Bone marrow derived mast cells injected into the osteoarthritic knee joints of mice induced by sodium monoiodoacetate enhanced spontaneous pain through activation of PAR2 and action of extracellular ATP.

Conditions that resemble osteoarthritis (OA) were produced by injection of sodium monoiodoacetate (MIA) into the knee joints of mice. Bone marrow derived mast cells (BMMCs) injected into the OA knee joints enhanced spontaneous pain. Since no spontaneous pain was observed when BMMCs were injected into the knee joints of control mice that had not been treated with MIA, BMMCs should be activated within the OA knee joints and release some pain-inducible factors. Protease activated receptor-2 (PAR2) antagonist (FSLLRY-NH2) almost abolished the pain-enhancing effects of BMMCs injected into the OA knee joints, suggesting that tryptase, a mast cell protease that is capable of activating PAR2, should be released from the injected BMMCs and enhance pain through activation of PAR2. When PAR2 agonist (SLIGKV-NH2) instead of BMMCs was injected into the OA knee joints, it was also enhanced pain. Apyrase, an ATP degrading enzyme, injected into the OA knee joints before BMMCs suppressed the pain enhanced by BMMCs. We showed that purinoceptors (P2X4 and P2X7) were expressed in BMMCs and that extracellular ATP stimulated the release of tryptase from BMMCs. These observations suggest that ATP may stimulate degranulation of BMMCs and thereby enhanced pain. BMMCs injected into the OA knee joints stimulated expression of IL-1ß, IL-6, TNF-α, CCL2, and MMP9 genes in the infrapatellar fat pads, and PAR2 antagonist suppressed the stimulatory effects of BMMCs. Our study suggests that intermittent pain frequently observed in OA knee joints may be due, at least partly, to mast cells through activation of PAR2 and action of ATP, and that intraarticular injection of BMMCs into the OA knee joints may provide a useful experimental system for investigating molecular mechanisms by which pain is induced in OA knee joints.

Eicosapentaenoic acid and docosahexaenoic acid synergistically attenuate bile acid-induced hepatocellular apoptosis.

BACKGROUND: Clinical studies have demonstrated improvement of parenteral nutrition (PN)-associated liver disease (PNALD) with ω3 polyunsaturated fatty acid (ω3PUFA) supplementation containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Experiments were designed to test the following hypotheses: (1) therapeutic effects of ω3PUFA are due to attenuation of cellular apoptosis induced by hydrophobic bile acid exposure, which occurs in cholestasis, and (2) attenuation of apoptosis by EPA and DHA is additive or synergistic. METHODS: Cultured HepG2 cells were treated with 50-200 µM chenodeoxycholic acid (CDCA) in the presence and absence of EPA, DHA, or EPA + DHA. Apoptosis was evaluated using cell staining with fluorescence microscopy and the Apo-ONE Homogeneous Caspase-3/7 assay. Specific apoptotic mediators were evaluated with quantitative RT-PCR. RESULTS: Treatment with EPA alone and DHA alone resulted in 22% and 9% attenuation of caspase-3/7 activity, respectively. Caspase-3/7 activity was attenuated by 52% when cells were treated with a combination of EPA and DHA (P = .0034). Treatment with EPA alone, DHA alone, and the combination of EPA and DHA all resulted in equal attenuation of apoptotic mediator gene expression. CONCLUSIONS: The combination of EPA and DHA resulted in a synergistic attenuation of bile acid-induced hepatocellular apoptosis, as assessed by caspase-3/7 activity, compared to EPA and DHA separately. The combination of EPA and DHA did not result in a synergistic attenuation of the upregulation of Fas or TRAIL-R2. These data suggest that EPA and DHA may be working via multiple intracellular pathways to attenuate bile acid-induced apoptosis.

Effects of chenodeoxycholic and ursodeoxycholic acids on lipid metabolism and gallstone formation in the prairie dog.

The prevention of cholesterol cholelithiasis by dietary chenodeoxycholic and ursodeoxycholic acids was studied in the male prairie dog (Cynomys ludovicianus). Gallstones were induced by administration of a semisynthetic diet containing 0.4% cholesterol for a period of 8 weeks. Groups of 5 or 6 animals received the lithogenic diet with added chenodeoxycholic or ursodeoxycholic acid (0.03% "low dose" or 0.06% "high dose"). Under the conditions used, the incidence of gallstones was reduced with the high dose of chenodeoxycholic acid and the low dose of ursodeoxycholic acid, but cholesterol crystals were detected in the biles of 20 of the 22 animals fed these bile acids. A control group maintained on a low (0.08%) cholesterol semisynthetic diet exhibited neither crystals nor stones and was the only group with a lithogenic index below 1.0. The administered bile acids tended to reduce the accumulation of cholesterol in liver and plasma. The activity of hepatic HMG-CoA reductase was significantly inhibited with all cholesterol-supplemented diets. Cholesterol 7 alpha-hydroxylase activity was elevated 83% in prairie dogs fed 0.4% cholesterol, but tended to return to normal levels when bile acids were added to this diet. Histologically, the livers of all animals on the semisynthetic (cholesterol-supplemented) diet exhibited bile duct proliferation, as well as portal fibrosis and inflammatory infiltration. These morphologic alterations were ameliorated by low dose supplementation with either chenodeoxycholic or ursodeoxycholic acid, but high dose bile acid supplementation failed to reduce these pathologic changes.