Rat Model Of Knee Osteoarthritis Research Articles

Exosomes derived from 3D-cultured MSCs alleviate knee osteoarthritis by promoting M2 macrophage polarization through miR-365a-5p and inhibiting TLR2/Myd88/NF-κB pathway

Exosomes (EXO) derived from mesenchymal stem cells (MSCs) are recently shown to have therapeutic effects in preclinical models of knee osteoarthritis (KOA). However, their potential for conversion into a clinical therapy are limited by several shortcomings such as low yield and suboptimal efficacy. This study developed 3D-EXO to improve exosome yield and efficacy through 3D-culturing of MSCs, with the aim of achieving overall better therapeutic effects compared to traditional 2D-cultured MSC-derived EXO (2D-EXO). In a rat KOA model, 3D-EXO led to better preservation of bone structure and protection from cartilage damage compared to 2D-EXO. Interestingly, in vitro experiments showed no significant differences between 3D-EXO and 2D-EXO in their direct effects on chondrocytes. To explain these findings, we looked into the possible effects of MSC-derived EXO on the immune microenvironment, specifically in modulating phenotypic expression in macrophages. In vitro investigations showed that 3D-EXO increased the expression of M2 phenotype markers in macrophages to a greater extent than 2D-EXO. RNA-seq analysis revealed that expression of miR-365a-5p was more upregulated in 3D-EXO than 2D-EXO. Through micro-RNA mimics transfection, we found that miR-365a-5p acted directly on TLR2 mRNA to inhibit TLR2 translation, indicating that 3D-EXO promoted M2 polarization of macrophages by inhibiting the TLR2/Myd88/NF-κB axis. The immunomodulatory effects of 3D-EXO were confirmed by the observation that the metabolic status of chondrocytes was significantly improved after co-culturing with 3D-EXO-treated macrophages. In summary, 3D-EXO can effectively promote M2 macrophage polarization through increased expression of miR-365a-5p, which in turn improves the metabolic state of chondrocytes and resulting in overall better in vivo therapeutic effects on KOA. The findings in this study imply the development of new therapies for KOA based on MSC-derived EXO.

The impact of sericin on inflammation, oxidative stress, and lipid metabolism in female rats with experimental knee osteoarthritis.

This study investigated the effects of sericin on inflammation, oxidative stress, and lipid metabolism in female rats with experimental knee osteoarthritis (KOA), focusing on evaluating its effectiveness via the sterol regulatory protein (SREBP)-1C and SREBP-2 pathways. The rats were randomly assigned to three experimental groups: the C group (control), the KOA group (KOA control), and the sericin group (KOA + sericin). The KOA model was created by injecting monosodium iodoacetate (MIA) into the knee joint. Sericin was administered intra-articularly to rats on days 1, 7, 14, and 21 (0.8g/kg/mL, 50 µL). After 21days, the rats were sacrificed, and serum samples were analyzed using an ELISA to measure tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-10, SREBP-1c, SREBP-2, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), cholesterol, triglyceride, and total oxidant-antioxidant status (TOS-TAS) levels. The KOA group exhibited higher serum TNF-α, IL-1β, TOS, SREBP-1C, ACC, FAS, triglyceride, SREBP-2, and cholesterol levels than the C group (P < 0.05). However, the levels of these cytokines, except cholesterol, were significantly lower in the sericin group than in the KOA group. The KOA group exhibited significantly lower serum TAS and IL-10 levels than the C group (P < 0.05). In the sericin group, there was a statistically significant increase (P < 0.05). Sericin shows promising potential for reducing inflammation, oxidative stress, and lipid metabolism in experimental models of KOA in rats. However, further clinical research is necessary to validate the potential of sericin as a therapeutic agent for treating KOA. Key Points • Sericin can reduce knee osteoarthritis (KOA) symptoms in an experimental rat model. • In particular, in the serum of an experimental KOA rat model, sericin specifically reduces the levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β), and increases the levels of anti-inflammatory cytokines, such as IL-10. • Sericin reduced lipid metabolism via the sterol regulatory protein (SREBP)-1C and SREBP-2 pathways and oxidative stress in the serum of the experimental KOA rat model. • The intra-articular administration of sericin has been shown to significantly reduce lipid metabolism, oxidative stress, and inflammation, as supported by biochemical analysis. These findings suggest its promising potential as an alternative treatment option for KOA.

Diagnostic biomarkers in knee osteoarthritis: Based on bioinformatics and experimental verification in vivo and in vitro.

Knee osteoarthritis (KOA) is a multifactorial whole-joint disease with a high rate of disability. Considering the complexity of KOA, there is an urgent need to discover new molecular pathological markers and multi-target treatment strategies. Two datasets, GSE51588 and GSE57218, were downloaded from the Gene Expression Omnibus database and screened for differentially expressed genes (DEGs) using the Gene Expression Omnibus 2R (GEO2R). Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were performed. A protein-protein interaction (PPI) network was constructed and hub genes were identified using Molecular Complex Detection (MCODE). Receiver-operating characteristic curves (ROC) were plotted for the genes, and their prognostic values were evaluated. The expression levels of the hub genes in the monosodium iodoacetate (MIA)-induced KOA rat model and lipopolysaccharide (LPS)-stimulated C28/I2 cells were verified using reverse transcription quantitative real-time PCR (RT-qPCR). Overall, 33 DEGs were up-regulated and 6 DEGs were down-regulated in the two datasets. A total of 12 hub genes were identified, including COL15A1, THY1, COL1A1, COL5A1, CTHRC1, MXRA5, FN1, COL1A2, COL3A1, SPARC, COL8A1, and COL2A1, which all could be used as biomarkers to differentiate KOA samples from healthy controls. More importantly, we found that THY1, CTHRC1, SPARC, and COL8A1 were significantly upregulated in vivo and in vitro compared with the controls (p < .01). The expression levels of THY1, CTHRC1, SPARC, and COL8A1 were elevated and had good prognostic values as biomarkers in KOA.

Effect of Low-Intensity Pulsed Ultrasound on Macrophage Properties and Fibrosis in the Infrapatellar Fat Pad in a Carrageenan-Induced Knee Osteoarthritis Rat Model.

In the progression of knee osteoarthritis (KOA), fibrosis of the infrapatellar fat pad (IFP) is a key pathological change. Low-intensity pulsed ultrasound (LIPUS) inhibits IFP fibrosis by decreasing the gene expression and activity of hypoxia-inducible factor (HIF-1α), which is a protein involved in IFP fibrosis in KOA rat models. On the other hand, macrophages play an important role in the progression of fibrosis in various tissues, and LIPUS irradiation suppresses macrophage infiltration and inflammatory cytokine secretion. However, whether LIPUS suppresses macrophage polarity and IFP fibrosis in KOA remains unclear. Therefore, we investigated the effect of LIPUS on macrophage polarity and IFP fibrosis. A KOA model was created by injecting carrageenin into the bilateral knee joints of Wistar rats (eight weeks old). Tissues were harvested over time for histological and molecular biological analysis. The KOA model was also subjected to LIPUS irradiation for two weeks following the injection of carrageenin. RM-4-positive cells were widely distributed in IFP two weeks after carrageenin administration, but M2 macrophages were significantly increased, and the Sirius red area was decreased in the LIPUS-irradiated group compared with those in the non-irradiated group. The gene expression of M1 macrophage markers was significantly decreased and that of M2 macrophage markers was significantly increased in the LIPUS-irradiated group. The expression of transforming growth factor-β (TGF-β) and type 1 collagen was also significantly decreased. These results suggest that LIPUS may serve as a novel approach for the treatment of KOA through its effect on M1 macrophages and suppression of TGF-β expression.

Vagotomy accelerates the onset of symptoms during early disease progression and worsens joint-level pathogenesis in a male rat model of chronic knee osteoarthritis

ObjectiveLow vagal tone is common in osteoarthritis (OA) comorbidities and results in greater peripheral inflammation. Characterizing vagal tone's role in OA pathogenesis may offer insights into OA's influences beyond the articular joint. We hypothesized that low vagal tone would accelerate onset of OA-related gait changes and worsen joint damage in a rat knee OA model. MethodsKnee OA was induced in male Sprague Dawley rats by transecting the medial collateral ligament and medial meniscus. Then, left cervical vagus nerve transection (VGX, n ​= ​9) or sham VGX (non-VGX, n ​= ​6) was performed. Gait and tactile sensitivity were assessed at baseline and across 12 weeks, with histology and systemic inflammation evaluated at endpoint. ResultsAt week 4, VGX animals showed limping gait characteristics through shifted stance times from their OA to non-OA limb (p ​= ​0.055; stance time imbalance ​= ​1.6 ​± ​1.6%) and shifted foot strike locations (p ​< ​0.001; spatial symmetry ​= ​48.4 ​± ​0.835%), while non-VGX animals walked with a balanced and symmetric gait. Also at week 4, while VGX animals had a mechanical sensitivity (50% withdrawal threshold) of 13.97 ​± ​7.70 compared to the non-VGX animal sensitivity of 29.74 ​± ​9.43, this difference was not statistically significant. Histologically, VGX animals showed thinner tibial cartilage and greater subchondral bone area than non-VGX animals (p ​= ​0.076; VGX: 0.80 ​± ​0.036 ​mm2; non-VGX: 0.736 ​± ​0.066 ​mm2). No group differences in systemic inflammation were observed at endpoint. ConclusionsVGX resulted in quicker onset of OA-related symptoms but remained unchanged at later timepoints. VGX also had thinner cartilage and abnormal bone remodeling than non-VGX. Overall, low vagal tone had mild effects on OA symptoms and joint remodeling, and not at the level seen in common OA comorbidities.

Curcumin regulates autophagy through SIRT3-SOD2-ROS signaling pathway to improve quadriceps femoris muscle atrophy in KOA rat model

Knee osteoarthritis (KOA) usually leads to quadriceps femoris atrophy, which in turn can further aggravate the progression of KOA. Curcumin (CUR) has anti-inflammatory and antioxidant effects and has been shown to be a protective agent for skeletal muscle. CUR has been shown to have a protective effect on skeletal muscle. However, there are no studies related to whether CUR improves KOA-induced quadriceps femoris muscle atrophy. We established a model of KOA in rats. Rats in the experimental group were fed CUR for 5 weeks. Changes in autophagy levels, reactive oxygen species (ROS) levels, and changes in the expression of the Sirutin3 (SIRT3)-superoxide dismutase 2 (SOD2) pathway were detected in the quadriceps femoris muscle of rats. KOA led to quadriceps femoris muscle atrophy, in which autophagy was induced and ROS levels were increased. CUR increased SIRT3 expression, decreased SOD2 acetylation and ROS levels, inhibited the over-activation of autophagy, thereby alleviating quadriceps femoris muscle atrophy and improving KOA. CUR has a protective effect against quadriceps femoris muscle atrophy, and KOA is alleviated after improvement of quadriceps femoris muscle atrophy, with the possible mechanism being the reduction of ROS-induced autophagy via the SIRT3-SOD2 pathway.

Hypobaric hypoxia aggravates osteoarthritis via the alteration of the oxygen environment and bone remodeling in the subchondral zone.

A high prevalence of osteoarthritis (OA) has been observed among individuals living at high altitudes, and hypobaric hypoxia (HH) can cause bone mass and strength deterioration. However, the effect of HH on OA remains unclear. In this study, we aimed to explore the impact of HH on OA and its potential mechanisms. A rat knee OA model was established by surgery, and the rats were bred in an HH chamber simulating a high-altitude environment. Micro-computed tomography (Micro-CT), histological analysis, and RNA sequencing were performed to evaluate the effects of HH on OA invivo. A hypoxic co-culture model of osteoclasts and osteoblasts was also established to determine their effects on chondrogenesis invitro. Cartilage degeneration significantly worsened in the HH-OA group compared to that in the normoxia-OA (N-OA) group, 4 weeks after surgery. Micro-CT analysis revealed more deteriorated bone mass in the HH-OA group than in the N-OA group. Decreased hypoxia levels in the cartilage and enhanced hypoxia levels in the subchondral bone were observed in the HH-OA group. Furthermore, chondrocytes cultured in a conditioned medium from the hypoxic co-culture model showed decreased anabolism and extracellular matrix compared to those in the normoxic model. RNA sequencing analysis of the subchondral bone indicated that the glycolytic signaling pathway was highly activated in the HH-OA group. HH-related OA progression was associated with alterations in the oxygen environment and bone remodeling in the subchondral zone, which provided new insights into the pathogenesis of OA.

Polydatin protects against articular cartilage degeneration by regulating autophagy mediated by the AMPK/mTOR signaling pathway.

Knee osteoarthritis (KOA) is one of the leading causes of disability. Polydatin has a potential effect on KOA treatment but the therapeutic mechanism is not clear. This study aims to investigate the therapeutic action of polydatin in KOA and its mechanism in activating autophagy via the AMP-activated protein kinase (AMPK)/mTOR signaling pathway. After a KOA rat model was established by anterior cruciate ligament transection surgery, model rats were treated with polydatin 40 mg/kg for 30 days. Subsequently, cartilage tissues were collected, and hematoxylin-eosin (HE), Safranin-O, and TUNEL staining, and western blotting were performed to evaluate the pathological damage and autophagy-related protein expression. Then, human chondrocyte C28/I2 cells were stimulated with lipopolysaccharide (LPS), and the effects of polydatin on C28/I2 cell viability, apoptosis, and autophagy-related protein expression were detected by MTT, Flow Cytometry, and western blot. In addition, an AMPK inhibitor (Dorsomorphin 2HCl) was used to probe the cell proliferation and apoptosis of polydatin-administered C28/I2 cells. Polydatin ameliorated the pathological damage in rat cartilage tissues and inhibited cell apoptosis in KOA rats. Meanwhile, in C28/I2 cells, polydatin promoted viability and reduced apoptosis. In addition, the protein expression of collagen II, LC3II/LC3I, Beclin-1, and p-AMPK/AMPK were upregulated, and p62 and p-mTOR/mTOR were downregulated by polydatin treatment. Interestingly, relative results showed that the protective effect of polydatin in LPS-stimulated-C28/I2 cells was blocked by the AMPK/mTOR inhibitor, dorsomorphin 2HCl. Our research showed that polydatin reduced apoptosis and activated autophagy both in vivo and in vitro by the AMPK/mTOR signaling pathway to protect against KOA, which provided the basis for further investigation into the potential therapeutic impact of polydatin on KOA.

Extracellular vesicles derived from mesenchymal stem cells mediate extracellular matrix remodeling in osteoarthritis through the transport of microRNA-29a.

Knee osteoarthritis (KOA) is a common orthopedic condition with an uncertain etiology, possibly involving genetics and biomechanics. Factors like changes in chondrocyte microenvironment, oxidative stress, inflammation, and immune responses affect KOA development. Early-stage treatment options primarily target symptom relief. Mesenchymal stem cells (MSCs) show promise for treatment, despite challenges. Recent research highlights microRNAs (miRNAs) within MSC-released extracellular vesicles that can potentially promote cartilage regeneration and hinder KOA progression. This suggests exosomes (Exos) as a promising avenue for future treatment. While these findings emphasize the need for effective KOA progression management, further safety and efficacy validation for Exos is essential. To explore miR-29a's role in KOA, we'll create miR-29a-loaded vesicles, testing for early treatment in rat models. Extraction of bone marrow MSC-derived extracellular vesicles, preparation of engineered vesicles loaded with miR-29a using ultrasonication, and identification using quantitative reverse transcription polymerase chain reaction; after establishing a rat model of KOA, rats were randomly divided into three groups: Blank control group injected with saline, normal extracellular vesicle group injected with normal extracellular vesicle suspension, and engineered extracellular vesicle group injected with engineered extracellular vesicle suspension. The three groups were subjected to general behavioral observation analysis, imaging evaluation, gross histological observation evaluation, histological detection, and immunohistochemical detection to compare and evaluate the progress of various forms of arthritis. General behavioral observation results showed that the extracellular vesicle group and engineered extracellular vesicle group had better performance in all four indicators of pain, gait, joint mobility, and swelling compared to the blank control group. Additionally, the engineered extracellular vesicle group had better pain relief at 4 wk and better knee joint mobility at 8 wk compared to the normal extracellular vesicle group. Imaging examination results showed that the blank control group had the fastest progression of arthritis, the normal extracellular vesicle group had a relatively slower progression, and the engineered extracellular vesicle group had the slowest progression. Gross histological observation results showed that the blank control group had the most obvious signs of arthritis, the normal extracellular vesicle group showed signs of arthritis, and the engineered extracellular vesicle group showed no significant signs of arthritis. Using the Pelletier gross score evaluation, the engineered extracellular vesicle group had the slowest progression of arthritis. Results from two types of staining showed that the articular cartilage of rats in the normal extracellular vesicle and engineered extracellular vesicle groups was significantly better than that of the blank control group, and the engineered extracellular vesicle group had the best cartilage cell and joint surface condition. Immunohistochemical detection of type II collagen and proteoglycan showed that the extracellular matrix of cartilage cells in the normal extracellular vesicle and engineered extracellular vesicle groups was better than that of the blank control group. Compared to the normal extracellular vesicle group, the engineered extracellular vesicle group had a better regulatory effect on the extracellular matrix of cartilage cells. Engineered Exos loaded with miR-29a can exert anti-inflammatory effects and maintain extracellular matrix stability, thereby protecting articular cartilage, and slowing the progression of KOA.

Casticin ameliorates osteoarthritic cartilage damage in rats through PI3K/AKT/HIF-1α signaling

Knee osteoarthritis (KOA) is a chronic, disabling knee joint lesion in which degeneration and defects in articular cartilage are the most important features. Casticin (CAS) is a flavonoid extracted from the Chinese herb Vitex species that has anti-inflammatory and antitumor effects. The aim of this study was to investigate the therapeutic and mechanistic effects of CAS on cartilage damage in KOA. A KOA rat model was established by anterior cruciate ligament transection (ACLT), and cartilage morphological changes were assessed by histological analysis and micro-CT scans. Subsequently, chondrocytes were treated with 10 ng/mL IL-1β to establish an OA model. CCK-8 assays and EdU assays were performed to assess the viability of CAS-treated chondrocytes. Western blotting, flow cytometry and Hoechst 33342/PI Double Stain were used to detect chondrocyte apoptosis. Western blotting, qRT‒PCR and ELISA were used to detect changes in inflammatory mediators. In addition, cartilage matrix-related indices were detected by Western blotting, qRT‒PCR and immunofluorescence (IF) analysis. Immunohistochemistry (IHC) and Western blotting were performed to detect the expression of p-PI3K, p-AKT and HIF-1α in vivo and in vitro. Micro-CT, pathological sections and related scores showed that CAS improved the alterations in bony structures and reduced cartilage damage and osteophyte formation in the ACLT model. In vivo, CAS attenuated IL-1β-induced cartilage matrix degradation, apoptosis and the inflammatory response. In addition, CAS inhibited the expression of the PI3K/AKT/HIF-1α signaling pathway in the ACLT animal model and IL-1β cell model. CAS may ameliorate cartilage damage in OA by inhibiting the PI3K/AKT/HIF-1α signaling pathway, suggesting that CAS is a potential strategy for the treatment of OA.

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis.

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable structural damage of the joint. Investigations into the link between articular cartilage, muscles, synovium, and other tissues surrounding the knee joint in KOA are of great importance. Currently, managing KOA includes lifestyle modifications, exercise, medication, and surgical interventions; however, the elucidation of the intricate mechanisms underlying KOA-related pain is still lacking. Consequently, KOA pain remains a key clinical challenge and a therapeutic priority. Tuina has been found to have a regulatory effect on the motor, immune, and endocrine systems, prompting the exploration of whether Tuina could alleviate KOA symptoms, caused by the upregulation of inflammatory factors, and further, if the inflammatory factors in skeletal muscle can augment the progression of KOA. We randomized 32 male Sprague Dawley (SD) rats (180-220 g) into four groups of eight animals each: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D). For groups A, B, and C, we injected 25 µL of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 µL of sterile saline solution) into the right knee joint cavity, and for group D, the same amount of sterile physiological saline was injected. All the groups were evaluated using the least to most stressful tests (paw mechanical withdrawal threshold, paw withdrawal thermal latency, swelling of the right knee joint, Lequesne MG score, skin temperature) before injection and 2, 9, and 16 days after injection.

Altered Brain Functional and Effective Connectivity Induced by Electroacupuncture in Rats Following Anterior Cruciate Ligament Transection.

The chronic pain arising from knee osteoarthritis (KOA) is a prevalent clinical manifestation. As a traditional Chinese approach, electroacupuncture (EA) has a positive influence in relieving chronic pain from KOA. The study aims to explore functional connectivity (FC) and effective connectivity (EC) alterations induced by EA in anterior cruciate ligament transection (ACLT) rat model of KOA using resting-state functional magnetic resonance imaging (fMRI). After the establishment of ACLT, rats were randomly divided into the EA group and the sham-EA group. The EA group received EA intervention while the sham-EA group received sham-intervention for 3 weeks. Mechanical pain threshold (MPT) assessment was performed before and after intervention, and fMRI was conducted after intervention. EA intervention effectively relieved pain in post-ACLT rats. Results of rest-state functional connectivity (rs-FC) analysis revealed that compared with the sham-EA group, the EA group had higher FC between the right raphe and the left auditory cortex, the left caudate_ putamen and the left internal capsule (IC), as well as the right zona incerta (ZI) and the left piriform cortex, but lower FC between the right raphe and the left hippocampus ventral, as well as the right septum and the left septum. Furthermore, Granger causality analysis (GCA) found the altered EC between the right septum and the left septum, as well as the left IC and the right septum. The results confirmed the effect of EA on analgesia in post- ACLT rats. The alterations of FC and EC, mainly involving basal ganglia and limbic system neural connections, might be one of the neural mechanisms underlying the effect of EA, providing novel information about connectomics plasticity of EA following ACLT.

Exploring rutinoside's impact on inflammation in a rat knee OA model induced by monosodium iodoacetate (MIA)

Link of Video Abstract: https://youtu.be/Dl8sYvPfd6o Introduction: Osteoarthritis (OA) is a common joint disease characterized by persistent inflammation and cartilage breakdown. Monosodium iodoacetate (MIA) is often used to generate OA in rats. This study aims to explore the anti-inflammatory benefits of rutinoside, a natural flavonoid glycoside, in rats with MIA-induced knee OA. Methods: The male Wistar rats were divided into five groups (n = 6) and randomly allocated to one of five treatments: control, OA, rutinoside, glucosamine sulfate, and sodium diclofenac-treated group. 3 mg of monosodium iodoacetate (MIA) was injected intra-articularly into the knee joints of rats to induce osteoarthritis. Rutinoside, glucosamine sulfate, and sodium diclofenac were given daily for four weeks. Body weight, joint swelling, knee bend score, interleukin-1 (IL-1) levels, interleukin-8 (IL-8) levels, and C-terminal telopeptide type II collagen (CTX-II) levels were all assessed. Result: Rutinoside significantly decreased body weight loss, joint edema, and knee bend test scores compared to the untreated OA group. Furthermore, rutinoside was equally effective as glucosamine sulfate and sodium diclofenac in these parameters. The rutinoside, glucosamine sulfate, and sodium diclofenac groups had significantly lower IL-1, IL-8, and CTX-II levels than the untreated OA group. Conclusion: Rutinoside reduced inflammatory responses, lowered joint edema, and improved knee bend test scores in a rat model of MIA-induced osteoarthritis. Its efficacy in preventing illness was comparable to glucosamine sulfate and sodium diclofenac. More study is needed to better understand rutinoside's underlying mechanisms and long-term consequences for treating osteoarthritis.

Development of a Novel Rat Knee Osteoarthritis Model Induced by Medial Meniscus Extrusion.

The medial meniscus extrusion (MME) is associated with increased stress on the knee joint, which leads to cartilage degeneration. To evaluate the etiology of knee osteoarthritis, it is extremely important to create animal models of the disease that more closely resemble actual clinical conditions in terms of symptomatology, molecular biology, and histology. This study aimed to create a clinically relevant model of MME in rats. Behavioral, molecular biological, and histological changes in the newly developed rat MME model were compared with those in sham and medial meniscus transection and medial collateral ligament transection (MMT) models to examine the characteristics of this model. In the MME rat model, behavioral evaluation shows abnormalities in gait compared with the other 2 groups, and molecular biological evaluation of the infrapatellar synovia of rats shows that gene expression of inflammatory cytokines, matrix-degrading enzymes, and pain-related nerve growth factor was increased compared with the sham group. Furthermore, histological evaluation reveals that cartilage degeneration was the most severe in the MME group. The newly developed MME model reproduced the characteristic pathology of MME in clinical practice, such as severe pain, inflammation, and rapid progression of osteoarthritis. The MME model, which might more closely mimic human knee osteoarthritis (OA), could be a useful model for elucidating the pathophysiology and considering therapeutic management for knee OA.

Anti-Osteoarthritic Effects of Antarctic Krill Oil in Primary Chondrocytes and a Surgical Rat Model of Knee Osteoarthritis.

Osteoarthritis (OA) is characterized by progressive cartilage destruction and synovitis; however, there are no approved disease-modifying OA drugs. Krill oil (KO) has been reported to possess anti-inflammatory properties and alleviate joint pain in knee OA, indicating its potential to target the inflammatory mechanism of OA. Therefore, the anti-OA effects of KO were investigated in primary chondrocytes and a surgical rat model of knee OA. The oral administration of KO at 200 and 100 mg/kg for 8 weeks improved joint swelling and mobility in the animal model and led to increased bone mineral density and compressive strength in the cartilage. The oral KO doses upregulated chondrogenic genes (type 2 collagen, aggrecan, and Sox9), with inhibition of inflammation markers (5-lipoxygenase and prostaglandin E2) and extracellular matrix (ECM)-degrading enzymes (MMP-2 and MMP-9) in the cartilage and synovium. Consistently, KO treatments increased the viability of chondrocytes exposed to interleukin 1α, accompanied by the upregulation of the chondrogenic genes and the inhibition of the ECM-degrading enzymes. Furthermore, KO demonstrated inhibitory effects on lipopolysaccharide-induced chondrocyte inflammation. Histopathological and immunohistochemical analyses revealed that KO improved joint destruction and synovial inflammation, probably due to the anti-inflammatory, anti-apoptotic, and chondrogenic effects. These findings suggest the therapeutic potential of KO for knee OA.

Anti-Inflammatory Effects of Boric Acid in Treating Knee Osteoarthritis: Biochemical and Histopathological Evaluation in Rat Model.

This study aimed to examine the anti-inflammatory properties of boric acid (BA) in treating knee osteoarthritis (KOA) in rats, evaluating its biochemical and histopathological therapeutic effects.A KOA rat model was induced by injecting monosodium iodoacetate into the knee joint. Random assignment was performed for the experimental groups as follows: group-1(control), group-2(KOA control), group-3 (BA:4mg/kg, orally), group-4(BA:10mg/kg, orally), group-5(BA:4mg/kg, intra-articularly), and group-6(BA:10mg/kg, intra-articularly). The rats received 100 µL of BA intra-articularly on days 1, 7, 14, and 21 or 1mL orally once a day (5days/week) for 4weeks. Serum levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and activityofmatrix metalloproteinase-13 (MMP-13) were measured. Histopathological and immunohistochemical analyses were performed on knee joint samples using specific antibodies for IL-1β, TNF-α, MMP-13, and nitric oxide synthase-2 (NOS-2).Group-2 exhibited higher serum IL-1β and TNF-α levels and MMP-13 activity than group-1 (P < 0.05). However, IL-1β and TNF-α levels and MMP-13 activity were lower in all treatment groups than in group-2, with statistically significant reductions observed in groups-4, 5, and 6. Histopathologically, group-2 displayed joint space narrowing, cartilage degeneration, and deep fissures. Groups-5 and 6 demonstrated significant joint space enlargement, articular cartilage tissue regeneration, and immunostaining patterns similar to those in group-1. Immunohistochemically, group-2 showed significant increases in IL-1β, TNF-α, MMP-13, and NOS-2 expression. However, all treatment groups exhibited reductions in these expression levels compared to group-2, with statistically significant decreases observed in groups-5 and 6 (P < 0.01).BA shows potential efficacy in reducing inflammation in experimental KOA model in rats. It may be a promising therapeutic agent for KOA, warranting further clinical studies for validation.

Melatonin enhances the ability of M2 macrophages to secrete IL10 by inhibiting Erk5 signaling pathway

BackgroundMelatonin plays a role in repairing damaged cartilage and regulating immune cells. The anti-inflammatory effect of Melatonin involves multiple pathways and molecular activation, which directly or indirectly inhibits inflammatory reaction. M2 macrophages have the ability to anti-inflammatory response and repair damaged tissues, secrete IL10 and IL-4, and participate in tissue repair and remodeling. Erk5 is a recently discovered member of the MAPK family and one of the least studied members. It plays an important role in cell differentiation, proliferation, secretion and other functions. This experiment aims to study how Melatonin affects M2 Macrophage polarization and secretion through ERK5 signaling pathway. MethodsThe RAW 264.7 macrophages were used for cell culture. The cells were cultured according to the pre-experimental results. The effects of Melatonin on M2 macrophages were comprehensively evaluated by CCK8 activity detection, RT-PCR, ELISA, cellular immunofluorescence, and WB.SD mice were selected to evaluate the effect of Melatonin on cartilage damage in rats with knee Osteoarthritis through HE staining, immunohistochemistry and immunofluorescence. ResultsMelatonin cultivates RAW 264.7 macrophages. Without affecting the polarization ratio of M2 Macrophage polarization, Melatonin may reduce Erk5 gene expression, reduce Erk5 and p-Erk5 protein synthesis, and cooperate with BIX 02189 to enhance the secretion function of existing M2 macrophages and increase the secretion of cytokines IL10. Immunohistochemistry of rat knee Osteoarthritis model confirmed that the expression of IL10 was up-regulated and the synthesis of type II collagen was enhanced, but immunofluorescence found that the polarization of M2 Macrophage polarization in subchondral bone was not obvious. ConclusionMelatonin enhances the ability of M2 macrophages to secrete IL10 by inhibiting Erk5 signaling pathway, but has no effect on M2 Macrophage polarization.

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.