Periplogenin attenuates LPS-mediated inflammatory osteolysis through the suppression of osteoclastogenesis via reducing the NF-κB and MAPK Signaling Pathways

doi:10.21203/rs.3.rs-3189611/v1

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Periplogenin attenuates LPS-mediated inflammatory osteolysis through the suppression of osteoclastogenesis via reducing the NF-κB and MAPK Signaling Pathways

https://doi.org/10.21203/rs.3.rs-3189611/v1

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published 17 Feb, 2024

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The key target for treating inflammatory osteolysis is osteoclasts. In an inflammatory environment, osteoclast differentiation increases, and bone resorption is enhanced. Periplogenin (Ppg) is a traditional Chinese medicine. It has anti-inflammatory and antitumor effects, but its impact on inflammatory osteolysis is unknown. This study found that Ppg prevented LPS-induced skull osteolysis by inhibiting the expression of inflammatory cytokines and osteoclast production. In vitro, Ppg blocked the RANKL-induced generation of osteoclasts, the development of pseudopodia bands, and bone resorption. Ppg also attenuated the expression of NFATc1, c-Fos, CTSK, and Atp6v0d2 proteins by inhibiting the NFATc1 signaling pathway. Additionally, Ppg inhibited the expression of osteoclast-specific genes, including NFATc1, c-Fos, CTSK, Atp6v0d2, and Mmp9. Moreover, Ppg also inhibited NF-κB and MAPK pathways. In vivo, Ppg reduced the number of osteoclasts on the surface of the bone and suppressed LPS-induced osteolysis of the skull. These outcomes suggest that Ppg can serve as a new alternative therapy for treating inflammatory osteolysis by inhibiting inflammation and osteoclasts.

Biological sciences/Drug discovery/Drug screening/Phenotypic screening

Biological sciences/Cell biology/Mechanisms of disease

Periplogenin

Osteolysis

Inflammatory osteolysis

Maintaining the integrity of bone metabolic balance is coordinated by osteoclasts-mediated bone resorption and osteoblast-mediated bone formation (1). Inflammation after tissue damage is essential for the early phases of repair, and its imbalance throws off the delicate process of bone remodeling (2). Chronic inflammation suppresses osteoblast development, decreases bone production, and accelerates osteoclasts, causing increased bone resorption (3). Variable inflammatory mediators play a crucial function in the deterioration of bone induced by osteoclasts (4). When inflammation occurs near bones, some inflammatory osteolysis diseases arise. Examples include rheumatoid arthritis (5), psoriatic arthritis (6), periodontitis (7), periprosthetic osteolysis (8), and osteomyelitis (9), which can cause focal erosion, thus leading to severe consequences. Enhanced bone resorption and disruption of bone metabolic balance occur as a result of the activation and recruitment of osteoclasts, which is triggered by the production of inflammatory cytokines. This process is the primary cause of inflammatory osteolysis (10). Potential mechanism-based treatment targets to stop inflammatory bone loss may be identified by gaining a deeper understanding of the mechanisms underlying bone resorption by osteoclasts and the cytokines that control their development and activity.

The fusion of monocyte/macrophage precursor cells derived from myeloid progenitor cells in the bone marrow results in osteoclasts, multinucleated giant cells. The development and functioning of osteoclasts are regulated by macrophage colony-stimulating factor (M-CSF) and RANKL (11). Stimulated by RANKL, the NF-κB and MAPK pathways are triggered to activate NFATc1 to drive osteoclast differentiation (12). Additionally, signaling pathways such as calcium (13), PI3K-AKT (14), Notch (15), and Hippo (16) also promote osteoclast differentiation and activation. With the activation of signaling pathways, osteoclast-specific genes are also expressed, including, Ctsk, Atp6v0d2, and Mmp9 (17).

Although substantial medical advances have improved the treatment of inflammatory osteolytic disease, there are still certain drawbacks, such as the failure to repair broken bone and resistance to therapy in more than one-third of rheumatoid arthritis and psoriatic arthritis patients (18). Recently, plant-derived natural products and their derivatives have proven valuable sources for exploring new avenues for treating clinical diseases due to their unique pharmacological activity (19). Periplogenin (Ppg) is a natural furocoumarin in the root of Pycnogenol that has a highly effective anti-psoriasis effect (20){Zhang, 2016 #6} and anti-tumor (21) activity. Moreover, Ppg exhibits anti-inflammatory effects in psoriasis. Nevertheless, the impact of Ppg on inflammatory osteolysis triggered by osteoclasts is unknown. The current research proposed that Ppg may act as a novel therapeutic agent for inflammatory osteolysis by inhibiting osteoclastic formation. Therefore, the research aimed to investigate the possible effectiveness of Ppg in osteolytic bone conditions related to osteoclasts and to elucidate the potential molecular role of Ppg in osteoclastic formation.

Ppg suppressed RANKL-induced osteoclast differentiation in vitro

Ppg has been found to exhibit anti-inflammatory and anti-tumor properties but its effect on osteoclasts is unclear. The current research assessed the impact of Ppg on osteoclast differentiation. After a culture period of 5-7 days, it was observed that Ppg suppressed osteoclast differentiation in a dose-dependent manner (Figures 1C and 1D). Additionally, the CCK-8 experiment revealed that Ppg did not inhibit BMM proliferation for 96 hours at doses below 100 μM (Figure 1B), indicating that Ppg was non-toxic to osteoclast survival. It was also observed that the inhibitory effect of Ppg was stronger during the early and middle phases of osteoclast development (Figures 1E and 1F).

Ppg suppressed osteoclast resorption function.

Furthermore, the bone resorption test showed that Ppg inhibited the resorptive function of osteoclasts on bovine bone sections (Figures 2C-E). Ppg-treated cells significantly reduced osteoclast size and osteoclast area compared to untreated cells (Figures 2A and 2B). These results suggest that Ppg suppressed the resorptive function of osteoclasts.

Ppg suppressed LPS-induced inflammatory response in BMMs

Using a Real-time quantitative PCR assay, Ppg was found to inhibit the transcription of IL-1β and IL-6 in BMMs induced by LPS and RANKL (Figures 3A and 3B). In addition, Ppg also attenuated LPS and RANKL-mediated BMMs differentiation of osteoclasts (Figures 3C and 3D).

Ppg suppressed LPS-induced osteolysis in mice in vivo

To explore the impact of ppg on inflammatory osteolysis in vivo, an LPS-induced model of mouse skull osteolysis was used. The weight of the mice did not significantly alter over the course of the 9-day experiment (Figure 4A), and no mice died or suffered any negative side effects. A previous report has highlighted that inhibiting the production of inflammatory environment and inflammatory factors can effectively relieve inflammatory osteolysis (22). Based on the fact that DXMS can inhibit LPS-induced inflammation production, it was chosen as a positive control group in the current investigation (23). IL-1β levels was assessed to determine the anti-inflammatory action of Ppg in vivo by using immunohistochemical labeling of mouse skull slices (Figure 5E and 5F). Consistent with the findings obtained from in vitro experiments, the in vivo experiments also revealed that Ppg treatment lowered the levels of IL-1β protein expression (Figure 4B). Micro-CT scanning of the isolated mouse skulls showed that the skull osteolysis of mice after Ppg treatment was significantly reduced (Figures 4C-F). Consistently, histology analyses showed that the BV/TV volume of mice in the high-dose Ppg group was comparable to the sham groups (Figures 5A and 5B). Ppg dramatically decreased the TRACP-positive cell/bone surface ratio and the overall number of osteoclasts per field in comparison to the LPS group (Figures 5C and 5D). The overall findings of the current research imply that Ppg inhibits osteoclast formation, has anti-inflammatory effects in vivo, and might inhibit LPS-mediated inflammatory bone loss.

Ppg downregulated the expression of genes and proteins specific to osteoclasts during osteoclastic formation.

Ppg was found to inhibit the expression of genes specific to osteoclasts, including c-Fos, NFATc1, CTSK, Atp6v0d2, and MMP9 by Real time PCR (Figures 6A-E). Consistently, it was observed that Ppg also inhibited protein expression of c-Fos, NFATc1, Atp6v0d2, and CTSK during osteoclast differentiation (Figures 6F-J). The outcomes highlight that Ppg suppressed the expression of genes and proteins specific to osteoclasts, thereby suppressing the differentiation and functioning of osteoclasts.

Ppg inhibited RANKL-induced NF-κB and MAPK pathways in osteoclast differentiation

Next, the effect of Ppg on NF-κB and MAPK pathways during osteoclast differentiation was investigated. The results of the luciferase reporter assay showed that treatment with Ppg inhibited the activation of NF-κB (Figures 7A), and at the same time, Ppg significantly blocks P65 nuclear translocation(Figures 7B). Consistent, western blot analysis showed that p-P65 (Figures 7C and 7D) and degradation of IκBα were also significantly inhibited (Figures 7C and 7E). Similarly, p-ERK was substantially suppressed 20 minutes after stimulation of RANKL (Figures 7F and 7G). In comparison, Ppg had no significant effect on p-P38 and p-JNK on BMMs mediated by RANKL at 0-60 minutes (Figures 7F, 7H, and 7I). These data suggest that Ppg suppressed osteoclast production by regulating NF-kB and MAPK pathways.

The hunt for novel medications to limit osteoclast function is a research hotspot for osteoclast-related osteolytic disorders since osteoclasts are essential cells implicated in bone loss. As a result of inhibiting the pro-inflammatory cytokines IL-1 and IL-6 and deactivating the NF-κB and ERK pathways, Ppg was able to suppress osteoclast differentiation and function in vitro. Further, the ability of Ppg to dampen LPS-induced inflammatory osteolysis was also highlighted.

Ppg was isolated from the root of a traditional Chinese medicinal plant called Periploca sepium Bunge and is recognized as one of the key components of Cortex Periplocae (24). The root bark of Periploca sepium Bunge, often called Cortex Periploca (Xiangjiapi in Chinese), has been commonly utilized for treating rheumatoid arthritis and promoting bone strength (20). Ppg also safeguards against hyperthyroidism and associated cardiovascular illness, according to several pharmacological trials (25). In addition, there have been reports of anti-inflammatory activity and anticancer properties of Ppg in recent years (26). The current study revealed its novel effect on osteoclast differentiation and function via dampening RANKL-induced osteoclast production, podosome belt formation, and bone resorption.

The critical pathways such as MAPK and NF-κB, activated by RANKL during osteoclastogenesis, have been extensively studied (27, 28). These pathways were key downstream cascades of TRAF6 activated by RANKL/RANK axis (29). NF-κB is a significant factor in osteoclast development and is involved in osteolytic bone diseases(30, 31). Phosphorylation of IκBα by the classic pathway and its degradation by the proteasome during osteoclast differentiation result in the translocation of the NF-κB dimer (p50 and p65/relA) into the nucleus (32). Extracellular signal-regulatory kinases 1 and 2 (ERK1/2) in the MAPK pathway are triggered by the sequential activation of MKKK, MKK, and MAPK (33). Activation of ERK, especially ERK1, can promote phosphorylation of transcription factors NFATc1 and c-Fos, thereby increasing their transcriptional activity and promoting osteoclast differentiation (34).

It is worth noting that Ppg is also a STAT3 inhibitor that directly and specifically binds to STAT3, resulting in the downregulation of STAT3 phosphorylation at Tyr705, thereby reducing downstream cascade dimerization, translocation, and function (21). Activation of STAT3 has been shown to induce RANKL production and stimulate osteoclast formation (35). Further investigation should focus on the interaction between Ppg and STAT3 in osteoclasts.

Furthermore, NFATc1 is an indispensable transcription factor for regulating osteoclast differentiation (36). The cytoplasmic version of NFATc1 is persistently present and strongly phosphorylated. When it is triggered by RANKL or its downstream signaling pathway, its serine residues are dephosphorylated and moved into the nucleus (37, 38). Following its entry into the nucleus, NFATc1 collaborates with other transcription factors to increase the expression of genes unique to osteoclasts, including Ctsk, Atp6v0d2, and Mmp9, thereby boosting osteoclast differentiation and maturation (39). One of the key roles of c-FOS in osteoclast formation is to produce NFATc1, which can work together to start a transcriptional regulatory cascade that activates several target genes associated with osteoclast development and function (40). This research demonstrates that Ppg can dampen NFATc1 and c-Fos mRNA and protein expression, contributing to the inhibition of osteoclast formation and function.

Inflammation activates ERK and NF-κB signaling pathways during osteoclastogenesis (41). Bacterial endotoxins, including LPS, are thought to be the primary mediators of inflammatory osteolysis (42). LPS induces the production of osteoclast cytokines, including MCSF and the NF-κB ligand RANKL, which directly stimulate osteoclast formation and activity, resulting in catastrophic bone loss (43, 44). Moreover, LPS is a powerful inducer of inflammation and pro-inflammatory cytokines that stimulate macrophages, fibroblasts, and other cells for secreting multiple pathogenic cytokines, including IL-1β and IL-6 (45, 46). Therefore, this study verified that Ppg can inhibit the inflammatory factors IL-1β and IL-6 in vitro. The anti-osteolytic action of Ppg was then confirmed using a mouse model of LPS-induced skull osteolysis to imitate clinical inflammatory osteolysis. In line with our results, previous studies have found that Ppg is able to improve psoriasis-like skin inflammation induced by IMQ (20).

In conclusion, these findings offer encouraging evidence that Ppg is capable of inhibiting inflammation-mediated osteoclastogenesis and thus holds as a therapeutic candidate for treating inflammatory osteolytic illness associated with overactivation of osteoclasts.

Media and reagents

Ppg (HPLC98%) was provided by the Chengdu Must Biological Technology Co., Ltd. (China) for this study. Fetal bovine serum (FBS), penicillin-streptomycin (PS) solution, and alpha-modified minimum essential medium (α-MEM) were supplied by Gibco (Thermo Fisher Scientific, United States). Dimethyl sulfoxide (DMSO) was used to dissolve Ppg to a storage concentration of 100 mM before diluting Ppg to different concentrations by adding α-MEM. Moreover, Cell Counting Kit-8 (CCK-8) was supplied by MedChemExpress (MCE, China). Recombinant M-CSF and RANKL mice were supplied by R&D Systems (USA). Specific primary antibodies, such as p-P65, P65, phosphorylated (p)-ERK, ERK, p-JNK (c-Jun N-terminal kinase), JNK, p-P38, P38, β-actin, V-ATPase d2, and c-Fos, as well as secondary antibodies, such as rabbit and mouse, were supplied by Cell Signaling Technology (USA). Furthermore, Abcam (UK) provided the antibodies against CTSK, IκBα, and NFATc1.

Cell Culture and Osteoclast Differentiation Assay

The Animal Center of Guangxi Medical University (China) provided male C57BL/6J mice 6-8 weeks old. Primary bone marrow macrophages (BMMs) were obtained from the femur and tibia of mice via bone marrow lavage. Culturing of BMMs was done in a complete medium containing 10% FBS, 1% PS, and 25 ng/mL M-CSF in α-MEM at 5% CO₂, a temperature of 37°C, and placed in a T75 flask (USA). Following a 48-hour incubation, 7×10³ cells/well were inoculated into a 96-well plate. On the second day following cell attachment, osteoclast differentiation was induced by adding an osteoclast culture medium containing RANKL and M-CSF, as per a previous report (47). Subsequently, varying concentrations of Ppg (10, 20, 30, 40, 50, 60, 70, or 80 μM) were added within six days. BMMs were treated with Ppg (80 μM) at several stages of osteoclastogenesis for investigating the impact of Ppg on the differentiation of osteoclasts. BMMs were rinsed with PBS and fixed with 4% PFA, followed by staining with TRAcP reagents (Sigma, USA). Mature osteoclasts refer to fusion cells with a minimum of three nuclei. Images of the samples were captured using a Bio-tek microscope (VT, USA), and the osteoclasts were quantified by utilizing Image J (NIH, Bethesda, USA).

Cell Viability/Cytotoxicity Assay

Cytotoxicity of Ppg was detected with the aid of the CCK-8 kit. BMMs (7×10³ cells/well) were seeded into 96-well plates. The following day, cells were stimulated with varying concentrations of 20, 40, 60, 80, or 100 μM for 96 hours. Next, 10 μL of CCK-8 reagent was introduced into each well before incubating under 37 °C and 5% carbon dioxide for 2 hours. The OD value of each well was computed at 450 nm by employing a TriStar2 LB 942 Microplate Reader (Berthold Technologies Gmbh&Co.KG, Germany)

Staining test for podosome belt formation

Culturing and treatment of BMMs were done with Ppg (0, 40, and 80) in 96-well plates. As previously described (48), staining, detection, and imaging of mature osteoclasts were done using RGB and DAPI channels with the aid of a Biotek microscope.

Bone pits resorption assay

The control group was set up before placing the labeled bovine bone slices into a 96-well plate. BMMs were placed into the wells at 8×10³ cells/well density. RANKL (50 ng/mL) was used to induce BMMs until tiny osteoclasts started appearing in the control wells. After that, osteoclasts from individual wells were intervened with Ppg (0, 40, and 80 μM) for 48 hours. Bovine bone slices were fixed with 2.5% glutaraldehyde (Solarbio, China) and scanned using scanning electron microscopy (SEM) to determine the resorption area. Furthermore, 4% PFA cells were fixed in control wells for 10 minutes, followed by staining using TRAcP to analyze osteoclast differentiation. Quantification of bone resorption pit regions and TRAcP-positive cells was performed using image J.

LPS-induced calvarial osteolysis mice model

The Laboratory Animal Ethics Committee of Guangxi Medical University granted its approval for all in vivo experiments. The protocol for appropriate care and use of laboratory animals was followed while carrying out these experiments. Twenty five male C57/BL6 mice aged 8 weeks were classified into five groups: sham (PBS injection daily), dexamethasone (DXMS) (alternating LPS 5 mg/kg and DXMS daily), LPS (lipopolysaccharide (LPS) 5 mg/kg alternating and PBS alternately), and Ppg (alternating LPS 5 mg/kg and Ppg 40 and 80 mg/kg). For eight consecutive days, 100 μL of relevant reagent was injected subcutaneously every day around the V-suture of the skull. On day 9, mice were euthanized by cervical dislocation. Subsequently, the skulls were isolated and immobilized using 4% formalin for further analysis.

Micro-CT scanning

Mice skulls immobilized with 4% PFA were scanned at 50 kV and 500 μA using the Skyscan 1176 micro-CT system (Bruker, USA), with additional parameters including a 9 μm pixel size, a 0.5 mm AI filter, and a 180-degree rotation step. The SkyScan NRecon platform was utilized to reconstruct three-dimensional (3D) images, which were then analyzed using SkyScan CTAn software (Bruker).

Histological examination

The mouse skull was subjected to micro-CT scanning and subsequently placed in 14% EDTA for a duration of 21 days. Furthermore, the mouse skull was embedded in paraffin and cut into 5-micron sections before being exposed to various staining techniques including TRAcP activity staining, hematoxylin-eosin (HE) staining, and immunohistochemical staining for further observation and analysis.

Real-time quantitative PCR assay

BMMs were seeded into a 6-well plate at a density of 1.5×10⁵ cells/well, and cells were induced with different Ppg (0, 40, 80 μM) until osteoclasts appeared. The total RNA extraction for individual samples was done by utilizing Trizol. Reverse transcription was conducted to generate cDNA as per the provided instructions, as a template for Real-time quantitative PCR. The following cycle parameters were set on the LightCycler ®96 system (Roche, Switzerland): 94°C for 10 minutes, followed by 45 cycles at 95°C× 15 seconds and 60°C×60 seconds. All data were normalized to β-actin using comparative threshold cycling. The primers utilized in the current investigation are presented in Table 1.

Western blot

BMMs in each well were lysed on ice with radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF), 1% phosphatase, and 1% protease inhibitor for a minimum of 30 minutes. After collecting the lysate, it was subjected to centrifugation at a rate of 12,000 rpm for 10 minutes. The extracted protein was then quantified by utilizing the BCA Protein Assay Kit (Beyotime, China). After mixing the prepared protein sample with sodium dodecyl sulfate (SDS) loading buffer, it was heated at 98 °C for 20 minutes. Moreover, the protein samples were transferred to a nitrocellulose membrane through an electrical transfer process after separating them by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Following the blocking of non-specific binding immune response using 5% skim milk in 1×TBST, the nitrocellulose membrane was soaked in a primary antibody and kept in an incubator at 4 °C with gentle shaking for a period of 12-14 hours. Subsequently, the nitrocellulose membranes were washed thrice with 1×TBST, followed by soaking in the corresponding secondary antibody, and kept at room temperature for 1 hour. Lastly, images of protein bands were captured by employing the Odyssey® DLx Imaging System (LI-COR, Inc., USA) and quantified with image J.

Statistical analysis

Data were presented as mean ± SD for n=3. Statistical analyses were conducted using either one-way ANOVA or Student’s t-test. The significance level was established at P < 0.05.

Acknowledgments

We acknowledge all the people who provided helpful input to this study.

Conflict of Interest

The authors declare no competing interests.

Ethics

Experimental and surgical operations were carried out in accordance with Guangxi Medical University standards and the animal welfare program. The Animal Ethics Committee of Guangxi Medical University (China) granted approval for the animal experiments to be carried out (approval code: 202207003).

Author contributions

Kai Gan: writing-first draft, visualization. Haoyu Lian: data curation. Tao Yang: methodology. Jian Huang: investigation. Junchun Chen: formal analysis. Yuangang Su: software. Jinmin Zhao: project administration, funding acquisition. Jiake Xu: writing-review & editing. Qian Liu: supervision, writing-review & editing, funding acquisition.

Funding

This research was supported by Guangxi Natural Science Foundation (2023GXNSFDA026058) and Guangxi Science and Technology Base and Talent Special Project (Grant No. GuikeAD19254003)

Jin F, Zhu Y, Liu M, Wang R, Cui Y, Wu Y, et al. Babam2 negatively regulates osteoclastogenesis by interacting with Hey1 to inhibit Nfatc1 transcription. International journal of biological sciences. 2022;18(11):4482-96.
Loi F, Córdova LA, Zhang R, Pajarinen J, Lin TH, Goodman SB, et al. The effects of immunomodulation by macrophage subsets on osteogenesis in vitro. Stem cell research & therapy. 2016;7:15.
Luo P, Wang P, Xu J, Hou W, Xu P, Xu K, et al. Immunomodulatory role of T helper cells in rheumatoid arthritis : a comprehensive research review. Bone & joint research. 2022;11(7):426-38.
Zhang H, Li S, Lu J, Jin J, Zhu G, Wang L, et al. α-Cyperone (CYP) down-regulates NF-κB and MAPKs signaling, attenuating inflammation and extracellular matrix degradation in chondrocytes, to ameliorate osteoarthritis in mice. Aging. 2021;13(13):17690-706.
Deng C, Zhang Q, He P, Zhou B, He K, Sun X, et al. Targeted apoptosis of macrophages and osteoclasts in arthritic joints is effective against advanced inflammatory arthritis. Nature communications. 2021;12(1):2174.
Pattison E, Harrison BJ, Griffiths CE, Silman AJ, Bruce IN. Environmental risk factors for the development of psoriatic arthritis: results from a case-control study. Annals of the rheumatic diseases. 2008;67(5):672-6.
Hajishengallis G, Moutsopoulos NM, Hajishengallis E, Chavakis T. Immune and regulatory functions of neutrophils in inflammatory bone loss. Seminars in immunology. 2016;28(2):146-58.
Loi F, Córdova LA, Pajarinen J, Lin TH, Yao Z, Goodman SB. Inflammation, fracture and bone repair. Bone. 2016;86:119-30.
Kavanagh N, Ryan EJ, Widaa A, Sexton G, Fennell J, O'Rourke S, et al. Staphylococcal Osteomyelitis: Disease Progression, Treatment Challenges, and Future Directions. Clinical microbiology reviews. 2018;31(2).
Maruotti N, Grano M, Colucci S, d'Onofrio F, Cantatore FP. Osteoclastogenesis and arthritis. Clinical and experimental medicine. 2011;11(3):137-45.
Boyce BF. Advances in the regulation of osteoclasts and osteoclast functions. Journal of dental research. 2013;92(10):860-7.
Dong M, Zeng J, Yang C, Qiu Y, Wang X. Asiatic Acid Attenuates Osteoporotic Bone Loss in Ovariectomized Mice Through Inhibiting NF-kappaB/MAPK/ Protein Kinase B Signaling Pathway. Frontiers in pharmacology. 2022;13:829741.
Erkhembaatar M, Gu DR, Lee SH, Yang YM, Park S, Muallem S, et al. Lysosomal Ca(2+) Signaling is Essential for Osteoclastogenesis and Bone Remodeling. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2017;32(2):385-96.
Ma Y, Ran D, Zhao H, Song R, Zou H, Gu J, et al. Cadmium exposure triggers osteoporosis in duck via P2X7/PI3K/AKT-mediated osteoblast and osteoclast differentiation. The Science of the total environment. 2021;750:141638.
Yamada T, Yamazaki H, Yamane T, Yoshino M, Okuyama H, Tsuneto M, et al. Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells. Blood. 2003;101(6):2227-34.
Li C, Wang S, Xing Z, Lin A, Liang K, Song J, et al. A ROR1-HER3-lncRNA signalling axis modulates the Hippo-YAP pathway to regulate bone metastasis. Nature cell biology. 2017;19(2):106-19.
Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone. 2007;40(2):251-64.
Cekici A, Kantarci A, Hasturk H, Van Dyke TE. Inflammatory and immune pathways in the pathogenesis of periodontal disease. Periodontology 2000. 2014;64(1):57-80.
Zhou L, Hong G, Li S, Liu Q, Song F, Zhao J, et al. Fangchinoline protects against bone loss in OVX mice via inhibiting osteoclast formation, bone resorption and RANKL-induced signaling. International journal of biological sciences. 2020;16(2):309-19.
Zhang W-J, Song Z-B, Bao Y-L, Li W-L, Yang X-G, Wang Q, et al. Periplogenin induces necroptotic cell death through oxidative stress in HaCaT cells and ameliorates skin lesions in the TPA- and IMQ-induced psoriasis-like mouse models. Biochem Pharmacol. 2016;105:66-79.
Hu Y, Liu F, Jia X, Wang P, Gu T, Liu H, et al. Periplogenin suppresses the growth of esophageal squamous cell carcinoma in vitro and in vivo by targeting STAT3. Oncogene. 2021;40(23):3942-58.
Meng X, Zhang W, Lyu Z, Long T, Wang Y. ZnO nanoparticles attenuate polymer-wear-particle induced inflammatory osteolysis by regulating the MEK-ERK-COX-2 axis. Journal of orthopaedic translation. 2022;34:1-10.
Świerczek A, Jusko WJ. Pharmacokinetic/Pharmacodynamic Modeling of Dexamethasone Anti-Inflammatory and Immunomodulatory Effects in LPS-Challenged Rats: A Model for Cytokine Release Syndrome. The Journal of pharmacology and experimental therapeutics. 2023;384(3):455-72.
Zhang YW, Bao YL, Wu Y, Yu CL, Li YX. 17betaH-Periplogenin, a cardiac aglycone from the root bark of Periploca sepium Bunge. Acta Crystallogr Sect E Struct Rep Online. 2012;68(Pt 6):o1582-3.
Panda S, Kar A. Periplogenin, isolated from Lagenaria siceraria, ameliorates L-T₄-induced hyperthyroidism and associated cardiovascular problems. Horm Metab Res. 2011;43(3):188-93.
He J, Bo F, Tu Y, Azietaku JT, Dou T, Ouyang H, et al. A validated LC-MS/MS assay for the simultaneous determination of periplocin and its two metabolites, periplocymarin and periplogenin in rat plasma: Application to a pharmacokinetic study. J Pharm Biomed Anal. 2015;114:292-5.
Li Y, Zhuang Q, Tao L, Zheng K, Chen S, Yang Y, et al. Urolithin B suppressed osteoclast activation and reduced bone loss of osteoporosis via inhibiting ERK/NF-κB pathway. Cell proliferation. 2022;55(10):e13291.
Qiu J, Jiang T, Yang G, Gong Y, Zhang W, Zheng X, et al. Neratinib exerts dual effects on cartilage degradation and osteoclast production in Osteoarthritis by inhibiting the activation of the MAPK/NF-κB signaling pathways. Biochem Pharmacol. 2022;205:115155.
Xiao L, Zhong M, Huang Y, Zhu J, Tang W, Li D, et al. Puerarin alleviates osteoporosis in the ovariectomy-induced mice by suppressing osteoclastogenesis via inhibition of TRAF6/ROS-dependent MAPK/NF-κB signaling pathways. Aging. 2020;12(21):21706-29.
Liu Q, Wu H, Chim SM, Zhou L, Zhao J, Feng H, et al. SC-514, a selective inhibitor of IKKβ attenuates RANKL-induced osteoclastogenesis and NF-κB activation. Biochem Pharmacol. 2013;86(12):1775-83.
Xu J, Wu HF, Ang ES, Yip K, Woloszyn M, Zheng MH, et al. NF-kappaB modulators in osteolytic bone diseases. Cytokine & growth factor reviews. 2009;20(1):7-17.
Mediero A, Perez-Aso M, Cronstein BN. Activation of adenosine A(2A) receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation. British journal of pharmacology. 2013;169(6):1372-88.
Widmann C, Gibson S, Jarpe MB, Johnson GL. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiological reviews. 1999;79(1):143-80.
Lavoie H, Gagnon J, Therrien M. ERK signalling: a master regulator of cell behaviour, life and fate. Nature reviews Molecular cell biology. 2020;21(10):607-32.
Deng Z, Zhang R, Li M, Wang S, Fu G, Jin J, et al. STAT3/IL-6 dependent induction of inflammatory response in osteoblast and osteoclast formation in nanoscale wear particle-induced aseptic prosthesis loosening. Biomaterials science. 2021;9(4):1291-300.
Lorenzo J. The many ways of osteoclast activation. The Journal of clinical investigation. 2017;127(7):2530-2.
Kang JY, Kang N, Yang YM, Hong JH, Shin DM. The Role of Ca(2+)-NFATc1 Signaling and Its Modulation on Osteoclastogenesis. International journal of molecular sciences. 2020;21(10).
Guo J, Ren R, Sun K, He J, Shao J. PERK signaling pathway in bone metabolism: Friend or foe? Cell proliferation. 2021;54(4):e13011.
Fretz JA, Shevde NK, Singh S, Darnay BG, Pike JW. Receptor activator of nuclear factor-kappaB ligand-induced nuclear factor of activated T cells (C1) autoregulates its own expression in osteoclasts and mediates the up-regulation of tartrate-resistant acid phosphatase. Molecular endocrinology (Baltimore, Md). 2008;22(3):737-50.
Matsuo K, Galson DL, Zhao C, Peng L, Laplace C, Wang KZ, et al. Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos. The Journal of biological chemistry. 2004;279(25):26475-80.
Zhu L, Luo C, Ma C, Kong L, Huang Y, Yang W, et al. Inhibition of the NF-κB pathway and ERK-mediated mitochondrial apoptotic pathway takes part in the mitigative effect of betulinic acid on inflammation and oxidative stress in cyclophosphamide-triggered renal damage of mice. Ecotoxicology and environmental safety. 2022;246:114150.
Rietschel ET, Brade H. Bacterial endotoxins. Scientific American. 1992;267(2):54-61.
Henderson B, Nair SP. Hard labour: bacterial infection of the skeleton. Trends in microbiology. 2003;11(12):570-7.
Hong CY, Lin SK, Kok SH, Cheng SJ, Lee MS, Wang TM, et al. The role of lipopolysaccharide in infectious bone resorption of periapical lesion. Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology. 2004;33(3):162-9.
Liu J, Wang S, Zhang P, Said-Al-Naief N, Michalek SM, Feng X. Molecular mechanism of the bifunctional role of lipopolysaccharide in osteoclastogenesis. The Journal of biological chemistry. 2009;284(18):12512-23.
Islam S, Hassan F, Tumurkhuu G, Dagvadorj J, Koide N, Naiki Y, et al. Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells. Biochemical and biophysical research communications. 2007;360(2):346-51.
Xu J, Tan JW, Huang L, Gao XH, Laird R, Liu D, et al. Cloning, sequencing, and functional characterization of the rat homologue of receptor activator of NF-kappaB ligand. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2000;15(11):2178-86.
Xu Y, Song D, Lin X, Peng H, Su Y, Liang J, et al. Corylifol A protects against ovariectomized-induced bone loss and attenuates RANKL-induced osteoclastogenesis via ROS reduction, ERK inhibition, and NFATc1 activation. Free radical biology & medicine. 2023;196:121-32.

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You are reading this latest preprint version

Periplogenin attenuates LPS-mediated inflammatory osteolysis through the suppression of osteoclastogenesis via reducing the NF-κB and MAPK Signaling Pathways

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Journal Publication

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Abstract

Figures

Introduction

Results

Ppg suppressed RANKL-induced osteoclast differentiation in vitro

Ppg suppressed osteoclast resorption function.

Ppg suppressed LPS-induced inflammatory response in BMMs

Ppg suppressed LPS-induced osteolysis in mice in vivo

Ppg downregulated the expression of genes and proteins specific to osteoclasts during osteoclastic formation.

Ppg inhibited RANKL-induced NF-κB and MAPK pathways in osteoclast differentiation

Discussion

Materials and Methods

Media and reagents

Cell Culture and Osteoclast Differentiation Assay

Cell Viability/Cytotoxicity Assay

Staining test for podosome belt formation

Bone pits resorption assay

LPS-induced calvarial osteolysis mice model

Micro-CT scanning

Histological examination

Real-time quantitative PCR assay

Western blot

Statistical analysis

Declarations

References

Table

Additional Declarations

Supplementary Files

Status:

Journal Publication

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