( ) was used to incubate the mature OCs for 2 days in the presence or absence of Pra-B. Finally, the cells were bleached for and then removed from the wells to measure the bone-resorption area. Images of the absorption areas were captured by microscopy, and the OC-resorption region was analyzed by ImageJ software.
用羟基磷灰石吸收法测定 OC 的脱矿化功能。以 细胞/孔的密度将破骨细胞接种到涂有胶原蛋白的6孔板中，每两天更换一次含有新鲜GST-rRANKL 和M-CSF（ ）的培养基。然后，在给定的时间点提取细胞，并以每孔 细胞的密度转移到涂有羟基磷灰石的96孔板或骨片（CLS3989，Corning，NY，USA）上。然后，用含有新鲜 M-CSF （ ）和 GST-rRANKL （ ）的完全 -MEM 培养成熟的 OC，在有或没有 Pra-B 的情况下培养 2 天。最后，将细胞漂白 ，然后从孔中取出，测量骨吸收面积。用显微镜捕捉吸收区域的图像，并用 ImageJ 软件分析 OC 吸收区域。

BMMs were cultured in a 6 -well plate cells/well and incubated with GST-rRANKL (50 ng ) and M-CSF ( with or without Pra-B at different concentrations for 5 days. Next, TRIzol was applied to extract total RNA. Single-stranded cDNA was reverse transcribed from of total RNA using oligo-DT primers. The obtained cDNA was amplified by real-time PCR using specific primers and SYBR Green (Imgenex, Littleton, CO, USA). Target gene expression was normalized to expression of the housekeeping gene Hprt. The fold change and ratio compared with data from the control group were calculated by the Livak equation. The primers used are listed in Table 1.
在6孔板 细胞/孔 中培养BMM，并与不同浓度的GST-rRANKL（50纳克 ）和M-CSF（ 与或不与Pra-B一起培养5天。然后，用 TRIzol 提取总 RNA。使用寡聚-DT引物从总 RNA 的 中反向转录单链 cDNA。使用特异性引物和 SYBR Green（Imgenex，Littleton，CO，USA）对获得的 cDNA 进行实时 PCR 扩增。目标基因的表达与看家基因 Hprt 的表达进行归一化。用 Livak 方程计算与对照组数据相比的折叠变化和比率。所用引物见表 1。

Table 1 表 1

Primer sequences used for qRT-PCR.
用于 qRT-PCR 的引物序列。

To determine the effect of Pra-B on osteoclastogenesis-associated markers, BMMs were transferred to 6 -well plates cells/well). Then the cells were treated with or without Pra-B for 5 days after RANKL stimulation. After and 5 days, the cells were lysed with radioimmunoprecipitation (RIPA) lysis buffer DNase I, phosphatase inhibitor and PMSF) to extract the proteins. To detect changes in the signaling pathway over the short term, BMMs were inoculated in 6-well plates cells/well) and cultured overnight in complete medium with M-CSF. After of starvation, the cells which stimulated with RANKL were pretreated with PraB for . Proteins were obtained with RIPA lysis buffer at different timepoints including 0, 10, 20, 30 and , and RIPA lysis buffer was used to obtain the proteins and SDS-PAGE was used to separate proteins. Then, the proteins were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Five percent skim milk was used to block the membranes for , after which the membranes were incubated overnight with primary antibodies at . After Tris-buffered saline/ Tween (TBST) was used to wash the membranes 3 times for , the membranes were incubated with a specific secondary antibody bound to horseradish peroxidase (HRP) for . Following the manufacturer's instructions, the membranes were treated with enhanced chemiluminescence reagents (Amersham, Piscataway, NJ, USA), and images were acquired via ImageQuant LAS 4000 (GE Healthcare, Sydney, Australia).
为了确定Pra-B对破骨细胞生成相关标志物的影响，将BMM转移到6孔板 细胞/孔）。然后在 RANKL 刺激后用或不用 Pra-B 处理细胞 5 天。 和5天后，用放射免疫沉淀（RIPA）裂解缓冲液 DNase I、磷酸酶抑制剂和 PMSF）裂解细胞，提取蛋白质。为了检测信号通路在短期内的变化，将 BMMs 接种到 6 孔板（ 细胞/孔）中，在含有 M-CSF 的完全培养基中培养过夜。饥饿 后，用 PraB 预处理 用 RANKL 刺激的细胞。在不同的时间点（包括 0、10、20、30 和 ），用 RIPA 裂解缓冲液获得蛋白质，并用 SDS-PAGE 分离蛋白质。然后，将蛋白质转移到 PVDF 膜（Bio-Rad，Hercules，CA，USA）上。用百分之五的脱脂牛奶封闭膜 ，然后用一抗在 下孵育过夜。用三相缓冲盐水/吐温（TBST）洗膜 3 次 后，用与辣根过氧化物酶（HRP）结合的特异性二抗孵育膜 。按照制造商的说明，用增强化学发光试剂（Amersham，Piscataway，NJ，USA）处理膜，并通过 ImageQuant LAS 4000（GE Healthcare，Sydney，Australia）获取图像。

BMMs at cells/well in 6-well plates were cultured and cotransfected with of pGL6-NF-кB-Luc plasmid by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, United States) based on the manufacturer's instructions, and then the cultured overnight. After BMMs being pretreated with Pra-B in a series of concentrations including 1, 2.5, 5 and for another an hour, cells were stimulated with RANKL ) for , and then harvested for analyzing the luciferase activity normalizd to the internal control activity.
用 Lipofectamine 3000（Invitrogen，Carlsbad，CA，United States）按照生产商的说明，以 细胞/孔的数量在 6 孔板中培养 BMM，并共转染 pGL6-NF-кB-Luc 质粒 ，然后培养过夜。用 1、2.5、5 和 等一系列浓度的 Pra-B 预处理 BMM 一小时后，用 RANKL ）刺激细胞 ，然后收获细胞，分析荧光素酶活性与内部对照活性的归一化。

After being washed with PBS, the BMMs were blocked with paraformaldehyde for and then sealed with 3% BSA for another . Next, rhodamine-conjugated phalloidin probe (Sigma Aldrich, Lyon, France) was used to stain F-actin for . Primary and secondary anti-p65 antibodies (1:500) (Sigma Aldrich, Lyon, France) conjugated with Alexa Fluor-488 (Life Technologies, Saint Aubin, France) were added to each well and incubated for before the cell nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Santa Cruz Biotechnology, USA) for . The results were visualized by a confocal fluorescence microscope (Nikon, A1 PLUS, Tokyo, Japan).
用 PBS 冲洗 BMM 后，用 多聚甲醛阻断 ，然后用 3% BSA 封闭 。然后，用罗丹明连接的类磷脂酰蛋白探针（Sigma Aldrich，法国里昂）对 F-actin 进行染色， 。在细胞核用 4',6-二脒基-2-苯基吲哚二盐酸盐（DAPI）（Santa Cruz Biotechnology, USA）染色之前，在每个孔中加入与 Alexa Fluor-488 （Life Technologies, Saint Aubin, France）共轭的抗 p65 一抗和二抗（1:500）（Sigma Aldrich, Lyon, France）并孵育 。结果由共聚焦荧光显微镜（Nikon，A1 PLUS，日本东京）观察。

BIOVIA Discovery Studio Visualizer (2016) (Waltham, MA, USA) was applied to predict potential binding between proteins and their ligands.
应用 BIOVIA Discovery Studio Visualizer（2016 年）（美国马萨诸塞州沃尔瑟姆）预测蛋白质与其配体之间的潜在结合。

We obtained protein structure data from the PBD website (https:// www.rcsb.org/), and among the available structures, a structure acquired via X-ray diffraction with a resolution under was the preferred option.
我们从 PBD 网站（https:// www.rcsb.org/）上获取了蛋白质结构数据，在现有结构中，通过 X 射线衍射获得的分辨率在 以下的结构是首选。

Then, structural data for the chemical ligands were obtained through the PubChem website (https://pubchem.ncbi.nlm.nih.gov/), and we gained the two-dimensional structure of Pra-B and downloaded its corresponding SDF file. After the protein structure was trimmed and unrelated ions, extraneous structures and elements were removed, the semiflexible LibDock operation was performed to determine the active site of the protein and analyze its binding potential with Pra-B. The nonbonding interactions and hydrogen bonds between Pra-B and related adjacent amino acid residues were analyzed.
然后，我们通过 PubChem 网站（https://pubchem.ncbi.nlm.nih.gov/）获得了化学配体的结构数据，并获得了 Pra-B 的二维结构，下载了其相应的 SDF 文件。在对蛋白质结构进行修剪并去除无关离子、无关结构和元素后，我们进行了半灵活的 LibDock 操作，以确定蛋白质的活性位点并分析其与 Pra-B 的结合潜力。分析了 Pra-B 与相关相邻氨基酸残基之间的非键相互作用和氢键。

BMMs cells/well) were seeded in 96 -well plates and cultured overnight. BMMs were transfected with siRNAs (GenePharma, Shanghai, China) under the use of the siRNA transfection reagent GPtransfection-Mate (GenePharma) based on the manufacturer's instructions.
BMM 细胞/孔）接种于 96 孔板并培养过夜。使用 siRNA 转染试剂 GPtransfection-Mate（GenePharma，中国上海），按照生产商的说明用 siRNA 转染 （GenePharma，中国上海）BMM。

First, the cells were cultured with an siRNA mixture for in -MEM containing FBS. To assess whether the transfection was successful, the expression of mRNA after 2 days and protein after 3days were detected. Further experiments were performed after the transfected BMMs were stimulated with Pra-B , RANKL and M-CSF (25 ng/mL).
首先，在含有 FBS的 -MEM中用 siRNA混合物培养 细胞。为了评估转染是否成功，2 天后检测了 mRNA 的表达，3 天后检测了蛋白质的表达。在用 Pra-B 、RANKL 和 M-CSF（25 ng/mL）刺激转染的 BMM 后，进行了进一步的实验。

BMMs ( cells/well) were placed in 96 -well plates and stimulated with RANKL for another . The mRNA transcription was blocked by actinomycin D ( ) (Sigma, St. Louis, MO, United States) for and then stimulated with Pra-B for . Finally cells were collected to extracted total RNA after and of RANKL ( ) treatment. Then, qRT-PCR was applied to analyze the transcription and remain of target mRNAs.
将 BMM（ 细胞/孔）置于 96 孔板中，用 RANKL 刺激 。用放线菌素 D （ ）（Sigma，St. Louis，MO，United States）阻断 mRNA 转录 ，然后用 Pra-B 刺激 。最后收集细胞，提取RANKL（ ）处理 和 后的总RNA。然后，应用 qRT-PCR 分析目标 mRNA 的转录和残留情况。

An immunoprecipitation experiment was performed according to previous literature. BMMs at cells/well in 6 -well plates were pretreated with peiminine for and then stimulated with RANKL for . The lysate was centrifuged at at for 20 . An anti-GSH antibody was added to the supernatant and incubated overnight at , followed by incubation with protein Sepharose beads (Invitrogen, Carlsbad, CA, USA) for another at . Western blotting was used to analyze the results.
根据以前的文献，进行了免疫沉淀实验。将 细胞/孔的BMMs放入6孔板中，用培米宁预处理 ，然后用 RANKL刺激 。裂解液在 离心20 。在上清液中加入抗GSH抗体 ，在 下孵育过夜，然后与蛋白 分离胶珠（Invitrogen, Carlsbad, CA, USA）在 下再孵育 。用 Western 印迹法分析结果。

To assess the effect of the drug on the osteoblast differentiation, MC3T3-E1 cells were cultured in -MEM and 10% FBS for 5 days. Then, MC3T3-E1 cells cells/well were transferred to osteogenic medium containing -glycerophosphate, and L-ascorbic acid 2-phosphate and treated with Pra-B at a concentration of and 10 or without it.
为了评估药物对成骨细胞分化的影响，MC3T3-E1细胞在 -MEM和10% FBS中培养了5天。然后，将MC3T3-E1细胞 /孔 转移到含有 -甘油磷酸酯和 L-抗坏血酸2-磷酸酯的成骨培养基中，并用浓度为 和10 的Pra-B处理或不处理。

To test the activity of ALP on the 7th day, the ALP staining kit (Promega, Fitchburg, WI, USA) was applied based on the manufacturer's suggested protocols and the number of positive cells was determined. To test the mineralization function of OBs on 21st day Alizarin red staining kit (Sigma-Aldrich) was applied, and the cells were incubated with paraformaldehyde for as well as Alizarin Red S solution with 1 per well for another
在第7天检测ALP的活性时，根据生产商建议的方法使用ALP染色试剂盒（Promega, Fitchburg, WI, USA），并测定阳性细胞的数量。第 21 天，应用茜素红染色试剂盒（Sigma-Aldrich）检测 OB 的矿化功能，并将细胞与 多聚甲醛培养 以及每孔 1 茜素红 S 溶液培养 。

All in vivo experiments were approved by the Institutional Animal Ethics Committee of Wenzhou Medical University (ethical approval No. wydw2019 -0247).
所有体内实验均经温州医科大学动物伦理委员会批准（伦理批准号：wydw2019 -0247）。

Twelve-week-old female C57BL/6 J mice were purchased from the Animal Center of the Chinese Academy of Science (Shanghai, China) and placed in isolated ventilated cages in a specific pathogen-free (SPF) room to adapt their new surroundings for 1 week before surgery. Then, all the mice were randomly divided into three groups: the sham group,
12周龄的雌性C57BL/6 J小鼠购自中国科学院动物研究所（中国上海），手术前将其放入无特定病原体（SPF）房间的隔离通风笼中适应新环境1周。然后，所有小鼠被随机分为三组：假组、
ovariectomized (OVX) group, and OVX + Pra-B treatment group. For the OVX and OVX + Pra-B groups, all mice received bilateral ovariectomy while those in the sham group underwent sham surgery [26]. After 7 days, mice in the OVX + Pra-B treatment group were intraperitoneally injected with Pra-B every 2 days for 6 weeks. The mice in the sham groups and OVX groups were injected with the same amount of PBS at the same time points, and Pra-B was dissolved in a saline solution with 5% DMSO for intraperitoneal injection. After the mice were euthanized, their tibias were removed and underwent microscopic computed tomography (micro-CT) imaging and histological analysis.
卵巢切除（OVX）组和 OVX + Pra-B 治疗组。卵巢切除组和卵巢切除+Pra-B治疗组的所有小鼠都接受了双侧卵巢切除术，而假手术组的小鼠则接受了假手术[26]。7天后，OVX + Pra-B治疗组的小鼠腹腔注射Pra-B ，每2天一次，连续注射6周。假组和OVX组的小鼠在相同的时间点注射等量的PBS，并将Pra-B溶解在含5% DMSO的生理盐水中进行腹腔注射。小鼠安乐死后，取出胫骨并进行显微计算机断层扫描（micro-CT）成像和组织学分析。

To assess the metabolic level of osteoclast and osteoblast, blood samples were collected, and centrifuged at 17,000 rpm for and their supernatant was stored at until use. The serum samples were stored in for before use and then, the enzyme-linked immunosorbent assay (ELISA) kits were applied to determine the Acp5, -CTX, PINP and BALP levels according to manufacturer's instructions (Cusabio, Wuhan, China).
为了评估破骨细胞和成骨细胞的新陈代谢水平，采集了血液样本，在17000转/分的转速下离心 ，上清液在 下保存至使用。血清样本在 中保存 后，根据生产商的说明（Cusabio，武汉，中国），应用酶联免疫吸附试验（ELISA）试剂盒测定Acp5、 -CTX、PINP和BALP的水平。

After the right tibias and lumbar vertebra were immobilized in paraformaldehyde for 1 day, micro-CT (SkyScan 1176; Bruker, Kontich, Belgium) was applied to analyze the tibias and the fifth lumbar vertebra [27].
将右侧胫骨和腰椎在 多聚甲醛中固定一天后，应用微型计算机断层扫描（SkyScan 1176；布鲁克公司，比利时孔蒂奇）对胫骨和第五腰椎进行分析[27]。
Then, images were obtained with a scan processing protocol from a previous study with the following settings: an isotropic pixel size of 9 ' a source current of , an aluminum filter thickness of 0.5 , and an X-ray tube voltage of . NRecon Reconstruction software was used for image reconstruction.
然后，使用先前研究的扫描处理程序获取图像，设置如下：各向同性像素大小为 9 '源电流为 ，铝滤光片厚度为 0.5 ，X 射线管电压为 。NRecon Reconstruction 软件用于图像重建。

Based on recommendations, the areas below the growth plate and in height were analyzed to estimate bone loss by determining the trabecular number (Tb.N), bone volume/tissue volume ratio (BV/TV), trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th).
根据建议，对生长板以下 区域和高度 区域进行分析，通过测定骨小梁数量（Tb.N）、骨量/组织体积比（BV/TV）、骨小梁分离度（Tb.Sp）和骨小梁厚度（Tb.Th）来估算骨量损失。

After we immobilized the tibias in 4% paraformaldehyde for 1 day, the tibias were decalcified in EDTA for 10 days. The sagittal sections of the paraffin-embedded tibias at a thickness of were stained with anti-TRAcP antibody (1:500) (Sigma Aldrich, Lyon, France) and hematoxylin and eosin (H&E). Images of the sections were scanned and processed using a uScope MXII digital microscope slide scanner (Microscopes International, Lubbock, TX, USA). The number of OCs on each bone surface (N.Oc/BS) was calculated with BIOQUANT OSTEO 2011 software.
将胫骨在 4% 多聚甲醛中固定 1 天后，在 EDTA 中脱钙 10 天。用抗TRAcP抗体（1:500）（Sigma Aldrich，法国里昂）和苏木精及伊红（H&E）对石蜡包埋的胫骨矢状切片进行染色，切片厚度为 。使用 uScope MXII 数码显微玻片扫描仪（Microscopes International，Lubbock，TX，USA）扫描和处理切片图像。使用 BIOQUANT OSTEO 2011 软件计算每个骨表面的 OC 数量（N.Oc/BS）。

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Fig. 1. Pra-B suppressed RANKL-induced osteoclastogenesis in vitro. (Specimen: BMMs), (A) The structure of Pra-B, (B-D) CCK-8 assay experiments were performed to evaluate the cytotoxicity of BMMs after treatment with Pra-B ( , and for 3,5 or 6days. (E) TRAcP staining experiments were performed to evaluate the osteoclastic differentiation of BMMs stimulated with Pra-B at different concentrations ( ) (magnification . Quantitative analysis of the number of TRAcP-positive OCs and number of nuclei in the cells in each group. (H)Representative images of TRAcP-positive cells after stimulation with Pra-B on the indicated days (magnification ). , and . The values and error bars are the means SEMs. All data were analyzed using unpaired Student's tests.
图 1.Pra-B 抑制了 RANKL 诱导的体外破骨细胞生成（标本：BMMs），（A）Pra-B 的结构，（B-D）CCK-8 检测实验评估了用 Pra-B 处理（ ，和 3、5 或 6 天）后 BMMs 的细胞毒性。(E) TRAcP 染色实验用于评估不同浓度（ ）的 Pra-B 刺激下 BMM 的破骨分化情况（放大 . 定量分析各组细胞中 TRAcP 阳性 OC 的数量和细胞核的数量。(H)在指定天数用 Pra-B 刺激后 TRAcP 阳性细胞的代表性图像（放大 ）。 和 。数值和误差为平均值 SEM。所有数据均采用非配对的学生 检验进行分析。

Data are all expressed as the mean SEM. The significance of the differences between groups was determined by one-way ANOVA. The effects of time and treatments on protein expression were examined by two-way ANOVA. Statistical significance was indicated when .
数据均以平均值 SEM表示。组间差异的显著性由单向方差分析确定。时间和处理对蛋白质表达的影响采用双因素方差分析。当 。

To explore the impact of Pra-B on RANKL-induced OC differentiation, we used various concentrations of Pra-B and in complete medium with M-CSF and RANKL to treat BMMs. We found that Pra-B inhibited OC differentiation in a dose-dependent manner. PraB at a concentration of had the most drastic inhibitory effect on OCs (Fig. 1E, F). We went into more detail about when osteoclastogenesis was suppressed by Pra-B. BMMs was stimulated by Pra-B at different times (days , and ). TRAcP staining showed that the disincentive to OC differentiation was largely in the early and middle stages (days ) and the adverse effect considerably abated in late stages (days 5-6) (Fig. 1 G, H). Next, we explored the potential cytotoxicity of Pra-B against primary BMMs with a cell viability assay.
为了探索Pra-B对RANKL诱导的OC分化的影响，我们在含有M-CSF和RANKL的完全培养基中使用了不同浓度的Pra-B 和 来处理BMMs。我们发现，Pra-B抑制OC分化的作用呈剂量依赖性。浓度为 的PraB对OC的抑制作用最强（图1E、F）。我们更详细地了解了Pra-B何时抑制破骨细胞生成。在不同时间（ 天和 天）用Pra-B刺激BMMs。TRAcP染色显示，Pra-B对OC分化的抑制作用主要发生在早期和中期（ 天），而在晚期（第5-6天），这种不利影响明显减弱（图1 G、H）。接下来，我们用细胞活力测定法探讨了 Pra-B 对原代 BMMs 的潜在细胞毒性。
CCK-8 assay experiments were performed to evaluate the cytotoxicity of BMMs after treatment with Pra-B , and for 3,5 or 6 days. The results in Fig. 1B-D suggest that Pra-B at concentrations below did not affect the viability of BMMs (Fig. 1B-D). These findings show that Pra-B attenuated OC differentiation during the whole process in a dose-dependent manner without any significant toxicity. Then, bone resorption was assessed, and immunofluorescent staining was performed to evaluate the function of OCs. BMMs were transplanted and cultured on hydroxyapatite-coated plates for 7 days, and mature OCs were harvested. Pra-B treatment inhibited bone resorption in a dose-dependent manner (Fig. 2D-E). Because formation of the F-actin belt is critical for the ability of OCs to dissolve bone, we also tested Factin ring formation via immunofluorescent staining. The results revealed that Pra-B treatment led to diminished actin ring formation, suggesting that the bone-resorption function of OCs was inhibited by Pra-B, and Pra-B at a concentration of had the most drastic inhibitory effect on both the differentiation and bone-resorption function of OCs (Fig. 2A-C). Thus, both the differentiation and function of OCs were suppressed by Pra-B.
CCK-8测定实验用于评估Pra-B 和 处理3、5或6天后对BMM的细胞毒性。图 1B-D 中的结果表明，浓度低于 的 Pra-B 不会影响 BMM 的活力（图 1B-D）。这些结果表明，Pra-B在整个过程中以剂量依赖的方式抑制了OC的分化，而没有明显的毒性。然后，对骨吸收进行评估，并进行免疫荧光染色以评价 OC 的功能。在羟基磷灰石涂层平板上移植并培养 BMMs 7 天后，收获成熟的 OCs。Pra-B 以剂量依赖的方式抑制了骨吸收（图 2D-E）。由于F-肌动蛋白带的形成对OCs溶解骨的能力至关重要，我们还通过免疫荧光染色检测了Factin环的形成。结果显示，Pra-B处理会导致肌动蛋白环形成减少，这表明OCs的骨吸收功能受到了Pra-B的抑制，而浓度为 的Pra-B对OCs的分化和骨吸收功能的抑制作用最为明显（图2A-C）。因此，Pra-B对OCs的分化和功能都有抑制作用。
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Fig. 2. Bone resorption was impaired by Pra-B. (Specimen: BMMs), (A) Immunofluorescence staining experiments were performed to show the formation of an Factin ring in BMMs stimulated with Pra-B (0, 5, ) and RANKL (scale bar ). (B, C) Quantitative analysis of the resorbed area in each group. (D) Bone absorption experiments were performed on hydroxyapatite-coated 96-well plates to detect the bone-resorption function of OCs treated with Pra-B at different concentrations ) (magnification ). (E) Quantitative analysis of the resorbed area in each group. , * , and . The values and error bars are the means SEMs. All data were analyzed using unpaired Student's tests.
图 2.Pra-B 影响骨吸收。(标本：BMMs），（A）免疫荧光染色实验显示了Pra-B（0，5， ）和RANKL（比例尺 ）刺激下的BMMs中Factin环的形成。(B、C）各组吸收面积的定量分析。(D) 在涂有羟基磷灰石的 96 孔板上进行骨吸收实验，以检测不同浓度的 Pra-B 处理 OCs 的骨吸收功能 )。(放大 ）。(E) 各组吸收面积的定量分析。 、* 和 。数值和误差为平均值 SEM。所有数据均采用非配对的学生 检验进行分析。

To further confirm the effect of Pra-B on OC formation and function, we examined mRNA expression of the OC marker genes Acp5, CTSK, NFATc1, and c-Fos. As shown by the qRT-PCR experiment, Pra-B suppressed NF-kB-induced OC-associated gene expression in a dosedependent manner, and Pra-B at a concentration of had the most drastic inhibitory effects (Fig. 3A-D). Moreover, we observed that Pra-B suppressed the bone resorption-linked genes CTSK and integrin . The protein expression of NFATc1 and c-Fos was also inhibited, especially on the fifth day (Fig. 3E-I). Thus, quantitative analysis supported the results of Western blot analysis. These results demonstrated the negative influence of Pra-B on both OC formation and bone absorption at the protein and gene expression levels.
为了进一步证实Pra-B对OC形成和功能的影响，我们检测了OC标记基因Acp5、CTSK、NFATc1和c-Fos的mRNA表达。qRT-PCR实验表明，Pra-B以剂量依赖的方式抑制了NF-kB诱导的OC相关基因的表达，其中 浓度的Pra-B的抑制作用最为显著（图3A-D）。此外，我们还观察到Pra-B抑制了骨吸收相关基因CTSK和整合素 。NFATc1和c-Fos的蛋白表达也受到抑制，尤其是在第五天（图3E-I）。因此，定量分析支持了 Western 印迹分析的结果。这些结果表明，Pra-B 在蛋白和基因表达水平上对 OC 的形成和骨吸收都有负面影响。

Next, we investigated whether Pra-B modulates RANKL-induced NFкB signaling pathways, which are key to osteoclastogenesis. qRT-PCR experiments were performed to probe the mRNA levels of the NF-kB family proteins including p65, p50 and p52(Fig. 4A-C). The result suggested that Pra-B inhibited the mRNA expression of p65 and p50, the classical dimer partner. Also, western blotting experiments were performed to examine the specific mechanism by which Pra-B affects NF-kB transport. The data showed that Pra-B downregulated the RANKLinduced phosphorylation of P65, suggesting that NF-kB signaling activation was inhibited by Pra-B (Fig. 4D-E). Besides, luciferase experiments were carried out. Luciferase activity analysis showed that Pra-B significantly inhibited NF-kB transcriptional activity compared with RANKL group (Fig. 4F). Then, to verify this finding, immunofluorescence experiments were carried out which showed that Pra-B suppressed the nuclear translocation of p65 (Fig. 4G). From the immunofluorescence staining results, we found that the p65-stained area of the nucleus was significantly lower after Pra-B stimulation compared with that of the control group. (Fig. 4G). These results suggested that both the activation of NF-kB signaling and the nuclear transport of p65 were downregulated by Pra-B.
接下来，我们研究了Pra-B是否调节了RANKL诱导的NFкB信号通路，该信号通路是破骨细胞生成的关键。结果表明，Pra-B 抑制了经典二聚体伙伴 p65 和 p50 的 mRNA 表达。此外，为了研究Pra-B影响NF-kB转运的具体机制，还进行了Western印迹实验。数据显示，Pra-B 下调了 RANKL 诱导的 P65 磷酸化，表明 NF-kB 信号激活受到了 Pra-B 的抑制（图 4D-E）。此外，还进行了荧光素酶实验。荧光素酶活性分析表明，与 RANKL 组相比，Pra-B 能显著抑制 NF-kB 的转录活性（图 4F）。为了验证这一结果，还进行了免疫荧光实验，结果显示Pra-B抑制了p65的核转运（图4G）。从免疫荧光染色结果中我们发现，与对照组相比，Pra-B 刺激后细胞核中 p65 染色面积明显减少。(图 4G）。这些结果表明，Pra-B 对 NF-kB 信号的激活和 p65 的核转运均有下调作用。

To dissect the molecular mechanisms underlying the effect of Pra-B on OC differentiation, computational docking was used, and the results indicated that Pra-B strongly binds GSTP1. Through Discovery Studio, we obtained the high-affinity Pra-B-binding site in GSTP1 (Fig. 5 A). Then, the nonbonding interactions between Pra-B and GSTP1 were analyzed, as shown in Fig. 5B, and found to include intermolecular forces and conjugated systems. More specifically, hydrogen bonds between Pra-B and GSTP1 were analyzed. The presence of hydrogen bonds enhanced the binding possibility and affinity of Pra-B for GSTP1 (Fig. 5 C). Therefore, the molecular docking assay indicated the high affinity between GSTP1 and Pra-B, suggesting that GSTP1 is a possible target of Pra-B. Concomitantly, to explore the effect of Pra-B on GSTP1, western blotting experiments showed that Pra-B activated GSTP1 expression during RANKL-induced osteoclastogenesis(Fig. 5E, F). In PCR experiment, when the transcription was blocked by actinomycin , the gene stability of GSTP1 was promoted by Pra-B compared to control group, indicating that Pra-B is a possible agonist of GSTP1 whose main function is mediated by up-regulating the protein expression and mRNA stability of GSTP1 (Fig. 5D).
为了剖析Pra-B对OC分化作用的分子机制，我们采用了计算对接的方法，结果表明Pra-B能与GSTP1强结合。通过Discovery Studio，我们得到了GSTP1的高亲和性Pra-B结合位点（图5 A）。然后，我们分析了 Pra-B 与 GSTP1 之间的非键相互作用（如图 5B 所示），发现其中包括分子间作用力和共轭体系。更具体地说，分析了 Pra-B 和 GSTP1 之间的氢键。氢键的存在提高了 Pra-B 与 GSTP1 结合的可能性和亲和力（图 5 C）。因此，分子对接分析表明 GSTP1 与 Pra-B 之间具有很高的亲和力，表明 GSTP1 可能是 Pra-B 的靶标。同时，为了探讨Pra-B对GSTP1的影响，Western印迹实验表明Pra-B在RANKL诱导的破骨细胞生成过程中激活了GSTP1的表达（图5E，F）。在PCR实验中，当放线菌素 阻断转录时，与对照组相比，Pra-B促进了GSTP1的基因稳定性，表明Pra-B可能是GSTP1的激动剂，其主要功能是上调GSTP1的蛋白表达和mRNA稳定性（图5D）。
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Fig. 3. Pra-B inhibited the activation of NF-кB-induced OC-associated genes. (Specimen: BMMs), (A-D) Quantitative real-time PCR analysis was performed to evaluate expression of the NF, -induced OC marker genes Acp5, CTSK, Nfatc1 and c-Fos after treatment with RANKL (50 ng/mL) and Pra-B (0,5, . (E) Western blotting experiments were performed to evaluate the protein expression of integrin , CTSK, NFATc-1, and c-Fos in the nucleus after treatment with RANKL and Pra-B for , and 5 days. (F-J) Quantitative analysis of the protein expression of integrin , CTSK, NFATc-1, and c-Fos at the given time points. All the protein levels were normalized to the -actin level. , and . The values and error bars are the means SEMs. All data were analyzed using unpaired Student's tests.
图 3.Pra-B 抑制了 NF-кB 诱导的 OC 相关基因的激活。(标本：BMMs），（A-D）在RANKL（50 ng/mL）和Pra-B（0.5， ）处理后，进行定量实时PCR分析以评估NF， 诱导的OC标记基因Acp5、CTSK、Nfatc1和c-Fos的表达。(E）Western 印迹实验评估 RANKL 和 Pra-B 处理 和 5 天后细胞核中整合素 、CTSK、NFATc-1 和 c-Fos 的蛋白表达。(F-J）给定时间点整合素 、CTSK、NFATc-1 和 c-Fos 蛋白表达的定量分析。所有蛋白水平均归一化为 -actin 水平。 和 。数值和误差为平均值 SEM。所有数据均采用非配对的学生 检验进行分析。

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Fig. 4. Pra-B inhibited the RANKL-induced NF-кB signaling pathway. (Specimen: BMMs) (A-C) Quantitative real-time PCR analysis was performed to evaluate expression of NF-kB family members including like p65, p50 and P52 after treatment with RANKL ( ) and Pra-B (0, 5, ). (D) Western blotting experiments were performed to evaluate the protein expression of p-p65, p65 after treatment with RANKL ( and Pra-B (10 for and (E) Quantitative analysis of the levels of p65 phosphorylation at the given time points. (F) NF-KB luciferase activity in BMMs treated with Pra-B at different concentrations. (G) Immunofluorescence staining experiments were performed to show the distribution of p65 in BMMs stimulated with Pra-B (10 M) and RANKL (scale bar ). , and . The values and error bars are the means SEMs. All data were analyzed using unpaired Student's tests.
图 4.Pra-B 抑制了 RANKL 诱导的 NF-кB 信号通路。(标本：BMMs）（A-C）实时定量 PCR 分析评估了 RANKL （ ）和 Pra-B （0，5， ）处理后 NF-kB 家族成员如 p65、p50 和 P52 的表达。(D) 进行 Western 印迹实验，以评估经 RANKL ( ) 和 Pra-B （10 为 和 处理后 p-p65、p65 的蛋白表达 (E) 给定时间点 p65 磷酸化水平的定量分析。(F）不同浓度的 Pra-B 处理的 BMM 中 NF-KB 荧光素酶活性。(G)免疫荧光染色实验显示了Pra-B（10 M）和RANKL刺激的BMM中p65的分布（比例尺 ）。 和 。数值和误差条为平均值 SEM。所有数据均采用非配对的学生 检验进行分析。

To further verify the leading role of Pra-B, GSTP1-siRNA was used, and the knockdown efficiency of GSTP1 was also detected (S-Fig. 1C, D). As expected, GSTP1-siRNA reversed the Pra-B-mediated repression of RANKL-induced OC differentiation and osteolytic function, which was confirmed by TRAcP staining as well as bone absorption analysis (Fig. 6A-D).
为了进一步验证Pra-B的主导作用，使用了GSTP1-siRNA，并检测了GSTP1的敲除效率（图1C，D）。不出所料，GSTP1-siRNA 逆转了 Pra-B 介导的对 RANKL 诱导的 OC 分化和溶骨功能的抑制，这一点通过 TRAcP 染色和骨吸收分析得到了证实（图 6A-D）。
Next, we focused on the influence of Pra-B on NF-кB signaling mediated through GSTP1. The findings showed that the RANKL-induced nuclear p65 content was increased in BMMs transfected with GSTP1siRNA relative to that transfected with ctrl-siRNA. Also, compared with cells stimulated with only RANKL, BMMs transfected with GSTP1siRNA and stimulated with RANKL and Pra-B exhibited no significant difference in their nuclear p65 content, suggesting that Pra-B exerts its inhibitory effect on NF-кB signaling mainly by activating GSTP1 (Fig. 6E-F). To support this finding, immunofluorescence staining
接下来，我们重点研究了Pra-B对通过GSTP1介导的NF-кB信号转导的影响。研究结果表明，与转染ctrl-siRNA的细胞相比，转染GSTP1siRNA的BMM细胞中RANKL诱导的核p65含量增加。此外，与只用 RANKL 刺激的细胞相比，转染了 GSTP1siRNA 并用 RANKL 和 Pra-B 刺激的 BMM 细胞的核 p65 含量没有显著差异，这表明 Pra-B 主要通过激活 GSTP1 来发挥其抑制 NF-кB 信号转导的作用（图 6E-F）。为了支持这一发现，免疫荧光染色

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Fig. 5. Pra-B is predicted to be an agonist of GSTP1. (Specimen: BMMs), (A) The 3D protein structure of GSTP1 and the binding site between GSTP1 and Pra-B. (B) The nonbonding interactions between GSTP1 and Pra-B (CID: 5319259). (C) Hydrogen bond interactions between Pra-B and adjacent amino acids in the GSTP1 protein. (D) The fold changes in GSTP1 mRNA expression after treatment with or without Pra-B for and stimulation of RANKL for or were determined by real-time PCR to evaluate the mRNA stability of GSTP1. (E) Western blotting experiments were performed to evaluate the protein expression of GSTP1 upon treatment with RANKL (50 ng/mL) and Pra-B (10 ) for 0, 10, 20, 30, 45 and (F) Quantitative analysis of the protein expression of GSTP1 at the given time points. * . The values and error bars are the means SEMs. All data were analyzed using unpaired Student's tests.
图 5.预测 Pra-B 是 GSTP1 的激动剂。(样本：BMMs），（A）GSTP1 的三维蛋白质结构以及 GSTP1 与 Pra-B 之间的结合位点。(B）GSTP1 与 Pra-B 之间的非键相互作用（CID：5319259）。(C) Pra-B 与 GSTP1 蛋白中相邻氨基酸之间的氢键相互作用。(D) 通过实时 PCR 检测 用或不用 Pra-B 处理以及 或 用 RANKL 刺激后 GSTP1 mRNA 表达的折叠变化，以评估 GSTP1 的 mRNA 稳定性。(E) 进行 Western 印迹实验，以评估 RANKL（50 ng/mL）和 Pra-B（10 ）处理 0、10、20、30、45 和 时 GSTP1 的蛋白表达 (F) 给定时间点 GSTP1 蛋白表达的定量分析。* 。数值和误差为平均值 SEM。所有数据均采用非配对的学生 检验进行分析。

experiments were performed, and we observed that the inhibitory effect of Pra-B on P65 nuclear transportation was blocked by GSTP1-siRNA (Fig. 6G). Therefore, GSTP1 is the key protein that regulates the inhibitory effect of Pra-B on OC differentiation.
实验中，我们观察到 GSTP1-siRNA 阻断了 Pra-B 对 P65 核运输的抑制作用（图 6G）。因此，GSTP1是调控Pra-B对OC分化抑制作用的关键蛋白。

3.6. Praeruptorin B mediates the upregulated expression of GSTP1 to promote its role in the -glutathionylation of IKK under conditions of RANKL-induced oxidative stress
3.6.在 RANKL 诱导的氧化应激条件下，Praeruptorin B 介导 GSTP1 的上调表达，促进其在 IKK 的 - 谷氨酰化中发挥作用

Severe oxidative stress is deemed to contribute to the activation of GSTP1's enzymatic activity and then promote its S-glutathionylation function. Such environment can also be detected in RANKL-stimulated OCs . Based on all evidence above, we hypothesized that Pra-B activated GSTP1 to promote the S-glutathionylation of IKK which inhibited the downstream NF-kB pathway during RANKL-induced osteoclastogenesis.
严重的氧化应激被认为有助于激活 GSTP1 的酶活性，进而促进其 S-谷胱甘肽化功能。在 RANKL 刺激的 OC 中也可以检测到这种环境 。基于上述证据，我们推测Pra-B激活GSTP1，促进IKK 的S-谷氨酰化，从而在RANKL诱导的破骨细胞生成过程中抑制下游NF-kB通路。
Considering the roles of GSTP1 in modulating RANKL-induced NF-kB signaling and in the catalysis of protein S-glutathionylation, we investigated whether the S-glutathionylation of IKK was reliant on GSTP1. Following exposure to RANKL, the level of IKK S-glutathionylation was very low. (Fig. 7C-D) Also, the application of GSTP1-siRNA was found to rescue the low S-glutathionylation of IKK , which indicating the role of GSTP1 on IKK in osteoclast. To further validate the role of Pra-B on GSTP1, IP was applied. A noticeable increase in IKK S-glutathionylation was detected upon Pra-B stimulation, which was related to a decrease in the phospho-P65 content and an increase in the I content. These parameters in Pra-B-treated BMMs transfected with GSTP1-siRNA were similar to those only treated with RANKL (Fig. 7CH). To define GSTP1-mediated glutathionylation as the key to its impact on downstream NF-kB mechanisms, GSH inhibitor DL-ButhionineSulfoximine (BSO) was applied, and the inhibited protein expression of IKK , IкB and p-p65 have been rescued (S-Fig. 2). Consequently, Pra-
考虑到GSTP1在调节RANKL诱导的NF-kB信号传导和催化蛋白质S-谷氨酰化中的作用，我们研究了IKK 的S-谷氨酰化是否依赖于GSTP1。暴露于 RANKL 后，IKK 的 S-谷氨酰化水平非常低。(图 7C-D）此外，应用 GSTP1-siRNA 还能挽救 IKK 的低 S-谷氨酰化，这表明 GSTP1 对破骨细胞中 IKK 的作用。为了进一步验证 Pra-B 对 GSTP1 的作用，应用了 IP。在Pra-B刺激下，检测到IKK S-谷胱甘肽化明显增加，这与磷酸化-P65含量的减少和I 含量的增加有关。转染了 GSTP1-siRNA 的 BMM 经 Pra-B 处理后，这些参数与只用 RANKL 处理的 BMM 相似（图 7CH）。为了确定GSTP1介导的谷胱甘肽化是其影响下游NF-kB机制的关键，应用了GSH抑制剂DL-ButhionineSulfoximine（BSO），IKK 、IкB 和p-p65被抑制的蛋白表达得到了挽救（S-图2）。因此，Pra-

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Fig. 6. Pra-B activated the expression of GSTP1 to suppress downstream NF-kB signaling and inhibit osteoclastogenesis. (Specimen: BMMs), (A) Bone absorption experiments were performed with bone slices to detect the bone-resorption function of OCs transfected with GSTP1-siRNA and treated with Pra-B (10 (magnification ). (B) Quantitative analysis of the resorbed area in each group. (C) Bone absorption experiments were performed with bone slices to detect the bone-resorption function of OCs transfected with GSTP1-siRNA and treated with Pra-B ) (magnification . (D) Quantitative analysis of the resorbed area in each group. (E) Western blotting experiments were performed to evaluate the protein expression of p65 in the nucleus after treatment with siRNAs and Pra-B for . (F) Quantitative analysis of the protein expression of p65 in the nucleus with the given treatments. (G) Immunofluorescence staining experiments were performed to show the distribution of p65 in BMMs treated with siRNA and stimulated with Pra-B ( ) and RANKL (scale bar ). *P , and **P . The values and error bars are the means SEMs. All data were analyzed using unpaired Student's tests.
图 6.Pra-B 激活 GSTP1 的表达，抑制下游 NF-kB 信号传导，抑制破骨细胞生成。(标本：BMMs），（A）用骨切片进行骨吸收实验，检测转染 GSTP1-siRNA 并用 Pra-B 处理（10 （放大 ）的 OCs 的骨吸收功能。(B）各组吸收面积的定量分析。(C) 用骨片进行骨吸收实验，检测转染 GSTP1-siRNA 并用 Pra-B 处理的 OC 的骨吸收功能。）(D) 各组骨吸收面积的定量分析。(E) 用 siRNAs 和 Pra-B 处理 后，进行 Western 印迹实验以评估 p65 在细胞核中的蛋白表达。 (F) 定量分析给定处理后 p65 在细胞核中的蛋白表达。(G）免疫荧光染色实验显示了经 siRNA 处理并受 Pra-B ( ) 和 RANKL 刺激的 BMM 中 p65 的分布（比例尺 ）。*P ，和 **P 。数值和误差条为平均值 SEM。所有数据均采用非配对的学生 检验进行分析。