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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 .

Kebin Xu , Ziyi Chen , Jialong , Chenlin Dong , Chengge Shi , Linglin ,
Kebin Xu , Ziyi Chen , Jialong , Chenlin Dong , Chengge Shi , Linglin
Zhixian Huang , Ge Shen , Te Wang , Yan Zhou a Department of Pharmacy, HwaMei Hospital, University of Chinese Academy of Sciences, Ningbo, Zhejiang 315010, China
a 中国科学院大学华美医院药学部,浙江宁波 315010
Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo, Zhejiang 315010, China
中国科学院大学宁波生命健康产业研究院,浙江宁波 315010
c The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
c 温州医科大学附属第二医院和育英儿童医院,浙江温州 325035

A R T I C L E I N F O

Keywords: 关键词:

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

Abstract 摘要

A B S T R A C T Osteoporosis a common disease in postmenopausal women which contains significant impact on the living quality of women. With the aging of the population, the number of patients suffer from osteoporosis has shown a significant increase. Given the limitations of clinical drugs for the treatment of osteoporosis, natural extracts with small side effects have a great application prospect in the treatment of osteoporosis. Praeruptorin B (Pra-B), is one of the main components found in the roots of Peucedanum praeruptorum Dunn and exhibits antiinflammatory effects. However, there is no research on the influence of Pra-B on osteoporosis. Here, we showed that Pra-B can dose-dependently suppress osteoclastogenesis without cytotoxicity. Receptor activator of nuclear factor kappa-B (NF-kB) ligand (RANKL)-induced the nuclear import of P65 was inhibited by Pra-B, which indicated the suppressive effect of Pra-B on NF-кB signaling. Further, Pra-B enhanced the expression of Glutathione S-transferase Pi 1 (GSTP1) and promoted the S-glutathionylation of IKK to inhibit the nuclear translocation of P65. Moreover, in vivo experiments showed that Pra-B considerably attenuated the bone loss in ovariectomy (OVX)-induced mice. Collectively, our studies revealed that Pra-B suppress the NF-kB signaling targeting GSTP1 to rescued RANKL-induced osteoclastogenesis in vitro and OVX-induced bone loss in vivo, supporting the potential of Pra-B for treating osteoporosis in the future.
A B S T R A C T 骨质疏松症是绝经后妇女的常见病,对妇女的生活质量有很大影响。随着人口老龄化的加剧,骨质疏松症患者的数量也在大幅增加。鉴于临床药物治疗骨质疏松症的局限性,副作用小的天然提取物在治疗骨质疏松症方面具有广阔的应用前景。Praeruptorin B(Pra-B)是牡丹根中的主要成分之一,具有抗炎作用。然而,目前还没有关于 Pra-B 对骨质疏松症影响的研究。在这里,我们研究发现,Pra-B 能在无细胞毒性的情况下,剂量依赖性地抑制破骨细胞的生成。Pra-B能抑制受体激活因子卡巴-B(NF-kB)配体(RANKL)诱导的P65核导入,这表明Pra-B对NF-кB信号转导有抑制作用。此外,Pra-B还能增强谷胱甘肽S-转移酶Pi 1(GSTP1)的表达,促进IKK 的S-谷胱甘肽化,从而抑制P65的核转位。此外,体内实验表明,Pra-B能显著减轻卵巢切除术(OVX)诱导的小鼠骨质流失。总之,我们的研究发现,Pra-B能抑制以GSTP1为靶点的NF-kB信号转导,从而挽救体外RANKL诱导的破骨细胞生成和体内OVX诱导的骨质流失,支持了Pra-B在未来治疗骨质疏松症的潜力。

1. Introduction 1.导言

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。

2. Materials and methods
2.材料和方法

2.1. Materials and reagents
2.1.材料和试剂

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)。

2.2. Cell culture and cytotoxicity assays
2.2.细胞培养和细胞毒性试验

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 M-CSF的完全 -MEM培养基中,在 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 -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 ,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]。

2.3. TRAcP staining 2.3.TRAcP 染色

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。

2.4. Bone resorption assay
2.4.骨吸收试验

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。

2.5. Quantitative real-time PCR analysis
2.5.实时定量 PCR 分析

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。
Gene Forward Reverse
Nfatc1 GGAGAGTCCGAGAATCGAGAT TTGCAGCTAGGAAGTACGTCT
C-fos GCGAGCAACTGAGAAGAC TTGAAACCCGAGAACATC
Acp5 TGTGGCCATCTTTATGCT GTCATTTCTTTGGGGCTT
Ctsk CCAGTGGGAGCTATGGAAGA AAGTGGTTCATGGCCAGTTC
Hprt GTTGGGCTTACCTCACTGCT TAATCACGACGCTGGGACTG

2.6. Western blot analysis
2.6.Western 印迹分析

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 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。

2.7. Luciferase reporter gene assay
2.7.荧光素酶报告基因检测

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。

2.8. Immunofluorescence staining
2.8.免疫荧光染色

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。

2.9. Computational docking
2.9.计算对接

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。

2.10. Cell transfection 2.10.细胞转染

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。

2.11. Determination of mRNA stability
2.11.测定 mRNA 的稳定性

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 后的总RNA。然后,应用 qRT-PCR 分析目标 mRNA 的转录和残留情况。

2.12. Immunoprecipitation
2.12.免疫沉淀

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。

2.13. Osteoblastogenesis assay
2.13.成骨细胞生成试验

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

2.14. Mouse ovariectomy procedure
2.14.小鼠卵巢切除术

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。

2.15. Micro-CT scanning 2.15.显微 CT 扫描

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。

2.16. Histological and histomorphometric analyses
2.16.组织学和组织形态计量学分析

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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Pra-B (10pM)
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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 (