Rat Model Of Knee Osteoarthritis Research Articles

Injectable hydrogel microspheres experimental research for the treatment of osteoarthritis

To prepare a novel hyaluronic acid methacrylate (HAMA) hydrogel microspheres loaded polyhedral oligomeric silsesquioxane-diclofenac sodium (POSS-DS) patricles, then investigate its physicochemical characteristics and in vitro and in vivo biological properties. Using sulfhydryl POSS (POSS-SH) as a nano-construction platform, polyethylene glycol and DS were chemically linked through the "click chemistry" method to construct functional nanoparticle POSS-DS. The composition was analyzed by nuclear magnetic resonance spectroscopy and the morphology was characterized by transmission electron microscopy. In order to achieve drug sustained release, POSS-DS was encapsulated in HAMA, and hybrid hydrogel microspheres were prepared by microfluidic technology, namely HAMA@POSS-DS. The morphology of the hybrid hydrogel microspheres was characterized by optical microscope and scanning electron microscope. The in vitro degradation and drug release efficiency were observed. Cell counting kit 8 (CCK-8) and live/dead staining were used to detect the effect on chondrocyte proliferation. Moreover, a chondrocyte inflammation model was constructed and cultured with HAMA@POSS-DS. The relevant inflammatory indicators, including collagen type Ⅱ, aggrecan (AGG), matrix metalloproteinase 13 (MMP-13), recombinant A disintegrin and metalloproteinase with thrombospondin 5 (Adamts5), and recombinant tachykinin precursor 1 (TAC1) were detected by immunofluorescence staining and real-time fluorescence quantitative PCR, with normal cultured chondrocytes and the chondrocyte inflammation model without treatment as control group and blank group respectively to further evaluate their anti-inflammatory activity. Finally, by constructing a rat model of knee osteoarthritis, the effectiveness of HAMA@POSS-DS on osteoarthritis was evaluated by X-ray film and Micro-CT examination. The overall particle size of POSS-DS nanoparticles was uniform with a diameter of about 100 nm. HAMA@POSS-DS hydrogel microspheres were opaque spheres with a diameter of about 100 μm and a spherical porous structure. The degradation period was 9 weeks, during which the loaded POSS-DS nanoparticles were slowly released. CCK-8 and live/dead staining showed no obvious cytotoxicity at HAMA@POSS-DS, and POSS-DS released by HAMA@POSS-DS significantly promoted cell proliferation (P<0.05). In the chondrocyte anti-inflammatory experiment, the relative expression of collagen type Ⅱ mRNA in HAMA@POSS-DS group was significantly higher than that in control group and blank group (P<0.05). The relative expression level of AGG mRNA was significantly higher than that of blank group (P<0.05). The relative expressions of MMP-13, Adamts5, and TAC1 mRNA in HAMA@POSS-DS group were significantly lower than those in blank group (P<0.05). In vivo experiments showed that the joint space width decreased after operation in rats with osteoarthritis, but HAMA@POSS-DS delayed the process of joint space narrowing and significantly improved the periarticular osteophytosis (P<0.05). HAMA@POSS-DS can effectively regulate the local inflammatory microenvironment and significantly promote chondrocyte proliferation, which is conducive to promoting cartilage regeneration and repair in osteoarthritis.

Platelet-rich plasma alleviates knee arthritis in rats by inhibiting p65.

Knee osteoarthritis (KOA) is a chronic joint disease characterized by the degeneration of articular cartilage. In this study, we explored the potential therapeutic effects of platelet-rich plasma (PRP) and identified molecular targets for treating KOA. A rat model of KOA was established via the Hulth method and primary knee joint chondrocytes were isolated to evaluate the effects of PRP and shRNA targeting p65 (sh-p65). ELISA was used to detect inflammatory factors, including IL-6, IL-1β, and TNF-α. HE staining, Safranin O/Fast Green staining and Masson staining were performed to evaluate the morphology of articular cartilage, followed by detection of p65, COL2A1, ACAN, MMP13, and ADAMTS5 expression. The proliferation and apoptosis of primary knee chondrocytes were detected by the CCK-8 assay and TUNEL staining, respectively. Treatment with either PRP or sh-p65 decreased IL-6, IL-1β, and TNF-α levels in the peripheral blood of KOA rats and chondrocyte culture supernatants, increased COL2A1 and ACAN levels, and decreased MMP13 and ADAMTS5 expression. Furthermore, administration of PRP or sh-p65 exerted protective effects on articular cartilage, enhanced the vitality of knee joint chondrocytes, and inhibited apoptosis. Collectively, PRP inhibited inflammation, promoted chondrocyte proliferation and cartilage matrix secretion, and induced cartilage regeneration by suppressing p65 expression; these effects allow PRP to alleviate KOA progression. P65-based targeted therapy administered in combination with PRP might be a promising strategy for treating KOA.

System pharmacology analysis to decipher the effect and mechanism of active ingredients combination from Duhuo Jisheng decoction on osteoarthritis in rats

Ethnopharmacology relevanceDuhuo Jisheng decoction is a traditional Chinese formula that has been widely used in clinical practice to treat osteoarthritis, which has the effects of removing invaded cold and dampness, relieving joint pain. However, it is difficult to determine the effective substances and mechanisms due to assorted herbs and components, and further research is needed. Aim of the studyThis study was designed to explore and verify the mechanism and targets of DHJSD in the treatment of OA via network analysis and experiments. MethodIn this study, the active ingredients of DHJSD were qualitatively analyzed by UPLC-QDA. Network analysis was used to identify common targets and pathways. Next, we explored the therapeutic mechanism of DHJSD through a rat model of knee osteoarthritis. HE staining was used to judge the establishment of the animal model. ELISA and Western blotting were used to verify the expression of key pathway proteins. ConclusionIn this study, seventeen chemical constituents in DHJSD were identified. According to the network analysis, we obtained the potential associated pathways of action. Then, molecular docking and SPR experiments showed that the sixteen identified components had high binding energies to IL-6. HE staining showed that the high-dose group of DHJSD had an obvious therapeutic effect on model rats. Compared with the model group, the levels of IL-1β, TNF-α, IL-6, MMP3, MMP13, ADAMTS4 and ADAMTS5 in serum and the expression of STAT3 and p-STAT3 protein in administration groups were significantly decreased. This result indicated that the IL-6/STAT3 signaling pathway was one of the important pathways regulated by DHJSD to improve OA.

Modulation of Brain Network Topological Properties in Knee Osteoarthritis by Electroacupuncture in Rats.

Osteoarthritis is a chronic, ongoing disease that affects patients, and pain is considered a key factor affecting patients, but the brain changes during the development of osteoarthritis pain are currently unclear. In this study, we used electroacupuncture (EA) to intervene the rat model of knee osteoarthritis and analyzed the changes in topological properties of brain networks using graph theory. Sixteen SD rat models of right-knee osteoarthritis with anterior cruciate ligament transection (ACLT) were randomly divided into electroacupuncture intervention group and control group. The electroacupuncture group was intervened on Zusanli (ST36) and Futu (ST32) for 20 min each time, five times a week for 3 weeks, while the control group was applied sham stimulation. Both groups were measured for pain threshold. The small-world properties and node properties of the brain network between the two groups after the intervention were statistically analyzed by graph theory methods. The differences are mainly in the changes in node attributes between the two groups, such as degree centrality, betweenness centrality, and so on in different brain regions (P<0.05). Both groups showed no small-world characteristics in the brain networks of the two groups. The mechanical thresholds and thermal pain thresholds were significantly higher in the EA group than in the control group (P<0.05). The study demonstrated that electroacupuncture intervention enhanced the activity of nodes related to pain circuit and relieved pain in osteoarthritis, which provides a complementary basis for explaining the effect of electroacupuncture intervention on pain through graphical analysis of changes in brain network topological properties and helps to develop an imaging model for pain affected by electroacupuncture.

No Evidence for Cognitive Impairment in an Experimental Rat Model of Knee Osteoarthritis and Associated Chronic Pain

Although chronic pain states have been associated with impaired cognitive functions, including memory and cognitive flexibility, the cognitive effects of osteoarthritis (OA) pain remain to be clarified. The aim of this study was to measure cognitive function in the mono-iodoacetate (MIA) rat model of chronic OA-like knee pain. We used young adult male Lister hooded rats, which are well-suited for cognitive testing. Rats received either a unilateral knee injection of MIA (3 mg/50 µL) or saline as control. Joint pain at rest was assessed for up to 12 weeks, using weight-bearing asymmetry, and referred pain at a distal site, using determination of hindpaw withdrawal thresholds. The watermaze delayed-matching-to-place test of rapid place learning, novel object recognition memory assay, and an operant response-shift and -reversal task were used to measure memory and behavioral flexibility. Open-field locomotor activity, startle response, and prepulse inhibition were also measured for comparison. MIA-injected rats showed markedly reduced weight-bearing on the injured limb, as well as pronounced cartilage damage and synovitis, but interestingly no changes in paw withdrawal threshold. Rearing was reduced, but otherwise, locomotor activity was normal and no changes in startle and prepulse inhibition were detected. MIA-injected rats had intact watermaze delayed-matching-to-place performance, suggesting no substantial change in hippocampal function, and there were no changes in novel object recognition memory or performance on the operant task of behavioral flexibility. Our finding that OA-like pain does not alter hippocampal function, unlike other chronic pain conditions, is consistent with human neuroimaging findings. PerspectiveYoung adult rats with OA-like knee pain showed no impairments in hippocampal memory function and behavioral flexibility, suggesting that OA pain impacts cognitive functions less than other chronic pain conditions. In patients, OA pain may interact with other factors (e.g., age, socio-economic factors, and medication) to impair cognition.

Clinical efficacy of Yiqi Yangxue formula on knee osteoarthritis and unraveling therapeutic mechanism through plasma metabolites in rats.

Objective: To observe the clinical efficacy and safety of Yiqi Yangxue formula (YQYXF) on knee osteoarthritis (KOA), and to explore the underlying therapeutic mechanism of YQYXF through endogenous differential metabolites and their related metabolic pathways. Methods: A total of 61 KOA patients were recruited and divided into the treatment group (YQYXF, 30 cases) and the control group (celecoxib, Cxb, 31 cases). Effects of these two drugs on joint pain, swelling, erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) were observed, and their safety and adverse reactions were investigated. In animal experiments, 63 SD rats were randomly divided into normal control (NC) group, sham operation (sham) group, model (KOA) group, Cxb group, as well as low-dose (YL), medium-dose (YM), and high-dose groups of YQYXF (YH). The KOA rat model was established using a modified Hulth method. Ultra-high-performance liquid chromatography/Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass (UHPLC-QE-MS)-based metabolomics technology was used to analyze the changes of metabolites in plasma samples of rats. Comprehensive (VIP) >1 and t-test p < 0.05 conditions were used to screen the disease biomarkers of KOA, and the underlying mechanisms of YQYXF were explored through metabolic pathway enrichment analysis. The related markers of YQYXF were further verified by ELISA (enzyme-linked immunosorbent assay). Results: YQYXF can improve joint pain, swelling, range of motion, joint function, Michel Lequesen index of severity for osteoarthritis (ISOA) score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, ESR, and CRP. No apparent adverse reactions were reported. In addition, YQYXF can improve cartilage damage in KOA rats, reverse the abnormal changes of 16 different metabolites, and exert an anti-KOA effect mainly through five metabolic pathways. The levels of reactive oxygen species (ROS) and glutathione (GSH) were significantly decreased after the treatment of YQYXF. Conclusion: YQYXF can significantly improve the clinical symptoms of KOA patients without obvious adverse reactions. It mainly improved KOA through modulating lipid metabolism-related biomarkers, reducing lipid peroxidation and oxidative stress.

PPAR-γ Activation Alleviates Osteoarthritis through Both the Nrf2/NLRP3 and PGC-1α/Δψ m Pathways by Inhibiting Pyroptosis.

Osteoarthritis (OA) is a common degenerative joint disease with a gradually increasing morbidity in the aging and obese population. Emerging evidence has implicated pyroptosis in the etiology of OA and it may be recognized as a therapeutic target in OA. We have previously reported regarding another disease that peroxisome proliferator-activated receptor gamma (PPAR-γ) activation exerts an anti-inflammatory effect by suppressing the nucleotide-binding and oligomerization domain-like receptor containing protein (NLRP) 3 inflammasome. However, the relationship between PPAR-γ and NLRP3-mediated pyroptosis in OA cartilage and its underlying mechanisms is fully unclear. In this study, we found that the level of NLRP3-mediated pyroptosis in severe lateral femoral condyle cartilage wear in the knee of an OA patient was significantly higher than that in the mild lateral femoral condyle cartilage wear areas. Moreover, in lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced primary chondrocytes and knee OA rat models, we demonstrated that activation of PPAR-γ by pioglitazone (Piog) attenuated LPS/ATP-induced chondrocyte pyroptosis and arthritis. These effects were partially counteracted by either blocking the nuclear factor erythroid-2-related factor (Nrf2)/NLRP3 or PGC1-α/Δψ m signaling pathway. Simultaneous depression of these two signaling pathways can completely abrogate the protective effects of Piog on OA and chondrocytes. Taken together, Piog protects OA cartilage against pyroptosis-induced damage by simultaneously activating both the Nrf2/NLRP3 and PGC-1α/Δψ m pathways, which enhances antioxidative and anti-inflammatory responses as well as mitochondrial biogenesis. Therefore, Piog may be a promising agent for human OA cartilage damage in future clinical treatments.

The effect of postmenopausal osteoporosis on subchondral bone pathology in a rat model of knee osteoarthritis

This study aimed to investigate the additional effect of ovariectomy-induced osteoporosis (OP) on the pathology of knee osteoarthritis (OA) in a rat meniscectomized model, particularly focusing on subchondral bone changes and pain behaviour. Rats were divided into four groups, sham, OP, OA, OP plus OA, and assessed for histology, osteoclast activity, subchondral bone microstructure, and pain-related behaviour. Rats with OP plus OA had significantly increased calcified cartilage and subchondral bone damage scores, increased densities of subchondral osteoclasts in the weight-bearing area, and more porous subchondral trabecular bone compared with rats with OA. Loss of tidemark integrity was observed most frequently in rats with OP plus OA. The density of subchondral osteoclasts correlated with the calcified cartilage and subchondral bone damage score in rats with OA (OA and OP plus OA). No significant differences in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) expression ratio in subchondral bone and pain-related behavioural tests were observed between rats with OA and rats with OP plus OA. In rats with OA, coexisting OP potentially aggravated OA pathology mainly in calcified cartilage and subchondral trabecular bone by increasing subchondral osteoclast activity.

Comparing Effectiveness of Hyaluronic Acid-Chitosan Nanoparticles Encapsulation Versus Hyaluronic Acid Monotherapy in Osteoarthritis Rat Model: Microarray Screening for miR-140.

Osteoarthritis is a debilitating, progressive joint disease linked to lower quality of life and higher health care costs. This study compared hyaluronic acid-chitosan nanoparticle encapsulation to hyaluronic-acid monotherapy in a rat model of knee osteoarthritis. Four groups of 40 adult male albino rats were designed. Group (Gp) I: control; Gp II (osteoarthritis model): intra-articular injection of monoiodoacetate; Gp III (hyaluronic acid-treated): intra-articular injections of hyaluronic-acid on days 14 and 21 after monoiodoacetate injection; and Gp IV (hyaluronic acid-chitosan nanoparticle-treated): intra-articular injections of hyaluronic acid-chitosan nanoparticle on days 14 and 21 after monoiodoacetate injection. After 28 days, knee joints were examined using H&E, Safranin O, and immunohistochemistry for nuclear factor kappa beta (NF-κB), inducible nitric oxide synthase (iNOS), and matrix metalloproteinase (MMP)-13. Quantification for gene expression of collagen-II, aggrecan, and micro-RNA-140; ELISA for interleukin (IL)-1β and IL-8; and western blotting for IKBα and NF-κB was estimated. Osteoarthritis-knee joints showed a severe cartilage damage and synovial inflammation with increased NF-κB, iNOS, and MMP-13 immunostaining, decreased miR-140, collagen II, and aggrecan levels, and increased inflammatory markers' gene expressions. The hyaluronic acid-chitosan nanoparticle significantly improved knee joint structure and reduced inflammatory cytokines compared to hyaluronic acid monotherapy. Intra-articular injection of hyaluronic acid-chitosan nanoparticle encapsulation revealed a significant improvement in the knee joint structure compared to hyaluronic-acid in a rat model of osteoarthritis.

Electroacupuncture ameliorates knee osteoarthritis in rats via inhibiting NLRP3 inflammasome and reducing pyroptosis.

Objective: Knee Osteoarthritis (KOA), is the most common joint disease worldwide. The pathogenesis of KOA is complex and electroacupuncture (EA) is an effective therapy for KOA, but the mechanism remains unclear. In this study, we aim to investigate the potential therapeutic effect of EA on the rat model of KOA induced by monosodium iodoacetate (MIA) and its relationship with NLRP3 inflammasome by immunohistochemistry and western blot. Methods: KOA was induced by intra-articular injection of MIA (3mg/50μL) into the right knee joint of rats. Forty-five male rats weighing 250-300g were randomly divided into 3 groups: control group, KOA group, and KOA + electroacupuncture group (KOA+EA). EA treatment lasted for 2weeks (6 times a week). Paw withdrawal threshold tests were used to assess mechanical allodynia once a week. Safranin O/Fast Green and hematoxylin and eosin (H&E) staining were used to assess the damage to cartilage, synovium, and subpatellar fat pad (IFP). Immunohistochemistry was used to observe NLRP3 inflammasome-associated protein-positive cells in the same field of view and western blot was used to detect the expression of the associated protein in cartilage tissue. Results: The KOA group showed mechanical hyperalgesia, joint inflammation, and significant cartilage tissue destruction. Safranin O/Fast Green and H&E staining revealed that EA alleviated the joint pathological changes caused by KOA and had a protective effect on cartilage, synovium, and IFP destruction. Mechanical allodynia pain and joint swelling were reduced in KOA rats after EA treatment. Immunohistochemistry and western blot showed significant inhibition of NLRP3 inflammasome-associated protein. Conclusion: The results indicate that EA can inhibit NLRP3 inflammasome and reduce pyroptosis, which results in the protection of cartilage tissue and the treatment of KOA. It provides reliable evidence for the development of EA in the treatment of KOA and the clinical application of acupuncture.

Mechanical stress protects against chondrocyte pyroptosis through TGF-β1-mediated activation of Smad2/3 and inhibition of the NF-κB signaling pathway in an osteoarthritis model

Osteoarthritis (OA) is a degenerative disease that is difficult to cure owing to its complicated pathogenesis. Exercise therapy has been endorsed as a primary treatment option. However, it remains controversial how exercise intensity regulates OA progression. Here, a declining propensity for TGF-β1 was predicted via bioinformatics analysis of microarray GSE57218 and validated in cartilage samples obtained from arthroplasty. Based on this, cyclic tensile strain or TGF-β1 intervention was performed on human OA chondrocytes, and we found that moderate-intensity mechanical loads protected chondrocytes against pyroptosis. During this process, the elevation of TGF-β1 is mechanically stimuli-dependent and exerts an inhibitory effect on chondrocyte pyroptosis. Moreover, we elucidated that TGF-β1 activated Smad2/3 and inhibited the NF-κB signaling pathway to suppress chondrocyte pyroptosis. Furthermore, we established a rat knee OA model by intra-articular injection of monosodium iodoacetate and performed treadmill exercises of different intensities. Similar to the in vitro results, we demonstrated that moderate-intensity treadmill exercise had an outstanding chondroprotective effect. An inappropriate intensity of mechanical stimulation may aggravate OA both in vivo and in vitro. Overall, our findings demonstrated that activation of the TGF-β1/Smad2/Smad3 axis and inhibition of NF-κB coordinately inhibit chondrocyte pyroptosis under mechanical loads. This study sheds light on the future development of safe and effective exercise therapies for OA.

Effect of Bushen Huoxue Decoction on Apoptosis-related Protein and Serum Inflammatory Factors in Rat Model of Knee Osteoarthritis

Effect of Bushen Huoxue Decoction on Apoptosis-related Protein and Serum Inflammatory Factors in Rat Model of Knee Osteoarthritis

Boiogito, a Japanese Traditional Herbal Medicine, Inhibits the Osteoclast Differentiation and Proliferation in the Subchondral Bone of an In Vivo Knee Osteoarthritis Rat Model

Boiogito (BO), a Japanese traditional herbal medicine, has been reported to prevent knee osteoarthritis (KOA) development in in vivo studies. In the early stage of KOA, osteoclasts proliferate in the subchondral bone. This study aimed to investigate the preventive effect of BO on osteoclast proliferation, which remains unclear, in a KOA-induced rat model. KOA was induced in 12-week-old male Wistar rats using surgical destabilization of the medial meniscus (DMM). BO was mixed with powdered chow, applying 1%, 3%, and 5% of the total feed, and administered to KOA-induced rats. The rats were divided into 6 groups: control, sham, DMM, DMM + BO 1%, DMM + BO 3%, and DMM + BO 5%. Rotarod tests were performed each week to assess the locomotor function, and the right knees were harvested 28 days after surgery for histological analysis. Oral administration of BO significantly inhibited the decrease in the latency to fall off in the rotarod test, which was aggravated in the DMM group. Furthermore, KOA development was significantly prevented in the BO-administrated groups as assessed by the Osteoarthritis Research Society International score. The number of multinucleated activated osteoclasts in the subchondral bone was decreased in the BO-treated groups, which was increased in the DMM group. Therefore, oral administration of BO may reduce articular cartilage degeneration, osteoclast differentiation and proliferation in the KOA patients.

Ebselen, an Active Seleno-Organic Compound, Alleviates Articular Cartilage Degeneration in a Rat Model of Knee Osteoarthritis.

Osteoarthritis (OA) is a prevalent articular disease mainly characterized by extracellular matrix degradation, apoptosis, and inflammation, which lead to cartilage destruction and abnormal bone metabolism. With undesirable side effects, current limited symptomatic treatments are aimed at relieving pain and improving joint mobility in patients with OA. Intra-articular (IA) hyaluronic acid (HA) injection, as a nonsurgical therapy, is commonly used in the clinical management of knee OA, but the efficacy of this therapeutic option remains controversial. Ebselen has tremendous pharmacological importance for some diseases due to its antioxidant, antiapoptotic, and anti-inflammatory features. However, there is no research examining the therapeutic effect of Ebselen in OA using the rat OA model. Therefore, we aimed to investigate the therapeutic effect of Ebselen on cartilage degeneration and its role in bone morphogenetic protein 2 (BMP2) and nuclear factor kappa B (NF-κB) signaling in the molecular pathogenesis of OA. We induced a knee OA model in rats with an IA injection of monosodium-iodoacetate (MIA). After the treatment of Ebselen, we evaluated its chondroprotective effects by morphological, histopathological, and immunohistochemical methods and an enzyme-linked immunosorbent assay. We report for the first time that Ebselen treatment alleviated articular cartilage degeneration in the rat knee OA model and reduced MIA-induced BMP2 and NF-κB expressions. In addition, our results unveiled that Ebselen decreased IL-β and IL-6 levels but did not affect COMP levels in the rat serum. Ebselen could be a promising therapeutic drug for the prevention and treatment of OA by alleviating cartilage degeneration and regulating BMP2 and NF-κB expressions.

MiR-219a-5p inhibits the pyroptosis in knee osteoarthritis by inactivating the NLRP3 signaling via targeting FBXO3.

This work was to identify the function and mechanism of miR-219a-5p in regulating knee osteoarthritis (KOA). Rat fibroblast-like synoviocytes (FLSs) were isolated to construct KOA cell model by lipopolysaccharide and adenosine triphosphate treatment. miR-219a-5p and FBXO3 expression in FLSs was modulated by transfection. Flow cytometry was executed to research FLSs apoptosis. Caspase-1 and IL-1β expression in FLSs was researched by immunofluorescence. The binding between miR-219a-5p and FBXO3 was identified by dual luciferase reporter gene assay. KOA rat model and miR-219a-5p up-modulation KOA rat model were constructed. Step size of rats was analyzed. Knee joints of rats were experienced Safranin O-fast green staining to evaluate the knee joint injury. FBXO3, pyroptosis-associated proteins, and IL-1β and IL-18 expression in FLSs and articular cartilage tissues of rats were assessed by Western blot, qRT-PCR and Enzyme-linked immunosorbent assay. KOA cell model had higher apoptosis percentage, expression of pyroptosis-associated proteins, and IL-1β and IL-18 level. miR-219a-5p up-modulation decreased the above indicators, whereas miR-219a-5p down-modulation increased the above indicators. FBXO3 expression was directly repressed by miR-219a-5p. Loss of FBXO3 suppressed the above indicators. FBXO3 counteracted the suppression of miR-219a-5p on the above indicators. miR-219a-5p agomir attenuated knee joint injury, increased step size of KOA rats, and reduced FBXO3, pyroptosis-associated proteins and level of IL-1β and IL-18 in the articular cartilage tissues of KOA rats. miR-219a-5p suppressed the pyroptosis in KOA by inactivating the NLRP3 signaling via targeting FBXO3, which might be a promising target for ameliorating KOA in the clinic.

Celastrol ameliorates osteoarthritis via regulating TLR2/NF-κB signaling pathway.

Objectives: Osteoarthritis (OA) is a joint disease characterized by degeneration of joint cartilage and is a significant cause of severe joint pain, physical disability, and impaired quality of life in the aging population. Celastrol, a Chinese herbal medicine, has attracted wide interests because of its anti-inflammatory effects on a variety of diseases. This study aimed to investigate the effect of celastrol on OA as well as the mechanisms in vivo and in vitro. Methods: A rat knee OA model was established using “medial collateral ligament transection (MCLT) + partial meniscectomy (pMMT)”. Eight weeks after surgery, the OA rats started to receive intra-articular injection of celastrol (1 mg/kg) once a week. Safranin O-fast green (S&F) and hematoxylin and eosin (H&E) staining were used to estimate histopathological changes. Micro-CT was used to evaluate bone volume of the subchondral bone of the knee joint. Chondrocytes were isolated from the knee cartilage of rats and OA patients. Enzyme linked immunosorbent assay (ELISA), Western Blot (WB), Polymerase Chain Reaction (PCR), and Immunohistochemistry (IHC) were used to detect the expression of inflammatory factors and stromal proteins, respectively. Results: We found that celastrol treatment significantly delayed the progression of cartilage damage with a significant reduction in osteophyte formation and bone resorption in OA rat model. In IL-1β-stimulated rat chondrocytes, celastrol significantly suppressed the production of inflammatory factors such as cyclooxygenase-2 (COX2), interleukin-6 (IL-6), and prostaglandin E2 (PEG2), and reduced IL-1β-induced matrix degradation by down-regulating the expression of matrix metalloproteinase 13 (MMP13). In addition, we found that toll-like receptor 2 (TLR2) was up-regulated in OA patients and rat knee OA models, while celastrol inhibited TLR2 signal and its downstream nuclear factor-kappa B (NF-κB) phosphorylation. Conclusion: In summary, celastrol may improve OA by inhibiting the TLR2/NF-κB signaling pathway, which provides innovative strategies for the treatment of OA.

Inhibitory effect of low‑intensity pulsed ultrasound on the fibrosis of the infrapatellar fat pad through the regulation of HIF‑1α in a carrageenan‑induced knee osteoarthritis rat model.

Fibrotic changes in the infrapatellar fat pad (IFP) are involved in the pathogenesis of knee osteoarthritis (KOA). HIF-1α is a transcription factor that is activated during hypoxia and is suggested to play a role in fibrosis in various organs. However, its participation in the fibrotic changes in IFP remains unclear. Therefore, we investigated the role of HIF-1α in IFP fibrosis using a carrageenan-induced KOA rat model and evaluated the potential of low-intensity pulsed ultrasound (LIPUS) as a novel treatment for KOA. A rat model was prepared by intra-articular injection of 0.5% carrageenan (50 µl) using 8-week-old male Wistar rats. Fibrosis of the IFP was evaluated histologically by hematoxylin and eosin and Sirius Red staining at 1 and 2 weeks after intra-articular injection. The mRNA expression levels of HIF-1α and fibrosis-related molecules, CTGF and VEGF, were analyzed using reverse transcription-quantitative PCR, and the DNA binding activity of HIF-1α was assessed using a binding assay. In addition, the effect of irradiation with LIPUS on the fibrosis of IFP was verified. Histological studies demonstrated a significant increase in the fibrosis of IFP 1 and 2 weeks after intra-articular injection of carrageenan, accompanied by overexpression of CTGF and VEGF, which was followed by upregulation of transcriptional activation of HIF-1α. Moreover, intervention with LIPUS for 2 weeks after injection of carrageenan attenuated fibrosis of IFP, accompanied by a significant reduction in the transcriptional activation of HIF-1α and decreased the gene expression levels of HIF-1α, CTGF, and VEGF. The present study demonstrated that activation of HIF-1α promoted fibrosis of IFP in carrageenan-induced arthritis in rats and that intervention with LIPUS decreased the activity of HIF-1α and inhibited fibrosis. These results suggest that LIPUS may serve as a novel approach for the treatment of KOA, through its modulation of HIF-1α.

BMSCs-derived Mitochondria Improve Osteoarthritis by Ameliorating Mitochondrial Dysfunction and Promoting Mitochondrial Biogenesis in Chondrocytes

Bone marrow mesenchymal stem cells (BMSCs)-derived exosomes and microvesicles can effectively improve knee osteoarthritis. We found that microvesicles performed a superior effect on improving mitochondrial function in chondrocytes than exosomes, which may be related to the ability of microvesicles carrying active mitochondria to replace damaged ones in chondrocytes. This study investigated the therapeutic effect of direct mitochondrial transplantation (MT) on knee osteoarthritis. IL-1β stimulated the osteoarthritis phenotype of rat chondrocytes, and the effect of BMSCs-derived mitochondria transplantation was observed in vitro. Knee osteoarthritis rat model was established by collagenase induction to observe the effect of intra-articular injection of mitochondria. Results showed that the mitochondria of BMSCs could be ingested by rat chondrocytes via co-incubation in vitro, and significantly improved osteoarthritis phenotype and mitochondrial function, and inhibited chondrocytes apoptosis. In vivo, BMSCs-derived mitochondria could be ingested by cartilage via intra-articular injection, ameliorated pathological cartilage injury, suppressed inflammation, inhibited chondrocytes apoptosis, and improved osteoarthritis phenotype. In addition, MT promoted mitochondrial biogenesis in chondrocytes by activating PGC-1α signaling. All above results suggest that BMSCs-derived mitochondria transplantation ameliorates knee osteoarthritis by improving chondrocytes mitochondrial dysfunction and promoting mitochondrial biogenesis.

Curcumin exerts chondroprotective effects against osteoarthritis by promoting AMPK/PINK1/Parkin-mediated mitophagy

Osteoarthritis (OA), a chronic degenerative disease with heterogeneous properties, is difficult to cure due to its complex pathogenesis. Curcumin possesses excellent anti-inflammatory and antioxidant properties and may have potential therapeutic value in OA. In this study, we investigated the action targets of curcumin and identified potential anti-OA targets for curcumin. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analyses were performed to evaluate these targets. Furthermore, we established a sodium monoiodoacetate-induced rat knee OA model and IL-1β induced OA chondrocyte model to verify the effect and mechanism of curcumin against OA. The GO and KEGG analyses screened seven hub genes involved in metabolic processes and the AMPK signaling pathway. Curcumin can significantly attenuate OA characteristics according to Osteoarthritis Research Society International (OARSI) and Mankin scores in OA rats. Additionally, curcumin is notably employed as an activator of mitophagy in maintaining mitochondrial homeostasis (ROS, Ca2+, ATP production, and mitochondrial membrane potential). The expression levels of mitophagy-related proteins were increased not only in articular cartilage but also in chondrocytes with curcumin intervention. Combining validation experiments and network pharmacology, we identified the importance of mitophagy in the curcumin treatment of OA. The chondroprotective effects of curcumin against OA are mediated by the AMPK/PINK1/Parkin pathway, and curcumin may serve as a potential novel drug for OA management.

Low-Intensity Pulsed Ultrasound Enhances the Efficacy of Bone Marrow-Derived MSCs in Osteoarthritis Cartilage Repair by Regulating Autophagy-Mediated Exosome Release.

ObjectiveThe present study explored whether low-intensity pulsed ultrasound (LIPUS) enhances the therapeutic efficacy of mesenchymal stem cells (MSCs) in osteoarthritis (OA) cartilage repair by regulating autophagy-mediated exosome release.DesignMSCs were isolated from the rat bone marrow and treated with rapamycin, 3-methyladenine, or LIPUS. The mechanism of the LIPUS-stimulated exosome release by MSCs was analyzed by inhibiting autophagy. In addition, the MSCs were co-cultured with OA chondrocytes and stimulated by LIPUS, with or without exosome release inhibitor intervention. The exosome release was detected through transmission electron microscopy (TEM), nanoparticle tracking analysis, and biomarker expression analysis. Autophagy was analyzed through TEM, autophagy-related gene expression analysis, and immunofluorescence analysis in vitro. Furthermore, a rat knee OA model was constructed and treated with MSCs, GW4869, and LIPUS. The cartilage repair was assessed through histopathological analysis and extracellular matrix protein expression analysis.ResultsThe in vitro results indicated that LIPUS promoted MSC exosome release by activating autophagy. The in vivo results demonstrated that LIPUS significantly enhanced the positive effects of MSCs on OA cartilage. These effects were significantly blocked by GW4869, an inhibitor of exosome release.ConclusionsLIPUS can enhance the therapeutic efficacy of MSCs in OA cartilage repair, and the underlying mechanism is related to the increase in autophagy-mediated exosome release.