DSS-induced inflammation is significantly enhanced in TNF-alpha-/- mice compared to TNF-alpha+/+ mice. These data suggest that persistent and marked blockage of TNF-alpha bioactivity may provide a detrimental effect on acute intestinal inflammation.

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ABSTRACT:We investigated whether N-acetylcysteine (NAC), a precursor of glutathione, could protect rabbit articular chondrocytes against nitric oxide (NO)-induced apoptosis and could prevent cartilage destruction in an experimental model of osteoarthritis (OA) in rats. Isolated chondrocytes were treated with various concentrations of NAC (0-2 mM). Apoptosis was induced by 0.75 mM sodium nitroprusside (SNP) dehydrate, which produces NO. Cell viability was assessed by MTT assay, while apoptosis was evaluated by Hoechst 33342 and TUNEL staining. Intracellular reactive oxygen species (ROS) and glutathione levels were measured, and expression of p53 and caspase-3 were determined by Western blotting. To determine whether intraarticular injection of NAC prevents cartilage destruction in vivo, cartilage samples of an OA model were subjected to H&E, Safranin O, and TUNEL staining. NAC prevented NO-induced apoptosis, ROS overproduction, p53 up-regulation, and caspase-3 activation. The protective effects of NAC were significantly blocked by buthionine sulfoximine, a glutathione synthetase inhibitor, indicating that the apoptosis-preventing activity of NAC was mediated by glutathione. Using a rat model of experimentally induced OA, we found that NAC also significantly prevented cartilage destruction and chondrocyte apoptosis in vivo. These results indicate that NAC inhibits NO-induced apoptosis of chondrocytes through glutathione in vitro, and inhibits chondrocyte apoptosis and articular cartilage degeneration in vivo. ß

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