The objective of the present study was to determine whether the expression of connexin 43 (Cx43) effected on inflammatory conditions in rat fibroblast-like synoviocytes (FLS) and on rat model of rheumatoid arthritis (RA). The expression of Cx43 in rat FLS stimulated with lipopolysaccharide (LPS) was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The effects of small-interfering RNA targeting Cx43 (siCx43) on pro-inflammatory cytokines and chemokine were assessed by real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA). The therapeutic and side effects of siCx43 in a rat model of collagen-induced arthritis (CIA) were examined by in vivo electroporation method. LPS markedly enhanced Cx43 gene expression in rat FLS, with transfection of siCx43 suppressing the over-expression of pro-inflammatory cytokines and the chemokine. Treatment of CIA rats with siCx43 significantly ameliorated paw swelling, and significantly reduced histological arthritis scores and radiographic scores. In histological appearance of rat ankle joints, siCx43 treatment significantly decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive (osteoclast-like) cells. These findings indicated that siCx43 had anti-inflammatory effects in rat FLS and efficiently inhibited the development of CIA. Cx43 may play an important role in the pathophysiology of RA, and may be a potential target molecule for novel RA therapies. Keywords: connexin 43; gap junction; pro-inflammatory cytokine; rheumatoid arthritis; collagen-induced arthritis Rheumatoid arthritis (RA) is one of the most common articular diseases, characterized by hyperproliferation of synovial cells and bone destruction. Pro-inflammatory cytokines and chemokines, including tumor necrosis factor (TNF)-a, interleukin (IL)-6, IL-1b, and stromal cell-derived factor 1 (CXCL12), have been associated with the progression of RA and may play pathogenic roles in the establishment of rheumatoid synovitis. 1,2Biological preparations targeting these pro-inflammatory cytokines are widely used clinically in the treatment of RA.3 However, there are caveats in using the biological preparations, including risk of infections such as tuberculosis, high cost, and individual variations in efficacy.4 Therefore, further investigations are needed to achieve remission of arthritis.Gap junctions are cell-cell communication channels, consisting of multimeric proteins called connexins, that allow the exchange of ions, second messengers, and metabolites between adjacent cells.5 To date, 21 human genes encoding connexins have been identified. The most widely expressed connexin gene is connexin 43 (Cx43), a protein expressed in the synovial cells and tissue.6 Cx43 plays an important role in the regulation of various immune processes. 7 In the nervous system, silencing of Cx43 downregulates the inflammatory response. 8,9 These reports suggest that Cx43 may be involved in RA pathophysiology, and that inhibition of Cx43 may decrease synovitis in RA.Therefore, we ...
ABSTRACT:We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF-1a)-dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl 2 , which induces HIF-1a, to simulate hypoxia, or transfected with siRNAs targeting HIF-1a (si-HIF-1a) and HSP70 (si-HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF-1a under hypoxia or simulated hypoxia, but not in the presence of si-HIF-1a. Hypoxia-induced overexpression of ECM genes was significantly suppressed by si-HIF-1a or si-HSP70. Cell viability positively correlated with hypoxia, but transfection with si-HIF-1a or si-HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside-treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si-HIF-1a or si-HSP70 almost completely blocked these effects. These findings indicated that HIF-1a-induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF1a may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression. Keywords: hypoxia-inducible factor-1a; heat shock protein 70; hypoxia; extracellular matrix; apoptosis Articular cartilage is an avascular tissue that derives its nutrition and oxygen supply via diffusion from synovial fluid and subchondral bone. It has been estimated that chondrocytes at the articular surface are exposed to approximately 6-10% oxygen (O 2 ), whereas chondrocytes in the deepest layers of the articular cartilage are exposed to only 1-5% O 2 . 1 Several studies have shown that chondrocytes can survive hypoxic conditions by adjusting their metabolism. 2,3 Oxygen is an important modulator of gene expression, with hypoxia-inducible factor (HIF) being the primary regulatory factor responding to variations in O 2 levels. 4,5 The transcription factor HIF-1alpha (HIF-1a) is important in maintaining proper cellular functions under hypoxic conditions. 5 In addition, HIF1a is essential for chondrogenesis, including chondrocyte growth arrest, survival, maturation, and apoptosis. 6 HIF-1a also promotes the synthesis of relevant extracellular matrix (ECM) components. 2 However, these HIF-1a-dependent anabolic pathways in chondrocytes remain poorly understood.Among the genes regulated via the HIF-1a pathway under hypoxic conditions are the highly conserved heat shock proteins (HSPs), which act as cellular chaperones for proteins misfolded by cellular stress. 7,8 For example, hypoxic conditions in the growth plate of tibial dyschondroplasia increases HIF-1a expression, which, in turn, induces increased levels of heat shock protein 70 (HSP70). 9 We also reported that HSP70 overexpression promotes the metabolic activity of chondrocyte...
The objective of the present study was to investigate the effects of heat stimulation and glutamine (Gln) on the expression of extracellular matrix genes and heat shock protein 70 (HSP70) in rat articular cartilage in vivo and to determine whether HSP70 expression achieved with a combination of microwave (MW) and Gln suppresses osteoarthritis (OA) progression in a rat OA model. Stimulation at 40 W was assumed to be appropriate in the present study, and the effects of heat treatment at this intensity were evaluated. Articular cartilage was collected at 8 h after heat stimulation and/or intraarticular Gln administration, and total RNA was extracted. The expression of HSP70, aggrecan, and type II collagen was quantified using real-time RT-PCR. Cartilage samples from the OA model were subjected to hematoxylin and eosin (HE) and safranin O staining. HSP70 and aggrecan expression was greatest in a group receiving both MW and Gln. In the rat OA model, the severity of OA was significantly milder in a group receiving MW and Gln than in the control group. HSP70, stimulated by the combination of MW heat and Gln, may be involved in the suppression of OA progression. ß
Simple bone cysts (SBCs) are benign bone tumors. However, the treatment of SBCs remains controversial because of their healing rate and the invasiveness of surgery. The purpose of the present study was to evaluate the treatment of SBCs using a cannulated hydroxyapatite (HA) pin.A total of 43 patients (35 males, 8 females; mean age 12.1 years; age range, 5–22 years) with SBCs were treated with continuous decompression by inserting ceramic HA pins between 1989 and 2014. The SBCs were located in the calcaneus in 23, the humerus in 15, the femur in 3, and the pelvis in 2 cases. In all patients, minimal fenestration of the cyst wall and curettage and multiple drilling in the cyst wall were performed, followed by insertion of the HA pin. The mean follow-up period was 26.6 months. Operating time, healing period, risk factors for recurrence, and the cure rate were evaluated.Healing was achieved without intervention in 38 patients after a mean of 6.4 months. Two patients had persistent small residual cysts, which had no changes after 1 year at the latest follow-up. There were 5 patients with recurrences (humerus 4, femur 1), who were cured by curettage and artificial bone grafting. The final healing rate by cannulation only using an HA pin was 88.2%. On Fisher exact test, age, site of SBCs, and distance from the physis were found to be significantly associated with SBC recurrence (P < 0.05).In the present study, cannulation using an HA pin for SBCs was found to be a useful technique, particularly for calcaneal cysts, because it is a minimally invasive procedure with a high cure rate. In patients <10 years, involvement of the humerus and contact with the growth plate were significant risk factors for SBC recurrence.
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