Is RANKL a potential molecular target in osteoarthritis?

OBJECTIVE: Osteoarthritis (OA) is a disease of joints, in which the bone under the articular cartilage undergoes increased remodelling activity. The question is whether a better understanding of the causes and mechanisms of bone remodelling can predict disease-modifying treatments. DESIGN: This review summarises the current understanding of the aetiology of OA, with an emphasis on events in the subchondral bone (SCB), and the cells and cytokines involved, to seek an answer to this question. RESULTS: SCB remodelling across OA changes the microstructure of the SCB, which alters the load-bearing properties of the joint and seems to have an important role in the initiation and progression of OA. Bone remodelling is tightly controlled by numerous cytokines, of which Receptor Activator of NFκB ligand (RANKL) and osteoprotegerin are central factors in almost all known bone conditions. In terms of finding therapeutic options for OA, an important question is whether controlling the rate of SCB remodelling would be beneficial. The role of RANKL in the pathogenesis and progression of OA and the effect of its neutralisation remain to be clarified. CONCLUSIONS: This review further makes the case for SCB remodelling as important in OA and for additional study of RANKL in OA, both its pathophysiological role and its potential as an OA disease target.

Secukinumab and Black Garlic Downregulate OPG/RANK/RANKL Axis and Devitalize Myocardial Interstitial Fibrosis Induced by Sunitinib in Experimental Rats.

Sunitinib has been associated with several cardiotoxic effects such as cardiac fibrosis. The present study was designed to explore the role of interleukin (IL)-17 in sunitinib-induced myocardial fibrosis (MF) in rats and whether its neutralization and/or administration of black garlic (BG), a form of fermented raw garlic (Allium sativum L.), could extenuate this adverse effect. Male Wistar albino rats received sunitinib (25 mg/kg three times a week, orally) and were co-treated with secukinumab (3 mg/kg, subcutaneously, three times total) and/or BG (300 mg/kg/day, orally) for four weeks. Administration of sunitinib induced significant increase in cardiac index, cardiac inflammatory markers, and cardiac dysfunction that were ameliorated by both secukinumab and BG, and to a preferable extent, with the combined treatment. Histological examination revealed disruption in the myocardial architecture and interstitial fibrosis in cardiac sections of the sunitinib group, which were reversed by both secukinumab and BG treatments. Both drugs and their co-administration restored normal cardiac functions, downregulated cardiac inflammatory cytokines, mainly IL-17 and NF-κB, along with increasing the MMP1/TIMP1 ratio. Additionally, they attenuated sunitinib-induced upregulation of the OPG/RANK/RANKL axis. These findings highlight another new mechanism through which sunitinib can induce interstitial MF. The current results propose that neutralizing IL-17 by secukinumab and/or supplementation with BG can be a promising therapeutic approach for ameliorating sunitinib-induced MF.

Equol exerts a protective effect on postmenopausal osteoporosis by upregulating OPG/RANKL pathway.

BACKGROUD: Estrogen deficiency is the leading cause of postmenopausal osteoporosis(PMOP) and phytoestrogens soy isoflavones (SI) have been shown to improve PMOP. Equol (Eq), an in vivo metabolite of phytoestrogens soy isoflavones (SI), has a more stable structure and stronger biological activity than its parent compound and has the greatest estrogenic activity. However, there are few studies on the therapeutic effect of Eq on PMOP. PURPOSE: To explore the therapeutic effect and mechanisms of Eq on POMP. METHODS: Osteoblast-like cells ROS1728 were cultured with different doses of Eq, estradiol (E2), separately. The effect of Eq on the proliferation, apoptosis, cell cycle of osteoblasts were detected by CCK-8 and flow cytometry, and the expression of OPG/RANK/RANKL signaling pathway of osteoblasts was detected by Quantitative real-time PCR (qRT-PCR) and Western blot (WB), and RNA silencing technology were carried out to explore the receptors through which Eq plays a role. Then PMOP rat model was established and treated by Eq or E2 to further verification of the effect and mechanism of Eq on PMOP. RESULT: Eq promoted the proliferation and inhibited the apoptosis of osteoblasts and increased the proportion of osteoblasts in the S phase and G2/M phase in a dose-dependent manner. Mechanistically, Eq treatment upregulated the expression of OPG and OPG/RANKL ratio in osteoblasts and this regulatory effect was mainly mediated through the ERß receptor. Furthermore, in vivo study, Eq improved microstructure and BMD of the femur of PMOP rat model, which imitated the osteoprotective effect of E2. Moreover, the Eq or E2 treatment increased serum levels of Ca, 1,25(OH)2D3, bone Gla-protein(BGP), and Type I procollagen (PC1), and reduced serum levels of phosphorus (P), parathyroid hormone(PTH), pyridinol (PYD), tartrate-resistant acid phosphatase (TRAP) and urinary level of deoxypyridinoline (DPD) in the treatment OVX group compared with the untreated OVX group. Meanwhile, Eq or E2 markedly induced the mRNA and protein expression of OPG and OPG/RANKL ratio. CONCLUSION: Eq can combine with ERß and exert a protective effect on PMOP by upregulating OPG/RANKL pathway.

Positive effect of compound amino acid chelated calcium from the shell and skirt of scallop in an ovariectomized rat model of postmenopausal osteoporosis.

BACKGROUND: Osteoporosis has become an important public health issue with the increase of aging population, and afflicts millions of people worldwide, particularly elderly or postmenopausal women. In the present study, we prepared compound amino acid chelated calcium (CAA-Ca) from processing by-products of Chlamys farreri, and evaluated its effect on postmenopausal osteoporosis with an ovariectomized (OVX) rat model. RESULTS: A 60-day treatment of OVX rats with CAA-Ca significantly enhanced the bone mineral density (BMD) and the bone calcium content. Meanwhile, some bone morphometric parameters, trabecular bone number (Tb.N), trabecular bone volume fraction (BV/TV), trabecular bone thickness (Tb.Th) and cortical bone wall thickness (Ct.Th), were also increased by 8.20%, 118.18%, 32.99% and 19.10%, respectively. In addition, the alkaline phosphatase (ALP) levels in serum were significantly reduced after CAA-Ca treatment, while the blood calcium levels were increased. Mechanistically, CAA-Ca down-regulated the levels of receptor activator of nuclear factor-κB (RANK) and receptor activator of nuclear factor-κB ligand (RANKL), and up-regulated osteoprotegerin (OPG) levels in osteoclasts, inhibiting bone resorption and bone loss. Meanwhile, CAA-Ca treatment raised ß-catenin levels and lowered Dickkopf1 (DKK1) levels in the Wnt signaling pathway of osteoblasts, which can promote calcium absorption and bone formation. CONCLUSION: The results suggested that CAA-Ca promoted bone formation, inhibited bone resorption and improved bone microstructure. Therefore, this study contributes to the potential application of CAA-Ca as a functional food resource in the treatment of postmenopausal osteoporosis. © 2021 Society of Chemical Industry.

LncRNA NORAD promotes bone marrow stem cell differentiation and proliferation by targeting miR-26a-5p in steroid-induced osteonecrosis of the femoral head.

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is a devastating orthopedic disease, which seriously affects the quality of life of patients. The study aims to investigate the effects of LncRNA NORAD on SONFH. METHODS: Human bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated from the proximal femur of patients during routine orthopedic surgery and then cultured with dexamethasone (Dex) and transfected with NORAD overexpression vector, siRNA-NORAD and miR-26a-5p mimics. The mRNA expression of NORAD, miR-26a-5p, OPG, RANK, and RANKL was detected by RT-qPCR. Cell proliferation and apoptosis was measured by CCK-8 assay and flow cytometry, respectively. The protein expression of RUNX2, OPG, RANK, and RANKL was detected by western blot. The dual-luciferase reporter gene assay was performed to confirm the binding between NORAD and miR-26a-5p. RESULTS: NORAD expression was downregulated in SONFH tissues, while miR-26a-5p expression was upregulated. Overexpression of NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis in hBMSCs, while knockdown of NORAD led to the opposite results. Moreover, NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis by regulation of miR-26a-5p in hBMSCs. CONCLUSIONS: NORAD expression was downregulated in SONFH tissues, while miR-26a-5p expression was upregulated. NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis by regulation of miR-26a-5p in hBMSCs.

Oral Exposure to ZnO Nanoparticles Disrupt the Structure of Bone in Young Rats via the OPG/RANK/RANKL/IGF-1 Pathway.

PURPOSE: To evaluate the effects of ZnO NPs on bone growth in rats and explore the possible mechanisms of action. MATERIALS AND METHODS: Three-week-old male rats received ultrapure water or 68, 203, and 610 mg/kg zinc oxide nanoparticles (ZnO NPs) for 28 days, orally. RESULTS: The high-dosage groups caused significant differences in weight growth rate, body length, and tibia length (P<0.05), all decreasing with increased ZnO NP dosage. There were no significant differences in body mass index (BMI) (P>0.05). The zinc concentration in liver and bone tissue increased significantly with increased ZnO NP dosage (P<0.05). Clearly increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were observed in the 610 mg/kg ZnO NP group (P>0.05), whereas alkaline phosphatase (ALP) increased in the 610 mg/kg ZnO NP group (P<0.05). Significant differences in insulin-like growth factor type 1 (IGF-1) levels and a decrease in calcium (Ca) levels were observed in 203 and 610 mg/kg ZnO NP groups (P<0.05). Phosphorus (P) levels increased and the Ca/P ratio decreased in the 610 mg/kg ZnO NP group (P<0.05). Micro-computed tomography (micro-CT) of the tibia demonstrated signs of osteoporosis, such as decreased bone density, little trabecular bone structure and reduced cortical bone thickness. Micro-CT data further demonstrated significantly decreased bone mineral density (BMD), trabecular number (Tb.N), and relative bone volume (BV/TV) with increasing dosage of ZnO NPs. Osteoprotegerin (OPG) expression and the ratio of OPG to receptor activator of nuclear factor-κB ligand (RANKL) were statistically lower in the 610 mg/kg ZnO NP group (P<0.05), whereas RANKL expression did not change significantly (P>0.05). CONCLUSION: We infer that ZnO NPs affect bone growth in young rats directly or indirectly by altering IGF-1 levels. Overall, the results indicate that ZnO NPs promote osteoclast activity and increase bone loss through the OPG/RANK/RANKL/IGF-1 pathway.

Therapeutic effects of triptolide from Tripterygium wilfordii Hook. f. on interleukin-1-beta-induced osteoarthritis in rats.

Osteoarthritis (OA) is a common yet destructive disease affecting the articular cartilage, and is a major cause of immense suffering and disability for millions of people. Previous studies have shown that triptolide (TPL), an active compound derived from Tripterygium wilfordii, has potent immunosuppressive and anti-inflammatory activities useful for treating chronic diseases. However, whether TPL has immunosuppressive activity against OA is not known. In this study, we assessed the therapeutic effects of TPL on interleukin-1-beta (IL-1ß)-induced OA in rats. Histological and protein analyses revealed that TPL not only could inhibit interleukin-6 (IL-6) and cyclooxygenase-2 (COX2) protein expression in cells and disrupt inflammation, but it also reduced the expression of matrix metalloproteinase (MMP)-3 and 13. Our results also supported the ability of TPL to suppress the osteoprotegerin/receptor activator of nuclear factor kappa-beta (NF-κB)/receptor activator of NF-κB ligand (OPG/RANK/RANKL) and NF-κB signaling pathways induced by IL-1ß. Together these data suggest that TPL may be a potentially valuable treatment for OA, regulating associated inflammation and pain.

Proper mechanical stress promotes femoral head recovery from steroid-induced osteonecrosis in rats through the OPG/RANK/RANKL system.

BACKGROUND: Long-term use of steroid may lead to osteonecrosis of the femoral head (ONFH). Mechanical stress may help bone formation and remodeling. This study aimed to probe the role of mechanical stress in the femoral head recovery in rats. METHODS: Rat models with ONFH were induced by steroid. Rats were subjected to different levels of mechanical stress (weight-bearing training), and then the morphology and bone density of femoral head of rats were measured. The mRNA and protein levels of the OPG/RANK/RANKL axis in rat femoral head were assessed. Gain- and loss-of function experiments of OPG were performed to identify its role in femoral head recovery following stress implement. The ex vivo cells were extracted and the effects of stress and OPG on osteogenesis in vitro were explored. RESULTS: Steroid-induced ONFH rats showed decreased bone density and increased bone spaces, as well as necrotic cell colonies and many cavities in the cortical bones and trabeculars. Proper mechanical stress or upregulation of OPG led to decreased RANK/RANKL expression and promoted femoral head recovery from steroid-induced osteonecrosis. However, excessive mechanical stress might impose too much load on the femurs thus leading even retard femoral head recovery process. In addition, the in vitro experimental results supported that proper stress and overexpression of OPG increased the osteogenesis of ex vivo cells of femoral head. CONCLUSION: This study provided evidence that proper mechanical stress promoted femoral head recovery from steroid-induced osteonecrosis through the OPG/RANK/RANKL system, while overload might inhibit the recovery process. This study may offer novel insights for ONFH treatment.

Effects of dietary calcium to available phosphorus ratios on bone metabolism and osteoclast activity of the OPG /RANK/RANKL signalling pathway in piglets.

Hydroxyapatite, a mineral form of calcium (Ca) and phosphorus (P) that gives bones their rigidity, is the major and essential component of bones and teeth in the human and animal body. A suitable ratio of Ca and P is vital for bone growth. The aim of this study was to explore the effects of dietary calcium to available phosphorus ratios (Ca/AP) on bone metabolism and osteoclast activity of the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL) signalling pathway in piglets. At days 15 and 29, the piglets were assessed for growth performance, blood indicators, cytokines and the OPG/RANK/RANKL signalling pathway. Our results showed that piglets fed a dietary Ca/AP ratio of 2:1 increases growth performance and regulates blood indicators and cytokines (parathyroid hormone (PTH), calcitonin (CT), vitamin D3 (VD3 ), insulin-like growth factor-1 (IGF-1), transforming growth factor-ß (TGF-ß), interleukin-1 (IL-1), interleukin-6 (IL-6), carboxyterminal propeptide of type I procollagen (PICP), tartrate-resistant acid phosphatase (TRACP), alkaline phosphatase (ALP) and osteocalcin (OCN) content). We also demonstrated that this ratio affects hormone secretion and further bone metabolism through the OPG/RANK/RANKL signalling pathway of osteoclasts. These results indicate that a suitable dietary Ca/AP ratio is vital for bone growth and reduce the incidence of bone diseases such as osteoporosis, providing a practical basis for the raising of piglets.

[Electroacupuncture Intervention Improves Cartilage Degeneration and Subchondral Bone Osteoporosis of Knee-joint Possibly by Adjusting OPG/RANK/RANKL Signaling in Ovariectomized Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on histopathological changes of cartilage and subchondral bone and osteoprotegerin (OPG)，receptor activator of nuclear factor-κB (RANK), and RANK ligand (RANKL) (OPG/RANK/RANKL) signaling and the expression of matrix metalloproteinase-13 (MMP-13) in ovariectomized(OVX)rats, so as to explore its mechanism underlying improvement of osteoporosis. METHODS: Three-month female SD rats were randomly divided into sham operation, model and EA groups (n＝8 in each group). The ovoariectomy model was established by resection of bilateral ovaries. Rats of the sham group were treated by simple removal of a piece of adipose tissue around the bilateral ovaries. EA (3 Hz/15 Hz, 1 mA) was applied to bilateral "Sanyinjiao" (SP 6), "Yanglingquan" (GB 34), "Yinlingquan" (SP 9) and "Zusanli" (ST 36) for 30 min, once daily (except the weekends) for 12 weeks. The histopathological changes of the subchondral bone of the right knee-joint were observed after Saffron O dyeing and evaluated by Mankin's score, and its anatomical structure including the bone volume fraction (bone volume/tissue volume, BV/TV), trabecular bone number (Tb. N) and trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) was observed by using Micro CT imaging. The urine C-terminal cross-linking telopeptide of type Ⅰ collagen (CTX-Ⅰ), CTX-Ⅱ (two bone resorption markers) and serum estrogen (E 2) contents were assayed by ELISA, and the expression levels of OPG, RANKL and MMP-13 mRNAs in the cartilage tissue of the left knee-joint were detected by quantitative RT-PCR. RESULTS: Following modeling, the BV/TV, Tb. N and Tb.Th levels were significantly decreased (P<0.01) and Tb.Sp and Mankin's score obviously increased in the model group compared with the sham group (P<0.01), suggesting a formation of osteoporosis and degeneration of the cartilage tissue. The serum E 2 content and OPG mRNA level in the cartilagetissue were significantly down-regulated (P<0.01), and urine CTX-Ⅰ and CTX-Ⅱ contents and RANKL mRNA and MMP-13 mRNA expression levels cartilagetissue were considerably up-regulated in the model group relevant to the sham group (P<0.01). After EA intervention, modeling-induced decrease of BV/TV, Tb.N, Tb.Th, E 2 and OPG mRNA levels and OVX-induced increase of Tb.Sp, Mankin's score, CTX-Ⅰ， CTX-Ⅱ， RANKL mRNA and MMP-13 mRNA levels were all completely reversed in the EA group relevant to the model group (P<0.01，P<0.05)．. CONCLUSION: EA intervention can inhibit subchondral bone osteoporosis and articular cartilage degeneration of knee-joint in OVX rats, which is closely associated with its effects in inhibiting the down-regulation of serum E 2 and OPG mRNA expression and up-regulation of CTX-Ⅰ， CTX-Ⅱ， RANKL mRNA and MMP-13 mRNA levels, including adjusting OPG/RANK/RANKL signaling.

(5R)-5-hydroxytriptolide (LLDT-8) prevents collagen-induced arthritis through OPG/RANK/RANKL signaling in a rat model of rheumatoid arthritis.

(5R)-5-hydroxytriptolide (LLDT-8) extracts from Tripterygium have anti-inflammatory, antineoplastic and immunity adjustment functions. The present study used a collagen-induced arthritis (CIA) model to evaluate whether LLDT-8 prevents collagen-induced arthritis, and investigated the signaling underlying this. Male Sprague-Dawley rats were induced to generate CIA, mimicking rheumatoid arthritis (RA). The presence of arthritis was determined using RA progression scores. The inflammatory cytokines interleukin (IL)-1ß, IL-6 and nuclear factor-κB were detected using enzyme-linked immunosorbent assay kits. Induced nitric oxide synthase (iNOS) and matrix metalloprotease (MMP)-13 protein expression were measured using western blot analysis. Lastly, reverse transcription-quantitative polymerase chain reaction was used to evaluate osteoprotegerin (OPG) and receptor activator of nuclear factor κB (RANK) gene expression. LLDT-8 improved RA progression scores and reduced the incidence and severity of CIA. Furthermore, LLDT-8 administration inhibited collagen-induced inflammation and iNOS protein expression in arthritic rats. The current data indicated that MMP-13 production was suppressed and OPG/RANKL expression was increased by LLDT-8 treatment in the arthritic rat. The present results suggest that LLDT-8 attenuates CIA through OPG/RANK/RANK ligand signaling in a rat model of RA.

The influence of corticosteroid treatment on the OPG/RANK/RANKL pathway and osteocalcin in patients with pemphigus.

INTRODUCTION: Pemphigus is a rare autoimmune blistering disease, which requires prolonged administration of corticosteroids at high doses. Although this therapy improves the health and lives of patients, it may have various side effects, for example osteoporosis. AIM: To assess the concentration of osteoprotegerin (OPG), the soluble receptor activator of nuclear factor-κß ligand (sRANKL) and osteocalcin in patients with pemphigus. MATERIAL AND METHODS: The study comprised a group of 29 patients with pemphigus (17 women and 12 men) aged between 23 years and 75 years treated from 1994 to 2009 in the Department of Dermatology and Venereology, Medical University of Lodz, as well as 24 healthy volunteers matched appropriately in terms of gender and age. RESULTS: In patients with pemphigus, the mean osteoprotegerin concentration was up to 16.46% higher than in the control group. The average RANKL concentration in serum of patients with pemphigus was 26.88% higher. However, the patient group demonstrated a significantly lower concentration of osteocalcin by up to 18.03%. CONCLUSIONS: Under corticosteroid treatment, RANKL, which is released by osteoblasts, links with the RANK specific osteoclast receptor and stimulates osteoclastogenesis. This reaction can be blocked by osteoprotegerin, which is a competitive inhibitor to the same receptor site. A decreased osteoblast activity stimulates bone loss. The reduced level of osteocalcin, which is regarded as a marker for bone formation, and a simultaneously elevated RANK level reveal the promotion of osteoclast proliferation in patients treated with corticosteroids.

Biochemical markers of bone metabolism in children with cow's milk allergy.

INTRODUCTION: Patients with cow's milk allergy (CMA) and following a cow milk protein-free diet for a long time are potentially at risk of developing bone abnormalities. To assess the balance between bone formation and resorption processes, we determined serum concentrations of osteocalcin (OC), bone alkaline phosphatase (BALP), C-terminal telopeptide of type I collagen (CTX), fetuin-A, osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in children with CMA. MATERIAL AND METHODS: The study included 50 prepubertal children with diagnosed cow's milk allergy, who were under systematic medical and nutritional care at the Institute of Mother and Child and 40 healthy counterparts as a control group. The concentrations of bone metabolism markers were determined by immunoenzymatic assays. RESULTS: The diets of all investigated children were correct in terms of phosphorus and magnesium contents but deficient in terms of calcium and vitamin D. Serum OC and CTX as well as fetuin-A concentrations were similar in both studied groups. The BALP activity was significantly (p < 0.05) higher in children with cow's milk allergy than in the controls. Serum OPG concentration was comparable in both groups, but the RANKL level was higher (p < 0.05) in CMA children than in healthy ones. Hence, the ratio of OPG/RANKL was lower in children with CMA. CONCLUSIONS: Our study demonstrates slight disturbances in the profile of bone metabolism markers in growing children with CMA. The increase in RANKL level and decrease in OPG/RANKL ratio may contribute to intensification of bone resorption in these patients.