Synovial Tissue Of Knee Joint Research Articles

Effect of acupuncture on arthritic pathological injury in rats with type Ⅱ collagen-induced rheumatoid arthritis.

To observe the effect of acupuncture stimulation of "Yanglingquan"(GB34), "Zusanli"(ST36) and "Xuanzhong" (GB39) on arthritis index (AI), joint synovial membrane pathology, serum-related immunoinflammatory factors, and expressions of tumor suppressor gene mt-p53, nuclear factor kappa B (NF-κB) and peroxisome proliferator activated receptor gamma (PPARγ) in knee joint synovial tissue of rats with type Ⅱ collagen-induced arthritis (CIA), so as to explore its possible mechanisms underlying improvement of rheumatoid arthritis (RA). Male SD rats were used in the present study. The CIA model was established by subcutaneous injection of collagen emulsion (200 μL/rat) in the tail root region on the first day and repeat (100 μL/rat) once on the 9th day. Eighteen successful CIA rats were randomized into model, medication and acupuncture groups, with 6 rats in each group. Other 6 normal rats were used as the normal control group. For rats of the medication group, leflunomide (1.9 mg/kg) was administrated by gavage, once a day, and for rats of the acupuncture group, manual acupuncture stimulation was applied to bilateral GB34, ST36, GB39 for 30 min, once a day, for 12 weeks. The arthritis index (AI) score (0-4 points) was evaluated once every week. The contents of IL-6, IL-17 and TNF-α in the serum were determined by ELISA. Histopathological changes of the ankle joint were observed by H.E. staining. The protein and mRNA expression levels of mt-p53, NF-κB p65, and PPARγ in the knee joint synovial tissue were determined by Western blot and quantitative real time PCR, separately. Compared with the normal control group, the AI scores at different time-points after modeling, contents of serum TNF-α, IL-6 and IL-17, expression levels of mt-p53, NF-κB p65, PPARγ proteins and mRNAs were significantly increased in the model group (P<0.01, P<0.05). In comparison with the model group, the AI scores at the 10th week in the medication group and at the 3rd, 9th and 10th week in the acupuncture group, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of mt-p53 and NF-κB p65 proteins in both medication and acupuncture groups, as well as mt-p53 and NF-κB p65 mRNAs in the medication group were apparently decreased (P<0.01, P<0.05), while the expression levels of PPARγ protein in both medication and acupuncture group and PPARγ mRNA in the medication group were significantly up-regulated (P<0.05, P<0.01). No significant differences were found between the acupuncture and medication groups in down-regulating the AI score and serum TNF-α, IL-6 and IL-17 contents. The effect of acupuncture was weaker than that of medication in down-regulating the expression of mt-p53 and NF-κB p65 proteins and mRNAs and in up-regulating PPARγ mRNA (P<0.01). H.E. results showed ankle cartilage hyperplasia, reduced joint cavity, mild fibroproliferation and inflammatory cell infiltration in the surrounding soft tissue of the ankle joint in rats of the model group, which was milder in both medication and acupuncture groups. Acupuncture stimulation can improve the degree of joint inflammation and swelling in CIA rats, which may be related to its effects in inhibiting the overexpression of immunoinflammatory factors in serum and regulating expression of mt-p53, NF-κB p65, PPARγ mRNAs and proteins in the synovial tissue.

Identification of CaMK4 as a sex-difference-related gene in knee osteoarthritis.

Osteoarthritis (OA) is a common degenerative joint disease with a higher prevalence in females than in males. Sex may be a key factor affecting the progression of OA. This study aimed to investigate critical sex-difference-related genes in patients with OA and confirm their potential roles in OA regulation. OA datasets GSE12021, GSE55457, and GSE36700 were downloaded from the Gene Expression Omnibus database to screen OA-causing genes that are differentially expressed in the two sexes. Cytoscape was used to construct a protein-protein interaction network and determine hub genes. Synovial tissues of patients (male and female) with OA and female non-OA healthy controls were obtained to confirm the expression of hub genes and screen the key genes among them. Destabilization of the medial meniscus (DMM)-induced OA mice model was established to verify the screened key genes. Hematoxylin and eosin (HE) staining and Safranin O-fast green dye staining were employed to observe synovial inflammation and pathological cartilage status. The abovementioned three datasets were intersected to obtain 99 overlapping differentially expressed genes, of which 77 were upregulated and 22 were downregulated in female patients with OA. The hub genes screened were EGF, AQP4, CDC42, NTRK3, ERBB2, STAT1, and CaMK4. Among them, Ca2+/calmodulin-dependent protein kinase-4 (CaMK4) was identified as a key sex-related gene for OA. It was significantly higher in female OA patients than in the cases of male patients. Moreover, CaMK4 was significantly increased in female patients with OA compared with the female non-OA group. These results suggest that CaMK4 plays an important role in the progression of OA. OA mouse models demonstrated that CaMK4 expression in the mice knee joint synovial tissue elevated after DMM, with aggravated synovial inflammation and significant cartilage damage. Cartilage damage improved after intraperitoneal administration of the CaMK4 inhibitor KN-93. CaMK4 is a key sex-related gene influencing the progression and pathogenesis of OA and may be considered as a new target for OA treatment.

RNA-Seq Reveals the mRNAs, miRNAs, and lncRNAs Expression Profile of Knee Joint Synovial Tissue in Osteoarthritis Patients

Osteoarthritis (OA) is a chronic disease common in the elderly population and imposes significant health and economic burden. Total joint replacement is the only currently available treatment but does not prevent cartilage degeneration. The molecular mechanism of OA, especially the role of inflammation in disease progression, is incompletely understood. We collected knee joint synovial tissue samples of eight OA patients and two patients with popliteal cysts (controls), measured the expression levels of lncRNAs, miRNAs, and mRNAs in these tissues by RNA-seq, and identified differentially expressed genes (DEGs) and key pathways. In the OA group, 343 mRNAs, 270 lncRNAs, and 247 miRNAs were significantly upregulated, and 232 mRNAs, 109 lncRNAs, and 157 miRNAs were significantly downregulated. mRNAs potentially targeted by lncRNAs were predicted. Nineteen overlapped miRNAs were screened based on our sample data and GSE 143514 data. Pathway enrichment and functional annotation analyses showed that the inflammation-related transcripts CHST11, ALDH1A2, TREM1, IL-1β, IL-8, CCL5, LIF, miR-146a-5p, miR-335-5p, lncRNA GAS5, LINC02288, and LOC101928134 were differentially expressed. In this study, inflammation-related DEGs and non-coding RNAs were identified in synovial samples, suggesting that competing endogenous RNAs have a role in OA. TREM1, LIF, miR146-5a, and GAS5 were identified to be OA-related genes and potential regulatory pathways. This research helps elucidate the pathogenesis of OA and identify novel therapeutic targets for this disorder.

Effect of heat-reinforcing needling on expression of serum inflammatory factors and autophagy of knee joint synovial tissue in rheumatoid arthritis rabbits with cold syndrome

To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis (RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA. Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups (n=10 rabbits in each group). The model of RA with cold syndrome was established by Freund's adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at "Zusanli" (ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor (TNF) -α, interleukin (IL)-1β, IL-6 and C-creactive protein (CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1), microtubule-associated protein light chain 3B (LC3B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3 (NLRP3) and nuclear factor-κB (NF-κB). Compared with the normal group, the circumference of the knee joint was increased (P<0.01), the skin temperature was decreased (P<0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1β, IL-6, and CRP were increased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3BⅡ/LC3BⅠof synovial tissue were significantly decreased (P<0.01), the mRNA expressions of NLRP3 and NF-κB were increased (P<0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased (P<0.01), whlie the skin temperature was increased (P<0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1β, IL-6 and CRP were decreased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3B Ⅱ/LC3B Ⅰ were increased (P<0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased (P<0.01) in the heat-reinforcing needling and agonist groups. Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1β, IL-6 and CRP.

Effect of electroacupuncture on pyroptosis of synovial tissues in rats with knee osteoarthritis

To observe the effect of electroacupuncture (EA) on pyroptosis-related proteins in synovium of knee joint in rats with knee osteoarthritis(KOA), in order to explore its mechanism underlying improvement of KOA. Forty male SD rats were randomly divided into control， model, EA and medication groups, with 10 rats in each group. The KOA model was established by injecting 0.2 mL 4% papain solution into the right intra-articular cavity, followed by repeating the injection again on day 4 and 7 after the first injection. After successful modeling, rats of the EA group received EA stimulation of "Neixiyan" (EX-LE4) and "Dubi"(ST35) on the right limb for 15 min, once every day, 6 days per week, for a total of 4 weeks, and those of the medication group received gavage of celecoxib 24 mg/kg, once every day, 6 days per week, for a total of 4 weeks. The severity of dysfunction of the right knee was assessed by using Lequesne's score. Serum interleukin(IL)-1β and IL-18 contents were detected by ELISA. Histopathological changes of the synovium tissue of the right knee joint were observed to give score (synovial pathological score) after H.E. staining. The expression position and intensity of Nod-like receptor pyrin domain 3 (NLRP3) in syno-vial tissue were observed by immunohistochemistry. The expression levels of NLRP3, apoptosis-associated speck-like protein containing card (ASC), Caspase-1, Gasdermin D(GSDMD), IL-1β and IL-18 mRNAs and proteins (including GSDMD-N) in the synovial tissue of the right knee joint were detected by real-time fluorescence quantitative PCR and Western blot, separately. Compared with the control group, the model group had a significant increase in the Lequesne's score, synovial pathological score, serum IL-1β and IL-18 contents, and the expression levels of NLRP3, ASC, Caspase-1, GSDMD, IL-1β, IL-18 mRNAs and proteins and GSDMD-N protein (P<0.01). Whereas relevant to the model group, both the EA and medication groups had marked lower levels of Lequesne's score and synovial pathological score, serum IL-1β and IL-18 contents, and expression levels of NLRP3, ASC, Caspase-1, IL-1β, IL-18 mRNAs and proteins, GSDMD mRNA and GSDMD-N protein (P<0.01, P<0.05). Comparison between two intervention groups showed that the contents of serum IL-1β and IL-18, and the expression levels of IL-1β mRNA and protein were significantly higher in the EA group than in the medication group (P<0.05, P<0.01). No significant differences were found between the EA and medication groups in the Lequesne's score, synovial pathological score, NLRP3, ASC, Caspase-1 and IL-18 mRNAs and proteins, as well as GSDMD mRNA (P>0.05). EA can alleviate the inflammatory response of synovial tissues of knee joints in KOA rats, which may be related to its function in down-regulating the expression levels of synovial NLRP3, ASC, Caspase-1, IL-1β and IL-18 mRNAs and proteins, and GSDMD mRNA and GSDMD-N proteins, reducing the occurrence of pyroptosis.

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.

Effect of electroacupuncture on JAK2/STAT3 pathway in synovial tissues of rats with rheumatoid arthritis

To observe the effect of electroacupuncture (EA) on Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway in knee joint synovial tissues of rats with rheumatoid arthritis (RA) and to explore the action mechanism of EA on RA. Twelve of the 48 SPF male Sprague-Dawley (SD) rats were assigned to a normal group by the random number table method. The remaining 36 rats were subjected to RA model preparation by intradermal injection of the Freund’s complete adjuvant into the right hind foot pad of each rat under sterile conditions. After the model was successfully prepared, rats were then divided into a model group, a drug group and an EA group according to a random number table method (n=12). Rats in the drug group were treated with 2 mL aqueous solution of tripterygium glycosides [8.1 mg/(kg·bw)]; rats in the EA group were treated with EA at bilateral Yanglingquan (GB 34) and Zusanli (ST 36), for 30 min each time; rats in the normal group and the model group were placed in a special rat fixation tank for 30 min each time, and received the same dose of normal saline as those in the drug group. Rats in all groups received intervention once a day for 4 weeks. Diameter of rat ankle joint and rat arthritis index were measured before and after the intervention. At the end of the experiment, the expressions of phospho-JAK2 and phospho-STAT3 were determined by immunohistochemistry. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect JAK2 and STAT3 mRNAs expressions. After the model was produced, the arthritis index 0.01); immunohistochemical staining cells with phospho-JAK2 and phospho-STAT3 were significantly decreased (all P>0.01); the expression levels of JAK2 and STAT3 mRNAs were decreased with statistical differences (all P>0.01). There were no significant differences between the EA group and the drug group (all P<0.05). EA can alleviate the inflammatory response of RA rats, improve their pathological conditions, reduce the expressions of phospho-JAK2 and phospho-STAT3 in the synovial tissue of knee joint, and decrease the expressions of JAK2 and STAT3 mRNAs. The therapeutic effect of EA is comparable to that of the tripterygium glycosides. The mechanism of EA treatment may be related to the inactivation of the JAK2/STAT3 pathway.

Effect of Electroacupuncture on Synovial M 1/M 2 Macrophage Polarization in Rats with Acute Gouty Arthritis

To explore the effect of electroacupuncture (EA) on the expression of synovial AMP-activated protein kinase (AMPK) protein α， arginase-1 mRNA, nitric oxide synthase 2 (NOS 2) mRNA, NOD-like receptor protein 3 (NLRP 3) mRNA, and interleukin-1 β (IL-1 β) mRNA in acute gouty arthritis (AGA) rats, so as to explore its mechanisms underlying improvement of AGA via M 1/M 2 macrophage polarization. Male Wistar rats were randomly divided into normal control, model, medication (colchicine) and EA groups (n＝15 rats in each group). The AGA model was established by injection of sodium urate crystal (MSU) suspension (0.2 mL) into the articular cavity of the left knee. The rats of the normal control group received articular injection of normal saline (0.2 mL) of the left knee, and those of the medication group were treated by gavage of the colchicine (0.3 mg•kg－1•d－1) once daily for 7 days. EA (2 Hz/10 Hz, 1.0 mA) was applied to "Zusanli"(ST 36) and "Sanyinjiao" (SP 6) of the left hind limb for 10 min, once daily for 7 days. The inflammatory conditions of the synovial membrane tissue of the left knee joint were observed by H．E. staining. The expression levels of phosphorylated AMPKα (p-AMPKα) protein, and arginase-1 (a maker of M 2 macrophages) mRNA, NOS 2 (a maker of M 1 macrophages) mRNA, NLRP 3 mRNA, and IL-1 β mRNA in the knee joint synovial tissue were detected by Western blot and quantitative real-time PCR, respectively. Compared with the normal group, the inflammatory cell infiltration of the synovial tissue was more severe, the expression of p-AMPKα protein was significantly decreased (P<0.01), and the expression levels of arginase-1, NOS 2, IL-1 β and NLRP 3 mRNAs were considerably increased in the model group (P<0.01). The expression levels of p-AMPKα protein and arginase-1 mRNA were significantly up-regulated, and those of NOS 2, IL-1 β and NLRP 3 mRNAs obviously down-regulated in both EA and medication groups relevant to the model group (P<0.01, P<0.05), suggesting an increase of M 2 macrophage and a decrease of M 1 macrophage activity after EA. No significant differences were found between the EA and medication groups in up-regulating p-AMPKα expression and in down-regulating NOS 2, IL-1 β and NLRP 3 mRNA expression (P>0.05), except higher up-regulation of arginase-1 mRNA in the medication group (P<0.05)．. EA intervention can up-regulate the expression of arginase-1 mRNA and p-AMPKα protein, and down-regulate the expression of NOS 2, IL-1 β and NLRP 3 mRNAs in synovial tissues in AGA rats, which may contribute to its anti-inflammatory effect by promoting conversion of macrophages from M 1 pro-inflammatory phenotype to M 2 anti-inflammatory phenotype.

Inhibition of Tubulin β-chain May Play a Regulatory Role in the Development of Rheumatoid Arthritis

AbstractPathological characteristics of Rheumatoid arthritis (RA), a systemic autoimmune disease, includes abnormal proliferation of synovial fibroblasts and increased angiogenesis.The study assessed the effect of tubulin β-chain gene (TBB) gene knockdown on the behaviour of rheumatoid arthritis (RA) fibroblast-like synoviocytes (SFs), which is significantly affected by the synthesis of tubulin. Cultured SFs were isolated from the mixed knee-joint synovial tissue of five RA patients and treated with siRNA targeting TBB. Knockdown efficiency and expression of relevant genes were detected by RT-PCR following 24 h and 36 h of transfection. MTT, Transwell, and wound scratch assays and flow cytometric analysis were used to assess cell proliferation and invasive and migratory capacity following TBB knockdown. The expression of TBB was detected after inhibition of the ERK, STAT3 and NF-kB pathways. The expression of IL-17, TNF-α, IL-1α, IL1-β, and IL-6 was quantified following TBB knockdown. The proliferation, invasion, and migration of RASFs were significantly decreased following TBB knockdown. ELISA showed a significant decrease in interleukin 1β (IL-1β) secretion and insignificantly reduced secretion of TNF-α, IL-17, and IL-1α after TBB knockdown. The expression of TBB in RASFs decreased with the increasing concentration of a STAT3 pathway inhibitor while TBB expression increased after down regulation of the ERK pathway. These results support a protective role for TBB knockdown in suppressing RASFs, laying a foundation for further studies on the pathogenic mechanisms of RA.

Pharmacodynamic mechanism of modified Ganlu Yaoyu San intreatment of rheumatoid arthritis based on MAPK signaling pathway

According to the findings, modified Ganlu Yaoyu San has a good anti-inflammatory activity, and can significantly alleviate the degree of arthritis. Its therapeutic effect for rheumatoid arthritis may be related to the regulation of MAPK pathway of synovial cells. In the study, the rat adjuvant arthritis(AA) model was established to further investigate the pharmacodynamic mechanism for regulating MAPK pathway of synovial cells. Enzyme-linked immune assay was used to determine the serum TNF-α level of AA rats administered with drug for two weeks, synovial tissue protein kinases ERK1/2 and p38 content were determined by immunohistochemistry, synovial tissue JNK1, ERK1, p38 gene(mRNA) expression were detected with fluorescence quantitative PCR(RT-PCR) method. According to the results, after administration for two weeks, the levels of serum TNF-α of AA rat was significantly decreased(P<0.05). After administration for four weeks, the protein expressions of p38 and ERK1/2 in synovial tissue were reduced(P<0.05 or P<0.01), the gene expressions of JNK1, p38 and ERK1 in knee joint synovial tissue were reduced(P<0.05 or P<0.01). In conclusion, modified Ganlu Yaoyu San can effectively treat rheumatoid arthritis. Its mechanism might be related to the reduction of TNF-α levels in serum, protein expression of p38 and ERK1/2 in synovial tissue, and JNK1, p38 and ERK1 gene expressions, and regulation of MAPK pathway.

Longterm professional athletic activity results in increased structural knee joint synovial tissue pathology

Longterm professional athletic activity results in increased structural knee joint synovial tissue pathology

Reduction of monocyte chemoattractant protein-1 expression in rheumatoid arthritis rat joints with light-emitting diode phototherapy.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that involves inflammation and pain of the joints. Light-Emitting Diode (LED) irradiation is being evaluated for treating RA; however, the mechanism is unclear. Monocyte chemotaxis protein (MCP)-1 is a key chemokine in the inflammatory status of RA, and MCP-1 levels in plasma are described as a marker for joint inflammation in RA. To understand the mechanism of the anti-inflammatory effect of LED irradiation on RA, the expression of MCP-1 was examined in the knee joints of collagen-induced arthritis (CIA) rats. The rats were immunized with type II collagen and CIA development was confirmed. CIA rat joints were irradiated with LED energy (3 sessions/week, 2 weeks. 840 nm, 2 J/cm(2)). Total RNA was isolated from the rat knee joint tissues and the MCP-1 mRNA levels were monitored with the reverse transcription polymerase chain reaction (RT-PCR) technique and real-time PCR. MCP-1 production in the rat knee joints was analyzed immunohistochemically. RT-PCR analysis demonstrated that MCP-1 mRNA levels had increased in CIA animals when compared to controls, and LED irradiation significant reduced the gene expression in CIA rats. Real-time PCR analysis confirmed a significant reduction in MCP-1 gene expression. The immunohistochemical analysis demonstrated strong MCP-1 staining in CIA rat joint synovial membrane tissue, and LED irradiation significantly reduced the staining. Since MCP-1 has been identified as an important chemokine in the pathogenesis of RA, the reduction of MCP-1 expression would appear to be one of the mechanisms in the reduction of inflammation by LED irradiation. LED irradiation reduced RA-related inflammation through the reduction of MCP-1 gene expression in CIA rat knee joint synovial tissue.

Coculture of Synovium-Derived Stem Cells and Nucleus Pulposus Cells in Serum-Free Defined Medium With Supplementation of Transforming Growth Factor-β1

A coculture of synovium-derived stem cells (SDSCs) and nucleus pulposus cells (NPCs) in a serum-free pellet system was treated with varying doses of transforming growth factor beta (TGF-beta). Cultures of either SDSCs or NPCs alone served as controls. The aim was to assess the feasibility of using SDSCs to supplement and replenish NPC population for disc regeneration. SDSCs have been proven to be a tissue-specific type of mesenchymal stem cell capable of chondrogenesis. NPCs are chondrocyte-like cells with a high ratio of aggrecan. However, the capacity of SDSCs to complement the NPC population is not known. SDSCs were negatively isolated from porcine knee joint synovial tissue and NPCs were isolated from porcine lumbar spines (L1-L5). SDSCs and NPCs were cocultured (50:50) in a serum-free pellet system with the supplementation of varying doses (0, 3, 10, and 30 ng/mL) of TGF-beta1 for 14 days. SDSCs or NPCs cultured alone served as controls. Chondrogenic differentiation markers were evaluated by histology, immunohistochemistry, biochemistry, and TaqMan PCR. The coculture of SDSCs and NPCs in a pellet system displayed comparable differentiation properties (high levels of collagen II, aggrecan and Sox 9, a low level of collagen I, and no collagen X detectable) to NPCs alone when treated with high doses of TGF-beta1. Moreover, the coculture and NPCs alone shared a similar higher ratio of aggrecan to collagen II. Hypoxia-inducible factor 1alpha (HIF-1alpha) was also observed to be up-regulated in coculture pellets at day 7 and had decreased at day 14 with the time of pellet tissue maturation. SDSCs may act as a potential mesenchymal stem cell candidate for NP regeneration. Further studies are needed to evaluate the in vivo effect of SDSCs on disc regeneration.

A10 TRANSFORMING GROWTH FACTOR-BETA INDUCES PERSISTENT SYNOVIAL FIBROSIS WHILE CONNECTIVE TISSUE GROWTH FACTOR-INDUCED FIBROSIS IS REVERSIBLE. ROLE FOR LYSYS HYDROXYLASE

Purpose: The main characteristics of osteoarthritis are cartilage damage, osteophyte formation and synovial fibrosis. Synovial fibrosis can contribute to the loss of the function of an OA joint. TGF-b is considered one of the main players in fibrotic diseases. However, CTGF, which can be induced by TGF-b, has been reported to be an important player as well, stated to contribute to maintenance of fibrotic tissue. We investigated the underlying mechanism in that leads to the observed difference in fibrosis persistence between TGF-band CTGF. Methods: We injected C57Bl/6 mice intra-articularly with Ad-TGF-b or Ad-CTGF. After 3, 7 and 21 days knee joint synovial tissue was isolated for RNA isolation or whole knee joints for histology. With Q-RT-PCR relative mRNA levels were determined of matrix molecules, proteases, protease inhibitors, and growth factors. To analyze the enzymes involved in collagen cross link formation, mRNA expression of lysyl hydroxylase (LH) was determined. CTGF-levels induced by TGF-b were measured by ELISA in 24 hour patella-synovial wash-outs of C57Bl/6 mice intra-articularly injected with Ad-TGF-b (3, 7 and 14 days). Results: TGF-b and CTGF induced synovial fibrosis in murine knee joints as shown by histology (as observed by an increase in synovial width). However, TGF-b-induced fibrosis was very persistent, whereas CTGFinduced fibrosis resorbed by day 28. TGF-b induced elevated collagen type I and very high levels of aggrecan expression and hardly any changes in collagen type II and III. CTGF induced no clear changes in collagen type I, II and III. CTGF even decreased aggrecan expression (day 21 only). CTGF induced no changes in MMP3, -9, -13, ADAMTS-4 or -5, whereas TGF-b increased MMP-3, -13 and ADAMTS4. Thus elevated levels of matrix degrading enzyme expression cannot explain the less persistent CTGF-induced fibrosis. TIMP1 (associated with matrix accumulation in fibrotic disorders), was highly up regulated by TGF-b (all days) and only slightly up regulated by CTGF (day 7). TGF-b also induced elevated mRNA levels of TGF-b1 and CTGF. Only TGF-b induced high levels of LH expression, especially LH2b, which has been implicated in hard-to-degrade collagen linkage and was found up regulated in fibrotic lesions. As TGF-b can induce CTGF and up regulated CTGF mRNA in our experiments, we measured CTGF levels in 24 hour patella-synovial wash outs and found that TGF-b induced 64 ng CTGF/ml after 3 days and that CTGF levels were still elevated by day 14. Conclusions: TGF-b induced increased collagen type I and aggrecan mRNA levels in synovial tissue, but also high levels of degrading enzymes. The latter does not explain the persistent nature of TGF-b versus CTGFinduced synovial fibrosis. Lack of collagen type I mRNA induced by CTGF corresponds to findings of other groups. Despite this fact, fibrosis is found. Only TGF-b induced high levels of TIMP1 expression, which was previously found associated with strain dependent fibrosis sensitivity in mice and implicated in accumulation of ECM in fibrotic disorders. TGF-b, not CTGF, induced high levels of LH which are crucial for ECM cross linking. Moreover, LH2b was reported by van der Slot et al. to induce harder-to-degrade crosslinks compared to LH2a. The 2b splicing variant was highly expressed upon stimulation with TGF-b. Since TGF-b clearly up regulated MMP expression a dominant role for TIMP appears unlikely. The strong induction of LH2b by TGF-b compared to CTGF, and consequently harder to degrade cross links, appears to be the most likely cause of the induction of irreversible fibrosis by TGF-b. A11 EFFICACY OF A SINGLE ULTRASOUND GUIDED INJECTION IN HIP OSTEOARTHRITIS

Unexpected finding of a marked non-neuronal cholinergic system in human knee joint synovial tissue

The cholinergic anti-inflammatory pathway is a newly discovered pathway. Another recent concept is the existence of a non-neuronal cholinergic system that has, so far, been defined for human tendons, intestine, airways and urinary bladder. The existence of such a system in joint synovial tissue is yet to be described. We therefore aimed to investigate the expression of choline acetyltransferase (ChAT) at both the protein and mRNA level using immunohistochemistry and in situ hybridisation, in human knee synovial tissue from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. The biopsy samples were collected from patients undergoing knee prosthetic surgery. Our results show that both ChAT protein and mRNA is expressed in fibroblast-like and mononuclear-like cells, and to some extent in blood vessel walls in the synovial tissue. The mononuclear-like cells showing ChAT expression were scattered throughout the synovial tissue or located in association with lymphoid aggregates. Thus, we present the first evidence of the existence of a marked non-neuronal cholinergic system in human synovial tissue. The existence of this system could lead to the development of alternative medications to those currently in use. The system might function as a cholinergic anti-inflammatory pathway in synovial tissue. Our observations show that synovial tissue of patients with marked RA or OA, a tissue in which cholinergic innervation is not proven to exist, is supplied with acetylcholine via production in non-neuronal cells within the tissue.

Endothelins modulate inflammatory reaction in zymosan-induced arthritis: participation of LTB4, TNF-α, and CXCL-1

Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET-1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan-induced arthritis. Intra-articular (i.a.) administration of selective ET(A) or ET(B) receptor antagonists (BQ-123 and BQ-788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 microg/cavity) markedly reduced knee-joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ET(A) or ET(B) receptor blockade suppressed zymosan-induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ET(A)/ET(B) with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ-123 or BQ-788 (i.a.; 15 pmol/cavity) also decreased zymosan-induced TNF-alpha production within 6 h, keratinocyte-derived chemokine/CXCL1 production within 24 h, and leukotriene B(4) at both time-points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan-induced inflammation, i.a. injection of ET-1 (1-30 pmol/cavity) or sarafotoxin S6c (0.1-30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee-joint synovial tissue expressed ET(A) and ET(B) receptors. These findings suggest that endogenous ETs contribute to knee-joint inflammation, acting through ET(A) and ET(B) receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.

Histopathological studies of the acute inflammation in synovial tissue of rat knee joint following intra-articular injection of PLA 2 from Chinese Cobra ( Naja naja atra) venom

A phospholipase A 2 was purified from Chinese Cobra Naja naja atra by a two-step procedure: gel filtration on Superdex 75 and reverse-phase high-performance liquid chromatography (HPLC) on a NUCLEOSIL 5 C18 column. Purified phospholipase A 2 was homogeneous, as indicated by capillary electrophoresis and electrospray mass spectrometry. It was a basic protein (p I=8.2±0.03) with a molecular mass of 13,258 Da. Amino acid sequence analysis of the N-terminal demonstrated a high degree of homology with other related PLA 2 from elapid venoms. The histopathological effects of the purified PLA 2 on synovial tissue of knee joint were studied in Wistar rats. Rats were injected intra-articularly with 100 μl solution of PLA 2 of different concentrations. Synovial tissue samples with patella were taken for light microscope study. Histopathological evaluation revealed a significant induction of acute inflammation in synovial tissue after injection, as indicated by synovial lining-cell hyperplasia, subsynovial cellular infiltration, and peri-articular soft-tissue cellular infiltration. Marked inflammatory and proliferative changes in synovial tissue were evident after repeated intra-articular injections of 100 μg PLA 2. This study failed to show any significant histological changes in cartilage of patella as well as in the surrounding muscle tissue of the knee joints. These results suggest that PLA 2 purified from Chinese Cobra venom induce time- and dose-dependent inflammatory changes in the synovial tissue of rat knee joint.

NF-kappa B activation in human knee-joint synovial tissue during the early stage of joint inflammation.

Conference Article| August 01 1997 NF-κB activation in human knee-joint synovial tissue during the early stage of joint inflammation Vanessa Gilston; Vanessa Gilston 1Inflammation Research Group, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 25–29 Ashfield Street, London E1 2AD, UK Search for other works by this author on: This Site PubMed Google Scholar Hugh W. Jones; Hugh W. Jones 1Inflammation Research Group, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 25–29 Ashfield Street, London E1 2AD, UK Search for other works by this author on: This Site PubMed Google Scholar Catherine C. Soo; Catherine C. Soo 1Inflammation Research Group, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 25–29 Ashfield Street, London E1 2AD, UK Search for other works by this author on: This Site PubMed Google Scholar Adam Coumbe; Adam Coumbe 1Inflammation Research Group, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 25–29 Ashfield Street, London E1 2AD, UK Search for other works by this author on: This Site PubMed Google Scholar Selina Blades; Selina Blades 1Inflammation Research Group, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 25–29 Ashfield Street, London E1 2AD, UK Search for other works by this author on: This Site PubMed Google Scholar Christian Kaltschmidt; Christian Kaltschmidt *Institute for Biochemistry and Molecular Biology, Albert-Ludwigs-University Freiburg D-791104, Germany Search for other works by this author on: This Site PubMed Google Scholar Patrick A. Baeuerle; Patrick A. Baeuerle +Tularik Inc., South San Francisco, CA 94080, USA Search for other works by this author on: This Site PubMed Google Scholar Chris J. Morris; Chris J. Morris 1Inflammation Research Group, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 25–29 Ashfield Street, London E1 2AD, UK Search for other works by this author on: This Site PubMed Google Scholar David R. Blake; David R. Blake 1Inflammation Research Group, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 25–29 Ashfield Street, London E1 2AD, UK Search for other works by this author on: This Site PubMed Google Scholar Paul G. Winyard Paul G. Winyard 1Inflammation Research Group, St. Bartholomew's and the Royal London School of Medicine and Dentistry, 25–29 Ashfield Street, London E1 2AD, UK Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1997) 25 (3): 518S. https://doi.org/10.1042/bst025518s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation Vanessa Gilston, Hugh W. Jones, Catherine C. Soo, Adam Coumbe, Selina Blades, Christian Kaltschmidt, Patrick A. Baeuerle, Chris J. Morris, David R. Blake, Paul G. Winyard; NF-κB activation in human knee-joint synovial tissue during the early stage of joint inflammation. Biochem Soc Trans 1 August 1997; 25 (3): 518S. doi: https://doi.org/10.1042/bst025518s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1997 Biochemical Society1997 Article PDF first page preview Close Modal You do not currently have access to this content.

Streptozotocin-induced diabetes decreases substance P levels in experimental arthritis in the rat knee

Diabetic patients with sensory neuropathy are predisposed to disorders of the musculoskeletal system. It has been postulated that altered neurogenic inflammation, involving the neuropeptide substance P, may play a part in this phenomenon. We investigated the effect of streptozotocin-induced (STZ) diabetes on the development of an antigenic (mBSA) monoarthritis in the rat with particular reference to changes in substance P levels in dorsal root ganglia (DRG) and knee joint synovium. We found that STZ-induced diabetes of 24 weeks duration reduced the substance P content of L 4/L 5 DRG and knee joint synovial tissue. Induction of mBSA arthritis in diabetic rats resulted in diminished increases in synovial substance P and knee joint swelling compared to non-diabetic arthritic controls. The results show that chronic STZ diabetes reduces neurogenic inflammatory responses in the rat knee which may render the joint more susceptible to arthritic attack.

Incorporation of [35S]sulphate and [3H]thymidine into isolated bovine articular chondrocytes in vitro. Effect of autogenous conditioned synovial medium.

The present work is a part of a study series concerned with the metabolic events taking place in the synovial joint tissues. We have previously reported that synovial products not only enhance the degradation of articular cartilage proteoglycans but also inhibit the synthesis of articular cartilage proteoglycans, and that these cellular interactions may be partly modulated by arachidonic acid metabolites. In the experiments reported here we show that the addition of conditioned synovial medium to confluent monolayer cultures of articular chondrocytes not only inhibits the synthesis of proteoglycans, measured as the incorporation of radiosulphate into cartilage proteoglycans but also inhibits the synthesis of DNA, measured as the incorporation of radiothymidine into the chondrocytes, in a dose-dependent manner. In our culture system, calf knee-joint synovial tissue produces PGE, and it has been reported that previous exposure to PGE2 affects the ability of the cell to respond to that same hormone. In these experiments, previous exposure to conditioned synovial medium affected neither the inhibition of the incorporation of radiosulphate nor the inhibition of the incorporation of radiothymidine.