[A protective effect of acetaminophen on ibuprofen-induced small intestinal damage in rats via the suppression of MMP-13].

BACKGROUND & AIMS: Capsule endoscopy has revealed that nonsteroidal anti-inflammatory drugs may cause damage not only to the stomach but also to the small intestine, which has become one of the most serious issues in gastroenterology. However, few studies have reported the effect of ibuprofen (IBP), which is widely prescribed worldwide, on the small intestine, and it remains unclear whether IBP can cause small intestinal damage. We have previously shown that acetaminophen (APAP), which is used as an antipyretic/analgesic drug, inhibits IBP-induced gastric damage by suppressing matrix metalloprotease-13 (MMP-13) gene expression. In this study, we investigated the ability of IBP to induce small intestinal damage and the efficacy of APAP against IBP-induced small intestinal damage in rats. MAIN METHODS: Nonfasted male Sprague-Dawley rats were orally administered with IBP (200mg/kg) and then euthanized at various time points (0, 4, 8, 16, and 24h) after the administration. The small intestine, jejunum, and ileum were removed, and intestinal lesions were measured. To elucidate the efficacy of APAP against IBP-induced small intestinal damage, the rats were treated with IBP (200mg/kg) with or without APAP (200mg/kg), and small intestinal damage was evaluated 24h after the administration. Moreover, the expression levels of GAPDH, TNFα, iNOS, and MMP-13 genes were determined at various time points (8, 16, and 24h) by RT-qPCR. KEY FINDINGS: The oral administration of IBP induced obvious small intestinal damage, which was found to be significant at 24h (p<0.05 vs 0h, Dunnett's test). The coadministration of APAP significantly prevented IBP-induced damage (p<0.05, Student's t-test). In addition, the expression levels of TNFα and iNOS genes were significantly increased by IBP (p<0.01 and p<0.05 vs. vehicle, respectively, Tukey-Kramer test), whereas the cotreatment with APAP suppressed the increases at 8h. Moreover, compared with the vehicle, the IBP treatment significantly increased the expression level of the MMP-13 gene (p<0.01) at each time point (8, 16, and 24h, Tukey-Kramer test), whereas the APAP cotreatment significantly suppressed the increase (p<0.01 vs. IBP at 8h, p<0.05 vs. IBP at 16 and 24h, Tukey-Kramer test). CONCLUSIONS: This study suggested that a single administration of IBP was associated with the risk of inducing small intestinal ulcers in rats, and APAP could prevent IBP-induced small intestinal damage by suppressing the MMP-13 gene expression.

[The Analgesic Effect of Aspirin on Inflammatory Pain in Rats Is Affected by Pain Intensity and Administration Timing].

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely known as painkillers. The analgesic action of NSAIDs is attributable to the inhibition of prostaglandin synthesis that occurs in response to blocking cyclooxygenase activity. The effective dose of NSAIDs can vary depending on pain intensity and administration timing; however, there are few studies on this. This study aimed to elucidate whether the analgesic effect of NSAIDs changes depending on the situation in which they are taken and we focused on the NSAID, aspirin (ASP). In a rat model of brewer's yeast-induced inflammation, pain caused by 20% (w/v) brewer's yeast-treatment was defined as "strong pain" and that caused by 2.5% (w/v) was defined as "weak pain". The analgesic effect of ASP (low-dose; 44 mg/kg or high-dose; 66 mg/kg) against strong pain was dose-dependent, but that against weak pain was the same. Furthermore, we defined drug administration after 3 h of brewer's yeast-treatment as "late administration" and that after 20 min as "early administration". In the case of strong pain, the analgesic effect of "late ASP administration" was dose-dependent, but that of "early ASP administration" was the same. These results suggest that low-dose NSAIDs have an analgesic effect against weak pain or when taken early.

Sustained Activation of the Unfolded Protein Response Induces Cell Death in Fuchs' Endothelial Corneal Dystrophy.

Purpose: The unfolded protein response (UPR) is believed to play a role in the pathogenesis of Fuchs' endothelial corneal dystrophy (FECD). The purpose of this study was to investigate whether unfolded proteins accumulate in the corneal endothelium in FECD and if they are involved in triggering cell death. Methods: Descemet's membranes with corneal endothelial cells (CECs) were obtained during keratoplasty, and expression of aggresomes, type 1 collagen, fibronectin, and agrin was evaluated. Endoplasmic reticulum (ER) stress of immortalized human CECs from non-FECD subjects and from FECD patients (iHCEC and iFECD, respectively) were evaluated. The effect of MG132-mediated aggresome formation on the UPR and intrinsic pathway and the effect of mitochondrial damage on UPR were also examined. The effect of CHOP knockdown on the ER stress-mediated intrinsic pathway was also evaluated. Results: Aggresome formation was higher in iFECD than in iHCEC and was colocalized with type 1 collagen, fibronectin, and agrin. GRP78, phosphorylated IRE1, PERK, and CHOP showed higher activation in iFECD than in iHCEC. MG132-mediated aggresome formation upregulated ER stress sensors, the mitochondrial membrane potential drop, cytochrome c release to the cytoplasm, and activation of caspase-9 and -3. By contrast, staurosporine-mediated mitochondrial damage did not induce ER stress. Knockdown of CHOP attenuated the ER stress-induced cleavage of caspase-9, which is caused by intrinsic pathway activation. Conclusions: Excessive synthesis of extracellular matrix proteins induced unfolded protein accumulation in FECD. Prolonged ER stress-mediated cell death, occurring via the intrinsic apoptotic signaling pathway, therefore might be associated with the pathogenesis of FECD.

Activation of TGF-ß signaling induces cell death via the unfolded protein response in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a slowly progressive bilateral disease of corneal endothelium in which accumulation of extracellular matrix (ECM) and loss of corneal endothelial cells (CECs) are phenotypic features. The corneal endothelium maintains corneal transparency by regulating water hydration; consequently, corneal endothelial dysfunction causes serious vision loss. The only therapy for corneal haziness due to corneal endothelial diseases, including FECD, is corneal transplantation using donor corneas, and no pharmaceutical treatment is available. We provide evidence that the expression levels of transforming growth factor-ß (TGF-ß) isoforms and TGF-ß receptors are high in the corneal endothelium of patients with FECD. A cell model based on patients with FECD shows that TGF-ß signaling induced a chronic overload of ECM proteins to the endoplasmic reticulum (ER), thereby enhancing the formation of unfolded protein and triggering the intrinsic apoptotic pathway through the unfolded protein response (UPR). We propose that inhibition of TGF-ß signaling may represent a novel therapeutic target that suppresses cell loss as well as the accumulation of ECM in FECD.

Activation of the Rho/Rho Kinase Signaling Pathway Is Involved in Cell Death of Corneal Endothelium.

Purpose: Rho kinase (ROCK) pathways control fundamental cell functions, making ROCK an important therapeutic target in several pathophysiologic conditions. The purpose of this study was to investigate whether inhibition of ROCK can suppress apoptosis of the corneal endothelium and to determine the role of ROCK signaling in regulating apoptosis. Methods: The effects of inhibitors of ROCK or myosin light chain (MLC) were evaluated in cultured monkey corneal endothelial cells (MCECs) irradiated with ultraviolet (UV) (100 J/m2) to induce apoptosis. Annexin V and TUNEL staining and Western blot for apoptosis-related proteins and focal adhesion complexes were then performed. RhoA activation was further evaluated by pull-down assays. ROCK inhibitor and caspase inhibitor effects on apoptosis were also evaluated in MCECs treated with ethylene glycol tetraacetic acid (EGTA) to induce MLC phosphorylation. Results: ROCK or MLC inhibition suppressed the caspase-3 cleavage and Annexin V and TUNEL expression typically seen during UV-mediated apoptosis of MCECs. The apoptotic stimulus activated RhoA and then induced phosphorylation of MLC via ROCK activation. EGTA-mediated phosphorylation of MLC was sufficient to induce the loss of cell contact with the substrate and subsequent apoptosis. Western blot showed that ROCK inhibition upregulated the expression of the focal adhesion complex in adhered cells, following UV stress. Conclusions: Apoptotic stimuli activated Rho/ROCK/MLC phosphorylation in the corneal endothelium, and subsequent actomyosin contraction induced apoptosis by loss of cell adhesion. ROCK inhibition suppressed MLC phosphorylation and subsequent cell death, and it counteracted the loss of cell adhesion by activating the focal adhesion complex.