Knee Osteoarthritis Rats Research Articles

Are Stem Cells Derived from Synovium and Fat Pad Able to Treat Induced Knee Osteoarthritis in Rats?

Background Osteoarthritis (OA) is a chronic disease and a significant cause of joint pain, tenderness, and limitation of motion. At present, no specific treatment is available, and mesenchymal stem cells (MSCs) have shown promising potentials in this regard. Herein, we aimed to evaluate the repairing potentials of stem cells derived from the synovium and fat pad in the treatment of OA. Methods Twenty-eight male rats (220 ± 20 g, aged 10-12 weeks), were randomly divided into four groups (n = 7): C1: nontreated group, C2: Hyalgan-treated group, E1: adipose tissue-derived stem cell-treated group, and E2: synovial membrane-based stem cell-treated group. Collagenase type II was injected into the left knee; after eight weeks, OA was developed. Then, stem cells were injected, and rats were followed for three months. Afterward, specimens and radiological images were investigated. p value ≤ 0.05 was set as statistically significant. Results Compared to the C1 group, the E1 and E2 groups showed significantly better results in all six pathological criteria as well as joint space width and osteophytes of medial tibial, medial femoral, and medial fabellar condyles (p ≤ 0.001). Similarly, compared to the C2 group, the E1 and E2 groups had better scores regarding surface, matrix, cell distribution, and cell population viability (p < 0.05). E2 showed considerably higher scores compared to C2 regarding subchondral bone and cartilage mineralization (p < 0.05). The joint space width was similar between the C2 and E groups. Conclusion Treatment of OA with MSCs, particularly synovial membrane-derived stem cells, not only prevented but also healed OA of the knee to some extent in comparison to the Hyalgan and nontreatment groups.

Intra-articular delivery of celastrol by hollow mesoporous silica nanoparticles for pH-sensitive anti-inflammatory therapy against knee osteoarthritis

BackgroundCelastrol has been proven effective in anti-inflammatory but was limited in the clinic due to the poor solubility and side effects induced by low bioavailability. Osteoarthritis has acidic and inflammatory environment. Our aim was to load celastrol into HMSNs and capped with chitosan to construct a pH-responsive nanoparticle medicine (CSL@HMSNs-Cs), which is of high solubility for osteoarthritis intra-articular injection treatment.MethodsThe CSL@HMSNs-Cs were assembled and the characteristics were measured. The CSL@HMSNs-Cs was applied in vitro in the chondrocytes collected from rats cartilage tissue and in vivo in the MIA induced knee osteoarthritis rats via intra-articular injection. Cytotoxicity assay, pH-responsive release, pain behavior, MRI, safranin o fast green staining, ELISA and western blot analysis were applied to evaluate the bioavailability and therapeutic effect of CSL@HMSNs-Cs.ResultsCSL@HMSNs-Cs was stable due to the protection of the chitosan layers in alkaline environment (pH = 7.7) but revealed good solubility and therapeutic effect in acidic environment (pH = 6.0). The cytotoxicity assay showed no cytotoxicity at relatively low concentration (200 μg/mL) and the cell viability of chondrocytes stimulated by IL-1β was increased in CSL@HMSNs-Cs group. Paw withdrawal threshold in CSL@HMSNs-Cs group is increased, and MRI and Safranin O Fast Green staining showed improvements in articular surface erosion and joint effusion. The upregulated expression levels of IL-1β, TNF-α, IL-6, MMP-3 and MMP-13 and NF-κB signaling pathway of chondrocytes were inhibited in CSL@HMSNs-Cs group.ConclusionHollow mesoporous silica nanoparticles were an ideal carrier for natural drugs with poor solubility and were of high biocompatibility for intra-articular injection. These intra-articular injectable CSL@HMSNs-Cs with improved solubility, present a pH-responsive therapeutic strategy against osteoarthritis.

L-Theanine Reduced the Development of Knee Osteoarthritis in Rats via Its Anti-Inflammation and Anti-Matrix Degradation Actions: In Vivo and In Vitro Study.

The etiology of osteoarthritis (OA) is multifactorial, with no effective disease-modifying-drugs. L-theanine has been reported to inhibit inflammatory responses in some diseases and this study aimed to investigate the effect of L-theanine on Interleukin-1(IL-1)β-stimulated chondrocytes, and in an injury-induced OA rat model. Primary chondrocytes were stimulated by IL-1β (10 ng/mL) for 24 h and then co-cultured with L-theanine for 24 h. The effects of L-theanine on IL-1β-stimulated expression of pro-inflammatory cytokines and hydrolytic enzyme were analyzed using Western blotting, quantitative polymerase chain reaction (q-PCR) and enzyme-linked immunosorbent assay (ELISA) kits. An immunofluorescence assay was used to detect nuclear factor kappa B (NF-κB) phosphorylation. OA was induced by anterior cruciate ligament transection (ACLT) surgery in rats and celecoxib was used as a positive control. OA severity was measured using the Osteoarthritis Research Society International (OARSI) grading system to describe histological changes. The results showed that L-theanine decreased the expression of pro-inflammatory mediators, including cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE-2), inducible nitric oxide synthase (iNOS), and nitric oxide (NO), both in vivo and in vitro. L-theanine treatment inhibited IL-1β-induced upregulation of matrix metalloproteinases (MMP)-3 and MMP-13, as well as inhibited NF-κB p65 activation. In vivo animal model showed that L-theanine administration (200 mg/kg) significantly alleviated OA lesions and decreased OARSI score. Our data indicated that L-theanine decreased inflammatory cytokines and protected extracellular matrix degradation through inhibition of the NF-κB pathway, and L-theanine may be considered a promising therapeutic strategy in OA prevention.

Chrysin Attenuates the NLRP3 Inflammasome Cascade to Reduce Synovitis and Pain in KOA Rats.

PurposeOur recent reports have revealed that inhibiting NLRP3 activation reduces synovial inflammation and fibrosis in knee osteoarthritis (KOA). Synovial inflammation is involved the entire process of KOA and promotes the progression of KOA. Natural flavonoid Chrysin from Scutellariae Radix, a traditional Chinese medicine, exhibits multifarious biological activities and potentially has protective activity against osteoarthritis. However, the mechanism of Chrysin in the treatment of synovial inflammation remains elusive. The purpose of our research was to explore the anti-inflammatory effects of Chrysin on KOA, which was induced by monoiodoacetic acid (MIA) in rats by targeting the NLRP3 inflammasome in the hopes of identifying an effective drug to treat KOA.MethodsThe MIA-induced KOA model was used to evaluate the cold pain threshold and paw withdrawal threshold (PWT) of joints after MIA (40 mg/mL) injection into the knee joints. Microscopically, we used LPS (5 ug/mL) and ATP (4 mmol/L) to stimulate fibroblast-like synovial cells (FLSs) to explore the underlying mechanisms and effects of Chrysin. Two staining methods, H&E and Sirius Red, were applied to assess histopathological changes in synovial membranes. Cellular signal transduction was determined by qRT-PCR and WB. Cytokine expression (inflammatory cytokines and pain-related cytokines) was detected by ELISA. The degree of chronic inflammatory pain was evaluated by c-Fos immunofluorescence.ResultsThe results showed that Chrysin not only attenuated synovial inflammation but also reduced the secretion of pain-related factors and increased the PWT and cold pain threshold in rats. Chrysin also inhibited NLRP3 inflammasome activation and increased IL-1β levels to alleviate the synovitis.ConclusionChrysin can relieve knee synovial inflammation and improve pain behavior in KOA rats, which may be related to the ability of Chrysin to inhibit NLRP3 inflammasome activation. Therefore, Chrysin may be developed as a new drug for the treatment of KOA.

Casticin suppresses monoiodoacetic acid-induced knee osteoarthritis through inhibiting HIF-1α/NLRP3 inflammasome signaling

Knee osteoarthritis (KOA) is a disabling chronic inflammatory disease that is closely associated with synovium tissue hypoxia and synovial fibrosis. Casticin, a compound purified from the Chinese herb Viticis Fructus, has been proved effective in preventing inflammation and fibrosis in previous studies. However, the effect of casticin on synovial fibrosis in KOA is not clear. In present study, we aimed to investigate how did casticin affect synovial fibrosis on monoiodoacetic acid (MIA)-induced KOA in rats. The MIA-induced knee osteoarthritis model and lipopolysaccharide (LPS) stimulated primary synovial fibroblasts inflammation model were established. Pathological and morphological changes in synovial tissue were observed by H&E and sirius red staining. The hypoxia of synovium was detected by pimonidazole staining and immunohistochemistry of hypoxia-inducible factors 1α (HIF-1α). The levels of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome components, fibrogenic markers (TGF-β, COL1A1 and TIMP1) and inflammatory cytokines were examined by western blotting, qRT-PCR or ELISA in both KOA rat models and primary synovial fibroblasts. Our data suggested that casticin improved hypoxia and inflammation in synovium tissue, as well the synovial fibrosis in rats. Besides, casticin inhibited the activation of NLRP3 inflammasome in MIA-induced KOA rats and synovial fibroblasts. In conclusion, our findings demonstrated that casticin alleviated MIA-induced KOA by inhibiting of HIF-1α/NLRP3 inflammasome activation. Therefore, casticin could be a potential treatment strategy for KOA.

Modulation of Hedgehog Signaling by Kappa Opioids to Attenuate Osteoarthritis.

Inhibition of hedgehog (HH) signaling prevents cartilage degeneration and promotes repair in animal models of osteoarthritis (OA). This study, undertaken in OA models and in human OA articular cartilage, was designed to explore whether kappa opioid receptor (KOR) modulation via the inhibition of HH signaling may have therapeutic potential for achieving disease-modifying activity in OA. Primary human articular cartilage and synovial tissue samples from patients with knee OA undergoing total joint replacement and from healthy human subjects were obtained from the National Disease Research Interchange. For in vivo animal studies, a partial medial meniscectomy (PMM) model of knee OA in rats was used. A novel automated 3-dimensional indentation tester (Mach-1) was used to quantify the thickness and stiffness properties of the articular cartilage. Inhibition of HH signaling through KOR activation was achieved with a selective peptide agonist, JT09, which reduced HH signaling via the cAMP/CREB pathway in OA human articular chondrocytes (P = 0.002 for treated versus untreated OA chondrocytes). Moreover, JT09 markedly decreased matrix degeneration induced by an HH agonist, SAG, in pig articular chondrocytes and cartilage explants (P = 0.026 versus untreated controls). In vivo application of JT09 via intraarticular injection into the rat knee joint after PMM surgery significantly attenuated articular cartilage degeneration (60% improvement in the tibial plateau; P = 0.021 versus vehicle-treated controls). In JT09-treated rats, cartilage content, structure, and functional properties were largely maintained, and osteophyte formation was reduced by 70% (P = 0.005 versus vehicle-treated controls). The results of this study define a novel mechanism for the role of KOR in articular cartilage homeostasis and disease, providing a potential unifying mechanistic basis for the overlap in disease processes and features involving opioid and HH signaling. Moreover, this study identifies a potential novel therapeutic strategy in which KOR modulation can improve outcomes in patients with OA.

Alleviation of medial meniscal transection-induced osteoarthritis pain in rats by human adipose derived mesenchymal stem cells.

Knee osteoarthritis (KOA) is a degenerative joint disorder manifested with deformity, pain, and functional disability due to damage of the articular cartilage. Cell therapy with mesenchymal stem cells (MSCs) holds great promise to alleviate or even cure the degenerative diseases including KOA. However, the evidence of efficacy of human adipose tissue-derived MSCs (hAdMSCs) on KOA therapy remains limited. Here, we evaluate the therapeutic efficacy of hAdMSCs for KOA, using a medial meniscal transection (MMT) rat model. Our study demonstrated that intra-articular injection of 1.25×106 hAdMSCs significantly attenuated MMT-induced joint pain in a KOA rats model. The results of this study provide strong evidence that hAdMSCs-based therapy can be regarded as a prominent treatment option for patients with KOA.

Contribution of nerves within osteochondral channels to osteoarthritis knee pain in humans and rats

Subchondral bone may contribute to knee osteoarthritis (OA) pain. Nerve growth factor (NGF) can stimulate nerve growth through TrkA. We aimed to identify how sensory nerve growth at the osteochondral junction in human and rat knees associates with OA pain. Eleven symptomatic chondropathy cases were selected from people undergoing total knee replacement for OA. Twelve asymptomatic chondropathy cases who had not presented with knee pain were selected post-mortem. OA was induced in rat knees by meniscal transection (MNX) and sham-operated rats were used as controls. Twice-daily oral doses (30mg/kg) of TrkA inhibitor (AR786) or vehicle were administered from before and up to 28 days after OA induction. Joints were analysed for macroscopic appearances of articular surfaces, OA histopathology and calcitonin gene-related peptide-immunoreactive (CGRP-IR) sensory nerves in medial tibial plateaux, and rats were assessed for pain behaviors. The percentage of osteochondral channels containing CGRP-IR nerves in symptomatic chondropathy was higher than in asymptomatic chondropathy (difference: 2.5% [95% CI: 1.1-3.7]), and in MNX-than in sham-operated rat knees (difference: 7.8% [95%CI: 1.7-15.0]). Osteochondral CGRP-IR innervation was significantly associated with pain behavior in rats. Treatment with AR786 prevented the increase in CGRP-IR nerves in osteochondral channels and reduced pain behavior in MNX-operated rats. Structural OA was not significantly affected by AR786 treatment. CGRP-IR sensory nerves within osteochondral channels are associated with pain in human and rat knee OA. Reduced pathological innervation of the osteochondral junction might contribute to analgesic effects of reduced NGF activity achieved by blocking TrkA.

The Effect of Mesenchymal Stem Cell Wharton’s Jelly on Nuclear Factor Kappa Beta and Interleukin-10 Levels in Osteoarthritis Rat Model

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease in one or more joints characterized by changes in pathological structures such as cartilage, hypertrophy, and remodeling of the subchondral bone and secondary inflammation of the synovium membrane, causing changes in joint components such as cells, matrices, and molecular production. At the molecular level, an imbalance between catabolic and anabolic activities in joint cartilage results in OA. Nuclear factor kappa beta (NFκβ) is a cytokine that plays an important role in the signaling pathway of the pathogenesis of OA in causing an inflammatory reaction, whereas interleukin (IL)-10 is an anti-inflammatory cytokine that is involved in the pathogenesis of OA.&#x0D; AIM: This study aims to prove the influence before and after administration mesenchymal stem cells from Wharton’s jelly on the serum NFκβ and IL-10 levels in OA rat models.&#x0D; MATERIALS AND METHODS: This research is an experimental study with the design of post-test-only control group design. The sample consisted of 16 OA rats as a control group and 16 OA rats treated with MSC-WJ as a treatment group. OA induction is done by injection of monosodium iodoacetate (MIA) into the intra-articular right knee. Giving MSC-WJ is done in the 3rd week after MIA induction. The serum NFκβ and IL-10 levels were measured after 3 weeks treated with MSC-WJ using the ELISA method. The statistical test used is an independent t-test. p &lt; 0.05 was said to be statistically significant.&#x0D; RESULTS: From the research results obtained, serum levels of knee OA of rat knee OA treated with mesenchymal stem cell Wharton jelly are lower than serum NFκβ levels of knee OA of the rat that is not treated, but the difference in levels of NFκβ is not significant (p &gt; 0.05). The serum IL-10 level of rat OA of knee treated with mesenchymal stem cell Wharton jelly was higher than the serum IL-10 level of rat OA of the knee that was not treated, difference in levels of IL-10, is significant (p &lt; 0.05).&#x0D; CONCLUSION: This study concluded that MSC-WJ significantly decreased the serum NFκβ levels of OA rats and not significantly increased the serum IL-10 levels of OA rats.

Multiple umbilical cord–derived MSCs administrations attenuate rat osteoarthritis progression via preserving articular cartilage superficial layer cells and inhibiting synovitis

Background/objectivesArticular cartilage erosion probably plays a substantial role in osteoarthritis (OA) initiation and development. Studies demonstrated that umbilical cord–derived mesenchymal stem cells (UCMSCs) could delay chondrocytes apoptosis and ameliorate OA progression in patients, but the detailed mechanisms are largely uncharacterised. In this study, we aimed to study the effects of UCMSCs on monosodium iodoacetate (MIA)–induced rat OA model, and explore the cellular mechanism of this effect. MethodsIntra-articular injection of 0.3 ​mg MIA in 50 ​μL saline was performed on the left knee of the 200 ​g weight male Sprague-Dawley rat to induce rat knee OA. A single dose of 2.5 ​× ​105 undifferentiated UCMSCs one day after MIA or three-time intra-articular injection of 2.5 ​× ​105 UCMSCs on Days 1, 7 ​and 14 were given, respectively. Four weeks after MIA, joints were harvested and processed for paraffin sections. Safranine-O staining, haematoxylin and eosin staining ​and immunohistochemistry of MMP-13, ADAMTS-5, Col-2, CD68 ​and CD4 were performed to observe cartilage erosion and synovium. For in vitro ​studies, migration ability of cartilage superficial layer cells (SFCs) by UCMSCs were accessed by transwell assay. Furthermore, catabolism change of MIA-induced SFCs by UCMSCs was performed by real-rime polymerase chain reaction of Col-X and BCL-2 genes. CCK-8 assay was performed to check proliferation ability of SFCs by UCMSCs-conditioned media. ResultIn this study, we locally injected human UCMSCs, which is highly proliferative and noninvasively collectible, into MIA-induced rat knee OA. An important finding is on obviously ameliorated cartilage erosion and decreased OA Mankin score by repeated UCMSCs injection after MIA injection compared with single injection, both of which attenuated OA progression compared with vehicle. Interestingly, we observed significantly increased number of SFCs on the articular cartilage surface, probably related to elevated proliferation, mobilisation and inhibited catabolism marker: Col-X and BCL-2 gene expression of cultured SFCs by UCMSCs-conditioned media treatment in vitro. In addition to the change of unique SFCs, catabolism markers of ADAMTS-5 and MMP-13 were substantially upregulated in the whole cartilage layer chondrocytes as well. Strikingly, MIA-induced inflammatory cells infiltration, on both CD4+ Th cells and CD68+ macrophages, and hyperplasia of the synovium, which was alleviated by repeated UCMSCs injection. ConclusionOur study demonstrated a critical role of repeated UCMSCs dosing on preserving SFCs function, cartilage structure and inhibiting synovitis during OA progression, and thus provided mechanistic proof of evidence for the use of UCMSCs on OA patients in the future. The translational potential of this articleUCMSCs are a relatively “young” stem cell, and noninvasively collectible. In our study, we clearly demonstrated that it could effectively delay OA progression, possibly through reserving SFCs function and inhibiting synovitis. Therefore, it could be a new promising therapeutic cell source for OA after further clinical trials.

Two‐Phase Change in Cartilage Metabolism Indicated by Serum Biomarkers at Onset of Posttraumatic Osteoarthritis in a Medial Meniscus Destabilization Rat Model

PurposeOsteoarthritis (OA) is a chronic degeneration of articular cartilage from a deficit of supporting proteins. Surgical destabilization of the medial meniscus (DMM) experimentally leads to trauma‐induced knee OA in rats. Changes in cartilage function or joint remodeling associated with joint instability or disease may be reflected by changes in synthesis or degradation of extracellular matrix, assessed by measuring metabolites, serving as biomarkers, of cartilage synthesis/degradation. Aggrecan chondroitin sulfate 846 epitope (CS846) is associated with synthesis of cartilage ground substance and is elevated in patients with OA. Degradation of type I and II collagen is related to increased serum concentration of C‐terminus of 3/4 peptide, measured as C1,2C.MethodsFemale Lewis rats (n=40) were randomly assigned to DMM (n=20) or sham (n=20) surgery groups. Surgical intervention followed a 2‐week acclimation. The joint capsule was opened and the medial meniscotibial ligament was transected for DMM; no ligaments were cut for sham. Blood samples were collected by tail clipping 1 week prior (baseline) and 2‐weeks (acute phase, surgery effect) and 6‐weeks (late phase, OA effect) postsurgery. Clotted blood was centrifuged (3000 rpm, 30 minutes) and aliquots of serum stored (−80ºC) until analysis. CS846 and C1,2C were analyzed by enzyme‐linked immunosorbent assay kits.ResultsCS846 and C1,2C serum level changes were significant over time (mean [SD] ng/ml) in both groups, but the dynamics were different. CS846 was elevated 2‐weeks postsurgery (1334.7 [1786.6]) compared to baseline (417.8 [591], P=.012), but decreased 6‐weeks postsurgery (610 [1213.4], P=.066) in DMM. CS846 did not change from baseline to 2‐weeks or 6‐weeks postsurgery (P=.16 and .35) in sham. C1,2C decreased 2‐weeks postsurgery in DMM (4211 [1176], P&lt;.001) and sham (4580 [1238], P&lt;.001) compared to baseline (sham 6817 [1348], DMM 5918 [1211]), but the decrease was slightly less in DMM (P=.076). C1,2C continued to decrease between 2‐weeks and 6‐weeks postsurgery in sham (3447 [897], P&lt;.001), but not DMM (4111 [485], P=.360); the between‐groups difference was significant (P&lt;.001).ConclusionsBiomarker dynamics suggested a 2‐phase progression of OA in the posttraumatic model. During the acute phase (2 weeks after trauma), altered joint configuration resulted in increased pressure on articular cartilage causing destruction of ground substance, indicated by increased CS846. Surgical intervention caused collagen type I fiber damage in both groups. Wound healing caused products of collagen breakdown (C1,2C) to rapidly decrease in sham and to a lesser extent in DMM. In the late phase of OA (6 weeks after trauma), erosion of cartilage resulted in collagen type II breakdown, indicated by a lesser decrease in C1,2C in DMM than sham. Continued damage to articular cartilage in DMM caused C1,2C to remain relatively constant between 2‐weeks and 6‐weeks postsurgery. These data indicate that CS846, but not C1,2C, is more useful in the diagnosis of early stages of OA and that the opposite is true in the later phase.

Effects of undernatured collagen supplementation on rat knee osteoarthritis

Effects of undernatured collagen supplementation on rat knee osteoarthritis

Shea Nut Oil Extracts Enhance the Intra-Articular Sodium Hyaluronate Effectiveness on Surgically Induced OA Progression in Rats.

Osteoarthritis (OA) progression is associated with joint pain and stiffness. Intra-articular hyaluronic acid (IAHA) injection in knee OA restores the viscoelasticity of the joint and prevents cartilage damage. Shea nut oil extract (SNO) was shown to provide chondroprotection on surgically-induced OA progression in rats. Here we aim to examine IAHA injection supplemented with SNO diet for a synergetic evaluation on the disease progression in OA rats. We employed an anterior cruciate ligament transection plus medial meniscectomy-induced knee OA rat model with up to 12 weeks of sign/behavior observation (knee width, weight-bearing) and histological assessments of joint damage. We found both IAHA and SNO alone significantly attenuated histological changes of cartilage degeneration and synovial reactions in these knee OA rats. Nonetheless, oral SNO alone mitigated OA pain and inflammation while IAHA alone had no significant impact on the weight-bearing test and knee joint swelling. Moreover, with IAHA-treated rats fed with oral SNO diet, additional anti-inflammatory and anti-nociceptive effects were found, which further enhanced and maintained IAHA protection. Given the differential phenotype of oral SNO vs. IAHA, a regimen of IAHA coupled with SNO supplement provides a long-term effect of IAHA treatment. Taken together, the SNO supplement can be safely used as an adjuvant diet for chronic symptomatic relief of OA coupled with IAHA management.

Effect of fire-needle intervention on joint function, cartilage impairment and inflammatory response in knee osteoarthritis rats

To observe the effect of fire-needle stimulation of "Neixiyan"(EX-LE4) and "Dubi"(ST35) on changes of motor function, structure of cartilage degradation and inflammatory factors in knee osteoarthritis (KOA) rats, so as to explore its underlying mechanisms in improving KOA. Thirty-nine male SD rats were randomly divided into normal, model and fire-needle groups (n＝13 in each group). The KOA model was established by injection of Monoiodoacetate (MIA, 1 mg) into the lumen of the right knee joint. On the 7th day after successful modeling, fire-needle was applied to EX-LE4 and ST35, twice a week for 3 weeks. The rats' behavioral reactions of gait (0 to 3 points) and claw pressure (0 to 3 points) were scored, and histopathological changes scored by assessing the impairment grade (0 to 6) and stage (0 to 4) of the articular cartilage after safranin O-fast green staining. The contents of serum Interleukin (IL)-1α， Tumor necrosis factor α(TNF-α), IL-10, IL-37 and transforming growth factor β(TGF-β) were assayed by ELISA, and ultrastructural changes were observed under transmission electron microscope(TEM). After modeling, the gait and claw-pressure scores were significantly increased in the model group in comparison with the normal group (P<0.05), and the OA score and contents of serum IL-1α and TNF-α were also appa-rently increased in the model group relevant to the normal group (P<0.05，P<0.01), while the levels of serum IL-10, IL-37 and TGF-β remarkably decreased in the model group in contrast to the normal group (P<0.01). After the intervention, the increase of gait and claw-pressure and OA scores, as well as serum IL-1α and TNF-α contents, and the decrease of serum IL-10, IL-37 and TGF-β levels were all reversed in the fire needle group (P<0.05, P<0.01). Outcomes of TEM showed more and larger lipid droplets, swollen mitochondria with some vacuoles, and expanded, broken or dissolved rough endoplasmic reticulum in the model group, which was milder in the fire-needle group. Fire-needle can improve motor function and relieve impairment of articular cartilage of KOA rats, which may be related to its effects in reducing inflammatory factors and in increasing anti-inflammatory factor levels.

Circulating microRNA let‑7e is decreased in knee osteoarthritis, accompanied by elevated apoptosis and reduced autophagy.

Knee osteoarthritis (KOA) is a major cause of leg disability in the elderly population. Recently, the expression levels of circulating microRNA (miRNA) let-7e have been reported to be significantly reduced in KOA. The aims of the present study were to assess the feasibility of let-7e as a serum marker for detecting KOA and to explore the underlying mechanisms of its involvement. Based on previous studies and bioinformatics analysis, let-7e may regulate apoptosis and autophagy of articular chondrocytes. A total of 10 patients with KOA and 10 patients with trauma without KOA were recruited to examine the levels of let-7e in peripheral blood. Subsequently, KOA rat models were established, and the levels of let-7e in the cartilage and serum were examined, the expression of apoptotic proteins and autophagy-related proteins in the cartilage were investigated, and apoptotic and autophagic activities of primary cultured chondrocytes were also detected. In patients with KOA, let-7e levels in the peripheral serum were significantly decreased compared with the control group, and this result was confirmed in the peripheral serum and cartilage of KOA rats. In addition, the expression levels of proteins involved in the apoptotic pathway were increased in the cartilage of KOA rats, and apoptotic activity was increased. The expression of autophagy-related proteins beclin 1 and microtubule associated protein 1 light chain 3 β (LC3B) II/LC3BI in the articular cartilage of KOA rats was lower compared with the controls, and autophagy was decreased. Si-Miao-San (SMS) treatment restored the expression of let-7e and reversed the changes in apoptosis and autophagy. Therefore, the present study provided additional evidence that circulating let-7e may be a potential serum biomarker for the diagnosis and treatment of KOA. Elevated apoptosis levels and decreased autophagy levels of cartilage tissue are involved in KOA, and treatment with SMS may reverse these effects.

Effect of whole body vibration on HIF-2α expression in SD rats with early knee osteoarthritis.

To investigate the effect of different frequencies of whole body vibration (WBV) on articular cartilage of early knee osteoarthritis (OA) rats and determine whether WBV would influence the pathway of hypoxia-inducible factor-2α (HIF-2α) regulation-related genes after 8weeks of treatment. Forty 8-week-old OA rats were divided into five groups: sham control (SC); high frequency 60Hz (HV1); high frequency 40Hz (HV2); middle frequency 20Hz (MV) and low frequency 10Hz (LV). WBV (0.3g) treatment was given 40min/day and 5days/week. After 8weeks, rats were killed and knees were harvested. OA grading score: Osteoarthritis Research Society International (OARSI), and the expression of related genes: interleukin-1β (IL-1β), HIF-2α, matrix metalloproteinases-13 (MMP-13), and collagen type II alpha 1 (COL2A1), at both mRNA and protein levels were analyzed. After 8weeks of WBV, our data showed that lower frequency (10Hz) was more effective than the higher ones, yet they all suggested that WBV alleviates the erosion of knee articular cartilage in early OA. The expression of IL-1β, HIF-2α and MMP-13 decreased with frequency and reached the lowest level at 10Hz, the expression of COL2A1 increased with frequency and reached the highest level at 10Hz. This study demonstrates that WBV could alleviate the degeneration of knee joints in an early OA rat model. WBV regulates related gene expression at both mRNA and protein levels. HIF-2α could be a therapeutic target. The effect of WBV is frequency dependent; the lower frequency shows better effects.

Value of Vitamin D Administration Versus Suppressing Gut Microbiota in Modifying Metabolic Associated Osteoarthritis in Rats

Abstract Background: Metabolic syndrome (MetS) is considered as common risk factors for knee osteoarthritis (OA). Aim of Study: Comparing the effect of vit. D supplemen-tation and gut microbiota suppression on the development of metabolic knee OA in rats is the aim of the study. Material and Methods: Forty adult male rats were subdi-vided into control and high carbohydrate high fat (HCHF) fed rats that furtherly subdivided into: HCHF (positive control), HCHF-Vitamin D treated & HCHF-antibiotics treated. Body mass index (BMI), serum glucose, insulin, total cholesterol, triglycerides, HDL-C, LDL-C, inflammatory cytokines were estimated. Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated. Synovial cytokine, tissue degen-erative enzymes gene expression and knee histopathology were assessed. Results: HCHF induced significant increase in BMI, glucose, insulin, HOMA-IR, serum and synovial cytokines and degenerative enzymes with dyslipidemia and knee joint damage compared with controls. Vit. D improved all parameters in comparison to HCHF fed group. Antibiotic administration improved glucose, insulin, HOMA-IR, dyslipidemia and serum cytokines but induced insignificant changes in BMI, synovial cytokines, degenerative enzymes and knee joint damage compared with HCHF fed group. Conclusion: HCHF diet led to development of knee joint damage associated with changes in the metabolic profile in rats. Vit D supplementation dramatically improved knee joint damage, obesity insulin resistance, dyslipidemia and inflam-mation systemically and locally while controlling gut dysbiosis by antibiotics did not improve obesity, local inflammation and knee damage but improved the other parameters less than Vit D.

Warm acupuncture improves arthritic injury by down-regulating expression of skeleton proteins in rats with knee osteoarthritis

To investigate the effect of warm acupuncture on chondrocyte cytoskeleton protein Rho associa-ted protein kinase (ROCK)/ monopherine domain kinase 1 (LIMK1)/Cofilin signaling of synovial tissue of the knee-joint in knee osteoarthritis (KOA) rats, so as to explore its mechanisms underlying improvement of KOA. One hundred-twenty SD rats (half male and half female) were randomly divided into 5 groups: normal control, model, acupuncture, moxibustion and warm acupuncture, with 24 rats in each group. The KOA model was established by injection of 4% Papain (0.25 mL/kg) into the right knee cavity on day 1, 3 and 7. Rats in the acupuncture, moxibustion and warm acupuncture groups were treated with manual acupuncture, moxibustion and warm acupuncture stimulation of "Neixiyan"(EX-LE4), "Waixiyan"(EX-LE5) and "Zusanli"(ST36), respectively for 20 min, once a day for 21 days. The volume of the right knee-joint was measured by using drainage method and its width measured using a vernier caliper. The histopathological changes of the right knee cartilage were observed after H．E. stain, and scored (0 to 14 points) with reference to Markin's methods. The expression levels of ROCK, Cofilin, phospho-Cofilin, LIMK1 and phospho-LIMK1 proteins of the right knee synovial tissue were detected by Western blot. After modeling, the width and the volume since day 6 of the right knee-joint and Markin score of the cartilage, as well as the expression levels of ROCK, phospho-Cofilin, and phospho-LIMK1 proteins were significantly increased in the model group in contrast to the normal control group (P<0.05，P<0.01). Following the interventions, the width and the volume since day 12 of the right knee-joint and Markin score of the cartilage, as well as the expression levels of ROCK, phospho-Cofilin, and phospho-LIMK1 proteins were reversed in the three treatment groups (P<0.05). The effect of warm acupuncture was significantly superior to that of both simple acupuncture and simple moxibustion in decreasing the width and the volume since day 15 of the right knee-joint and Markin score of the cartilage, as well as in down-regulating the expression levels of ROCK, phospho-Cofilin and phospho-LIMK1 proteins (P<0.05). No significant differences were found between the acupuncture and moxibustion groups in deceasing all the aforementioned indexes (P>0.05)．. Acupuncture, moxibustion and warm acupuncture can reduce arthritic injury in KOA rats, which is closely associated with their effects in down-regulating the expression of chondrocyte cytoskeletal proteins ROCK, phospho-Cofilin and phospho-LIMK1. The efficacy of warm acupuncture is evidently superior to that of simple acupuncture and simple moxibustion.

Thirty days after anterior cruciate ligament transection is sufficient to induce signs of knee osteoarthritis in rats: pain, functional impairment, and synovial inflammation.

To compare the unilateral signs of knee osteoarthritis (KOA) 30 and 60days after anterior cruciate ligament transection (ACLT). Pain, gait function, synovial fluid inflammation, and histopathological changes in the synovial membrane were analyzed, as well as the interaction between the variables. Male Wistar rats (n = 32; 219.2 ± 18.6g) were randomly distributed into four groups of eight animals each. Two groups were submitted to unilateral ACLT surgery to induce KOA and analyzed after 30 (KOA30) and 60days (KOA60). Two control groups (without surgery) were also assessed after the same time periods (C30 and C60). All the groups were evaluated before ACLT from the least to most stressful tests (skin temperature, mechanical response threshold, gait test, thermal response threshold, and joint swelling), as well as 30 and 60days after surgery. After euthanasia, the synovial fluid and synovial membrane were collected. Thirty days after ACLT, KOA30 showed decrease paw print area and mechanical response threshold, higher joint swelling, skin temperature, leukocyte count, cytokine levels, and synovitis score. No differences were found between KOA30 and KOA60. Our data showed that 30days after ACLT is sufficient to induce signs of KOA in rats, such as pain, functional impairment, and synovial inflammation, suggesting that a shorter time period can be used as an experimental model.

Interventional effects of the direct application of \u201cSanse powder\u201d on knee osteoarthritis in rats as determined from lipidomics via UPLC-Q-Exactive Orbitrap MS

BackgroundOur previous clinical evidence suggested that the direct application of “Sanse powder” the main ingredient of “Yiceng” might represent an alternative treatment for knee osteoarthritis. However, the mechanism underlying its effect is poorly understood. In this study, we investigated the mechanism of the effect of direct “Sanse powder” application for the treatment of knee osteoarthritis (KOA) in rats by using lipidomics.MethodsKOA rats were established by cutting the anterior cruciate ligament, and the cold pain threshold and mechanical withdrawal threshold (MWT) of seven rats from each group were measured before modelling (0 days) and at 7, 14, 21 and 28 days after modelling. Histopathological evaluation of the synovial tissue was performed by haematoxylin and eosin (H&E) staining after modelling for 28 days. Interleukin-1β (IL-1β), pro-interleukin-1β (pro-IL-1β) and tumor necrosis factor-α (TNF-α) proteins in synovial tissue were measured by western blot, and the mRNA expression levels of IL-1β and TNF-α in synovial tissue were measured using Real-time reverse transcription polymerase chain reaction (qRT-PCR), the levels of IL-1β and TNF-α in rat serum were measured by enzyme-linked immunosorbent assay (ELISA), Serum lipid profiles were obtained by using ultra-performance liquid chromatography combined with quadrupole-Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap MS).ResultsThe results confirmed that the direct application of “Sanse powder” had a significant protective effect against KOA in rats. Treatment with “Sanse powder” not only attenuated synovial tissue inflammation but also increased the levels of the cold pain threshold and MWT. In addition, the lipidomics results showed that the levels of diacylglycerol (DAG), triacylglycerols (TAGs), lysophosphatidylcholine (LPC), phosphatidylcholine (PC), fatty acid esters of hydroxy fatty acids (FAHFAs), and phosphatidylethanolamine (PE) were restored almost to control levels following treatment.ConclusionsLipidomics provides a better understanding of the actions of direct application “Sanse powder” therapy for KOA.