Rat Cartilage Research Articles

Ozone induces autophagy by activating PPARγ/mTOR in rat chondrocytes treated with IL-1β

BackgroundOsteoarthritis (OA) is the main cause of older pain and disability. Intra-articular injections of ozone (O3) commonly have been found to have antioxidative and anti-inflammatory effects to reduce pain and improve function in knee osteoarthritis. It has been reported that reduced autophagy in chondrocytes plays an important role in the development of OA. This study aimed to probe the role of O3 on the autophagy in chondrocytes treated with IL-1β.MethodsPrimary chondrocytes were isolated from Wistar rats cartilage within 3 days. The OA chondrocytes model was induced via treatment with IL-1β for 24 h. Then the cells were treated with O3 and GW9662, the inhibitor of PPARγ. Cell viability was assessed by CCK-8. Further, the cells subjected to Western blot analysis, qRT-PCR and immunofluorescence assay. The numbers of autophagosomes were observed via transmission electron microscopy.Results30 μg/ml O3 improved the viability of chondrocytes treated with IL-1β. The decreased level of autophagy proteins and the numbers of autophagosomes improved in IL-1β-treated chondrocytes with O3 via activating PPARγ/mTOR. In addition, the qRT-PCR results showed that O3 decreased the levels of IL-6, TNF-α and MMP-3, MMP-13 in chondrocytes treated with IL-1β.Conclusions30 μg/ml O3 improved autophagy via activating PPARγ/mTOR signaling and suppressing inflammation in chondrocytes treated with IL-1β.

The presence and degradation of nerve fibers in articular cartilage of neonatal rats

PurposeTo investigate the presence and change of nerve fibers and neuropeptide during early development of articular cartilage in neonatal rats.MethodsArticular cartilage in distal-femoral epiphyses was collected from neonatal Sprague Dawley rats, which were 1-day, 5-day, and 10-day postnatal (P1, P5 and P10). Microscopy, immunofluorescence, transmission and scanning electron microscopy (TEM and SEM) were performed for detection of nerve fibers. Quantitative analysis for substance P (SP) and neuropeptide Y (NPY) was conducted using immunofluorescence and enzyme-linked immunosorbent assay (ELISA).ResultsTEM showed the existence of myelinated nerve fibers in the extracellular matrix of articular cartilage in both P1, P5 and P10 rats, and they formed synaptic contacts with chondrocytes. During this time, chondrocytes proceeded with their development, and the nerve fibers gradually degraded. The ELISA results showed significant increase of the sensory neuropeptide SP and the sympathetic neuropeptide NPY in the cartilage tissue. Immunofluorescence results showed the distribution of SP and NPY in the perichondrium, the cartilage canals, the plasma of chondrocytes, and extracellular matrix in the cartilage tissue.ConclusionsNerve fibers exist in the matrix of articular cartilage during early development of knee joints in neonatal rats. Nerve fibers form synaptic contacts with chondrocytes at the early stage and then degrade gradually in the course of chondrocyte development. SP and NPY significantly increase in articular cartilage during this very period. These results indicate that the nerve fibers and the neuropeptide they secrete may exert important effect on the development of articular cartilage.

Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway.

HMGB1 is a highly conserved nuclear protein that is rapidly released into the extracellular environment during infection or tissue damage. In osteoarthritis, HMGB1 acts as a pro-inflammatory cytokine inducing a positive feedback loop for synovial inflammation and cartilage degradation. The aim of this study was to explore the role of HMGB1 in inflammation and catabolism of temporomandibular joint osteoarthritis (TMJOA) and whether inhibition of HMGB1 affects TMJOA. Human synovial fibroblasts were incubated with HMGB1, the expression of pro-inflammatory cytokines and catabolic mediators were measured by Western blot and ELISA. NF-κB signaling pathway involvement was studied by the NF-κB inhibitor and detected by Western blotting and immunofluorescence staining. TMJOA was induced by an injection of complete Freund’s adjuvant (CFA) into anterosuperior compartment of rat’s joint. An anti-HMGB1 antibody was used to assess the effect to HMGB1 in the synovium and cartilage of the CFA-induced TMJOA rats by hematoxylin and eosin, Safranin O, Masson trichrome staining, immunohistochemistry and immunofluorescence. HMGB1 markedly increased the production of MMP13, ADAMTS5, IL-1β and IL-6 through activating NF-κB signaling pathway in human synovial fibroblasts. In vivo, application of the HMGB1 neutralizing antibody effectively ameliorated the detrimental extent of TMJOA. Furthermore, the HMGB1 neutralizing antibody reduced the expression of NF-κB, pro-inflammatory cytokines and catabolic mediators in the synovium and cartilage of CFA-induced TMJOA rats. HMGB1 inhibition alleviates TMJOA by reducing synovial inflammation and cartilage catabolism possibly through suppressing the NF-κB signaling pathway and may become a therapeutic method against TMJOA.

Effect of Buyi Pishen acupuncture on inflammatory factor and cartilage matrix in adjuvant arthritis rats

To observe the effect of Buyi Pishen acupuncture (acupuncture for invigorating spleen and kidney) on inflammatory factor and synovial cartilage matrix in adjuvant arthritis (AA) rats, and to explore the mechanism of acupuncture for rheumatoid arthritis (RA). A total of 60 clean male Wistar rats were randomized into a normal group, a model group, a tripterygium wilfordii polyglycoside tablet (TWP) group and an acupuncture group, 15 rats in each group. Rats in the model group, the TWP group and the acupuncture group received intradermal injection of Freund's complete adjuvant (FCA) at right hind foot pad to induce the AA model. TWP suspension of 8 mg/kg was given by gavage in the TWP group. Acupuncture was applied at "Shenshu" (BL 23), "Pishu" (BL 20) and right "Housanli" (ST 36), "Sanyinjiao" (SP 6), "Yanglingquan" (GB 34) in the acupuncture group, 15 min a time, once a day. The intervention was given 15 days in both TWP group and acupuncture group. The foot-pad swelling degree before modeling, before and after intervention and the arthritis index (AI) score before and after intervention were calculated; the serum levels of interleukin (IL)-1β, IL-4, IL-10 and tumor necrosis factor-α (TNF-α) were detected by ELISA method; the ultrastructure and histomorphological changes of synovium issue were observed by transmission electron microscope and HE staining; the positive expression of matrix metalloproteinase (MMP)-3 and MMP-9 in synovium issue was detected by immunohistochemistry method. Before intervention, foot-pad swelling degree of the model group, the TWP group and the acupuncture group was increased compared with the normal group (P<0.01). After intervention, foot-pad swelling degree and AI score were increased compared with the normal group (P<0.01), foot-pad swelling degree and AI scores in the TWP group and the acupuncture group were lower than the model group (P<0.05), and those in the acupuncture group were decreased compared with the TWP group (P<0.05). The model group exhibited unclear nuclear membrane of synovial cells, chromatin pyknosis, massive inflammatory cell infiltration and hyperplasia in synovial tissue; the TWP group and the acupuncture group exhibited clear and smooth nuclear membrane of synovial cells, inapparent chromatin pyknosis, less inflammatory cell infiltration and hyperplasia in synovial tissue, the acupuncture group exhibited less matrix destruction as well. Compared with the normal group, serum levels of IL-1β and TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were increased (P<0.01), while serum levels of IL-4 and IL-10 were decreased (P<0.01) in the model group. Compared with the model group, serum levels of IL-1β and TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were decreased (P<0.05, P<0.01), while serum levels of IL-4 and IL-10 were increased (P<0.05) in the TWP group and the acupuncture group; compared with the TWP group, serum level of TNF-α and positive expression of MMP-3 and MMP-9 in synovium issue were decreased (P<0.05), while serum levels of IL-4 and IL-10 were increased (P<0.05) in the acupuncture group. Buyi Pishen acupuncture can effectively improve the injury of articular cartilage in AA rats, its mechanism maybe related to reducing the inflammatory reaction in synovium and inhibiting the degradation of articular cartilage matrix.

A musculoskeletal finite element model of rat knee joint for evaluating cartilage biomechanics during gait.

Abnormal loading of the knee due to injuries or obesity is thought to contribute to the development of osteoarthritis (OA). Small animal models have been used for studying OA progression mechanisms. However, numerical models to study cartilage responses under dynamic loading in preclinical animal models have not been developed. Here we present a musculoskeletal finite element model of a rat knee joint to evaluate cartilage biomechanical responses during a gait cycle. The rat knee joint geometries were obtained from a 3-D MRI dataset and the boundary conditions regarding loading in the joint were extracted from a musculoskeletal model of the rat hindlimb. The fibril-reinforced poroelastic (FRPE) properties of the rat cartilage were derived from data of mechanical indentation tests. Our numerical results showed the relevance of simulating anatomical and locomotion characteristics in the rat knee joint for estimating tissue responses such as contact pressures, stresses, strains, and fluid pressures. We found that the contact pressure and maximum principal strain were virtually constant in the medial compartment whereas they showed the highest values at the beginning of the gait cycle in the lateral compartment. Furthermore, we found that the maximum principal stress increased during the stance phase of gait, with the greatest values at midstance. We anticipate that our approach serves as a first step towards investigating the effects of gait abnormalities on the adaptation and degeneration of rat knee joint tissues and could be used to evaluate biomechanically-driven mechanisms of the progression of OA as a consequence of joint injury or obesity.

The Level of Histone Deacetylase 4 is Associated with Aging Cartilage Degeneration and Chondrocyte Hypertrophy.

PurposeTo determine the role of histone deacetylase 4 (HDAC4)-controlled chondrocyte hypertrophy in the onset and development of age-related osteoarthritis (OA).MethodsMorphological analysis of human knee cartilages was performed to observe structural changes during cartilage degeneration. HDAC4 expression was deleted in adult aggrecan (Acan)-CreERT2; HDAC4fl/fl transgenic mice. The onset and development of age-related OA were investigated in transgenic and control mice using hematoxylin and eosin (H&E) and Safranin O staining. Furthermore, the progression of ACLT-induced OA following adenovirus-mediated HDAC4 overexpression was explored in rats. The expression levels of genes related to hypertrophy, cartilage matrix and its digestion, and chondrocyte proliferation were investigated using qPCR. Immunohistochemistry (IHC) was used to explore the mechanisms underlying HDAC4-controlled age-related changes in OA progression.ResultsIn human cartilage, we performed morphological analysis and IHC, the results showed that hypertrophy-related structural changes are related to HDAC4 expression. Age-related OA was detected early (OARSI scores 2.7 at 8-month-old) following HDAC4 deletion in 2-month-old mice. Furthermore, qPCR and IHC results showed changes in hypertrophy-related genes Col10a1, Runx2 and Sox9 in chondrocytes, particularly in the expression of Runt-related transcription factor 2 (Runx2, 13.29±0.99 fold). The expression of the main cartilage matrix-related genes Col2a1 and Acan decreased, that of cartilage matrix digestion-related gene MMP-13 increased, while that of chondrocyte proliferation-related genes PTHrP, Ihh and Gli1 changed. In contrast, rat cartilage’s qPCR and IHC results showed opposite outcomes after HDAC4 overexpression.ConclusionBased on the results above, we concluded that HDAC4 expression regulates the onset and development of age-related OA by controlling chondrocyte hypertrophy. These results may help in the development of early diagnosis and treatment of age-related OA.

Chsy1 deficiency reduces extracellular matrix productions and aggravates cartilage injury in osteoarthritis.

Osteoarthritis (OA) is a kind of degenerative joint disease marked by the destruction of articular cartilage due to the degeneration of chondrocytes. CHSY1, one of the glycosyltransferases, is involved in the synthesis of chondroitin sulfate. Herein, we found that the expression of Chsy1 was decreased in the knee cartilage of OA rats. In order to investigate the role of CHSY1 in chondrogenesis and OA, we established a Chsy1 stable knockdown cell line in mouse ATDC5 chondrocytes by lentivirus. It was found that Chsy1 deficiency resulted in a reduction of extracellular matrix production in chondrocytes and a promotion of endochondral osteogenesis, which was indicated by the decreased expression of early chondrocytes genes (Col2a1, Sox9), and the increased expression of cartilage hypertrophy genes (Col10a1, Runx2, Mmp13, Mmp3). The expression trend of these genes is considered to be the characteristic of osteoarthritis. In addition, knockdown of Chsy1 could upregulate BMP signaling in differentiated chondrocytes, whereas Chsy1 overexpression had opposite effects. The reduction of extracellular matrix production and the promotion of endochondral osteogenesis by Chsy1 knockdown could be rescued by BMP signaling inhibitor LDN193189. Furthermore, the abnormally enhanced BMP signaling and the high expression of OA biomarker Mmp3 in primary cells of OA rats could be rescued by either LDN193189 or Chsy1 overexpression. These results implicate a role for Chsy1 in regulating extracellular matrix production and endochondral osteogenesis through BMP signaling; and a lack of Chsy1 could aggravate the cartilage damage of osteoarthritis.

Piezo1 Affects Temporomandibular Joint Osteoarthritis by Influencing pSmad3.

Objective: The aim of this research was to study the expression of Piezo1 in a rat temporomandibular joint osteoarthritis animal model and to explore its mechanism for inducing inflammatory changes. Methods: A total of 24male SD rats aged approximately 8 weeks were randomly divided into three groups: the blank control group, complete Freund’s adjuvant group (CFA), and CFA + inhibitor (GsMTx4) group. After 3 weeks, the condylar heads of the rats were evaluated by micro-CT, HE, immunohistochemistry, safranin O staining, and other experimental techniques. Protein was extracted from the subchondral bone, and the changes in Piezo1, Smad3, and pSmad3 levels in each group were detected by Western blotting. p < 0.05 was considered to indicate statistical significance. Results: The degree of damage to the cartilage and subchondral bone in the Piezo1 inhibitor group was smaller than that in the CFA group. The expression level of Piezo1 in the CFA group was higher than that in the other groups, and the difference was statistically significant. The expression of pSmad3 in the CFA group was also higher than that in the other groups (p < 0.05). Conclusion: Piezo1 is expressed in the condylar cartilage and subchondral bone of rats, and the degree of condylar destruction can be improved by influencing the pSmad3 expression.

Inhibitory effect of artesunate on bone destruction in rheumatoid arthritis: an exploration based on AhR/ARNT/NQO1 signaling pathway

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 μmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 μmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.

Effects of swimming exercise and RLN2 induction on relaxin levels, MMP-13, TGFβ expression in the pubic cartilage of pregnant rats

Context: Swimming exercises can be used as an intervention to maintain the stability of the synthesis of the pubic symphysis cartilage. Aims: To evaluate the effects of swimming exercise on relaxin levels, MMP-13, TGFβ expression in pubic cartilage at pregnant rats with induction of RLN2 gene. Methods: Thirty-six female Wistar rats (six in each group) were impregnated with 24 male rats (1:1). Three groups had swimming exercises for 60 min per day, and the other groups were treated as the control group. Two groups were given subcutaneous RLN2 gene. Relaxin plasma was collected from the sinus retro-orbital and performed twice on the eighth day and the 20th of pregnancy. The pubic symphysis of the rats was collected on the 20th day of pregnancy, and the decalcification process was carried out before making histological preparations. Results: There was an increase in the amount of relaxin in the relaxin-induced group after 24 h induction (p=0.000). Relaxin levels of pregnant rats on the 20th day correlated with the expression of MMP-13, p=0.029; R2=0.447. In the treatment group, relaxin levels and MMP-13 expression were lower than the non-treatment group, p=0.005 and p=0.000, respectively. In contrast, there was a higher expression of TGFβ in the treatment group, p=0.000. Conclusions: Relaxin plasma levels have a significant correlation with the expression of MMP-13, and swimming exercise increases the expression of TGFβ and decreases relaxin levels and MMP-13 expression in the pubic cartilage.

Warm needling reduces oxidative damage and inflammation of cartilage in knee osteoarthritis rats

To observe the effect of warm needling on the expression of oxidative stress related factors and pro-inflammatory factors in cartilage of mono sodium iodoacetate (MIA) induced knee osteoarthritis (KOA) model rats, so as to explore its mechanisms underlying improvement of KOA. Sixty male Sprague Dawley rats were randomly divided into 5 groups: control, model, acupuncture, moxibustion,warm needling, with 12 rats in each group. Rats of the acupuncture, moxibustion,warm needling groups received manual acupuncture or moxibustion or both stimulation of "Zusanli" (ST36) for 15 minutes, once a day for 21 days beginning from the third day after modeling. The foot volume was measured by drainage method, and the plantar mechanical contraction reflex threshold (mechanical pain threshold, MPT) measured by using an electronic pain meter. After 21 days of treatment, the histopathological changes of knee joint were observed by HE staining, and Mankin score was calculated to evaluate the degree of cartilage destruction. The malondialdehyde (MDA) level was measured by colorimetry, and immunofluorescence staining was used to observe the expression of NOX2, SOD2 or IL-1β. Compared with the control group, the knee joint swelling volume from the 3rd day after modeling, Mankin score, MDA level, and the number of NOX2 and IL-1β positive cells were significantly increased (P<0.01, P<0.05), while the MPT from the 3rd day after modeling, and the number of SOD2 positive cells were considerably decreased (P<0.01) in the model group. After the interventions, the increased levels of the knee joint swelling volume from the 12th day after modeling, and the Mankin score, MDA level, NOX2 and IL-1β positive cells, and the levels of decreased MPT from the 9th day after modeling and SOD2 positive cell number were reversed in the acupuncture, moxibustion,warm needling groups (P<0.05, P<0.01), and the effects of warm needling were significantly superior to those of simple manual acupuncture and simple moxibustion in down-regulating knee joint volume, Mainkin score, MDA le-vel, and NOX2 and IL-1β positive cells, and in up-regulating MPT from the 12th day after modeling, and the number of SOD2 positive cells (P<0.05). No significant differences were found between the acupuncture and moxibustion groups in the levels of all the indexes mentioned above (P>0.05). HE staining showed rough and damaged articular surface, with subchondral neovascularization and moderate connective tissue hyperplasia, and abundant lymphocyte and monocyte infiltration in the model group, which was milder in the acupuncture, moxibustion groups particularly in the warm needling group after 21 days' interventions. Warm needling can relieve knee joint pain, swelling and inflammatory damage in KOA rats, which may be associated with its function in inhibiting oxidative stress and inflammation in the cartilage of KOA. The therapeutic effect of warm needling is better than that of manual acupuncture and moxibustion alone.

Folic acid-modified lysozyme protected gold nanoclusters as an effective anti-inflammatory drug for rapid relief of gout flares in hyperuricemic rats

BackgroundGout normally occurs when excess urate crystals accumulate in the joints that induce inflammation. The inflammations further result in gout flares, causing serious pain. Some anti-inflammatory drugs can relieve pain, but they have various side effects. Nanomedicines can reduce side effects but are mostly based on the delivery of additional organic drugs, which adds complexity and may cause other health problems. We developed folic acid (FA)-modified lysozyme (Lys)-protected gold nanoclusters (AuNCs) (i.e., FLA) by a one-pot method, and successfully used FLA as an effective anti-inflammatory drug for relief of gout flares in hyperuricemic rats. MethodsThe morphological changes of the cells were studied by Transmission electron microscopy (TEM) and fluorescence microscopy. Western blot was conducted to understand the inflammation factors. AutoDock Vina was used for molecular docking of FLA. ResultsFLA reduced the inflammation, oxidative stress level in vitro. By injecting FLA into gout rat models, inflammations, cartilage destructions and pain during gout flares were also significantly relieved. ConclusionCompared with complicated nano-delivery systems, FLA directly exhibits exciting therapeutic efficiency against gout flares, which will have considerable prospects in the clinic.

Effect of manipulation on cartilage in rats with knee osteoarthritis based on the Rho-associated protein kinase/LIM kinase 1/Cofilin signaling pathways.

To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase (ROCK)/LIM-kinase1 (LIMK1)/Cofilin signaling pathway. Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups ( = 8 each): blank group, model group, manipulation group, celecoxib group, and manipulation combined with celecoxib group (MC group). The osteoarthritis model was established by injecting 0.2 mL 4% papain into the articular disc of the rats. After successfully establishing the model, we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan (EX-LE4), Waixiyan (EX-LE5), Xuehai (SP10), Liangqiu (ST34), and Zusanli (ST36) acupoints for 10 min each time. Also, the celecoxib group was gavaged with 24 mg•kg•d celecoxib, while the MC group was treated using both of these two methods. After four weeks, the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue. The expressions of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in serum were observed using the enzyme-linked immunosorbent assay. Besides, we detected the expressions of ROCK, LIMK1, Phospho-LIM-kinase1 (Phospho-LIMK1), Cofilin, and Phospho-Cofilin by Western blot. Compared to the model group, the manipulation group, celecoxib group, and MC group all exhibited superior results concerning pathological morphologic changes of cartilage, as observed by hematoxylin-eosin staining and calculated using the Mankin score. Besides, in contrast to the blank group, the model group exhibited elevated serum levels of IL-1β and TNF-α ( 0.01), while the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage were all higher ( 0.01). Also, the serum levels of IL-1β and TNF-α in each treatment group were lower (0.01) than in the model group. Moreover, there were lower expressions of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the manipulation group and the MC group (< 0.01). Compared with the model group, the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different ( > 0.05). In this study, we established that manipulation has a better curative effect than celecoxib. Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.

Effects of transcutaneous auricular vagus nerve stimulation on bone and cartilage destruction in rats with rheumatoid arthritis

To observe the alleviating effect of transcutaneous auricular vagus nerve stimulation (taVNS) on articular cartilage and bone destruction in rats with collagen-induced arthritis (CIA), and explore the cellular and molecular mechanisms of taVNS against rheumatoid arthritis (RA). The male SD rats were randomly divided into normal control group (n=12), model group (n=12), and taVNS group (n=12). The CIA rat model was established by multi-point injection of emulsion prepared from type Ⅱ bovine collagen and Freund's incomplete adjuvant into the root of rat tail. The rats in the taVNS group were treated with taVNS at bilateral auricular conchae, 30 min per time, once a day, for consecutive 28 d. The cartilage destruction of the ankle joint was observed by safranin O-fast green staining, the production of osteoclasts in the joint tissue by tartrate-resistant acid phosphatase (TRAP) staining, and the bone erosion by X-ray and Micro-CT imaging. The protein expression levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG) in the synovial tissues were detected by Western blot. Compared with the normal control group, the CIA rats presented with typical RA symptoms and elevated arthritis index (AI,P<0.05). After intervention with taVNS, the AI remarkably declined in comparison with that in the model group (P<0.05). Compared with the control group, the model group displayed loss of cartilage matrix in the ankle joint, thinned cartilage layer, obvious cartilage damage, and increased number of osteo-clasts in the joint (P<0.01); the imaging results showed bone loss and three-dimensional structural destruction of ankle joint and aggravated bone erosion (P<0.01); the expression levels of MMP-1, MMP-3 and MMP-13, and RANKL/OPG ratio were significantly elevated in the synovial tissue of ankle joint (P<0.01, P<0.05), while the expression level of OPG was decreased (P<0.05). Compared with the model group, taVNS resulted in relatively intact cartilage layer of ankle joint, alleviated cartilage destruction, decreased number of osteoclasts (P<0.01), improved bone erosion, loss, and three-dimensional structural destruction (P<0.01), and diminished MMP-1, MMP-3, and MMP-13 expression and RANKL/OPG ratio in the synovial tissue of ankle joint (P<0.05, P<0.01), while the expression level of OPG was increased (P<0.05). taVNS effectively relieves bone and cartilage destruction in CIA rats, which might be related to its efficacy in reducing the production of osteoclasts in joint tissues and down-regulating the expression levels of MMP-1, MMP-3 and MMP-13, and RANKL/OPG ratio.

The RNA-binding protein SND1 promotes the degradation of GPX4 by destabilizing the HSPA5 mRNA and suppressing HSPA5 expression, promoting ferroptosis in osteoarthritis chondrocytes.

Heat shock protein family A member 5 (HSPA5), a recently identified suppressor of ferroptosis, was reported to potentially regulating osteoarthritis. However, the exact role of HSPA5 and how its expression was regulated in osteoarthritis are largely unclear. Rat primary chondrocytes were treated with 10ng/mL IL-1β for 24h and incubated with ferrostatin-1 (a ferroptosis inhibitor). Cell viability, production of TNF-α, ROS and MDA, expression levels of collagen II, MMP13, GPX4, and SND1, and Fe2+ concentration were detected. Gain- and loss-of-function manipulations were performed to investigate the effect of HSPA5 on chondrocyte functions, and SND1 shRNA (sh-SND1) was transfected into IL-1β-treated primary chondrocytes alone or together with sh-HSPA5. Furthermore, the interaction between HSPA5 and GPX4 and the regulation of HSPA5 on GPX4 were explored. Finally, SND1 was knocked down in the rats with osteoarthritis, and the histopathology, expression of HSPA5-GPX4 axis, and levels of oxidative stress markers were evaluated. IL-1β treatment could enhance extracellular matrix (ECM) degradation (collagen II reduced and MMP13 increased), promote ferroptosis, manifested by decreased cell viability, increased levels of TNF-α, ROS, MDA, and Fe2+ concentrations, and decreased level of GPX4 protein, and increase SND1 expression in chondrocytes, which could be reversed by ferrostatin-1. Knockdown of SND1 enhanced ECM degradation and suppressed ferroptosis IL-1β-treated chondrocytes, which could be eliminated by knockdown of HSPA5. SND1 bound with HSPA5 at the 3'UTR and destabilized the HSPA5 mRNA. HSPA5 protein directly bound with GPX4 protein and positively regulate its expression. HSPA5 overexpression suppressed IL-1β-induced chondrocyte ferroptosis, while this effect was counteracted by GPX4 silencing. Knockdown of SND1 upregulated HSPA5 and GPX4 in rat cartilage, inhibited inflammatory damage and ferroptosis, and alleviated OA progression. The RNA-binding protein SND1 promotes the degradation of GPX4 by destabilizing the HSPA5 mRNA and suppressing HSPA5 expression, promoting ferroptosis in osteoarthritis chondrocytes.

The Expression of MDM2 Gene Promoted Chondrocyte Proliferation in Rats with Osteoarthritis via the Wnt/β-Catenin Pathway.

This study aimed to investigate the regulatory mechanism of MDM2 gene expression on cartilage cell proliferation in Osteoarthritis (OA) rats. For this purpose, 22 SD rats were randomly divided into normal control (10 cases) and treated (12 cases) groups. Treated group was used for OA modelling with the modified Hulth method. After a week, RT-PCR was used to detect MDM2 in cartilage tissue of rats, Wnt 1, Wnt 3 a, Wnt 10 b and β-catenin genes mRNA expression. Rat chondrocytes were isolated and cultured, and the recombinant eukaryotic expression vector pcDNA3.1 myc-siRNA-MDM2-β-catenin and co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-β-catenin was used to transfect chondrocytes and the proliferation and related gene expression levels of the transfected chondrocytes were detected by MTT method and RT-PCR. The results showed that compared with the control group, MDM2, Wnt 1, Wnt 3 a, Wnt 10b and β-catenin genes in OA rat cartilage constructed by Hulth method were increased (p<0.05). The pcDNA3.1 myc-beta-catenin transfection slowed down the proliferation of OA chondrocytes, different from the non-transfected OA group (p<0.001), and increased Wnt 1, Wnt 3a, Wnt 10b and β-catenin genes expression compared with the Control group (p<0.05), but did not affect the expression of MDM2. The transfection of siRNA-MDM2 was opposite to pcDNA3.1 myc-β-catenin. The co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-beta-catenin transfection did not affect the proliferation of OA chondrocytes. In general, the high expression of MDM2 in OA rats restricts the proliferation of chondrocytes, which may be related to the main pathogenesis of the occurrence and development of OA in vivo, and the regulation of MDM2 on the proliferation of chondrocytes may be achieved through the Wnt/ β-catenin pathway.

Smart µ‐Fiber Hydrogels with Macro‐Porous Structure for Sequentially Promoting Multiple Phases of Articular Cartilage Regeneration

AbstractHydrogels are widely utilized for articular cartilage regeneration. However, traditional hydrogels with nano‐sized pores lead to poor cell and tissue infiltration. Furthermore, hydrogels that can stimulate each phase of cartilage regeneration to enhance cartilage repair are highly demanded. Herein, a macro‐porous hydrogel is fabricated by crosslinking mixed sodium alginate (SA) and hyaluronic acid (HA) solution into SA/HA micro‐fibers (µ‐fibers) first and subsequently crosslinking the µ‐fibers into hydrogel blocks. Besides, exosomes derived from NR8383 cells activated by lipopolysaccharides (LPS) and stimulated with bioglass (BG) ion extracts (LPS/BG‐exo) are incorporated into the hydrogel as they exhibit a strong ability to regulate inflammation homeostasis and recruit bone marrow mesenchymal stem cells (BMMSCs). Meanwhile, poly (lactic‐co‐glycolic acid) (PLGA) microspheres containing kartogenin (KGN) (PLGAKGN) are encapsulated within the hydrogel for inducing the chondrogenic differentiation of recruited BMMSCs when KGN is released after the LPS/BG‐exo. Thus, the obtained macro‐porous SA/HAexo‐PLGAKGN hydrogel can not only significantly improve the infiltration of tissues into the hydrogel but also sequentially deliver LPS/BG‐exo and KGN to enhance the cartilage regeneration in a rat cartilage defect model. Prospectively speaking, the SA/HAexo‐PLGAKGN hydrogel is a potential candidate for other tissue regeneration as the bioactive substances can be adjusted according to different purposes.

Pulsed electromagnetic field alleviates synovitis and inhibits the NLRP3/Caspase-1/GSDMD signaling pathway in osteoarthritis rats

ABSTRACT Low-grade inflammation is a key mediator of the pathogenesis of Osteoarthritis (OA). Pulsed electromagnetic field (PEMF) can improve the symptoms of OA and potentially acts as an anti-inflammatory. The aim of this study was to investigate the effect of the PEMF on OA and its relationship with the NLRP3/Caspase-1/GSDMD signaling pathway.18 Three-month-old Sprague-Dawley (SD) rats were randomly divided into three groups (n = 6 per group): 1) OA group, 2) OA+PEMF group (OA with PEMF exposure), 3) Control group (sham operation with placebo PEMF). Rats in the OA and OA+PEMF groups were subjected to bilateral anterior cruciate ligament transection and ovariectomy. PEMF scheme: Pulse waveform, 3.82 mT, 8 Hz, 40 min/day, 5 days a week, for 12 weeks. The expression levels of NLRP3, Caspase-1, GSDMD, IL-1β, and MMP-13 were detected by qRT-PCR and Western blot. The pathological structures of OA were monitored with Safranin O/fast green staining and hematoxylin eosin staining. Our results showed that PEMF alleviated the degree of inflammation and degeneration of cartilage in rats with OA, based on the histopathological changes and decline of the expression of IL-1β and MMP-13. Moreover, the over-expression of NLRP3, Caspase-1, and GSDMD in the cartilage of the OA rats decreased after PEMF treatment. These results suggested that PEMF could be a highly promising noninvasive strategy to slow down the progression of OA and inhibition of the NLRP3/Caspase-1/GSDMD signaling pathway might be involved in the beneficial effect of PEMF.

Cangxitongbi capsules protect the articular cartilage in the rat knee through the long non-coding RNA HOTAIR/p38MAPK pathway.

BackgroundKnee osteoarthritis (KOA) is a leading cause of chronic pain and disability, and as such, it poses a significant economic burden. Traditional Chinese medicine (TCM), as well as complementary and alternative medicine, can offer safe and effective treatments for KOA. Cangxitongbi (CXTB) capsule is a Chinese patented medicine for KOA treatment and has a remarkable curative effect. This article evaluated the effects and mechanisms of CXTB in protecting joint cartilage in vivo.MethodsThe KOA model was constructed in rats using the modified Hulth method. CXTB (35 mg/kg) was administered intragastrically for 4 weeks. Hematoxylin and eosin (HE) staining of the knee articular were performed to evaluate the efficiency of CXTB. Western blot analysis, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA) were used to investigate the protective mechanisms of CXTB in joint cartilage.ResultsCXTB effectively improved the morphological structure of the cartilage and bone in the knee joint by enhancing autophagy and regulating the expression of related protease and inflammatory factors. Furthermore, CXTB downregulated the expression of the long non-coding RNA (lncRNA) Hox transcript antisense intergenic RNA (HOTAIR) and inhibited the activation of the p38MAPK pathway. Conversely, overexpression of lncRNA HOTAIR suppressed the protective effects of CXTB on the knee joint.ConclusionsCXTB capsules can protect the knee articular cartilage in rats through the lncRNA HOTAIR/p38MAPK pathway.

Repair impact of vibration exercise with different frequencies on articular cartilage of rats with early knee osteoarthritis and its JNK/NF-κB, SOX9 mechanisms

Objective: To investigate the repair effect and JNK/NF-κB,SOX9 mechanisms of vibration exercise with different frequencies on articular cartilage in rats with early knee osteoarthritis. Methods: Forty-eight adult male SD rats were randomly divided into six groups(n=8):model control group(MC),high frequency vibration group 1 (GP1,60 Hz),high frequency vibration 2 group (GP2,40 Hz),medium frequency vibration group (ZP,20 Hz),minor frequency group(DP,10 Hz)and normal control group(NC). Except for NC group,the rats in each group were made into early knee osteoarthritis model after six weeks of knee joint cavity injection of papain solution and 2% mixture l-cysteine on the 1st,4 th and 7th day. Each exercise group was subjected vibration to 40 minutes a day with amplitude of 2～5 mm and 5 days a week. Four weeks later, the articular cartilage of the lateral femoral condyle of the both back leg knee joints were detected by HE staining,serine O staining and Mankin scores for morphological observation. The expression levels of JNK,NF-κB p65 and Sox9 mRNA in articular cartilage of the medial femoral condyle were detected by RT-qPCR,and the protein expressions of JNK,NF-κB p65 and Sox9 were detected by Western blot. Results: Compared with the NC group,the Mankin score in other groups was significantly higher (P＜0.01). Compared with the MC group,the Mankin score of each vibration group was significantly lower(P＜0.05),the mRNA and protein expressions of JNK and NF-κB p65 in each vibration training group were significantly lower (P＜0.01),the expressions of Sox9 mRNA and protein in vibration training group were increased significantly (P＜0.01). Compared with the higher frequency group,the Mankin score,the mRNA and protein expressions of JNK and NF-κB p65 of lower frequency group were significantly lower (P＜0.05 or P＜0.01). But the expressions of Sox9 mRNA and protein were significantly higher (P＜ 0.05 or P＜0.01). Conclusion: Vibration exercise of different frequencies may present varying degrees of cartilage repair impact in rats with early knee osteoarthritis,and the cartilage repair by low-frequency vibration training is better than that by high-frequency vibration. This can be one of the mechanisms on controlling collagen synthesis by down-regulating JNK/NF-κB expression and increasing SOX9 activity of OA articular cartilage.