Destabilization Of The Medial Meniscus Mice Research Articles

Thermoresponsive Polymeric Hydromorphone Prodrug Provides Sustained Local Analgesia without Apparent Adverse Effects.

The extensive use of opioids for chronic pain management has contributed significantly to the current opioid epidemic. While many alternative nonopioid analgesics are available, opioids remain the most potent analgesics for moderate to severe pain management. In addition to the implementation of multimodal analgesia, there is a pressing need for the development of more effective and safer opioids. In this study, we developed a thermoresponsive N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based hydromorphone (HMP) prodrug (ProGel-HMP, HMP content = 16.2 wt %, in base form). The aqueous solution of ProGel-HMP was free-flowing at 4 °C but became a hydrogel when the temperature was raised to ≥37 °C, allowing sustained local retention when administered in vivo. When tested in the destabilization of the medial meniscus (DMM) mouse model of osteoarthritis (OA), ProGel-HMP was retained after intra-articular injection in the OA knee joint for at least 2 weeks postinjection, with low extra-articular distribution. ProGel-HMP was not detected in the central nervous system (CNS). A single dose of ProGel-HMP produced rapid and sustained joint pain resolution for greater than 14 days when compared to saline and dose-equivalent HMP controls, likely mediated through peripheral μ-opioid receptors in the knee joint. Systemic analgesia effect was absent in the DMM mice treated with ProGel-HMP, as evident in the lack of difference in tail flick response between the ProGel-HMP-treated mice and the controls (i.e., Healthy, Saline, and Sham). Repeated dosing of ProGel-HMP did not induce tolerance. Collectively, these data support the further development of ProGel-HMP as a potent, safe, long-acting and nonaddictive analgesic for better clinical pain management.

CHRONIC STRESS RESULTS IN INCREASED PAIN PERCEPTION IN OSTEOARTHRITIS IN VIVO

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice.

A causative role for periarticular skeletal muscle weakness in the progression of joint damage and pain in OA

Although osteoarthritis (OA) is regarded as a disease of the articular cartilage, recent research has demonstrated alterations in periarticular muscles that surround the affected joint. Here, we investigated changes in periarticular muscle during the progression of OA, as well as the cause-and-effect relationship between muscle weakness and OA, in a mouse model of OA by destabilization of the medial meniscus (DMM). Pathological phenotypes in the periarticular muscles were assessed in the early and late stages of OA by DMM. OA pathology and pain behavior in the mice after DMM induction were examined in response to periarticular muscle weakness induced by multiple rounds of barium chloride (BaCl2) injections. The examinations were also performed in myostatin knockout mice with strengthened muscle phenotypes by muscle hypertrophy. Morphological alterations in the tibialis anterior (TA) and quadriceps muscles in DMM mice included variations in muscle-fiber size, aberrant extracellular matrix (ECM) deposition, inflammatory cell infiltration, and decreased muscle mass. Periarticular muscle fibers isolated from DMM mice showed reductions in the number of satellite cells and myogenic capacity of primary myoblast, as well as proliferation. DMM + muscle injury mice also showed exacerbated joint degeneration compared to the DMM vehicles. Myostatin knockout mice were characterized by attenuated OA and the complete abrogation of pain behavior after DMM. Our results suggest an association between muscle weakness and OA progression and pain.

Mir-25-3p in extracellular vesicles from fibroblast-like synoviocytes alleviates pyroptosis of chondrocytes in knee osteoarthritis.

Knee osteoarthritis (KOA) is defined as a joint disease that occurs mostly among elderly people. Fibroblast-like synoviocytes-derived extracellular vesicles (FLS-EVs) have impacts on the treatment of OA. This study elucidated the mechanism of miR-25-3p in pyroptosis of chondrocytes in KOA. FLSs and EVs were extracted from neonatal mice; destabilization of the medial meniscus (DMM) was used to simulate KOA in mice, followed by the evaluation of cartilage damage and the contents of MMP-3 and MMP-13 in KOA mice. Lipopolysaccharide (LPS) was used to induce inflammation damage in mouse chondrocytes ATDC5, and the cell viability and the expressions of NLRP3, Cleaved-Caspase-1, GSDMD-N, IL-18, and IL-1β were examined. We found that FLS-EV treatment mitigated the knee-joint damage and symptoms of KOA mice, decreased MMP-3 and MMP-13, and inhibited pyroptosis of chondrocytes in DMM mice and LPS-induced ATD5 cells. Then, Cy3-labeled miR-25-3p in mice chondrocytes was observed and the expressions and the binding relation of miR-25-3p and cytoplasmic polyadenylation element-binding protein 1 (CPEB1) were verified. It showed that FLS-EVs carried miR-25-3p into chondrocytes, and upregulated miR-25-3p expression while inhibited CPEB1 transcription, resulting in mitigation of pyroptosis of chondrocytes, and CPEB1 overexpression reversed the inhibition of FLS-EVs on pyroptosis of chondrocytes in KOA.

Ginsenoside compound K alleviates osteoarthritis by inhibiting NLRP3‑mediated pyroptosis.

Ginsenoside compound K (GCK) has been previously reported to be a potent antiarthritic and bone-protective agent. Therefore, the present study aimed to explore the potential effects of GCK on osteoarthritis and its regulatory effects on the pyroptosis of chondrocytes. Primary mouse chondrocytes (PMCs) were used for in vitro analysis. ELISA assays revealed that compared with the untreated cells, TNF-α induced a significant increase in IL-6, MMP13, A disintegrin and metalloproteinase with thrombospondin motifs 5 and MMP3 expression but induced a significant decrease in aggrecan and collagen II expression. By contrast, GCK reversed the aforementioned alterations in a dose-dependent manner. Experimental osteoarthritis was subsequently induced in mice through transection of the medial meniscotibial ligament and medial collateral ligament in the right knee [destabilization of the medial meniscus (DMM) mice]. GCK was found to reduce cartilage degradation in vivo in DMM mice, which was assessed using the Osteoarthritis Research Society International (OARSI) score, collagen II and MMP13 expression. Cartilage degradation is associated with higher OARSI score, decreased collagen II and increased MMP13 expression. In PMCs, TNF-α treatment stimulated an increase in the expression of NLR family pyrin domain containing 3 (NLRP3), Gasdermin D-N terminal (GSDMD-NT), cleaved caspase-1 and mature IL-1β, markers that indicate the occurrence of pyroptosis. However, GCK treatment suppressed the increase of the aforementioned proteins in a dose-dependent manner. Immunohistochemistry staining of the knee joint tissue sections from the DMM mice confirmed that GCK attenuated the NLRP3 and GSDMD-NT expression that was induced by DMM surgery. In conclusion, the present study revealed that GCK can reduce cartilage degradation in an osteoarthritis model by inhibiting the NLRP3-inflammasome activation and subsequent pyroptosis.

MiR-124-3p sabotages lncRNA MALAT1 stability to repress chondrocyte pyroptosis and relieve cartilage injury in osteoarthritis

BackgroundOsteoarthritis (OA) is a prevalent inflammatory joint disorder. microRNAs (miRNAs) are increasingly involved in OA.AimOur study is proposed to clarify the role of miR-124-3p in chondrocyte pyroptosis and cartilage injury in OA.MethodsOA mouse model was established via the treatment of destabilization of the medial meniscus (DMM), and the in vitro cell model was also established as mouse chondrocytes were induced by lipopolysaccharide (LPS). Mouse cartilage injury was assessed using safranin-O-fast green staining, hematoxylin–eosin staining, and OARSI grading method. Expressions of miR-124-3p, MALAT1, KLF5, and CXCL11 were determined. Cartilage injury (MMP-13, osteocalcin), inflammation (IL-6, IL-2, TNF-, IL-1β, and IL-18)- and pyroptosis-related factors (Cleaved Caspase-1 and GSDMD-N) levels were detected. Mechanically, MALAT1 subcellular localization was confirmed. The binding relationships of miR-124-3p and MALAT1 and MALAT1 and KLF5 were verified. MALAT1 half-life period was detected. Then, miR-124-3p was overexpressed using agomiR-124-3p to perform the rescue experiments with oe-MALAT1 or oe-CXCL11.ResultsmiR-124-3p was downregulated in DMM mice and LPS-induced chondrocytes where cartilage injury, and increased levels of inflammation- and pyroptosis-related factors were found. miR-124-3p overexpression relieved cartilage injury and repressed chondrocyte pyroptosis. miR-124-3p bounds to MALAT1 to downregulate its stability and expression, and MALAT1 bounds to KLF5 to enhance CXCL11 transcription. Overexpression of MALAT1 or CXCL11 annulled the repressive function of miR-124-3p in chondrocyte pyroptosis.ConclusionmiR-124-3p reduced MALAT1 stability and inhibited the binding of MALAT1 and KLF5 to downregulate CXCL11, thereby suppressing chondrocyte pyroptosis and cartilage injury in OA.

Inhibition of PGE2 in Subchondral Bone Attenuates Osteoarthritis.

Aberrant subchondral bone architecture is a crucial driver of the pathological progression of osteoarthritis, coupled with increased sensory innervation. The sensory PGE2/EP4 pathway is involved in the regulation of bone mass accrual by the induction of differentiation of mesenchymal stromal cells. This study aimed to clarify whether the sensory PGE2/EP4 pathway induces aberrant structural alteration of subchondral bone in osteoarthritis. Destabilization of the medial meniscus (DMM) using a mouse model was combined with three approaches: the treatment of celecoxib, capsaicin, and sensory nerve-specific prostaglandin E2 receptor 4 (EP4)-knockout mice. Cartilage degeneration, subchondral bone architecture, PGE2 levels, distribution of sensory nerves, the number of osteoprogenitors, and pain-related behavior in DMM mice were assessed. Serum and tissue PGE2 levels and subchondral bone architecture in a human sample were measured. Increased PGE2 is closely related to subchondral bone’s abnormal microstructure in humans and mice. Elevated PGE2 concentration in subchondral bone that is mainly derived from osteoblasts occurs in early-stage osteoarthritis, preceding articular cartilage degeneration in mice. The decreased PGE2 levels by the celecoxib or sensory denervation by capsaicin attenuate the aberrant alteration of subchondral bone architecture, joint degeneration, and pain. Selective EP4 receptor knockout of the sensory nerve attenuates the aberrant formation of subchondral bone and facilitates the prevention of cartilage degeneration in DMM mice. Excessive PGE2 in subchondral bone caused a pathological alteration to subchondral bone in osteoarthritis and maintaining the physiological level of PGE2 could potentially be used as an osteoarthritis treatment.

Therapeutic Effects of Non-Euphorigenic Cannabis Extracts in Osteoarthritis.

Introduction: Osteoarthritis (OA) is disabling and degenerative disease of the joints that is clinically characterized by pain and loss of function. With no disease-modifying treatment available, current therapies aim at pain management but are of limited efficacy. Cannabis products, specifically cannabinoids, are widely used to control pain and inflammation in many diseases with no scientific evidence demonstrating their efficacy in OA. Objective: We investigated the effects of non-euphorigenic cannabis extracts, CBD oil and cannabigerol oil (CBG oil), on pain and disease progression in OA mice. Methods and Results: Twelve-week-old male C57BL/6J mice received either sham or destabilization of the medial meniscus (DMM) surgery. DMM mice were treated with vehicle, CBD oil, or CBG oil. The gait of DMM mice was impaired as early as 2 weeks following surgery and continued deteriorating until week 8, which was restored by CBD oil and CBG oil treatments throughout the disease course. Mechanical allodynia developed in DMM mice, however, was not ameliorated by any of the treatments. On the other hand, both CBD oil and CBG oil ameliorated cold allodynia. In open field test, both oil treatments normalized changes in the locomotor activity of DMM mice. CBD oil and CBG oil treatments significantly reduced synovitis in DMM mice. Only CBG oil reduced cartilage degeneration, chondrocyte loss, and matrix metalloproteinase 13 expression, with a significant increase in the number of anabolic chondrocytes. Subchondral bone remodeling found in vehicle-treated DMM mice was not ameliorated by either CBD or CBG oil. Conclusions: Our results show evidence for the therapeutic efficacy of CBD oil and CBG oil, where both oils ameliorate pain and inflammation, and improve gait and locomotor activity in OA mice, representing clinical pain and function. Importantly, only CBG oil is chondroprotective, which may provide superior efficacy in future studies in OA patients.

Metformin alleviates osteoarthritis in mice by inhibiting chondrocyte ferroptosis and improving subchondral osteosclerosis and angiogenesis

BackgroundOsteoarthritis (OA) is the most common musculoskeletal disease, and it has a complex pathology and unknown pathogenesis. Chondrocyte ferroptosis is closely associated with the development of OA. As a common drug administered for the treatment of type 2 diabetes, metformin (Met) is known to inhibit the development of ferroptosis. However, its therapeutic effect in OA remains unknown. The present study aimed to explore the effects of Met on cartilage and subchondral bone in a mouse OA model and to explore the potential underlying mechanisms.MethodsA mouse OA model was induced using destabilization of the medial meniscus (DMM) surgery, chondrocyte ferroptosis was induced using an intra-articular injection of Erastin, and Met (200 mg/kg/day) was intragastrically administered for 8 weeks after surgery. H&E and Safranin O‑fast green staining were used to evaluate cartilage degeneration, and μ‑computed tomography was used to evaluate changes in subchondral bone microarchitecture. Moreover, immunohistochemical staining was performed to detect mechanistic metalloproteinases 13, type II collagen, glutathione peroxidase 4, acyl-CoA synthetase long-chain family member 4, solute carrier family 7 member 11 and p53. Runt-associated transcription factor 2 and CD31 were detected using immunofluorescent staining.ResultsMet protected articular cartilage and reversed the abnormal expression of ferroptosis-related proteins in the chondrocytes of DMM mice. Moreover, intra-articular injection of Erastin induced ferroptosis in mouse chondrocytes, and Met eliminated the ferroptosis effects induced by Erastin and protected articular cartilage. In addition, the results of the present study demonstrated that Met alleviated the microstructural changes of subchondral osteosclerosis and reduced heterotypic angiogenesis in DMM mice.ConclusionMet alleviates the pathological changes of OA by inhibiting ferroptosis in OA chondrocytes, alleviating subchondral sclerosis and reducing abnormal angiogenesis in subchondral bone in advanced OA.

Can CD200R1 Agonists Slow the Progression of Osteoarthritis Secondary to Injury?

Post-traumatic knee osteoarthritis is characterized by cartilage degeneration, subchondral bone remodeling, osteophyte formation, and synovial changes. Therapeutic targeting of inflammatory activity in the knee immediately post injury may alter the course of osteoarthritis development. This study aimed to determine whether CD200R1 agonists, namely the protein therapeutic CD200Fc or the synthetic DNA aptamer CCS13, both known to act as anti-inflammatory agents, are able to delay the pathogenesis of injury-associated knee osteoarthritis in a murine model. Ten week old male C57BL/6 mice were randomized and surgical destabilization of the medial meniscus (DMM) to induce knee arthritis or sham surgery as a control were performed. CCS13 was evaluated as a therapeutic treatment along with CD200Fc and a phosphate-buffered saline vehicle control. Oligonucleotides were injected intra-articularly beginning one week after surgery, with a total of six injections administered prior to sacrifice at 12 weeks post-surgery. Histopathological assessment was used as the primary outcome measure to assess cartilage and synovial changes, while µCT imaging was used to compare the changes to the subchondral bone between untreated and treated arthritic groups. We did not find any attenuation of cartilage degeneration or synovitis in DMM mice with CD200Fc or CCS13 at 12 weeks post-surgery, nor stereological differences in the properties of subchondral bone. The use of CD200R1 agonists to blunt the inflammatory response in the knee are insufficient to prevent disease progression in the mouse DMM model of OA without anatomical restoration of the normal joint biomechanics.

β2-Adrenoceptor Deficiency Results in Increased Calcified Cartilage Thickness and Subchondral Bone Remodeling in Murine Experimental Osteoarthritis.

PurposeRecent studies demonstrated a contribution of adrenoceptors (ARs) to osteoarthritis (OA) pathogenesis. Several AR subtypes are expressed in joint tissues and the β2-AR subtype seems to play a major role during OA progression. However, the importance of β2-AR has not yet been investigated in knee OA. Therefore, we examined the development of knee OA in β2-AR-deficient (Adrb2-/- ) mice after surgical OA induction.MethodsOA was induced by destabilization of the medial meniscus (DMM) in male wildtype (WT) and Adrb2-/- mice. Cartilage degeneration and synovial inflammation were evaluated by histological scoring. Subchondral bone remodeling was analyzed using micro-CT. Osteoblast (alkaline phosphatase - ALP) and osteoclast (cathepsin K - CatK) activity were analyzed by immunostainings. To evaluate β2-AR deficiency-associated effects, body weight, sympathetic tone (splenic norepinephrine (NE) via HPLC) and serum leptin levels (ELISA) were determined. Expression of the second major AR, the α2-AR, was analyzed in joint tissues by immunostaining.ResultsWT and Adrb2-/- DMM mice developed comparable changes in cartilage degeneration and synovial inflammation. Adrb2-/- DMM mice displayed elevated calcified cartilage and subchondral bone plate thickness as well as increased epiphyseal BV/TV compared to WTs, while there were no significant differences in Sham animals. In the subchondral bone of Adrb2-/- mice, osteoblasts activity increased and osteoclast activity deceased. Adrb2-/- mice had significantly higher body weight and fat mass compared to WT mice. Serum leptin levels increased in Adrb2-/- DMM compared to WT DMM without any difference between the respective Shams. There was no difference in the development of meniscal ossicles and osteophytes or in the subarticular trabecular microstructure between Adrb2-/- and WT DMM as well as Adrb2-/- and WT Sham mice. Number of α2-AR-positive cells was lower in Adrb2-/- than in WT mice in all analyzed tissues and decreased in both Adrb2-/- and WT over time.ConclusionWe propose that the increased bone mass in Adrb2-/- DMM mice was not only due to β2-AR deficiency but to a synergistic effect of OA and elevated leptin concentrations. Taken together, β2-AR plays a major role in OA-related subchondral bone remodeling and is thus an attractive target for the exploration of novel therapeutic avenues.

Pharmacological characterization of GLPG1972/S201086, a potent and selective small-molecule inhibitor of ADAMTS5

A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is a key enzyme in degradation of cartilage in osteoarthritis (OA). We report the pharmacological characterization of GLPG1972/S201086, a new, potent and selective small-molecule ADAMTS5 inhibitor. Potency and selectivity of GLPG1972/S201086 for ADAMTS5 were determined using fluorescently labeled peptide substrates. Inhibitory effects of GLPG1972/S201086 on interleukin-1α-stimulated glycosaminoglycan release in mouse femoral head cartilage explants and on interleukin-1β-stimulated release of an ADAMTS5-derived aggrecan neoepitope (quantified with ELISA) in human articular cartilage explants were determined. In the destabilization of the medial meniscus (DMM) mouse and menisectomized (MNX) rat models, effects of oral GLPG1972/S201086 on relevant OA histological and histomorphometric parameters were evaluated. GLPG1972/S201086 inhibited human and rat ADAMTS5 (IC50±SD: 19±2nM and <23±1nM, respectively), with 8-fold selectivity over ADAMTS4, and 60->5,000-fold selectivity over other related proteases in humans. GLPG1972/S201086 dose-dependently inhibited cytokine-stimulated aggrenolysis in mouse and human cartilage explants (100% at 20μM and 10μM, respectively). In DMM mice, GLPG1972/S201086 (30-120mg/kg b.i.d) vs vehicle reduced femorotibial cartilage proteoglycan loss (23-37%), cartilage structural damage (23-39%) and subchondral bone sclerosis (21-36%). In MNX rats, GLPG1972/S201086 (10-50mg/kg b.i.d) vs vehicle reduced cartilage damage (OARSI score reduction, 6-23%), and decreased proteoglycan loss (∼27%) and subchondral bone sclerosis (77-110%). GLPG1972/S201086 is a potent, selective and orally available ADAMTS5 inhibitor, demonstrating significant protective efficacy on both cartilage and subchondral bone in two relevant in vivo preclinical OA models.

Asporin regulated by miR-26b-5p mediates chondrocyte senescence and exacerbates osteoarthritis progression via TGF-β1/Smad2 pathway.

This study aimed to investigate the role and mechanism of asporin in modulating chondrocyte senescence in OA pathology. Asporin and senescence-related hallmark expression were examined in human and experimental OA mouse cartilage samples. Twelve-week-old male C57 mice were administered with recombinant protein (rm-asporin)- or asporin-siRNA-expressing lentiviruses via intra-articular injection once a week after destabilization of the medial meniscus (DMM) surgery to induce OA. Cartilage damage was measured using the Osteoarthritis Research Society International score. Senescence-associated β-galactosidase (SA-β-Gal) staining, γH2AX, p21 and p16INK4a were analysed by immunofluorescence staining and western blot to assess the specific role of asporin in chondrocyte senescence. The TGF-β1-Smad2 signalling pathway and miR-26b-5p were further evaluated to explore the mechanism of asporin in OA. Asporin was upregulated in articular chondrocytes of OA patients and DMM mice and accompanied by accumulation of senescent cells. Asporin overexpression exaggerated OA progression, whereas silencing asporin restored chondrocyte homeostasis and deferred chondrocyte senescence, leading to markedly attenuated DMM-induced OA. Cellular and molecular analyses showed that asporin can be inhibited by miR-26b-5p, which was significantly downregulated in OA cartilage, leading to exacerbation of experimental OA partially through inhibition of TGF-β1-Smad2 signalling in chondrocytes. Our findings indicate that asporin plays an essential role in chondrocyte senescence and OA pathogenesis. Upregulated by miR-26b-5p, asporin inhibits the TGF-β1-Smad2 pathway to accelerate chondrocyte senescence and exacerbate cartilage degeneration. Targeting the miR-26b-5p-asporin-Smad2 axis may serve as a practical therapeutic strategy to delay chondrocyte senescence and OA development.

Low-intensity pulsed ultrasound inhibits fibroblast-like synoviocyte proliferation and reduces synovial fibrosis by regulating Wnt/β-catenin signaling.

ObjectiveSynovial fibrosis is a characteristic symptom of osteoarthritis (OA), which is closely associated with joint pain and stiffness. Previous studies have reported that low-intensity pulsed ultrasound (LIPUS) can alleviate cartilage degradation in OA. However, the functions and mechanisms of LIPUS in OA synovial fibrosis are still unknown.MethodsThe destabilization of the medial meniscus (DMM) mouse model of OA was established in C57 male mice and fibroblast-like synoviocytes (FLS) were isolated from synovial tissue of OA patients. The knee joint diameter, Masson's trichrome (MT) and Hematoxylin-eosin (HE) staining were used to evaluate synovial fibrosis and hyperplasia. The Immunohistochemistry (IHC) staining was performed to detected the expression of synovial fibrosis makers and the activation of Wnt/β-catenin signaling in vivo. FLS were treated with TGF-β1 to serve as an in vitro model of synovial fibrosis, Wnt3a was used to activate the Wnt/β-catenin signaling in cells. Cell proliferation was detected by using EdU assay, cell viability was performed by CCK8 assay. The protein levels of α-SMA, CTGF, Col Ⅰ, β-catenin, active β-catenin, c-Myc and cyclin D1 were examined by western blot and immunofluorescence staining.ResultsTwo weeks after the LIPUS treatment, the synovial fibrosis, synovial hyperplasia and synoviocyte proliferation in the DMM model were significantly decreased. In vitro, LIPUS directly inhibited the TGF-β1-induced fibrotic response and proliferation of FLS. Meanwhile, LIPUS suppressed Wnt/β-catenin signaling in the synovium of DMM mice and cultured FLS. More importantly, we found that the synovial fibrosis makers, Wnt/β-catenin pathway downstream proteins and FLS proliferation were significantly decreased in Wnt3a-stimulated FLS following LIPUS treatment.ConclusionsOur results present a novel role of LIPUS in OA-related synovial fibrosis, which is associated with its ability to repress Wnt/β-catenin signaling in FLS.The translational potential of this articleThis study provides new insight into the clinical application of LIPUS as a therapeutic option to manage synovial fibrosis in OA.

MicroRNA-183 attenuates osteoarthritic pain by inhibiting the TGFα-mediated CCL2/CCR2 signalling axis.

AimsMicroRNA-183 (miR-183) is known to play important roles in osteoarthritis (OA) pain. The aims of this study were to explore the specific functions of miR-183 in OA pain and to investigate the underlying mechanisms.MethodsClinical samples were collected from patients with OA, and a mouse model of OA pain was constructed by surgically induced destabilization of the medial meniscus (DMM). Reverse transcription quantitative polymerase chain reaction was employed to measure the expression of miR-183, transforming growth factor α (TGFα), C-C motif chemokine ligand 2 (CCL2), proinflammatory cytokines (interleukin (IL)-6, IL-1β, and tumour necrosis factor-α (TNF-α)), and pain-related factors (transient receptor potential vanilloid subtype-1 (TRPV1), voltage-gated sodium 1.3, 1.7, and 1.8 (Nav1.3, Nav1.7, and Nav1.8)). Expression of miR-183 in the dorsal root ganglia (DRG) of mice was evaluated by in situ hybridization. TGFα, CCL2, and C-C chemokine receptor type 2 (CCR2) levels were examined by immunoblot analysis and interaction between miR-183 and TGFα, determined by luciferase reporter assay. The extent of pain in mice was measured using a behavioural assay, and OA severity assessed by Safranin O and Fast Green staining. Immunofluorescent staining was conducted to examine the infiltration of macrophages in mouse DRG.ResultsmiR-183 was downregulated in tissue samples from patients and mice with OA. In DMM mice, overexpression of miR-183 inhibited the expression of proinflammatory cytokines (IL-6, IL-1β, TNF-α) and pain-related factors (TRPV1, Nav1.3, Nav1.7, Nav1.8) in DRG. OA pain was relieved by miR-183-mediated inhibition of macrophage infiltration, and dual luciferase reporter assay demonstrated that miR-183 directly targeted TGFα.ConclusionOur data demonstrate that miR-183 can ameliorate OA pain by inhibiting the TGFα-CCL2/CCR2 signalling axis, providing an excellent therapeutic target for OA treatment.Cite this article: Bone Joint Res 2021;10(8):548–557.

Osteoking Decelerates Cartilage Degeneration in DMM-Induced Osteoarthritic Mice Model Through TGF-β/smad-dependent Manner.

Osteoarthritis (OA) is a common disease characterized by cartilage degeneration. In recent years much attention has been paid to Traditional Chinese Medicine (TCM) since its treatments have shown efficacy for ameliorating cartilage degradation with mild side effects. Osteoking is a TCM prescription that has long been used in OA treatment. However, the exact mechanism of Osteoking are not fully elucidated. In the current study, destabilization of the medial meniscus (DMM)-induced OA mice was introduced as a wild type animal model. After 8 weeks of administration of Osteoking, histomorphometry, OARSI scoring, gait analysis, micro-CT, and immunohistochemical staining for Col2, MMP-13, TGFβRII and pSmad-2 were conducted to evaluate the chondroprotective effects of Osteoking in vivo. Further in vitro experiments were then performed to detect the effect of Osteoking on chondrocytes. TGFβRIICol2ER transgenic mice were constructed and introduced in the current study to validate whether Osteoking exerts its anti-OA effects via the TGF-β signaling pathway. Results demonstrated that in wild type DMM mice, Osteoking ameliorated OA-phenotype including cartilage degradation, subchondral bone sclerosis, and gait abnormality. Col2, TGFβRII, and pSmad-2 expressions were also found to be up-regulated after Osteoking treatment, while MMP-13 was down-regulated. In vitro, the mRNA expression of MMP-13 and ADAMTS5 decreased and the mRNA expression of Aggrecan, COL2, and TGFβRII were up-regulated after the treatment of Osteoking in IL-1β treated chondrocytes. The additional treatment of SB505124 counteracted the positive impact of Osteoking on primary chondrocytes. In TGFβRIICol2ER mice, spontaneous OA-liked phenotype was observed and treatment of Osteoking failed to reverse the OA spontaneous progression. In conclusion, Osteoking ameliorates OA progression by decelerating cartilage degradation and alleviating subchondral bone sclerosis partly via the TGF-β signaling pathway.

Losartan protects against osteoarthritis by repressing the TGF-β1 signaling pathway via upregulation of PPARγ.

ObjectiveLosartan and activation of the peroxisome proliferator-activated receptor-γ (PPARγ) have been previously reported to alleviate the progression of osteoarthritis (OA). However, the nature of the interaction between losartan and PPARγ in OA remains elusive. Therefore, we aimed to investigate the mechanism of the regulation of PPARγ by losartan in the context of OA.MethodsClinical samples of OA patients were collected and the chondrocytes were further isolated, and used to construct OA chondrocyte model via induction with IL-1β. An OA mouse model was developed by the surgical destabilization of the medial meniscus (DMM). OA chondrocytes were treated with losartan, PPARγ siRNA and the PPAR-γ agonist GW1929 alone or in combination. Furthermore, the OA mice were treated with varying doses of losartan to determine the best mode of administration and treatment dose. Subsequently, the DMM mice were treated with losartan and GW9662. Expression of PPARγ, key proteins of the transforming growth factor-beta1 (TGF-β1) signaling pathway and the markers of OA degeneration were evaluated by the Western blot analysis, while effects on OA inflammatory factors were determined by ELISA.ResultsThe downregulation of PPARγ and the upregulation of TGF-β1 signaling pathway were detected in the OA cartilage tissues and chondrocytes. Losartan treatment or PPARγ activation contributes to reduced levels of IL-6, IL-1β, TNF-α, and COX-2, expression of TGF-β1, MMP-13, ADAMTS-4, ADAMTS-5, HtrA1, and iNOS, along with reduced Smad2 and Smad3 phosphorylation, but elevated PPARγ and Collagen II expression in vivo and in vitro. Additionally, the intraarticular injection of losartan into the knee joint proved to be the best mode of administration, and 10 ​mg/mL being the optimal treatment concentration.ConclusionOur results show that losartan could arrest the progression of OA by upregulating PPARγ expression and inactivating the TGF-β1 signaling pathway.The translational potential of this article: Our results provide a biological rationale for the use of losartan as a potential candidate for OA treatment.

Metformin attenuates traumatic osteoarthritis in obese mice induced by high-fat diet partly by reducing leptin

Purpose: Obesity is one of the main risk factors for osteoarthritis (OA), and evidence is accumulating that proinflammatory adipokines such as leptin play an important role in OA. Metformin (Met) has been shown to reduce weight and relieve inflammation. Here, we combine surgical joint damage with high-fat diet (HFD) in mice to investigate the effect of metformin on OA and its potential mechanism. Methods: Mice were fed with HFD or normal diet (ND) from 3 weeks old and destabilization of the medial meniscus (DMM) surgery was performed at 12 weeks old. Metformin was administrated in drinking water immediately after DMM and the joint structural changes were observed at 5 and 10 weeks after the surgery. OA was evaluated by Osteoarthritis Research Society International (OARSI) score, synovitis score, and osteophyte volume reconstructed by micro-computed tomography. Macrophage infiltration in synovium and leptin expression in adipose tissue were detected by immunofluorescence. In vitro, the apoptosis of primary human chondrocytes and the subtypes of macrophages were detected by flow cytometry. Murine chondrogenic cell line ATDC5 and macrophage cell line RAW264.7 were incubated in fat conditioned medium (FCM) from mouse abdominal fat, and the metabolic changes were detected by western blot and ELISA. Results: The OARSI score, synovitis score and osteophyte volume of ND + DMM + Met mice and HFD + DMM + Met mice were significantly lower than that of ND + DMM mice and HFD + DMM mice, respectively. Importantly, the interaction between diet and Met on OARSI score was statistically significant at 5 weeks (F = 6.364, P = 0.020) and 10 weeks (F = 4.949, P = 0.038) after DMM. The mean difference of OARSI score in HFD mice (2.08 at 5 weeks and 3.17 at 10 weeks) was greater than that in ND mice (0.8 at 5 weeks and 2 at 10 weeks). Metformin reduced the expression of ADAMTS-5 and MMP13 in articular chondrocytes, and increased the expression of autophagy marker LC3. The M1 macrophage marker iNOS was decreased and the M2 macrophage marker CD206 was increased in synovium of metformin- treated mice. Interestingly, metformin reduced the leptin levels in abdominal fat and articular chondrocytes in HFD mice but not ND mice. The expression of MMP-13, ADAMTS-5 and p-S6 of ATDC5 cells in HFD + DMM + Met FCM was significantly lower than that in HFD + DMM FCM. The content of TNF-α and IL-1β of RAW264.7 cells in HFD + DMM + Met FCM was significantly lower than that in HFD + DMM FCM. Furthermore, the expression of ADAMTS-5 and p-S6 in ATDC5 and the secretion of TNF-α and IL-1β in RAW264.7 was significantly reduced after neutralization of leptin in HFD + DMM FCM. Conclusions: Metformin showed a therapeutic effect on OA both in ND mice and in HFD mice. The therapeutic effect of metformin on OA in HFD mice is better than that in ND mice, which may be related to decreasing leptin levels.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Hydrogen (H2) Alleviates Osteoarthritis by Inhibiting Apoptosis and Inflammation via the JNK Signaling Pathway.

BackgroundOsteoarthritis (OA) is a very common condition and leads to joint pain, disability, and price tag all over the world. Pathogenesis of OA is closely related to numerous inflammatory and apoptosis cytokines. Hydrogen (H2) reportedly exhibits a diversity of effects such as anti-apoptotic, anti-inflammatory, and anti-oxidative properties via the JNK pathway. However, it is unknown whether H2 has a protective effect against OA via the JNK signaling pathway. Therefore, the aim of this study was to figure out whether hydrogen has protective effect on chondrocyte and further explore the possible underlying mechanism.MethodsThe chondrocytes were obtained from the human cartilage tissues. Cells were stimulated by TBHP and treated with hydrogen. In vitro treatment effects were evaluated by Western blot assay, real-time PCR, immunofluorescence and TUNEL method. We conducted mice model of destabilization of the medial meniscus (DMM) and treated with hydrogen. In vivo treatment effects were evaluated by X-ray imaging assay, safranin O (SO) staining, TUNEL staining and immunohistochemical assay.ResultsOur results showed that hydrogen can inhibit inflammatory factors (ADAMTS5 and MMP13) and apoptosis factors (cleaved caspase-3, cytochrome c, and Bax) in TBHP-induced chondrocytes. Furthermore, hydrogen can suppress the activation of JNK signaling pathway, whereas the effect of hydrogen can be abolished by anisomycin (a JNK activator). In vivo results showed that hydrogen can down-regulate the expression of p-JNK and cleaved caspase-3 expression.ConclusionWe uncovered that hydrogen (H2) could alleviate apoptosis response and ECM degradation in human chondrocytes via inhibiting the activation of the JNK signaling pathway. Meanwhile, in the surgically-induced DMM mice model, treatment with hydrogen (H2) performed a significant role in OA progression.

BMP5 silencing inhibits chondrocyte senescence and apoptosis as well as osteoarthritis progression in mice.

In this study, we using the in vivo destabilization of the medial meniscus (DMM) mouse model to investigate the role of bone morphogenetic protein 5 (BMP5) in osteoarthritis (OA) progression mediated via chondrocyte senescence and apoptosis. BMP5 expression was significantly higher in knee articular cartilage tissues of OA patients and DMM model mice than the corresponding controls. The Osteoarthritis Research Society International scores based on histological staining of knee articular cartilage sections were lower in DMM mice where BMP5 was knocked down in chondrocytes than the corresponding controls 4 weeks after DMM surgery. DMM mice with BMP5-deficient chondrocytes showed reduced levels of matrix-degrading enzymes such as MMP13 and ADAMTS5 as well as reduced cartilage destruction. BMP5 knockdown also decreased chondrocyte apoptosis and senescence by suppressing the activation of p38 and ERK MAP kinases. These findings demonstrate that BMP5 silencing inhibits chondrocyte senescence and apoptosis as well as OA progression by downregulating activity in the p38/ERK signaling pathway.