Shuji Nakagawa

Enhanced intestinal inflammation induced by dextran sulfate sodium in tumor necrosis factor-alpha deficient mice.

Background and Aims: Tumor necrosis factor‐α (TNF‐α) is a potent pro‐inflammatory cytokine thought to be involved in the pathogenesis of inflammatory bowel disease. To further define the role of TNF‐α in intestinal inflammation, we studied the effects of dextran sulfate sodium (DSS) administration in mice with targeted deletions of TNF‐α gene.

N-acetylcysteine prevents nitric oxide-induced chondrocyte apoptosis and cartilage degeneration in an experimental model of osteoarthritis.

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.

Doubling time of serum CA 19-9 in the clinical course of patients with pancreatic cancer and its significant association with prognosis.

Pancreatic cancer is generally a disease with a poor prognosis, and relationship between change of serum CA 19‐9 level and progression of this disease was investigated with regard to clinical pace of disease and tumor growth.

Free radical-scavenging activity of Crassostera gigas extract (JCOE)

In the present study, we evaluate the free radical-scavenging activity of JCOE (Japan clinic oyster extract), a powder extracted from Crassostera gigas by a spin-trapping method using electron paramagnetic resonance (EPR), and also estimate the protective effect against gastric mucosal cell injury induced by hydrogen peroxide. The EPR study demonstrated that JCOE directly scavenged superoxide radical as well as hydroxyl radical in a concentration-dependent manner. After exposure to hydrogen peroxide for 4 h in Hanks balanced buffered solution, cell viability of rat gastric mucosal cells (RGM-1) was measured by modified MTT assay. Hydrogen peroxide-induced injury was not reversed by 1-h preincubation with 100 to 1,000 micrograms/mL JCOE solution which has high reactivity to hydroxyl radicals, indicating that the active ingredients, including taurine of JCOE on scavenging action of hydroxyl radical, did not penetrate cell membranes easily. Twenty-four hour pretreatment with the JCOE solution significantly reversed the decrease in cell viability induced by hydrogen peroxide, indicating the possibility that JCOE solution may stimulate the endogenous eliminating system against hydrogen peroxide.

Enhancement of lipid peroxidation and of the antitumor effect of hyperthermia upon combination with oral eicosapentaenoic acid

The present study was designed to determine the effect of eicosapentaenoic acid (EPA) on the susceptibility of tumor cells to treatments that kill the cells by lipid peroxidation. Using AH109A carcinoma, a rat liver cancer, we measured EPA content, levels of antioxidants, and degree of lipid peroxidation in tumor tissue and normal liver tissue after oral administration of EPA. In the control group treated with distilled water, EPA in tumor tissue was lower than in normal liver tissue, suggesting that its content of polyunsaturated fatty acids (the substrates for lipid peroxidation) was inherently low. Levels of antioxidants also tended to be lower in tumor tissue. EPA level increased in both tumor and normal tissues after oral administration of EPA. At the same time, glutathione peroxidase (GSH-Px) increased in normal tissue, whereas tumor tissue displayed no increase in antioxidants; instead GSH decreased. The EPA-induced change in balance between substrates for lipid peroxidation and antioxidants suggested that tumor tissue might become more susceptible to lipid peroxidation than normal liver tissue. In fact, hyperthermia treatment did enhance lipid peroxidation and antitumor action. Our results indicate that oral EPA specifically increases the susceptibility of liver tumor tissue to lipid peroxidation, and hence enhance the antitumor effect of hyperthermia and prolongs survival.

Methylprednisolone inhibits neutrophil-endothelial cell interactions induced by interleukin-1 β under flow conditions

The effects of methylprednisolone (m-PSL) on IL-1beta-induced neutrophil-endothelial cell interactions, which are normally mediated by increased expression of both intercellular adhesion molecule-1 (ICAM-1) and E-selectin on endothelial cells, were examined using an in vitro flow system. Human neutrophilic polymorphonuclear leukocytes (PMN) were perfused at a shear stress of 1 dyne/cm2 on human umbilical vein endothelial cells (HUVEC) pretreated with IL-1beta (20 U/mL) for 4 hours. Many PMN adhered to IL-1-stimulated HUVEC and then migrated beneath endothelial cell monolayers. Treatment of HUVEC with m-PSL inhibited adherence and migration of PMN in a dose dependent manner. M-PSL also inhibited IL-1beta-induced upregulation of E-selectin and ICAM-1 on HUVEC in a dose dependent manner. These results suggest that m-PSL works as an anti-inflammatory agent through inhibiting PMN-endothelial cell interactions.

HIF-1α-induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions.

We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF‐1α)‐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 CoCl2, which induces HIF‐1α, to simulate hypoxia, or transfected with siRNAs targeting HIF‐1α (si‐HIF‐1α) and HSP70 (si‐HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF‐1α under hypoxia or simulated hypoxia, but not in the presence of si‐HIF‐1α. Hypoxia‐induced overexpression of ECM genes was significantly suppressed by si‐HIF‐1α or si‐HSP70. Cell viability positively correlated with hypoxia, but transfection with si‐HIF‐1α 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‐1α or si‐HSP70 almost completely blocked these effects. These findings indicated that HIF‐1α‐induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF‐1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression.

Combined microwave irradiation and intraarticular glutamine administration-induced HSP70 expression therapy prevents cartilage degradation in a rat osteoarthritis model.

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.

Small interfering RNA targeting CD81 ameliorated arthritis in rats

CD81 belongs to a family of cell-surface protein (tetraspanin) known as one of the up-regulated elements in rheumatoid arthritis synoviocytes. In this study, the therapeutic effect of small interfering RNA targeting CD81 (siCD81) was examined by in vivo electroporation method. Treatment with siCD81 significantly ameliorated paw swelling of collagen-induced arthritic (CIA) rats. In histological examination, hypertrophy of synovium, bone erosion, and degeneration of articular cartilage were milder in rats treated with siCD81 than in the control group and the non-specific siRNA group. Expression of synoviolin, a rheumatoid regulator, was suppressed by siCD81. Thus, therapeutic intervention by targeting CD81 may be used in the treatment of rheumatoid arthritis.

A New Arthroscopic-Assisted Drilling Method Through the Radius in a Distal-to-Proximal Direction for Osteochondritis Dissecans of the Elbow

We developed a new arthroscopic-assisted drilling method through the radius in a distal-to-proximal direction for osteochondritis dissecans (OCD) of the elbow. Only 1 drill hole is created in the radius by use of a single 1.8-mm K-wire inserted from the shaft of the radius approximately 3 cm distal to the humeroradial joint into the joint, which allows drilling of the entire OCD lesion. The forearm is supinated so that the tip of the K-wire is at the lateral side of the lesion in the humeral capitellum, and drilling is performed at 30 degrees elbow flexion. The flexion angle is changed from 30 degrees to 60 degrees to 90 degrees to 120 degrees while maintaining supination, to drill in 4 sites (1 site for each angle of flexion) of the lateral side of the OCD lesion. Next, we move the forearm from supination to pronation so that the tip of the K-wire is placed in the medial side of the lesion in the humeral capitellum, and as with the lateral side, drilling is performed in 4 sites. With this technique, the entire OCD lesion can be vertically drilled under arthroscopic guidance. This method is minimally invasive, and an early return to sports could be possible.