Knee Osteoarthritis Model Research Articles

MiR-467b alleviates lipopolysaccharide-induced inflammation through targeting STAT1 in chondrogenic ATDC5 cells.

Osteoarthritis (OA) is one of the most common degenerative joint diseases worldwide. Chondrocytes are activated in OA patients, accompanied by excessive chondrogenic proliferation and production of inflammatory cytokines. MiR-467b is implicated in the regulation of artherosclerosis and pro-inflammatory cytokine secretion. However, the precise role of miR-467b in OA remains unclear. In the present study, we induced inflammation in chondrogenic ATDC5 cells using lipopolysaccharide (LPS). LPS treatment significantly elevated the production of interleukin-6 (IL-6), IL-1β and tumour necrosis factor-α (TNF-α) in ATDC5 cells, accompanied by decreased miR-467 level. Then, we over-expressed miR-467b using its specific mimics in ATDC5 cells, and LPS-induced inflammation was significantly inhibited as evidenced by decreased IL-6, IL-1β and TNF-α levels. MiR-467b agomir also alleviated inflammation in rat knee osteoarthritis (KOA) model. In addition, we validated that signal transducer and activator of transcription 1 (STAT1) was a downstream target of miR-467b. LPS treatment significantly increased the STAT1 expression while miR-467b mimic transfection partially reversed this effect. Moreover, STAT1 knockout reversed the increased contents of IL-6, IL-1β and TNF-α. Furthermore, miR-467b over-expression significantly decreased the production of IL-6, IL-1β and TNF-α induced by LPS treatment, which was partially reversed by further STAT1 over-expression. In summary, our findings demonstrated that miR-467b alleviated LPS-induced inflammation through targeting STAT1, and this miR-467b/STAT1 regulation axis may provide a new therapeutic target for OA clinical management.

Effect of warm needle moxibustion on morphological changes of cartilage and subchondral bone in knee osteoarthritis rabbits

To observe the effect of warm needle with moxibustion (WNM) on morphological changes of articular cartilage and subchondral bone tissues in knee osteoarthritis (KOA) rabbits, so as to explore whether WNM intervention can delay the development of KOA. Forty male New Zealand rabbits were randomly divided into blank control, model, WNM and medication (Alendronate sodium) groups, with 10 rabbits in each group. A KOA model was established by immobilizing the right hind limb of rabbits with orthopedic casting tape for 6 weeks. The rabbits of the WNM group received WNM stimulation at "Neixiyan"(EX-LE4) "Waixiyan"(ST35) and "Heding"(EX-LE2) for 15 min, once a day for 4 weeks and those of the medication group received gavage of Alendronate sodium (150 μg·kg-1·d-1) once a day for 4 weeks. X-ray examination and magnetic resonance imaging (MRI) scanning were performed to observe the structure of the knee joints. Outcomes of X-ray examination were used to assess the degree of bone hyperplasia and joint space stenosis which were scaled according to Kellgren and Lawrence (K-L) standards, and those of MRI used to evaluate the degree of cartilage damage and bone marrow edema degrees which were respectively scored according to Recht scaling standards and semi-quantitative whole-organ MRI score (WORMS). Histopathological changes (degeneration degrees) of the articular cartilage were observed after H.E. staining, and given Mankin score. The ultrastructure of the cartilage surface and chondrocytes was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. Compared with the blank control group, the K-L grade, Recht grade, WORMS and Mankin score of the model group were significantly increased (P<0.05,P<0.01). After the intervention and in comparison with the model group, the K-L grade, Recht grade, WORMS and Mankin score of both WNM and medication groups were significantly decreased (P<0.05,P<0.01). Results of SEM showed severe defect, bulge and uneven surface of the cartilage with irregular growth and regeneration, and those of TEM showed degeneration and swelling of chondrocytes, margination of endonuclear chromatin, reduction in the number of organelles with disordered arrangement, extreme expansion of endoplasmic reticulum with detachment of the ribosome, unclear mitochondria, and disordered distribution of collagen fibers in the extracellular matrix in the model group, which was relatively milder in both the WNM and medication groups. Compared with the medication group, the Mankin score in the WNM group was evidently lower (P<0.05). WNM can effectively slow down the degeneration of cartilage and subchondral bone of KOA rabbits, delay the development of KOA.

Effects of "knot-loosing" of acupotomy on motor function and morphological changes of knee joint in knee osteoarthritis rabbits

To observe the effect of "knot-loosing method" of acupotomy on morphological changes in knee osteoarthritis (KOA) rabbits, so as to provide experimental basis for clinical acupotomy treatment of KOA. Male New Zealand rabbits were randomly divided into blank control, model and acupotomy groups, with 8 rabbits in each group. By using the modified Videman method, the left hindlimb was immobilized for 6 weeks with a plaster cast to replicate the KOA model. The knot-loosing manipulation of acupotomy was applied to "Hedingci" (located at the attachment of the positive upper margin of the quadriceps tendon and the patella),"Binneixia"（at "Neixiyan" ［EX-LE4］, the medial side of ligamentum patella）, "Binwaixia" （at "Waixiyan" ［ST35］, the lateral side of patellar ligament）, "Chengfeijian" (at the lateral collateral ligament of the knee in the la-teral joint space), "Weiyangci"(at the medial margin of the biceps femoris and the lateral side of the transverse striation), and "Yinlingci"(at the end-point of the medial tibia of the goose foot tendon), respectively, once a week for 4 times. One week after the last intervention, the left knee joint dysfunction severity (pain, maximum walking distance, and some activities of daily living) was evaluated by using modified Lequesne Index score. X-ray and magnetic resonance imaging(MRI)examinations were conducted to assess the articular space size, effusion and cartilage smoothness of the left knee for which the MRI Osteoarthritis Knee Score (MOAKS) was given according to their severity. The histopathologic changes of the cartilage of the left knee were observed by Hematoxylin-eosin (H.E.) staining and light microscope, which Mankin score was given. In addition, the surface of the cartilage was also examined by macroscopic observation. ①Following modeling, the Lequesne Index score, MOAKS, macroscopic observation score and Mankin score were significantly increased in the model group (P<0.05). X-ray and MRI imaging showed a narrower joint space, more effusion, rough cartilage surface; and H.E. staining displayed rough cartilage surface, and disordered arrangement and occasional clusters of the chondrocytes in the left knee joint. ②In comparison with the blank control group, the Lequesne Index, MOAKS, macroscopic observation and Mankin scores were significantly down-regulated in the acupo-tomy group (P<0.05). The abovementioned injury changes shown by X-ray, MRI, macroscopic and microscopic observations were relatively milder in the acupotomy group. Acupotomy loosing based on the "Jingjin theory" of traditional Chinese medicine can effectively improve the motor ability of knee joint and repair pathological changes of the cartilage in KOA rabbits.

Vanillic Acid Reduces Pain-Related Behavior in Knee Osteoarthritis Rats Through the Inhibition of NLRP3 Inflammasome-Related Synovitis

Objectives: Synovitis plays an important role in knee osteoarthritis (KOA) pain. The activation of the NOD-like receptor protein 3 (NLRP3) inflammasome in fibroblast-like synoviocytes (FLSs) promotes KOA development. In this study, we aimed to investigate whether vanillic acid (VA), a monomer derived from Chinese herbal medicines, could target NLRP3 inflammasome-related synovitis to reduce pain. Methods: Rats in the KOA and KOA + VA groups were injected with monosodium iodoacetate (MIA) in the knee to induce KOA. From day 14, the KOA + VA group was given VA at 30 mg/kg every day via gastric intubation. FLSs were collected from the synovial tissues. We examined both the protein and gene expression of caspase-1, apoptosis-associated speck-like protein with a caspase recruitment domain (ASC), NLRP3, components of the NLRP3 inflammasome, and interleukin-1β (IL-1β) and IL-18 in vivo and in vitro. Results: The upregulation of caspase-1, ASC, and NLRP3 in the KOA model were reduced by VA. VA also lowered the level of IL-1β and IL-18 in the KOA model. In addition, VA relieved pain-related behavior of KOA model rats and downregulated the pain mediators CGRP, NGF, and TrkA in FLSs. Interestingly, we also observed reduced synovial fibrosis in the animal experiments. Conclusion: Our research showed that VA reduces synovitis and pain-related behaviors in a rat model of KOA, which provides the basis for further investigations into the potential therapeutic impact of VA in KOA.

LncPVT1 promotes cartilage degradation in diabetic OA mice by downregulating miR-146a and activating TGF-β/SMAD4 signaling.

To investigate the role of LncRNA PVT1 (plasmacytoma variant translocation 1) in hyperglycemia-triggered cartilage damage using the diabetic osteoarthritis (OA) mice model. Streptozotocin (STZ) was used to induce mouse diabetes. Knee OA model was induced through transection of anterior cruciate ligament (ACLT). Severity of arthritis was assessed histologically by Safranin O-Fast Green Staining using Mankin Scores. LncRNA PVT1 and miR-146a were detected by real-time polymerase chain reaction (PCR) in cartilage tissue. Moreover, the interaction among PVT1, miR-146a, and SMAD4 was examined by luciferase reporter assays. Mice were injected intra-articularly with ad-siRNA-PVT1 and ad-siRNA scramble control. Articular concentrations of TNF-α, IL-1, IL-6 and TGF-β1 were determined using enzyme-linked immunosorbent assay. Levels of type II Collagen (COL2A1), TGF-β1, p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4 and nuclear SMAD4 were detected by western blot analysis. PVT1 expression was significantly increased, whereas miR-146a was markedly decreased in diabetic OA mice than in non-diabetic OA and control. Increased PVT1 expression in diabetic OA mice was significantly associated with Mankin score and reduced miR-146a as well as Collagen alpha-1(II) (COL2A1) expressions. In vivo, intra-articular injection of ad-siRNA-PVT1 efficiently increased miR-146a and COL2A1 expressions, alleviated joint inflammation, decreased the expression of pro-inflammatory mediators, and suppressed TGF-β/SMAD4 pathway in diabetic OA mice. Our results demonstrate LncRNA PVT1 is involved in cartilage degradation in diabetic OA and correlated with disease severity. Efficiency of ad-siRNA-PVT1 in controlling joint inflammation in diabetic OA mice is associated with the suppression of the expression of miR-146a, pro-inflammatory cytokines and activation of TGF-β/SMAD4 pathway.

Honey reverses disease progression, has anti-nociceptive and anti-inflammatory effects in a rat model of knee osteoarthritis induced by monosodium iodoacetate

<h2>Summary</h2><h3>Background</h3> Clinically, osteoarthritis manifests as joint pain with concomitant loss of joint function, which may ultimately result in a substantially reduced quality of life for the patient. Although, a lot is known about the symptom of the disease, the pathophysiology behind the structural changes is complex and poorly understood. By understanding the mechanisms driving joint tissue destruction in osteoarthritis and identifying the key factors involved, new targets for therapy are emerging that will go beyond symptomatic relief to slowing or stopping the progression of osteoarthritis. <h3>Aim</h3> Thus, the aim of this study was to evaluate the effect of honey on disease progression, pain perception and inflammation in monosodium iodoacetate (MIA)-induced knee osteoarthritis in female Wistar rats. <h3>Methods</h3> Thirty, twelve-month old female Wistar rats, weighing between 200 g & 250 g, were randomly divided into five groups of six animals each. Animals in group one were not induced and served as the control, while animals in groups two to five were injected with monosodium iodoacetate in the right knee. In addition, animals in group two received normal saline (1 ml/kg b.w.), group three received arthocare (glucosamine/chondroitin sulphate 6.67/8.33 mg/kg b.w.), group four received low dose honey (250 mg/kg b.w.) while group five received high dose honey (1,000 mg/kg b.w.) and were treated for twenty one days. All animals were subjected to assessment of tactile allodynia (von Frey test), acute inflammation (knee edema), and serum biomarkers: tumour necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF), prostaglandin E₂ (PG E₂) & cartilage oligomeric matrix protein (COMP) as well as histo-pathological assessment of the right knee joint. <h3>Results</h3> Honey (at high and low doses), significantly (p < 0.05) reduced tactile allodynia on von Frey test (60 ± 20g) in animals subjected to experimental knee osteoarthritis induced by MIA. Knee edema was also significantly (p < .05) reduced by both high and low doses of honey. Low dose honey significantly (p < .05) reduced he serum levels of TNF-α (61.5 ± 22 pg/ml), VEGF (31 ± 6.1 pg/ml) and COMP (41 ± 14 ng/ml) but, had no effect on the serum level of PG E₂. High dose honey on the other hand, only significantly reduced the serum level of TNF-α (87 ± 22 pg/ml) but, had no effect on the serum levels of VEGF, COMP and PG E₂. However, the administration of honey did not show any significant effect on histo-pathological features of the induced osteoarthritis. <h3>Conclusion</h3> Honey reversed disease progression and reduced pain perception as well as inflammation in MIA-induced knee osteoarthritis in female Wistar rats. However, honey had no effect on the histo-pathology of the knee joint.

Effect of intra-articular dexmedetomidine on experimental osteoarthritis in rats.

Pharmacological treatment of osteoarthritis is still inadequate due to the low efficacy of the drugs used. Dexmedetomidine via the intra-articular (i.a.) route might be an option for the treatment of osteoarthritis-associated pain. The present study assessed the analgesic and anti-inflammatory effects of dexmedetomidine administered via the i.a. route in different doses in an experimental model of rat knee osteoarthritis induced with monosodium iodoacetate. Rats were allocated to four groups with 24 animals in each group. The OA (osteoarthritis), DEX-1 (dexmedetomidine in dose of 1μg/kg) and DEX-3 (dexmedetomidine in dose of 3μg/kg) groups were subjected to induction of osteoarthritis through injection of monosodium iodoacetate (MIA) via the i.a. route on the right knee; the control group was not subjected to osteoarthritis induction. Clinical assessment was performed on day 0 (before osteoarthritis induction) and then on days 5, 10, 14, 21 and 28 after induction. Treatment was performed on day 7 via the i.a. route, consisting of dexmedetomidine in doses of 1 and 3 μg/kg, while group OA received 0.9% normal saline. The animals were euthanized on days 7, 14, 21 and 28. Samples of the synovial membrane were collected for histopathological analysis, and the popliteal lymph nodes were collected for measurement of cytokines (interleukin [IL] IL-6, tumor necrosis factor alpha [TNF-α]). Dexmedetomidine (1 and 3 μg/kg) significantly reduced the animals' weight distribution deficit during the chronic-degenerative stage of osteoarthritis and improved the pain threshold throughout the entire experiment. Histological analysis showed that dexmedetomidine did not cause any additional damage to the synovial membrane. The TNF-α levels decreased significantly in the DEX-3 group on day 28 compared with the OA group. Dexmedetomidine reduced pain, as evidenced by clinical parameters of osteoarthritis in rats, but did not have an anti-inflammatory effect on histological evaluation.

MicroRNA-199-3p up-regulation enhances chondrocyte proliferation and inhibits apoptosis in knee osteoarthritis via DNMT3A repression.

Studies have pivoted on the position of microRNAs (miRNAs) in knee osteoarthritis (KOA) but not the more specific function of miR-199-3p. Thus, this study is to uncover the mechanism of miR-199-3p in KOA. Rats KOA models were established by modified Hulth method. miR-199-3p expression was observed in cartilage of KOA rats. The binding sites of miR-199-3p were predicted by bioinformatics analysis and the potential interaction between DNA methyltransferase 3A (DNMT3A) and miR-199-3p was verified by dual-luciferase reporter gene assay. Rats were injected with miR-199-3p agomir or antagomir and DNMT3A siRNA into the knee joint. Inflammatory response factors in serum and cartilage tissues, cell apoptosis, and pathological status of cartilage tissues were detected. Chondrocytes were isolated from KOA cartilages and treated with miR-199-3p mimicorinhibitor and DNMT3A siRNA. Chondrocyte proliferation and apoptosis were detected. miR-199-3p expression was suppressed in cartilage of KOA rats. Dual-luciferase reporter gene assay proved that a miR-199-3p-binding site was located in the 3'UTR of DNMT3A mRNA. Inflammation, chondrocyte apoptosis and cartilage pathological changes were improved by miR-199-3p agomir but aggravated by miR-199-3p antagomir. The effects of miR-199-3p antagomir on KOA rats were partially reversed by DNMT3A siRNA. miR-199-3p mimic or DNMT3A siRNA decreased KOA chondrocytes apoptosis and promoted proliferation. miR-199-3p inhibitor showed the opposite functions to miR-199-3p mimic. The effects of miR-199-3p inhibitor on chondrocytes were reversed by DNMT3A siRNA. This study highlights that miR-199-3p up-regulation or down-regulation of DNMT3A induces chondrocyte proliferation and inhibits apoptosis in KOA, which may widen our eyes to treat patients with KOA.

Expression of Piezo mRNA is unaffected in a rat model of knee osteoarthritis

Osteoarthritis of the knee impairs activities of daily living of those affected. Its irreversible degenerative changes to the knee joint induce functional disturbance and unpleasant arthralgia. The pain has inflammatory components and often is manifested with mechanical allodynia and hyperalgesia. Sustained weight bearing and joint movements increase pain sensitivity in knee osteoarthritis. Understanding the mechanisms underlying the mechanical allodynia and hyperalgesia might provide a therapeutical target for pain relief in patients with such symptoms. Piezo channel is a mechanically activated ion channel that may be involved in mechanical transduction in the articular cartilage. Although it has been shown that inflammation potentiates Piezo channel current induced by mechanical stimulation, whether Piezo expression levels are influenced by knee osteoarthritis has remained unknown. We measured Piezo mRNA in knee joints and dorsal root ganglia after establishing a model of knee osteoarthritis in rats using monosodium iodoacetate and found Piezo mRNA level is not upregulated. This finding raises a question as whether and how Piezo channels may be involved in mechanically induced pain in osteoarthritis.

Therapeutic effect of Huzhangoside D in rats with knee osteoarthritis induced by anterior cruciate ligament transection

Background: Knee osteoarthritis (KOA) is an age-related disease. Huzhangoside D is a saponin isolated from genus Clematis L. (Ranunculaceae). The aim of this study is to explore the anti-inflammatory, apoptotic, and autophagy regulation effects of huzhangoside D on KOA in a rat model. Materials and Methods: The KOA model was established by an anterior cruciate ligament transection surgery. Huzhangoside D was administered for 4 weeks. The weight-bearing assay, morphology observation, and intrinsic mechanism exploration were performed. Results: After administration, the weight-bearing assay showed that huzhangoside D promoted joint function recovery. Hematoxylin-eosin and safranin O-Fast green staining indicated that huzhangoside D ameliorated the structural damage. The Mankin scores were decreased in the huzhangoside D groups. Huzhangoside D enhanced cartilage thickness. Enzyme-linked immunosorbent assay study revealed that huzhangoside D downregulated the proinflammatory cytokine (tumor necrosis factor alpha, interleukin-6, and interleukin-1β) levels, while it upregulated the anti-inflammatory cytokine (interleukin-10) level in rat serum. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that huzhangoside D downregulated the apoptosis ratio of cartilage cells. Immunohistochemical staining showed that huzhangoside D upregulated the autophagy-related protein beclin-1, ATG5, ATG7, and light chain 3 levels and downregulated the p62 level. Moreover, the AKT and mTOR signaling pathway activities were downregulated. The 3-MA combination with huzhangoside D downregulated the weight-bearing function and morphology of the knee and upregulated the proinflammatory cytokines, which showed the role of autophagy as a protective mechanism in the effect of huzhangoside D. Conclusion: This study revealed that huzhangoside D is a promising agent in KOA treatment.

Extracts of Aurantiochytrium alga improves symptoms in knee osteoarthritis and muscle atrophy models

Extracts of <i>Aurantiochytrium</i> alga improves symptoms in knee osteoarthritis and muscle atrophy models

7 T-MRI および micro-CT を用いた変形性膝関節症モデルの経時的生体評価

This study aims to investigate inflammatory changes and osteophyte formation in a rat osteoarthritis (OA) model longitudinally by using 7 T-magnetic resonance imaging (MRI) and micro-computed tomography (CT). This OA model is induced surgically by removing the medial collateral ligament and medial meniscus of the right knee joint. Using 7 T-MRI, we compared the relative signal value of the medial collateral site and the area (mm2) of the upper end of the tibia at the right knee joint to those of the left knee joint on T2WI. In addition, we compared statistically the coefficient of variance (CV) of signal intensity on the subchondral bone, the area (mm2), the major axis (mm) and the minor axis (mm) of the upper end of the tibia by the use of micro-CT images. In MRI experiment, the relative signal value was significantly higher at 2, 6, and 10 weeks postoperatively in the medial part of right knee joint than that in the left one. In micro-CT experiment, CV was significantly higher from 6 weeks postoperatively in the subchondral bone of surgical side. Rough and irregular surface at the medial tibia was also observed by 3D images. Using 7 T-MRI and micro-CT, we're able to observe the knee osteoarthritis model rat longitudinally.

Patellar Inward Pushing Method Relieves Knee Osteoarthritis Via Regulating Cytokines

Background and Objective: Knee osteoarthritis (KOA) is a chronic degenerative disease characterized by pain, morning stiffness and swelling in the knee joints. And KOA is common in the elderly and seriously affects the exercise function and physical health of patients. This study aimed to explore the curative effects of patellar inward pushing method (PIPM) on KOA. Methods: In this study, we established rabbit animal models of KOA for the research by using the New Zealand white rabbits. A total of n=30 rabbits were divided into 5 groups by random number table method: blank group, model group, glucosamine hydrochloride (GH) group, PIPM group and PIPM combined with GH group. The rabbits were then modeled. Results: After 9-weeks cultured in groups, 5 ml blood was collected from the heart, and cytokines were detected. The result suggested that iNOS, NO and TNF-α were the pathogenic inflammatory factor of KOA, and aggravated cartilage damage and degeneration. Besides, this study indicated that PIPM combined with GH treatment significantly reduced the activity of inflammatory cytokines in serum and joint fluid of KOA models in rabbits. In addition, PIPM combined with GH therapy exhibited the best therapeutic effect among these treatments, which was working on KOA better than PIPM treatment alone or GH treatment alone.

Patellar Inward Pushing Method Relieves Knee Osteoarthritis Via Regulating Cytokines

&lt;p&gt;Background and Objective: &amp;nbsp;Knee osteoarthritis (KOA) is a chronic degenerative disease characterized by pain, morning stiffness and swelling in the knee joints. And KOA is common in the elderly and seriously affects the exercise function and physical health of patients. This study aimed to explore the curative effects of patellar inward pushing method (PIPM) on KOA.&lt;/p&gt;&#x0D; &lt;p&gt;Methods: &amp;nbsp;In this study, we established rabbit animal models of KOA for the research by using the New Zealand white rabbits. A total of n=30 rabbits were divided into 5 groups by random number table method: blank group, model group, glucosamine hydrochloride (GH) group, PIPM group and PIPM combined with GH group. The rabbits were then modeled.&lt;/p&gt;&#x0D; &lt;p&gt;Results: &amp;nbsp;After 9-weeks cultured in groups, 5 ml blood was collected from the heart, and cytokines were detected. The result suggested that iNOS, NO and TNF-&amp;alpha; were the pathogenic inflammatory factor of KOA, and aggravated cartilage damage and degeneration. Besides, this study indicated that PIPM combined with GH treatment significantly reduced the activity of inflammatory cytokines in serum and joint fluid of KOA models in rabbits. In addition, PIPM combined with GH therapy exhibited the best therapeutic effect among these treatments, which was working on KOA better than PIPM treatment alone or GH treatment alone.&lt;/p&gt;&#x0D; &lt;p&gt;Conclusion: &amp;nbsp;PIPM could effectively treat KOA via regulating cytokines, and the PIPM combined with GH therapy could be a novel therapeutic strategy for KOA.&lt;/p&gt;

CD11b Signaling Prevents Chondrocyte Mineralization and Attenuates the Severity of Osteoarthritis.

Osteoarthritis (OA) is a progressive joint disease that is strongly associated with calcium-containing crystal formation (mineralization) by chondrocytes leading ultimately to cartilage calcification. However, this calcification process is poorly understood and treatments targeting the underlying disease mechanisms are lacking. The CD11b/CD18 integrin (Mac-1 or αMβ2), a member of the beta 2 integrin family of adhesion receptors, is critically involved in the development of several inflammatory diseases, including rheumatoid arthritis and systemic lupus erythematosus. We found that in a collagen-induced arthritis, CD11b-deficient mice exhibited increased cartilage degradation compared to WT control animals. However, the functional significance of CD11b integrin signaling in the pathophysiology of chondrocytes remains unknown. CD11b expression was found in the extracellular matrix and in chondrocytes in both healthy and damaged human and murine articular cartilage. Primary murine CD11b KO chondrocytes showed increased mineralization when induced in vitro by secondary calciprotein particles (CPP) and quantified by Alizarin Red staining. This increased propensity to mineralize was associated with an increased alkaline phosphatase (Alp) expression (measured by qRT-PCR and activity assay) and an enhanced secretion of the pro-mineralizing IL-6 cytokine compared to control wild-type cells (measured by ELISA). Accordingly, addition of an anti-IL-6 receptor antibody to CD11b KO chondrocytes reduced significantly the calcification and identified IL-6 as a pro-mineralizing factor in these cells. In the same conditions, the ratio of qRT-PCR expression of collagen X over collagen II, and that of Runx2 over Sox9 (both ratio being indexes of chondrocyte hypertrophy) were increased in CD11b-deficient cells. Conversely, the CD11b activator LA1 reduced chondrocyte mineralization, Alp expression, IL-6 production and collagen X expression. In the meniscectomy (MNX) model of murine knee osteoarthritis, deficiency of CD11b led to more severe OA (OARSI scoring of medial cartilage damage in CD11b: 5.6 ± 1.8, in WT: 1.2 ± 0.5, p < 0.05, inflammation in CD11b: 2.8 ± 0.2, in WT: 1.4 ± 0.5). In conclusion, these data demonstrate that CD11b signaling prevents chondrocyte hypertrophy and chondrocyte mineralization in vitro and has a protective role in models of OA in vivo.

Exploration on effect mechanism of Miao medicinal acupuncture therapy in treatment of knee osteoarthritis based on TRPV ion channel

To observe the effect of Miao medicinal acupuncture therapy on transient receptor potential vanilloid (TRPV) channel in knee joint synovial tissue of the rabbits with knee osteoarthritis (KOA) model and to explore the mechanism of Miao medicinal acupuncture therapy in treatment of KOA. Of 34 New Zealand male rabbits, 6 rabbits were selected randomly as the normal group. KOA model was established in the rest rabbits by injecting a mixture of papain and L-cysteine in right knee joints. The 24 successfully modeled rabbits were randomized into a model group, a Miao medicinal acupuncture therapy group, a dermal needle group and a smearing group, 6 rabbits in each one. In the Miao medicinal acupuncture therapy group, Miao medicinal acupuncture therapy was adopted, in which, the roller type of dermal needle was used on the surface of right knee joint [a rectangle shape formed by "Xuehai" (SP 10), "Liangqiu" (ST 34), "Yanglingquan" (GB 34) and "Yinlingquan" (SP 9)], rolling in a "" shape, on which, Miao medicinal solution was smeared in advance. In the dermal needle group, the rolling stimulation was exerted on the right the right knee joint surface with the roller type of dermal needle. In the smearing group, Miao medicinal solution was smeared on the right knee joint surface. The intervention was given once every two days, 3 times weekly and the intervention was exerted consecutively for 4 weeks. Successively, on day 1, 21, 28, 35, 42 and 49 of experiment, paw withdrawal threshold (von Frey threshold) after mechanical stimulation was detected in the rabbits. HE staining was adopted to observe the histomorphological changes of the right knee joint cartilage in the rabbits. ELISA was used to determine the contents of interleukin-1 (IL-1β) and tumor necrosis factor-α (TNF-α) in the right knee synovial fluid. Western blot method and real-time PCR were used to determine the relative expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue of the rabbits. Compared with the normal group, on day 49 of experiment, von Frey threshold was reduced significantly in the rabbits of the model group (P<0.01), the integrety of cartilage surface of knee joint was seriously damaged, the structural layers were disordered, the chondrocytes were clustered, the tide lines were distorted and the matrix staining disappeared. The contents of IL-1β and TNF-α of the right knee synovial fluid were increased significantly (P<0.01), and the relative expressions of protein and mRNA of TRPV1 and TRPV4 in the synovial tissue of the right knee were increased significantly (P<0.01). Compared with the model group, on day 49 of experiment, von Frey threshold was increased significantly in each of the Miao medicinal acupuncture therapy group, the dermal needle group and the smearing group (P<0.01). The right knee joint cartilage was complete in morphology, the structure clear was in layer, the cells were arranged in order and the matrix staining was uniform. The contents of IL-1β and TNF-α of the right knee synovial fluid were reduced significantly (P<0.01, P<0.05), and the relative expressions of protein and mRNA of TRPV1 and TRPV4 in the synovial tissue of the right knee were reduced significantly (P<0.01). Compared with either the dermal needle group or the smearing group, on day 49 of experiment, von Frey threshold was increased significantly in the Miao medicinal acupuncture therapy group (P<0.01). The right knee joint cartilage surface was even, the structure layers were clear. The contents of IL-1β and TNF-α of the right knee synovial fluid were reduced significantly (P<0.05), and the relative expressions of protein of TRPV1 and TRPV4 in the synovial tissue were reduced (P<0.01, P<0.05). Compared with the smearing group, the relative expression of TRPV1 mRNA in the synovial tissue was reduced significantly in the Miao medicinal acupuncture therapy group (P<0.05). Miao medicinal acupuncture therapy plays a role in treatment of KOA probably through inhibiting the expressions of IL-1β and TNF-α of knee synovial fluid and down-regulating the expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue.

Relationship between the pyroptosis of fibroblast‑like synoviocytes and HMGB1 secretion in knee osteoarthritis.

High mobility group box 1 (HMGB1) is an important downstream product of pyroptosis in macrophages, and it serves a vital role in numerous inflammatory diseases. Previous studies have reported that HMGB1 is released by fibroblast-like synoviocytes (FLSs) that are activated by inflammatory cytokines in knee osteoarthritis (KOA); however, the mechanism via which FLS promotes HMGB1 secretion in KOA remains unknown. According to our previous study, pyroptosis occurs in FLSs of patients with KOA and is mediated by Nod-like receptor protein (NLRP)1 or NLRP3 inflammasomes. However, the specific relationship between HMGB1 secretion and FLS pyroptosis requires further investigation. In the present study, the association between HMGB1 secretion and FLS pyroptosis was investigated in vitro and in vivo. In this study, western blotting, ELISA and reverse transcription-quantitative PCR were used to measure expression levels of proteins and mRNA. Caspase-1 activity assay and Hoechst 33342/PI double staining were used to observe the pyroptosis of FLSs. Hematoxylin and eosin staining was used to observe the destruction of cartilage in KOA. Increased expression levels of pyroptosis-related proteins and HMGB1 in the synovium of rat anterior cruciate ligament transection-induced KOA models were identified, and these changes were significantly mitigated via the intra-articular injection of a caspase-1 inhibitor. In vitro, FLSs were treated with lipopolysaccharide (LPS) + ATP to induce pyroptosis, and HMGB1 secretion was subsequently measured. LPS + ATP significantly increased the expression levels of pyroptosis-related proteins and HMGB1 in FLSs, and these effects were significantly mitigated by small interfering RNAs targeting NLRP1, NLRP3, apoptosis-associated speck-like protein with a caspase-recruitment domain or caspase-1. Therefore, the present results indicated that NLRP1/NLRP3 inflammasome-mediated and caspase-1-dependent FLS pyroptosis increased HMGB1 secretion in KOA. These findings may provide a therapeutic strategy to decrease synovial inflammatory responses during KOA progression.

Synovial Mesenchymal Stem Cell-Derived EV-Packaged miR-31 Downregulates Histone Demethylase KDM2A to Prevent Knee Osteoarthritis

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have emerged as important mediators of intercellular communication in response to cartilage damage. In this study, we sought to characterize the inhibitory role of microRNA (miR)-31 encapsulated in synovial MSC (SMSC)-derived EVs in knee osteoarthritis (OA). The expression of miR-31, lysine demethylase 2A (KDM2A), E2F transcription factor 1 (E2F1), and pituitary tumor transforming gene 1 (PTTG1) was validated in cartilage tissues of knee OA patients. Following SMSC-EV extraction and identification, chondrocytes with the miR-31 inhibitor were added with SMSC-EVs, whereupon the effects of miR-31 on proliferation and migration of chondrocytes were assessed. The interaction among miR-31, KDM2A, E2F1, and PTTG1 in chondrocyte activities was probed in vitro, along with an in vivo mouse knee OA model. We identified downregulated miR-31, E2F1, and PTTG1 and upregulated KDM2A in cartilage tissues of knee OA patients. SMSC-EV-packaged miR-31 potentiated chondrocyte proliferation and migration as well as cartilage formation by targeting KDM2A. Mechanistically, KDM2A bound to the transcription factor E2F1 and inhibited its transcriptional activity. Enrichment of E2F1 in the PTTG1 promoter region activated PTTG1 transcription, accelerating chondrocyte proliferation and migration. SMSC-EVs and EVs from miR-31-overexpressed SMSCs alleviated cartilage damage and inflammation in knee joints in vivo. SMSC-EV-encapsulated miR-31 ameliorates knee OA via the KDM2A/E2F1/PTTG1 axis.

Effects of ultrasound on vascular endothelial growth factor in cartilage, synovial fluid, and synovium in rabbit knee osteoarthritis.

Ultrasound is commonly used to treat knee osteoarthritis (KOA), which has unique advantages with regard to relieving pain and inflammation as well as delaying cartilage degeneration, but the underlying mechanisms are less clear. The study aimed to investigate the therapeutic effects of ultrasound on vascular endothelial growth factor (VEGF) expression in cartilage, the synovium, and synovial fluid (SF) in a rabbit model of KOA. Twenty-four New Zealand rabbits were randomly divided into ultrasound (group A), sham ultrasound (group B) and no-ACLT control groups (group C). Six weeks after undergoing anterior cruciate ligament transection (ACLT), group A was treated with ultrasound and group B was treated with sham ultrasound. Two weeks thereafter, the morphology of the synovium and cartilage were observed. Cartilage and synovium were scored using the Mankin scale and Krenn V scores, respectively. VEGF expression in the cartilage, SF, and synovium of ACLT knee joints was analyzed via immunohistochemistry, western blotting, and RT-PCR. Cartilage degeneration and synovitis were the most severe in group B and the least severe in group C. Similarly, Mankin scores and Krenn V scores were highest in group B and lowest in group C (p<0.05). There were also significant differences in the VEGF IOD of cartilage or synovium, VEGF protein content in SF, and VEGF mRNA expression in cartilage or SF (p<0.05). Ultrasound can relieve synovitis and delay cartilage degradation, and the mechanisms of ultrasound for the treatment of KOA may involve inhibition of the expression of VEGF in the synovium, SF, and cartilage.

Intervention effect of Youguiwan on rats with knee osteoarthritis and its mechanism

Objective: To study the effects of Youguiwan on the osteoglycin (OGN), osteonectin (ON), fibrinogen 2 (FBN2) of articular cartilage tissue in the model of knee osteoarthritis (KOA). Methods: Sixty SD rats were randomly divided into six groups: sham control group, model group, glucosamine sulfate group, Youguiwan (high-dose, middle-dose and low-dose )group. The modified Hulth method was used to establish KOA models for 6 weeks. The sham control group and the model group were treated with normal saline. The rats in Youguiwan high-dose, middle-dose, low-dose groups were treated with Youguiwan at the doses of 4.8, 2.4, 1.2 g/kg by gavage respectively, and the glucosamine sulfate group was treated with glucosamine sulfate 0.17 g/kg. The rats were administrated for 8 weeks according to the dose. After intervening, articular cartilage of rats were obtained, the pathological changes were observed by using HE staining method, and Mankin score was evaluated. The expressions of OGN, ON and FBN2 in articular cartilage were detected by immunohistochemistry. The expression of GSK-3β in articular cartilage was detected by Western blot. Results: Compared with the sham control group, the Mankin score was obviously increased in the model group, the protein expression of FBN2 was increased significantly, yet the protein expressions of OGN, ON and GSK-3β were decreased significantly (P＜0.01), articular cartilage was seriously damaged, and chondrocytes were arranged in disorder. Compared with the model group, the Mankin score was declined obviously in the high-dose Youguiwan group, the protein expression of FBN2 was significantly decreased, but the protein expression of GSK-3β was significantly increased, the protein expressions of OGN and ON were significantly increased in the middle-dose and high-dose Youguiwan group (P＜0.05 or P＜0.01), cartilage structure was tended to be normal, the chondrocytes distribution was uneven, and articular cartilage surface was not smooth. Conclusion: Youguiwan can significantly improve the articular cartilage degeneration of KOA rats, its mechanism maybe raise OGN and ON protein expression level to promote the ossification and reconstruction of articular cartilage.