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Β-Ecdysterone Prevents LPS-Induced Osteoclastogenesis by Regulating NF-Κb Pathway in Vitro

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Β-Ecdysterone Prevents LPS-Induced Osteoclastogenesis by Regulating NF-Κb Pathway in Vitro β-Ecdysterone Prevents LPS-Induced Osteoclastogenesis by Regulating NF-κB Pathway in Vitro Yuling Li Aliated Hospital of North Sichuan Medical College Jing Zhang Aliated Hospital of North Sichuan Medical College Caiping Yan Aliated Hospital of North Sichuan Medical College Qian Chen Aliated Hospital of North Sichuan Medical College Chao Xiang Aliated Hospital of North Sichuan Medical College Qingyan Zhang Aliated Hospital of North Sichuan Medical College Xingkuan Wang Aliated Hospital of North Sichuan Medical College ke jiang ( [email protected] ) Aliated Hospital of North Sichuan Medical College Research article Keywords: β-ecdysterone, lipopolysaccharide, osteoclast, NF-κB Posted Date: November 24th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-112215/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/33 Abstract Background Lipopolysaccharide (LPS), a bacteria product, plays an important role in orthopedic diseases. Drugs that inhibit LPS-induced osteoclastogenesis are urgently needed for the prevention of bone destruction. Methods In this study, we evaluated the effect of β-ecdysterone (β-Ecd), a major component of Chinese herbal medicines derived from the root of Achyranthes bidentata BI on LPS-induced osteoclastogenesis in vitro and explored the mechanism underlying the effects of β-Ecd on this. Results We showed that β-Ecd inhibited LPS-induced osteoclast formation from osteoclast precursor RAW264.7 cells. The inhibition occurred through suppressing the production of osteoclast activating TNF-α, IL-1β, PGE2 and COX-2, which led to down-regulating expression of osteoclast-related genes including RANK, TRAF6, MMP-9, CK and CA. Besides, β-Ecd treatment can inhibit LPS-induced activation of NF-κB signaling pathway in RAW264.7 cells. Meanwhile, inhibition of NF-κB signaling pathway decreased the formation of osteoclasts and expression of pro-inammatory cytokines which LPS-induced. Collectively, β-Ecd can prevent LPS-induced osteoclast formation in vitro by regulating NF-κB signaling pathway. Conclusions These ndings provide evidences that β-Ecd might be benecial as a valuable choice for the prevention and treatment of bacteria-induced bone destruction disease, and give new insights for understanding its possible mechanism. Introduction The osteoclast is a large, multinucleated cell from the monocyte-macrophage lineage [1]. It can degrade bone tissue by secreting H+, Cl−, cathepsin K (CtsK) and matrix metalloproteinase (MMPs) in the resorption area [2] and plays a key physiological role in bone remodeling and a pathological role in bone destruction diseases including osteopetrosis, osteoporosis, rheumatoid arthritis, inammatory osteolysis [3], Paget’s disease, osteopetrosis and pycnodysostosis [4]. Inheritance of osteoclast differentiation from hematopoietic progenitors requires macrophage colony stimulating factor (M-CSF) and receptor activated nuclear factor kappa-B (NF-κB) ligand (RANKL) to monitor differentiation [5-7]. These factors are the key determinants in stimulating the differentiation and activation of osteoclast, and are very crucial in bone remodeling [8]. The lipopolysaccharide (LPS) is a central component of the outer membrane in gram-negative bacteria that plays a key role in host-pathogen interactions with the innate immune system [9]. LPS is a potent Page 2/33 endotoxin and is highly resistant to degradation by mammalian enzymes thus providing a persistent inammatory stimulus [10]. It can cause inammatory bone loss, leading to severe morbidity and large medical expenses and it is also considered to be a key pathogen of inammatory osteolytic diseases such as osteomyelitis, septic arthritis, periodontitis, and infection of orthopedic implants [11]. LPS- induced pro-inammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, prostaglandin E2 (PGE2) and IL-6, can directly stimulate osteoclast differentiation and ultimately lead to the destructive bone loss by means of increasing the expression of receptor activator for nuclear factor- κB ligand (RANKL) [12-14]. Furthermore, osteoclasts arise in the bone marrow from the fusion of haematopoietic cells of a monocyte/macrophage lineage after stimulation by M‐CSF and RANKL [15]. LPS is considered to be an important factor in osteogenesis and osteoclast metabolism disorder in infectious bone diseases, but there are still no relevant reports on how to effectively regulate the local bone growth metabolism disorder caused by LPS from the molecular mechanism, and there is also no effective response in the clinic. So, exploring the molecular mechanism on how to intervene and regulate the impact of LPS on bone metabolism disorders is of great signicance for understanding and treating infectious bone diseases. β-ecdysterone (β-Ecd), a major component of Chinese herbal medicines derived from the root of Achyranthes bidentata BI [16, 17]. β-Ecd has been reported to possess antiapoptotic and anti- inammatory pharmacological effects [16]. Besides, it has been demonstrated to exhibit a number of functions, including anabolic and hepatoprotective effects [18] and some studies have indicated that β‐ecdysterone may increase the synthesis of collagen protein and inhibit cell apoptosis by regulation of autophagy [18-20]. Related studies have shown that multiple signaling pathways involved MAPK [21, 22], Smad/BMP2 [23, 24], Wnt/β-catenin [25, 26], NF-κB [27] and other signaling pathways are involved in the regulation of bone tissue metabolism. Among these pathways, the nuclear factor-κB (NF-κB) signaling pathway is particularly closely related to the regulation of bone metabolism under inammatory conditions, and it plays an important role in regulating osteoclast differentiation, activation and osteoblast activity [28]. Previous studies found that β-Ecd can inhibit LPS-mediated osteoclast differentiation and fusion, but the specic signaling pathway involved is unclear. So we hypothesize that β-Ecd may inhibit LPS-induced osteoclastogenesis by regulating NF-κB pathway in vitro. Indeed, the ndings presented in this study perfectly support the hypothesis. This study provides a reference and direction for the correct evaluation of the pharmacological effects of these natural plant drugs on bone tissue under inammatory conditions and the application of natural plant drugs in the regulation of bone metabolism in infectious bone diseases. Materials And Methods Cell culture and treatment Page 3/33 Osteoclast precursor RAW264.7 cells were obtained from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s Modied Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 100 units/mL penicillin, and 100 μg/mL streptomycin maintained at 37 ℃ with a 5 % CO2 atmosphere. Cultured RAW264.7 cells were pretreated for 1 h with β-Ecd at various concentrations followed by exposure to LPS (100 ng/mL) at indicated times. RAW264.7 cells were used to assess if β- Ecd had toxic effect on them. For inhibitor studies, NF-κB pathway-specic inhibitor was added to cell culture 30 min before treatmented with β-Ecd and maintained throughout the experiment. Assessment of cell viability by CCK-8 assay The assay cell counting kit-8 (CCK-8) was used to measure whether the β-Ecd had toxic effects on the RAW264.7 cells. RAW264.7 cells were cultured to the logarithmic growth phase, and after digestion, 1×105/mL cell suspension was prepared with the pre-warmed medium and inoculated into a 96-well cell culture plate with a volume of 100 μl per well. After the cells were adhered, the original medium was aspirated, and RAW264.7 cells were added at concentrations of 0 μM, 5 μM, 10 μM, 20 μM and 40 μM β- Ecd, PBS as a control. The next day, the CCK-8 solution was added 10 μl per well, and after another 4 hours’ culture, the absorbance of each well was measured at 450 nm. This experiment was in quadruplicate. Osteoclastogenesis assay in vitro To assess whether β-ecdysterone inhibits LPS-induced osteoclast formation, a total of 1×103 RAW264.7 cells/well were seeded in a 96-well plate and treated with or without LPS at a concentration of 100 ng/mL alone or in combination with different β-Ecd at concentrations of 5, 10, 20 and 40 μM. That is to say, the assay included six groups: PBS, LPS, LPS+5 μM β-Ecd, LPS+10 μM β-Ecd, LPS+20 μM β-Ecd and LPS+40 μM β-Ecd. Besides, to assess the effect of inhibiting NF-κB pathways on osteoclast formation in vitro, the assay was changed to four groups containing PBS, LPS, LPS+40 μM β-Ecd and LPS+sulfasalazine (NF- κB inhibitor). Cells were washed twice with phosphate buffered saline (PBS), xed in 4% paraformaldehyde (pH 7.4) at room temperature (r.t.) for 15 min and stained for TRAP using leukocyte Acid Phosphatase Kit 387-A according to the manufacturer’s protocol. TRAP-positive multinucleated cells (MNCs) containing three or more nuclei were counted as osteoclast under a IX70 microscope (Olympus, Japan). Western blot analysis RAW264.7 cells were cultured overnight. After treatment with or without β-Ecd (5, 10, 20 and 40 μM), the cells were stimulated with LPS to detect receptor activator of nuclear factor kappa B (RANK), TNF receptor associated factor 6 (TRAF6), matrix metalloproteinase 9 (MMP-9), cathepsin K (CK), carbonic anhydrsae II (CA) inhibitor of nuclear factor kappa B α (IκBα) or phosphorylation-IκBα (p-IκBα). The cellular lysates were prepared. The above proteins were separated by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically onto polyvinyl diuoride (PVDF)membranes. The blots were blocked with 4% bovine serum albumin (BSA) for 1 h at r.t. Page 4/33 and then probed with rabbit anti-mouse antibodies against RANK, TRAF6, MMP-9, CK, CA, IκBα, p-IκBα and β-actin at 4°C overnight. After washing three times, the blots were subsequently incubated with a goat horseradish peroxidase‐conjugated secondary antibody for 2 h at r.t. After four times washing for 10 min in Tris-buffered saline with Tween-20 (TBST), the membranes were detected using an chemiluminescence detection system.
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