The Influence of Prenatal Fumonisin Exposure on Bone Properties,as well as OPG and RANKL Expression and Immunolocalization,in Newborn Offspring Is Sex and Dose Dependent

The current study examined the effects of exposure of pregnant dams to fumonisins (FBs; FB1 and FB2), from the seventh day of pregnancy to parturition, on offspring bone metabolism and properties. The rats were randomly divided into three groups intoxicated with FBs at either 0, 60, or 90 mg/kg b.w. Body weight and bone length were affected by fumonisin exposure, irrespective of sex or dose, while the negative and harmful effects of maternal FBs’ exposure on bone mechanical resistance were sex and dose dependent. The immunolocalization of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-Β ligand (RANKL), in bone and articular cartilage, indicated that the observed bone effects resulted from the FB-induced alterations in bone metabolism, which were confirmed by the changes observed in the Western blot expression of OPG and RANKL. It was concluded that the negative effects of prenatal FB exposure on the general growth and morphometry of the offspring bones, as a result of the altered expression of proteins responsible for bone metabolism, were dose and sex dependent.     

An oligodeoxynucleotide with promising modulation activity for the proliferation and activation of osteoblast

The paper explored the regulatory role of oligodeoxynucleotides (ODNs) with specific sequences in the proliferation and activation of osteoblast, using human osteoblast-like cell line MG 63 as the model. Through the administration of ODNs to MG 63 cells at a concentration of 1.0 μg/mL, ODN MT01 with positive effects on proliferation and activation of osteoblast was selected from 11 different ODNs by methyl thiazolyl tetrazolium (MTT) assay and alkaline phosphatase (ALP) activity measurement. To get a deeper insight into the molecular mechanism, effects of ODN MT01 treatment on the expression level of Sp7, runx-2, collagen-I, osteoprotegerin (OPG) and RANK ligand (RANKL) were determined using quantitative real time PCR and Western blotting. Remarkably, the mRNA and protein expression levels of Sp7, runx-2, collagen-I and OPG were improved after ODN MT01 treatment. Meanwhile, the protein expression level of RANKL was dramatically decreased. These results suggested that ODN MT01 had a significant impact in facilitating osteogenic proliferation and activation, and provided a direct evidence for the notion that single strand ODN could regulate the balance of bone formation and resorption, and thus was of great potential in the rebuilding of alveolar bone.     

NLRP3 Inflammasome Negatively Regulates RANKL-Induced Osteoclastogenesis of Mouse Bone Marrow Macrophages but Positively Regulates It in the Presence of Lipopolysaccharides

In inflammatory bone diseases such as periodontitis, the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome accelerates bone resorption by promoting proinflammatory cytokine IL-1β production. However, the role of the NLRP3 inflammasome in physiological bone remodeling remains unclear. Here, we investigated its role in osteoclastogenesis in the presence and absence of lipopolysaccharide (LPS), a Gram-negative bacterial component. When bone marrow macrophages (BMMs) were treated with receptor activator of nuclear factor-κB ligand (RANKL) in the presence of NLRP3 inflammasome inhibitors, osteoclast formation was promoted in the absence of LPS but attenuated in its presence. BMMs treated with RANKL and LPS produced IL-1β, and IL-1 receptor antagonist inhibited osteoclastogenesis, indicating IL-1β involvement. BMMs treated with RANKL alone produced no IL-1β but increased reactive oxygen species (ROS) production. A ROS inhibitor suppressed apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) speck formation and NLRP3 inflammasome inhibitors abrogated cytotoxicity in BMMs treated with RANKL, indicating that RANKL induces pyroptotic cell death in BMMs by activating the NLRP3 inflammasome via ROS. This suggests that the NLRP3 inflammasome promotes osteoclastogenesis via IL-1β production under infectious conditions, but suppresses osteoclastogenesis by inducing pyroptosis in osteoclast precursors under physiological conditions.     

Bisphosphonates Reduce Smoking-Induced Osteoporotic-Like Alterations by Regulating RANKL/OPG in an Osteoblast and Osteoclast Co-Culture Model

Co-culture models have become mandatory for obtaining better insights into bone homeostasis, which relies on the balance between osteoblasts and osteoclasts. Cigarette smoking (CS) has been proven to increase the risk of osteoporosis; however, there is currently no proven treatment for osteoporosis in smokers excluding cessation. Bisphosphonates (BPs) are classical anti-osteoclastic drugs that are commonly used in examining the suitability of bone co-culture systems in vitro as well as to verify the response to osteoporotic stimuli. In the present study, we tested the effects of BPs on cigarette smoke extract (CSE)-affected cells in the co-culture of osteoblasts and osteoclasts. Our results showed that BPs were able to reduce CSE-induced osteoporotic alterations in the co-culture of osteoblasts and osteoclasts such as decreased matrix remodeling, enhanced osteoclast activation, and an up-regulated receptor activator of nuclear factor (NF)-kB-ligand (RANKL)/osteoprotegerin (OPG) ratio. In summary, BPs may be an effective alternative therapy for reversing osteoporotic alterations in smokers, and the potential mechanism is through modulation of the RANKL/OPG ratio.     

A Review on the Molecular Mechanisms of Action of Natural Products in Preventing Bone Diseases

The drugs used for treating bone diseases (BDs), at present, elicit hazardous side effects that include certain types of cancers and strokes, hence the ongoing quest for the discovery of alternatives with little or no side effects. Natural products (NPs), mainly of plant origin, have shown compelling promise in the treatments of BDs, with little or no side effects. However, the paucity in knowledge of the mechanisms behind their activities on bone remodeling has remained a hindrance to NPs’ adoption. This review discusses the pathological development of some BDs, the NP-targeted components, and the actions exerted on bone remodeling signaling pathways (e.g., Receptor Activator of Nuclear Factor κ B-ligand (RANKL)/monocyte/macrophage colony-stimulating factor (M-CSF)/osteoprotegerin (OPG), mitogen-activated protein kinase (MAPK)s/c-Jun N-terminal kinase (JNK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), Kelch-like ECH-associated protein 1 (Keap-1)/nuclear factor erythroid 2–related factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1), Bone Morphogenetic Protein 2 (BMP2)-Wnt/β-catenin, PhosphatidylInositol 3-Kinase (PI3K)/protein kinase B (Akt)/Glycogen Synthase Kinase 3 Beta (GSK3β), and other signaling pathways). Although majority of the studies on the osteoprotective properties of NPs against BDs were conducted ex vivo and mostly on animals, the use of NPs for treating human BDs and the prospects for future development remain promising.     

A High Body Mass Index and the Vacuum Phenomenon Upregulate Pain-Related Molecules in Human Degenerated Intervertebral Discs

Animal studies suggest that pain-related-molecule upregulation in degenerated intervertebral discs (IVDs) potentially leads to low back pain (LBP). We hypothesized that IVD mechanical stress and axial loading contribute to discogenic LBP’s pathomechanism. This study aimed to elucidate the relationships among the clinical findings, radiographical findings, and pain-related-molecule expression in human degenerated IVDs. We harvested degenerated-IVD samples from 35 patients during spinal interbody fusion surgery. Pain-related molecules including tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, calcitonin gene-related peptide (CGRP), microsomal prostaglandin E synthase-1 (mPGES1), and nerve growth factor (NGF) were determined. We also recorded preoperative clinical findings including body mass index (BMI), Oswestry Disability Index (ODI), and radiographical findings including the vacuum phenomenon (VP) and spinal instability. Furthermore, we compared pain-related-molecule expression between the VP (−) and (+) groups. BMI was significantly correlated with the ODI, CGRP, and mPGES-1 levels. In the VP (+) group, mPGES-1 levels were significantly higher than in the VP (−) group. Additionally, CGRP and mPGES-1 were significantly correlated. Axial loading and mechanical stress correlated with CGRP and mPGES-1 expression and not with inflammatory cytokine or NGF expression. Therefore, axial loading and mechanical stress upregulate CGRP and mPGES-1 in human degenerated IVDs, potentially leading to chronic discogenic LBP.     

An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis

To investigate the effect of oligodeoxynucleotides (ODNs) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts, in order to find a candidate ODN with potential for the treatment of periodontitis, a series of ODNs were designed and selected to test their effect on the promotion of the differentiation of BMSCs to osteoblasts in vitro and on the repair of periodontal tissue in rats with periodontitis. It was found that MT01, one of the ODNs with the sequences of human mitochondrial DNA, stimulated the proliferation of BMSCs, the differentiation of BMSCs to osteoblasts and mRNA expression of bone-associated factors including Runx2, Osterix, OPG, RANKL and collagen I in vitro. In vivo study showed that MT01 prevented the loss of alveolar bone in the rats with periodontitis and induced the production of proteins of OPG and Osterix in the bone tissue. These results indicated that MT01 could induce differentiation of BMSCs to osteoblasts and inhibit the alveolar bone absorption in rats with periodontitis.     

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.     

VIP/VPAC Axis Expression in Immune-Mediated Inflammatory Disorders: Associated miRNA Signatures

Few studies have considered immune-mediated inflammatory disorders (IMID) together, which is necessary to adequately understand them given they share common mechanisms. Our goal was to investigate the expression of vasoactive intestinal peptide (VIP) and its receptors VPAC1 and VPAC2 in selected IMID, analyze the effect of biological therapies on them, and identify miRNA signatures associated with their expression. Serum VIP levels and mRNA of VPAC and miRNA expression in peripheral blood mononuclear cells were analyzed from 52 patients with psoriasis, rheumatoid arthritis, Graves’ disease, or spondyloarthritis and from 38 healthy subjects. IMID patients showed higher levels of VIP and increased expression of VPAC2 compared to controls (p < 0.0001 and p < 0.0192, respectively). Receiver operating characteristic curve analysis showed that the levels of VIP or VPAC2 expression were adequate discriminators capable of identifying IMID. Treatment of IMID patients with anti-TNFα and anti-IL12/23 significantly affected serum VIP levels. We identified miRNA signatures associated with levels of serum VIP and VPAC2 expression, which correlated with IMID diagnosis of the patients. The results indicate that the expression of VIP/VPAC2 is able of identify IMIDs and open up a line of research based on the association between the VIP/VPAC axis and miRNA signatures in immune-mediated diseases.     

Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation.     

Calycosin Suppresses RANKL-Mediated Osteoclastogenesis through Inhibition of MAPKs and NF-κB

Calycosin, an isoflavonoid phytoestrogen, isolated from Radix Astragali, was reported to possess anti-tumor, anti-inflammation, and osteogenic properties, but its impact on osteoclast differentiation remains unclear. In this study, we examined the effects of calycosin on osteoclastogenesis induced by RANKL. The results showed that calycosin significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). Calycosin also dose-dependently suppressed the formation of bone resorption pits by mature osteoclasts. In addition, the expression of osteoclatogenesis-related genes, including cathepsin K (CtsK), tartrate-resistant acid phosphatase (TRAP), and MMP-9, was significantly inhibited by calycosin. Furthermore, the results indicated that calycosin down-regulated the expression levels of NFATc1 and c-Fos through suppressing the activation of NF-κB and MAPKs. Our results indicate that calycosin has an inhibitory role in the bone loss by preventing osteoclast formation, as well as its bone resorptive activity. Therefore, calycosin may be useful as a therapeutic reagent for bone loss-associated diseases.     

全长PTH(1-34)-HSA融合蛋白在毕赤酵母中的表达及其活性

目的在毕赤酵母中表达全长甲状旁腺激素(Parathyroid hormone,PTH)(1-34)与人血清白蛋白(Human serumalbumn,HSA)的融合蛋白PTH(1-34)-HSA,并检测其活性。方法在质粒pPIC9K-PTH(1-34)2-HSA的基础上设计引物,引入6His标签及肠激酶酶切位点,从该质粒中扩增6His-EK-PTH(1-34)-HSA基因,插入pPIC9K载体中,构建重组表达质粒pPIC9K-6His-EK-PTH(1-34)-HSA,转化毕赤酵母GS115,甲醇诱导表达;表达的6His-PTH(1-34)-HSA融合蛋白通过两步亲和层析纯化和肠激酶酶切,获得全长的PTH(1-34)-HSA融合蛋白;检测融合蛋白对UAMS-32P成骨细胞增殖活力、碱性磷酸酶活性以及RANKL和OPG基因转录水平的影响。结果重组表达质粒经测序证实构建正确;表达的6His-PTH(1-34)-HSA融合蛋白相对分子质量约70 000,可与PTH抗体和HSA抗体特异性结合,发酵获得的融合蛋白的浓度为200 mg/L;纯化的融合蛋白可与抗PTH、HSA和6His标签抗体特异性结合,经肠激酶酶切后,该蛋白失去了与6His抗体反应的能力,保持了与PTH和HSA抗体结合的能力;酶切后的全长PTH(1-34)-HSA融合蛋白保持了PTH的生物活性,能促进UAMS-32P成骨细胞的增殖活力,提高细胞的碱性磷酸酶的活性,上调细胞RANKL基因的转录水平以及抑制OPG基因的转录水平。结论成功在毕赤酵母中表达了全长PTH(1-34)-HSA融合蛋白,有望提高PTH的成药性。     

Osteoporosis Due to Hormone Imbalance: An Overview of the Effects of Estrogen Deficiency and Glucocorticoid Overuse on Bone Turnover

Osteoporosis is a serious health issue among aging postmenopausal women. The majority of postmenopausal women with osteoporosis have bone loss related to estrogen deficiency. The rapid bone loss results from an increase in bone turnover with an imbalance between bone resorption and bone formation. Osteoporosis can also result from excessive glucocorticoid usage, which induces bone demineralization with significant changes of spatial heterogeneities of bone at microscale, indicating potential risk of fracture. This review is a summary of current literature about the molecular mechanisms of actions, the risk factors, and treatment of estrogen deficiency related osteoporosis (EDOP) and glucocorticoid induced osteoporosis (GIOP). Estrogen binds with estrogen receptor to promote the expression of osteoprotegerin (OPG), and to suppress the action of nuclear factor-κβ ligand (RANKL), thus inhibiting osteoclast formation and bone resorptive activity. It can also activate Wnt/β-catenin signaling to increase osteogenesis, and upregulate BMP signaling to promote mesenchymal stem cell differentiation from pre-osteoblasts to osteoblasts, rather than adipocytes. The lack of estrogen will alter the expression of estrogen target genes, increasing the secretion of IL-1, IL-6, and tumor necrosis factor (TNF). On the other hand, excessive glucocorticoids interfere the canonical BMP pathway and inhibit Wnt protein production, causing mesenchymal progenitor cells to differentiate toward adipocytes rather than osteoblasts. It can also increase RANKL/OPG ratio to promote bone resorption by enhancing the maturation and activation of osteoclast. Moreover, excess glucocorticoids are associated with osteoblast and osteocyte apoptosis, resulting in declined bone formation. The main focuses of treatment for EDOP and GIOP are somewhat different. Avoiding excessive glucocorticoid use is mandatory in patients with GIOP. In contrast, appropriate estrogen supplement is deemed the primary treatment for females with EDOP of various causes. Other pharmacological treatments include bisphosphonate, teriparatide, and RANKL inhibitors. Nevertheless, more detailed actions of EDOP and GIOP along with the safety and effectiveness of medications for treating osteoporosis warrant further investigation.     

Finely-Tuned Calcium Oscillations in Osteoclast Differentiation and Bone Resorption

Calcium (Ca²⁺) plays an important role in regulating the differentiation and function of osteoclasts. Calcium oscillations (Ca oscillations) are well-known phenomena in receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption via calcineurin. Many modifiers are involved in the fine-tuning of Ca oscillations in osteoclasts. In addition to macrophage colony-stimulating factors (M-CSF; CSF-1) and RANKL, costimulatory signaling by immunoreceptor tyrosine-based activation motif-harboring adaptors is important for Ca oscillation generation and osteoclast differentiation. DNAX-activating protein of 12 kD is always necessary for osteoclastogenesis. In contrast, Fc receptor gamma (FcRγ) works as a key controller of osteoclastogenesis especially in inflammatory situation. FcRγ has a cofactor in fine-tuning of Ca oscillations. Some calcium channels and transporters are also necessary for Ca oscillations. Transient receptor potential (TRP) channels are well-known environmental sensors, and TRP vanilloid channels play an important role in osteoclastogenesis. Lysosomes, mitochondria, and endoplasmic reticulum (ER) are typical organelles for intracellular Ca²⁺ storage. Ryanodine receptor, inositol trisphosphate receptor, and sarco/endoplasmic reticulum Ca²⁺ ATPase on the ER modulate Ca oscillations. Research on Ca oscillations in osteoclasts has still many problems. Surprisingly, there is no objective definition of Ca oscillations. Causality between Ca oscillations and osteoclast differentiation and/or function remains to be examined.     

Lutein Prevents High Fat Diet‐Induced Atherosclerosis in ApoE‐Deficient Mice by Inhibiting NADPH Oxidase and Increasing PPAR Expression

Epidemiological and experimental studies provide supportive evidence that lutein, a major carotenoid, may act as a chemopreventive agent against atherosclerosis, although the underlying molecular mechanisms are not well understood. The main aim of this study was to investigate the effects of lutein on the alleviation of atherosclerosis and its molecular mechanisms involved in oxidative stress and lipid metabolism. Male apolipoprotein E knockout mice (n = 55) were fed either a normal chow diet or a high fat diet (HFD) supplemented with or without lutein for 24 weeks. The results showed that a HFD induced atherosclerosis formation, lipid metabolism disorders and oxidative stress, but noticeable improvements were observed in the lutein treated group. Additionally, lutein supplementation reversed the decreased protein expression of aortic heme oxygenase‐1 and increased the mRNA and protein expressions of aortic nicotinamide‐adenine dinucleotide phosphate oxidase stimulated by a HFD. Furthermore, the decreased mRNA and protein expression levels of hepatic peroxisome proliferator‐activated receptor‐α, carnitine palmitoyltransferase 1A, acyl CoA oxidase 1, low density lipoprotein receptors and scavenger receptor class B type I observed in mice with atherosclerosis were markedly enhanced after treatment with lutein. Taken together, these data add new evidence supporting the anti‐atherogenic properties of lutein and describing its mechanisms of action in atherosclerosis prevention, including oxidative stress and lipid metabolism improvements.     

Is Inflammation a Friend or Foe for Orthodontic Treatment?: Inflammation in Orthodontically Induced Inflammatory Root Resorption and Accelerating Tooth Movement

The aim of this paper is to provide a review on the role of inflammation in orthodontically induced inflammatory root resorption (OIIRR) and accelerating orthodontic tooth movement (AOTM) in orthodontic treatment. Orthodontic tooth movement (OTM) is stimulated by remodeling of the periodontal ligament (PDL) and alveolar bone. These remodeling activities and tooth displacement are involved in the occurrence of an inflammatory process in the periodontium, in response to orthodontic forces. Inflammatory mediators such as prostaglandins (PGs), interleukins (Ils; IL-1, -6, -17), the tumor necrosis factor (TNF)-α superfamily, and receptor activator of nuclear factor (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) are increased in the PDL during OTM. OIIRR is one of the accidental symptoms, and inflammatory mediators have been detected in resorbed roots, PDL, and alveolar bone exposed to heavy orthodontic force. Therefore, these inflammatory mediators are involved with the occurrence of OIIRR during orthodontic tooth movement. On the contrary, regional accelerating phenomenon (RAP) occurs after fractures and surgery such as osteotomies or bone grafting, and bone healing is accelerated by increasing osteoclasts and osteoblasts. Recently, tooth movement after surgical procedures such as corticotomy, corticision, piezocision, and micro-osteoperforation might be accelerated by RAP, which increases the bone metabolism. Therefore, inflammation may be involved in accelerated OTM (AOTM). The knowledge of inflammation during orthodontic treatment could be used in preventing OIIRR and AOTM.