Knee Joint Diameter Research Articles

Metallothionein-1 mitigates the advancement of osteoarthritis by regulating Th17/Treg balance

Osteoarthritis (OA) is a chronic inflammatory joint disorder characterized by cartilage degradation and bone remodeling. This study investigated the regulatory role of metallothionein 1 (MT1) in modulating immune responses and the balance between regulatory T cells (Treg) and T helper 17 cells (Th17) in OA. Peripheral blood mononuclear cells (PBMCs) from healthy individuals and OA patients were assessed for cytokine expression linked to Treg/Th17 homeostasis. OA was induced in wild-type (WT) and Mt1 knockout (MT1KO) mice via surgical destabilization of the medial meniscus. Clinical scores, pathological features, inflammatory cytokines, and Treg/Th17 balance were evaluated. MT1KO mice showed significantly elevated Mt1, pro-inflammatory cytokines (IL-1, IL-6, TNF-α) and exacerbated OA progression, characterized by increased knee joint diameter, inflammatory infiltration, and cartilage destruction. Mechanistically, disrupted Treg/Th17 balance played a pivotal role in OA exacerbation, with MT1KO promoting Th17 differentiation and reducing Treg populations. Additionally, the compensatory elevation of anti-inflammatory interleukin-10 (IL-10) in OA patients hinted at a nuanced immune regulatory mechanism. The study illuminates intricate interactions involving MT1, Treg/Th17 cells, and pro-inflammatory cytokines in OA pathogenesis, suggesting MT1′s potential as a pivotal regulatory factor and a therapeutic target for mitigating immune dysregulation in OA.

Dicoumarol attenuates NLRP3 inflammasome activation to inhibit inflammation and fibrosis in knee osteoarthritis.

Knee osteoarthritis (KOA) is a major cause of disability in elderly individuals. Dicoumarol is a coumarin‑like compound derived from sweet clover [Melilotus officinalis (L.) Pall]. It has been suggested that dicoumarol exhibits various types of pharmacological activities, including anticoagulant, antitumor and antibacterial effects. Due to its various biological activities, dicoumarol has a potential protective effect against OA. Therefore, the present study aimed to assess the effects of dicoumarol on knee osteoarthritis. In the present study, dicoumarol was found to protect rat synoviocytes from lipopolysaccharide (LPS)‑induced cell apoptosis. Western blot analysis showed that dicoumarol significantly reduced the protein expression levels of fibrosis‑related markers and inflammatory cytokines (Tgfb, Timp, Col1a, Il1b and Il18). The inhibitory rates of these proteins were all >50% (P<0.01) compared with those in the LPS and ATP‑induced group. Consistently, the mRNA expression levels of these markers and cytokines were decreased to normal levels by dicoumarol after the treatment of rat synovial fibroblasts with LPS and ATP. Mechanistic studies demonstrated that dicoumarol did not affect NF‑κB signaling, but it did directly interact with NOD‑like receptor protein 3 (NLRP3) to promote its protein degradation, which could be reversed by MG132, but not NH4Cl. The protein half‑life of NLRP3 was accelerated from 26.1 to 4.3 h by dicoumarol. Subsequently, dicoumarol could alleviate KOA in vivo; knee joint diameter was decreased from 11.03 to 9.93 mm. Furthermore, the inflammation and fibrosis of the knee joints were inhibited in rats. In conclusion, the present findings demonstrated that dicoumarol could impede the progression of KOA by inhibiting NLRP3 activation, providing a potential treatment strategy for KOA.

Treatment of rheumatoid arthritis by injection of sinomenine solid lipid nanoparticles under a fluorescence endoscopic laser confocal microscope

A fluorescence endoscopic laser confocal microscope(FELCM) was used to direct the injection of sinomenine solid lipid nanoparticles(Sin-SLN) into the joint, and the in vitro effectiveness of Sin-SLN in the treatment of rheumatoid arthritis(RA) was evaluated. Sin-SLN was prepared with the emulsion evaporation-low temperature curing method. The Sin-SLN prepared under the optimal conditions showed the encapsulation efficiency of 64.79%±3.12%, the drug loading of 3.84%±0.28%, the average particle size of(215.27±4.21) nm, and the Zeta potential of(-32.67±0.84) mV. Moreover, the Sin-SLN demonstrated good stability after sto-rage for 30 days. The rabbit model of RA was established by the subcutaneous injection of ovalbumin and complete Freund's adjuvant. Five groups were designed, including a control group, a model group, a Sin(1.5 mg·kg~(-1)) group, a Sin-SLN(1.5 mg·kg~(-1)) group, and a dexamethasone(positive drug, 1.0 mg·kg~(-1), ig) group. The control group and the model group only received puncture treatment without drug injection. After drug administration, the local skin temperature and knee joint diameter were monitored every day. The knee joint diameter and the local skin temperature were lower in the drug administration groups than in the model group(P&lt;0.05, P&lt;0.01). FELCM recorded the morphological alterations of the cartilage of knee joint. The Sin-SLN group showed compact tissue structure and smooth surface of the cartilage. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the serum le-vels of interleukin-1(IL-1) and tumor necrosis factor-α(TNF-α). The findings revealed that the Sin-SLN group had lower IL-1 and TNF-α levels than the model group(P&lt;0.05, P&lt;0.01). Hematoxylin-eosin(HE) staining was employed to reveal the pathological changes of the synovial tissue, which were significantly mitigated in the Sin-SLN group. The prepared Sin-SLN had uniform particle size and high stability. Through joint injection administration, a drug reservoir was formed. Sin-SLN effectively alleviate joint swelling and cartilage damage of rabbit, down-regulated the expression of inflammatory cytokines, and inhibited the epithelial proliferation and inflammatory cell infiltration of the synovial tissue, demonstrating the efficacy in treating RA.

Anti-Inflammatory Effects of Ripe Areca Nut Ethyl Acetate Fraction in Wistar Rats with Knee Joints Osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis. Meloxicam are one of NSAIDs commonly used that have many side effects. Areca nut (Areca catechu L), which contains flavonoids, can provide anti-inflammatory effects. This study aimed to determine the potential anti-inflammatory effect of ethyl acetate fraction from ripe areca nut in OA rat model. Twenty five of adults, male Rattus novergicus strain Wistar were induced to OA with Monosodium Iodoacetate (MIA), then divided into five groups that each group were given ethyl acetate fraction of ripe areca nut doses of 15mg/kgBW, 30mg/kgBW, and 60mg/kgBW, meloxicam 1.35mg/kgBW and aquadest as negative control for 14 days. Diameter of the knee joint is measured by digital metric kaliper to assess joint edema. The Histopathological examination with Haematoxyllin-Eosin stain to assess degree of inflammation on synovial. Among the areca groups, dose 30mg/kgBW had knee joint diameter reduced better and lower synovitis score. In conclusion, ethyl acetate fraction of the ripe areca nut group has anti inflammation effect in OA model but not dose-dependent.

The pro-inflammatory cytokine IL-Iβ alteration by deer (Rusa unicolor) antler extract on osteoarthritis rat model

Osteoarthritis is a disease associated with articular cartilage degradation, intra-articular area inflammation, and subchondral bone replacement. Cytokine IL-1β has a prominent function in the inflammations process that passes in the joints. The 70% ethanol extracts of deer antler (250 and 500 mg/kg BW) and glucosamine sulfate (250 kg/BW) were evaluated for four weeks in reducing cytokine IL-1β to rat model OA-induced Monosodium iodoacetate. Measurements of joint diameter in rat’s knee and hyperalgesia were performed on weeks 0, 1, 2, 3, 4, 5, 6, and 7. The presence of a significant difference in the stimulation thermal latency (p = 0.00) and the resulting increase in swelling of joint diameter (p = 0.00) are evidence that MIA has successfully induced the rat modeling of OA. A significant decrease in cytokine IL-Iβ levels was shown on week 3 after MIA injection (p = 0.00). Both concentrations of deer extracts significantly reduced knee joint diameter (p = 0.00), latency thermal stimulation (p = 0.00), and cytokine IL-1β levels (p = 0.00). Based on the results, it can be concluded that the 70% ethanol extract of deer antler is a potential medicine for OA therapy.

Pathological Characteristics of Monosodium Iodoacetate-Induced Osteoarthritis in Rats.

This study aimed to identify pain-related behavior and pathological characteristics of the knee joint in rats with monosodium iodoacetate (MIA)-induced osteoarthritis (OA). Knee joint inflammation was induced by intra-articular injection of MIA (4mg/50 µL, n = 14) in 6-week-old male rats. Knee joint diameter, weight-bearing percentage on the hind limb during walking, the knee bending score, and paw withdrawal to mechanical stimuli were measured to evaluate edema and pain-related behavior for 28 d after MIA injection. Histological changes in the knee joints were evaluated using safranin O fast green staining on days 1, 3, 5, 7, 14, and 28 after OA induction (n = 3, respectively). Changes in bone structure and bone mineral density (BMD) were examined 14 and 28 d after OA (n = 3, respectively) using micro-computed tomography (CT). The knee joint diameter and knee bending scores of the ipsilateral joint significantly increased 1 d after MIA injection, and the increased knee joint diameter and knee bending score persisted for 28 d. Weight-bearing during walking and paw withdrawal threshold (PWT) decreased from 1 and 5 d, respectively, and were maintained up to 28 d after MIA. Cartilage destruction started on day 1, and Mankin scores for bone destruction significantly increased for 14 d, as shown by micro-CT imaging. The present study demonstrated that histopathological structural changes in the knee joint due to inflammation started soon after MIA injection, which induced OA pain from inflammation-related acute pain to spontaneous and evoked associated chronic pain.

Retinoic Acid Receptor Gamma (RARγ) Promotes Cartilage Destruction through Positive Feedback Activation of NF-κB Pathway in Human Osteoarthritis.

Osteoarthritis (OA) is a severe inflammation-related disease which leads to cartilage destruction. The retinoic acid receptor gamma (RARγ) has been indicated to be involved in many inflammation processes. However, the role and mechanism of RARγ in cartilage destruction caused by inflammation in OA are still unknown. Here, we demonstrated that the RARγ was highly expressed in chondrocytes of OA patients compared with healthy people and was positively correlated with the damage degree of cartilage in OA. Cytokine TNF-α promoted the transcription and expression of RARγ through activating the NF-κB pathway in OA cartilage. In addition, the overexpression of RARγ resulted in the upregulation of matrix degradation and inflammation associated genes and downregulation of differentiation and collagen production genes in human normal chondrocyte C28/I2 cells. Mechanistically, overexpression of RARγ could increase the level of p-IκBα and p-P65 to regulate the expression of downstream genes. RARγ and IκBα also could interact with each other and had the same localization in C28/I2 cells. Moreover, the SD rats OA model induced by monosodium iodoacetate indicated that CD437 (RARγ agonist) and TNF-α accelerated the OA progression, including more severe cartilage layer destruction, larger knee joint diameter, and higher serum ALP levels, while LY2955303 (RARγ inhibitor) showed the opposite result. RARγ was also highly expressed in OA group and even higher in TNF-α group. In conclusion, RARγ/NF-κB positive feedback loop was activated by TNF-α in chondrocyte to promote cartilage destruction. Our data not only propose a novel and precise molecular mechanism for OA disease but also provide a prospective strategy for the treatment.

Resistin contributes perivascular adipose tissue dysfunction in a rheumatoid arthritis mouse model

BackgroundPatients with rheumatoid arthritis (RA) experience 50% more risk of mortality attributed to cardiovascular disease (CVD). PVAT dysfunction, which leads to vascular dysfunction, is driven by increased proinflammatory adipokines, and overactivation of immune cells. The proinflammatory adipokine resistin modulates vascular function, and circulating, synovial and serum resistin concentrations are increased in RA patients. We hypothesized that resistin causes PVAT dysfunction, inflammation and macrophage infiltration in a RA experimental model.MethodsAntigen‐induced arthritis (AIA) was induced in 12 weeks‐old C57BL/6 male mice. AIA immunized mice received mBSA (intraarterial injection, 10 µg in10 µl PBS/week) or PBS (10 µl) for 5 weeks. Disease activity was determined based on immune cells profile in lymph nodes, by flow cytometry, and measurement of the mediolateral knee joint diameter. Thoracic aorta, with or without PVAT, were isolated after five weeks of AIA onset for functional, cellular, and molecular assays. Data are represented as mean and standard error, and student´s T test (p&lt;0.05) was used for statistical analysis. All the experiments were approved by the Ethics Committee on Animal Research of the FMRP,USP (protocol nº 15/2020).ResultsInguinal lymph nodes of AIA showed increased CD4+/IL‐17 cells compared to control [(%) AIA: 10.4 ± 1.06 vs. CT 1.8 ± 1.06, n=6] and the mediolateral knee diameter was increased in AIA compared to control mice [(mm) AIA 4.38 ± 0.06 vs. CT 3.50 ± 0.04, n=6]. Aorta from AIA mice had a dysfunctional PVAT, decreased phenylephrine (Pe) maximum responses (Emax) and no changes in Pe logEC50 compared to CT [Emax (mN): CT ‐PVAT 10.6 ± 0.3 vs. CT +PVAT 8.7 ± 0.2; AIA ‐PVAT 6.8 ± 0.3 vs. CT +PVAT 7.0 ± 0.2, n=6‐8]. 40 ng/ml of resistin for 4 hours compromised aortic PVAT function [Emax (mN): WT–PVAT + Resistin = 5,8 ± 0,1 vs. WT+PVAT + Resistin = 6,0 ± 0,1; CT ‐PVAT= 10,6 ± 0,3 vs. CT +PVAT 8,7 ± 0,2, n=7‐8]. Resistin concentrations were increased in the PVAT, plasma, and knee of AIA mice vs. control [(pg/ml) Serum: AIA 899.2 ± 11 vs. CT 837.9 ± 18; PVAT: AIA 217.0 ± 24 vs. CT 121 ± 18; Knee: AIA 28.3 ± 1.7 vs. CT 19.5 ± 1.1, n=4‐8). mRNA genemarkers of type 1 (M1) macrophages, including monocyte chemoattractant protein‐1 (CCL2), interleukin‐1beta (IL‐1b), inducible nitric oxide synthase (iNOS), and tumor necrosis alpha (TNFα) were increased in AIA PVAT [(‐∆∆ct) CCL2: AIA 2.6 ± 0.3 vs. CT 0.82 ± 0.10; iNOS: AIA 2,5± 0,7 vs. CT 0,6 ± 0,1; IL‐1b: AIA 2.0 ± 0.4 vs. CT 1.0; TNFα: AIA 1,2 ± 0,1 vs. CT 0,5 ± 0,0; n=5‐8, 2]. mRNA gene markers of type 2 macrophages (M2) such as resistin‐like molecule alpha (Retnla), L‐arginase (Arg1), Mannose Receptor C‐Type 1 (Mrc1) was increased in PVAT from AIA mice vs. control [(‐∆∆ct) Arg1: AIA 3,4 ± 0,6 vs. CT 0,8 ± 0,1; Retna AIA 2,0 ± 0,6 vs. CT 1,0 ± 0,2; n=5‐7, 2]. Flow cytometry analysis confirmed increased M1 and M2 markers in the PVAT of AIA mice [(% of cells) M1:F4/80+CD11b+:AIA 11,8 ± 1,6 vs. Sham 6,0 vs. 1,1; M2: CD206+CD11b+ :AIA 20,4 ± 1,7 vs. Sham 11,8 ± 1,2; n=6‐8].ConclusionAIA‐associated PVAT dysfunction, i.e. loss of its anti‐contractile effect, is linked to increased resistin and an inflammation profile involving macrophages‐related cytokines and chemokines.

Investigation of the effects of therapeutic ultrasound or photobiomodulation and the role of spinal glial cells in osteoarthritis-induced nociception in mice.

Pain is the most common symptom of osteoarthritis, and spinal glia is known to contribute to this symptom. Therapeutic ultrasound and laser therapy have been used to effectively treat osteoarthritis, with few adverse effects. Thus, this study aimed to investigate the effects of ultrasound and photobiomodulation on the symptoms and evaluate the participation of spinal glia in osteoarthritis-induced nociception in mice. Male Swiss mice were subjected to osteoarthritis induction with a 0.1-mg intra-articular injection of monosodium iodoacetate. Additionally, the mice received chronic ultrasound or photobiomodulation treatment for 21days or a single treatment at day 14. Nociception was evaluated using von Frey filaments, and osteoarthritis symptoms were assessed by analysis of gait, joint temperature, and knee joint diameter. The role of spinal microglia and astrocytes on nociception was evaluated via an intrathecal injection of minocycline or fluorocitrate, and the spinal release of IL-1β and TNF-α was assessed by ELISA after chronic treatment with ultrasound or photobiomodulation. Our data showed that both single and chronic treatment with ultrasound or photobiomodulation attenuated the osteoarthritis-induced nociception. No differences in gait, knee joint temperature, or knee joint diameter were found. The intrathecal injection of minocycline and fluorocitrate decreased the osteoarthritis-induced nociception. There was an increase in the spinal levels of TNF-α, which was reverted by chronic ultrasound and laser treatments. These results suggest that osteoarthritis induces nociception and glial activation via spinal release of TNF-α and that the chronic treatment with ultrasound or photobiomodulation decreased nociception and TNF-α release.

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.

Losartan protects endothelium-dependent relaxation in vivo in a murine model of rheumatoid arthritis

Angiotensin II-type 1 receptor stimulation is recognised to promote inflammation, a state central to the development and maintenance of rheumatoid arthritis. Herein we examined the use of losartan, an angiotensin II-type 1 receptor antagonist, on vascular reactivity, knee joint diameter and behavioural assessment of pain in a Freund's complete adjuvant (FCA) mouse model of joint inflammation.Monoarthritis was induced via FCA in the presence or absence of losartan with naive mice serving as controls. Knee joint swelling, joint pain (assessed by dynamic weight bearing of limb use), knee joint artery reactivity (assessed ex vivo) and blood perfusion of the knee joint (assessed in vivo) were determined.FCA mediated a significant increase in knee joint diameter and reduced weight-bearing (a surrogate for pain sensation) of the affected limb. Notably, these phenomena were substantially reduced when mice were prophylactically treated with losartan. Assessment of arterial relaxation and blood perfusion with acetylcholine stimulation revealed that FCA resulted in significant vascular dysfunction, which was resolved to naïve levels with losartan treatment.Through the actions of losartan, these findings indicate that the angiotensin II-type 1 receptor is a likely therapeutic target of importance in the development of the physical changes, pain sensation and vascular dysfunction found in inflammatory arthritis.

Effects of Dipsacus asperoides Extract on Monosodium Iodoacetate-Induced Osteoarthritis in Rats Based on Gene Expression Profiling.

The root of Dipsacus asperoides C. Y. Cheng et T. M. Ai is traditionally used as an analgesic and anti-inflammatory agent to treat pain, rheumatoid arthritis, and bone fractures. However, neither its effects on osteoarthritis (OA) nor its effects on the arthritic cartilage tissue transcriptome have not been fully investigated. In this study, we used a rat model of monosodium iodoacetate- (MIA-) induced OA to investigate the therapeutic effects of a Dipsacus asperoides ethanolic extract (DAE, 200 mg/kg for 21 days). The study first assessed joint diameter, micro-CT scans, and histopathological analysis and then conducted gene expression profiling using RNA sequencing in articular cartilage tissue. We found that DAE treatment ameliorates OA disease phenotypes; it reduced the knee joint diameter and prevented changes in the structural and histological features of the joint, thereby showing that DAE has a protective effect against OA. Based on the results of gene expression profiling and subsequent pathway analysis, we found that several canonical pathways were linked to DAE treatment, including WNT/β-catenin signaling. Taken together, the present results suggest molecular mechanism, involving gene expression changes, by which DAE has a protective effect in a rat model of MIA-induced OA.

The Influence of the Demographic Factor on the Growth and Development of Younger School-Age Girls

In order to determine the difference in morphological characteristics, a survey was conducted on a sample of 120 fourth-grade elementary schoolgirls from different socio-economic backgrounds in Montenegro. The total sample of respondentswas divided into two sub-samples. One sub-group consisted of 60 schoolgirls from urban and the other 60 schoolgirls of the same age from rural environment. A sample of measuring instruments consisted of 12 measures (body height, arm length, leg length, shoulder width, elbow diameter, knee diameter, body mass, upper arm circumference, lower leg circumference, skin fold of the back, skin fold of the upper arm and skin fold of the abdomen). In the process of statistical data processing, descriptiveand canonical discriminatory analysis were applied in order to identify possible differences between sub-databases. The obtained research data generally indicate the existence of statistically significant differences between sub-samples. This difference is evident in indicators of longitudinal and transversal dimensionality of the skeleton, and in contrast, the difference was not evident in indicators of volume and body weight and subcutaneous fat tissue. Girls from the urban environment were taller and had more pronounced measures in the upper and lower extremities and girls from rural areas had more pronounced measures of knee joint diameter than their peers from urban life.

Sesquiterpene lactones; Damsin and neoambrosin suppress cytokine-mediated inflammation in complete Freund's adjuvant rat model via shutting Akt/ERK1/2/STAT3 signaling

Ethnopharmacologial relevanceAlthough Damsissa (Ambrosia maritima) is traditionally used as anti-inflammatory and diuretic, the biological activity and mechanism of action of its major constituents are to be elucidated. Aimto decipher the anti-arthritic potential of damsin (DMS) and neoambrosin (NMS) and to unfold their molecular signaling in complete Freund's adjuvant (CFA)-induced arthritis model. Materials and methodsthe right hind paw was inoculated with CFA (0.1 ml) at day 0 and 7 while treatments were started from the 14th day and continued for 2 weeks. Rats were randomly assigned into 4 groups; normal group (NRML), CFA-induced arthritis group, CFA-induced arthritis treated with DMS and NMS (10 mg/kg/day) as 3rd and 4th group; respectively. ResultsThroughout experimental period, treatments ameliorated the increase of paw volume, knee joint diameter and nociception tests as reflected in open field arena. Also, DSM and NMS suppressed phosphorylation of Akt, STAT-3, ERK1/2 which was further mirrored by inactivation of GSK3β and downregulation of MCP-1 together with CCN1 and NF-kβ in hind paw tissue. Concomitantly, inflammation markers; TNF-α, IL-6, -12 were lowered as confirmed microscopically during examination of hind paw tissue. ConclusionDSM and NMS-induced suppression of NF-kβ subdues clinical features of RA most probably through repression of Akt/ERK1/2/STAT3 pathway. Therefore, DMS and NMS can serve as safe and effective treatment for rheumatoid arthritis, one of the most disabling chronic, inflammatory and painful autoimmune disease.

Contribution of salidroside to the relieve of symptom and sign in the early acute stage of osteoarthritis in rat model

Ethnopharmacological relevanceThe genus Rhodiola has been used to treat cough, hemoptysis, fever, pain, bruise and other symptoms which are related to injury and inflammation over a thousand years in traditional Tibetan medicine. Salidroside (p-hydroxyphenethyl-β-D-glucoside) is one of the most potent bioactive ingredients of the genus Rhodiola. Aim of studyThe present study aimed to explore whether salidroside could alleviate the clinical symptom and sign in the early acute stage of osteoarthritis (OA) in monosodium iodoacetate (MIA) rat model, and its underlying mechanisms. Materials and methodsOsteoarthritis (OA) was induced in rat knees by intra-articular injection of MIA; simultaneously salidroside was administered by intravenous injection. Pain behaviors were evaluated by knee-bend test, hind limb weight-bearing asymmetry and hind paw mechanical withdrawal threshold. The joint swelling was determined by the difference of knee joint diameter. Inflammatory exudates in synovial fluid were evaluated by leukocyte counting and protein content. Cytokines, chemokines, reactive oxygen species (ROS) and reactive nitrogen species (RNS) markers were determined by Enzyme-linked immunosorbent assay (ELISA) and colorimetric assay in synovial fluid. Pro-inflammatory gene expressions in synovial tissue were detected by quantitative real time RT-PCR (qRT-PCR). Nuclear factor kappa-B (NF-κB) DNA binding assay and western blot were used to determine NF-κB activation and ROS marker protein expression in synovial tissue. Glycosaminoglycan (GAG) content in the cartilage was measured by dimethylmethylene blue method. Hematoxylin and eosin (H&E), Safranin O-fast green and a modified Mankin grading system were used to evaluate the histology of articular cartilage. ResultsSalidroside could alleviate pain and joint swelling in the early acute stage of OA in rat model, reduced the number of leukocytes, total protein content, proinflammatory mediators and ROS/RNS markers in synovial fluid, down regulated the expression of proinflammatory genes in synovium, inhibited the activation of NF- κ B and oxidative stress response in synovium, promoted the synthesis of cartilage GAG, prevented the loss of proteoglycan and chondrocyte degeneration. ConclusionsSalidroside effectively alleviates acute symptom and sign of OA in rat model by its anti-inflammatory and antioxidant affects to inhibit synovial inflammation, which provides a new strategy to prevent the onset and progression of OA.

Proteoglycan synthesis rate as a novel method to measure blood-induced cartilage degeneration in non-haemophilic and haemophilic rats.

IntroductionHaemophilic animal models are used to study blood‐induced cartilage damage, but quantitative and sensitive outcome measures are needed.AimTo develop a novel quantitative method for detecting early cartilage degeneration in a haemophilic rat model of blood‐induced joint damage.MethodsThe 35Sulphate incorporation (35SO4 2− assay) was applied to tibial and patellar cartilage of wild‐type rats to quantify baseline proteoglycan synthesis and to evaluate the effect of 4‐day blood exposure in vitro. Next, haemarthrosis was induced in 39 FVIII‐deficient rats and characterized by changes in knee joint diameter and development of bone pathology (using micro‐CT). Four‐ and 16‐day posthaemarthrosis proteoglycan synthesis rate (PSR) was assessed using the 35SO4 2− assay, with the contralateral knee as control.ResultsIn vitro, a decrease in PSR in tibial and patellar cartilage was demonstrated following blood exposure. In vivo, joint diameter and development of bone pathology confirmed successful induction of haemarthrosis. In the blood‐exposed knee, tibial and patellar PSR was inhibited 4 and 16 days after induced haemarthrosis. Interestingly, at day 16 the proteoglycan synthesis in the contralateral knee was also inhibited to an extent correlating with that of the blood‐exposed knee.ConclusionFor the first time, early changes in cartilage matrix synthesis upon blood exposure were quantified with the 35SO4 2− assay in a haemophilic rat model, establishing this assay as a novel method to study blood‐induced cartilage damage.

Attenuation of IL-1ß on the use of glucosamine as an adjuvant in meloxicam treatment in rat models with osteoarthritis.

Background Osteoarthritis (OA) is the most prevalent joint disease and a common cause of joint pain, functional loss, and disability. The severity of this disease is always associated with increased levels of proinflammatory cytokines, which play an important role in cartilage damage, synovitis, and other damage to joint tissues. The discovery that many soluble mediators such as cytokines or prostaglandins can increase the production of matrix metalloproteinases by chondrocytes led to the first steps of an inflammatory state. Several studies show that cytokines, such as interleukin 1ß, have a major role in the development of inflammation that occurs in these joints. The use of glucosamine as an adjuvant to meloxicam therapy is expected to inhibit the development of inflammatory OA. Methods The OA model in rat was induced by single injection of intraarticular monosodium iodoacetate (MIA). The development of OA was observed for 21 days. Furthermore, the evaluation of glucosamine potency as an adjuvant of meloxicam therapy for reducing IL-1ß was done by combined treatment at a low dose of meloxicam 1 mg/kg BW with glucosamine at a dose of 125, 250, or 500 mg/kg BW orally for 28 days. Response to hyperalgesia and knee joint diameter was measured on days 0, 7, 14, 21, 28, 35, 42, and 49. IL-1ß levels were measured on day 21 and day 49 after MIA injection. Results MIA injection successfully induced OA as marked by a significant difference in the time of latency to heat stimulus (p < 0.01) and a significant increase in joint diameter (p < 0.01). On day 21, IL-1ß levels showed a significant decrease in MIA injection (p = 0.05). The administration of meloxicam and glucosamine did not induce significant decrease in knee joint diameter (p > 0.10), but was able to significantly increase the latency time to heat stimulus (p < 0.01). IL-1ß levels also showed a significant decrease after administering a combination of glucosamine and meloxicam (p < 0.01). Conclusions Taken together, the use of glucosamine as an adjuvant in meloxicam therapy may be caused by the synergistic mechanism of meloxicam for the attenuation of OA development through systemically reducing IL-1ß.

The change of proinflammatory cytokine tumor necrosis factor α level in the use of meloxicam in rat model of osteoarthritis.

Background Osteoarthritis (OA) is a chronic disease in the joints. One of the proinflammatory cytokines that is thought to have a major role in the inflammatory process, the emergence of pain, and cartilage damage in OA is tumor necrosis factor α (TNF-α). Meloxicam is a nonsteroidal anti-inflammatory drug class of drugs that is relatively selective in inhibiting the activity of cyclooxygenase 2 (COX-2) formation. This study is conducted to prove the change in TNF-α level in the use of meloxicam with model in animals suffering from OA. Methods The OA rat model was induced with sodium monoiodoacetate intra-articularly. Rats were divided into 5 groups: negative control group, positive control group, and treatment groups with various doses of meloxicam. Hyperalgesia effect was evaluated using a warm plate test, and TNF-α level was determined using enzyme-linked immunosorbent assay. Results The treatment groups that received meloxicam at a dose of 1.0, 3.0, or 10.0 mg/kg body weight (BW) did not show significant differences in rat knee joint diameter (p = 0.99), but showed a significant difference in sensitivity to heat stimulation (p = 0.02) compared to the control group. Osteoarthritis rats experienced a significant reduction in TNF-α level after being given meloxicam at a dose of 10 mg/kg BW compared with the control group. This shows that the 10 mg/kg BW of meloxicam is a potential dose in reducing the TNF-α level in OA rat models. Conclusions Based on these data, it can be concluded that the inhibition of pain and the development of OA by meloxicam in animal models may be assigned to a decreased level of TNF-α.

EPCR Deficiency Prevents Development of Hemophilic Arthropathy

Recent studies from our laboratory showed that clotting factor VIIa (FVIIa), whose primary function is to initiate the coagulation cascade following its binding to procoagulant cofactor tissue factor (TF), also binds anticoagulant cofactor, endothelial cell protein C receptor (EPCR). EPCR plays a key role in the activated protein C (APC)-mediated anticoagulant pathway by promoting the activation of protein C bound to it. Pharmacological concentrations of rFVIIa could compete with protein C for the EPCR and downregulate APC generation by preventing protein C binding to the EPCR. Our recent studies showed that FVIIa binding to EPCR also induces barrier-protective and anti-inflammatory effects both in vitro and in vivo model systems. Bio-distribution of rFVIIa in mice revealed that a small fraction of rFVIIa administed to mice i.v. accumulates in knee joints and retained there for three to seven days. The present study was conducted to investigate the relevance of FVIIa binding to EPCR in pharmacological FVIIa treatment of joint disease in hemophilia. For this, we first generated hemophilia A mice lacking EPCR or overexpressing EPCR. To generate EPCR deficient FVIII-/- mice, Procrflox/flox and Procr+/floxMeox2+/cre mice were first backcrossed with FVIII-/- mice to generate Procrflox/flox/FVIII-/- and Meox2+/cre/FVIII-/- mice. EPCR deficient FVIII-/- mice (Procr-/-/FVIII-/-) were generated by breeding female Procrflox/flox/FVIII-/- with male Procr+/floxMeox2+/cre /FVIII-/- mice. EPCR- overexpressing FVIII-/- were generated by crossing Tie2-EPCR mice with FVIII-/- mice. We induced joint bleeding in FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice by needle puncture injury. Right knee joint intra articular space was pierced with 30G needle to induce joint bleeding; left knee joint served as an uninjured control. Injured mice were treated with a single dose of rFVIIa (1 mg/kg) at 20 min following the injury or three doses of rFVIIa (1 mg/Kg) at 20 min, 24 h and 72 h following the injury. In controls, saline was administered in place of rFVIIa. The knee injury was evaluated by measuring knee joint diameter and visual bleeding score. At the end of 14 days post injury, mice were euthanized, and the knee joints were excised and fixed for immunohistochemical analysis. Hemophilic arthropathy was evaluated by scoring a set of pathological parameters (e.g., synovial hyperplasia, neoangiogenesis, presence of blood, iron score, villus formation, and cartilage degeneration). A subset of mice was used to collect synovial fluid at 7 days post injury. Assessment of joint bleeding at 5 h following the needle injury, by extracting and measuring hemoglobin levels in joint tissues and monitoring hematocrit, showed an equal amount of bleeding in all three genotypes of mice (FVIII-/-, EPCR deficient FVIII-/-, and EPCR-overexpressing FVIII-/- mice). Administration of three doses of FVIIa was effective in preventing hemophilic arthropathy in FVIII-/- and EPCR-overexpressing FVIII-/- mice, whereas a single dose of rFVIIa had a minimal effect in reducing hemophilic arthropathy. Interestingly, a single dose of FVIIa fully corrected the needle injury-induced hemophilic arthropathy in EPCR-deficient FVIII-/- mice. More importantly, EPCR-deficient FVIII-/- mice failed to develop full-blown hemophiic arthropathy even in the absence of any treatment. Measurement of inflammatory cytokines in the synovial fluid showed a robust increase in IL-6 levels in FVIII-/- mice and a single dose of FVIIa substantially reduced IL-6 levels. Interestingly, IL-6 levels in the synovial fluid of injured EPCR-overexpressing FVIII-/- mice were markedly higher compared to injured FVIII-/- mice, three doses of rFVIIa was required to attain a significant reduction in IL-6 levels in these mice. In contrast to these data, we found very little IL-6 levels in the synovial fluids of injured EPCR-deficient FVIII-/- mice. Overall, these data indicate that EPCR levels profoundly influence hemophilic arthorpathy. EPCR-deficiency protects from development hemophilic arthropathy because down-regulation of APC generation in EPCR deficiency could allow sufficient thrombin generation in hemophilia to prevent joint bleeding and bleeding-associated inflammation. Disclosures Rao: Takeda: Research Funding.

Local Biological Reactions and Pseudotumor-Like Tissue Formation in relation to Metal Wear in a Murine In Vivo Model.

Metal wear debris and released ions (CoCrMo), which are widely generated in metal-on-metal bearings of hip implants, are also found in patients with metal-on-polyethylene bearings due to the mechanically assisted crevice corrosion of modular taper junctions, including head-neck and neck-stem taper interfaces. The resulting adverse reactions to metal debris and metal ions frequently lead to early arthroplasty revision surgery. National guidelines have since been published where the blood metal ion concentration of patients must consistently be monitored after joint replacement to prevent serious complications from developing after surgery. However, to date, the effect of metal particles and metal ions on local biological reactions is complex and still not understood in detail; the present study sought to elucidate the complex mechanism of metal wear-associated inflammation reactions. The knee joints in 4 groups each consisting of 10 female BALB/c mice received injections with cobalt chrome ions, cobalt chrome particles, and ultra-high-molecular-weight polyethylene (UHMWPE) particles or PBS (control). Seven days after injection, the synovial microcirculation and knee joint diameter were assessed via intravital fluorescence microscopy followed by histological evaluation of the synovial layer. Enlarged knee diameter, enhanced leukocyte to endothelial cell interactions, and an increase in functional capillary density within cobalt chrome particle-treated animals were significantly greater than those in the other treatment groups. Subsequently, pseudotumor-like tissue formations were observed only in the synovial tissue layer of the cobalt chrome particle-treated animals. Therefore, these findings strongly suggest that the cobalt chrome particles and not metal ions are the cause for in vivo postsurgery implantation inflammation.