Toshie Okada

Erythropoietin and its derivative protect the intestine from severe ischemia/reperfusion injury in the rat

OBJECTIVE To investigate the protective effect of erythropoietin (EPO) and its nonhematopoietic derivative (asialoEPO) against intestinal ischemia/reperfusion (I/R) injury in a rat model. METHODS The superior mesenteric artery of Wistar rats was clamped for 60 minutes and then released. The rats were divided into 4 groups (n = 15 in each group): sham operation (Sham), vehicle treatment (Vehicle), EPO treatment (EPO), and asialoEPO treatment (AsialoEPO). EPO and asialoEPO were administered subcutaneously at 1000 units/kg for 10 minutes before clamping, 30 minutes after the start of clamping, and just before declamping. This treatment was followed by determination of 72-hour survival rates, serum TNF-alpha and IL-6 levels, histologic evaluation of the small intestine, quantification of the number of apoptotic cells, and analysis of the antiapoptotic molecules Bcl-xL and XIAP by Western blotting. RESULTS The survival rates at 72 hours after I/R injury in the Sham, Vehicle, EPO, and AsialoEPO groups were 100%, 33%, 75%, and 83%, respectively (P < .05). Blood TNF-alpha and IL-6 were significantly more suppressed in the EPO and AsialoEPO groups than in the Vehicle group at 6 hours after I/R injury. Histologically, injury to villi in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. The number of apoptotic cells in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. Western blotting revealed that EPO and asialoEPO constitutively increased the expression of Bcl-xL. CONCLUSIONS EPO and asialoEPO exert a strong protective effect against intestinal I/R injury, possibly by inhibiting release of TNF-alpha and IL-6 and decreasing apoptosis.

Asialoerythropoietin has strong renoprotective effects against ischemia-reperfusion injury in a murine model

Background. The renoprotective effect of erythropoietin (EPO) and the nonhematopoietic EPO, asialoEPO was investigated in a murine ischemia-reperfusion injury (I/R) model. Methods. I/R was created by clamping the right renal pedicle for 60 min after left nephrectomy. Balb/c mice were divided into four groups (n=15 in each group): sham operation (Sham), vehicle treatment (Vehicle), EPO treatment (EPO), and asialoEPO treatment (AsialoEPO). EPO and asialoEPO were given at a dose of 500 IU/kg 30 min before I/R. Plasma creatinine (Cr), survival, and the number of apoptotic cells were analyzed. Protein expression was analyzed by Western blotting. Results. Plasma Cr level was not significantly different at 6 hr after I/R. At 24 hr after I/R, the Cr (mg/dL) levels in Sham, Vehicle, EPO, and asialoEPO were 0.13±0.01, 1.24±0.70, 0.24±0.08, and 0.25±0.13, respectively (P<0.05). The numbers of apoptotic cells in these groups were 0.1±0.1, 98.9±42.6, 3.3±0.7, and 2.9±1.6, respectively (P<0.05). Western blotting revealed that in kidney tissue of mice treated with EPO and asialoEPO, p38-MAPK and the proapoptotic molecule Bad was decreased, and the antiapoptotic molecules Bcl-xL and XIAP were increased. Survival rates at 7 days after I/R injury in the Sham, Vehicle, EPO, and AsialoEPO groups were 100%, 21.4%, 23.1%, and 53.8%, respectively (P=0.05). Conclusion. EPO and asialoEPO attenuated renal dysfunction caused by I/R in mouse kidney at the same level, but only asialoEPO improved survival.

New Antineoplastic Agent, MK615, from UME (a Variety of) Japanese Apricot Inhibits Growth of Breast Cancer Cells in vitro

Abstract:  MK615 is an extract mixture containing hydrophobic substances from Japanese apricot. In this study, the antineoplastic effects of MK615 against breast cancer cells were investigated. Two breast cancer cell lines, MDA‐MB‐468 (MDA) and MCF7, were cultured with (600, 300, and 150 μg/mL) or without MK615. After 72 hours of incubation, growth inhibition was evaluated by MTT assay. The cells were then cultured with MK615 (300 μg/mL) and morphological changes were studied by light and electron microscopy. Finally, the mechanism of the antineoplastic effect of MK615 was evaluated by cell cycle and apoptosis assay. MK615 inhibited the growth of MDA and MCF7 in a dose‐dependent manner. The percentage growth inhibition of MDA at dosages of 600, 300, and 150 μg/mL was 59.2%, 52.4%, and 23.3%, respectively, and that for MCF7 was 83.5%, 52.7%, and 16.6%, respectively. Morphological changes after MK615 treatment included massive vacuolization in the cytoplasm and apoptotic changes in the nucleus. These changes began to be apparent after at least 6 hours of incubation. Cell cycle analysis showed that MK615 increased the proportion of cells in the G2‐M phase in both MDA (7.8–11.7%) and MCF7 (8.1–18.7%), and finally both cell lines became apoptotic. The proportion of apoptotic cells increased with incubation time. MK615 effectively inhibits the growth of breast cancer cells in vitro, possibly by cell cycle modification and apotosis induction. MK615 should be further investigated as a promising anti–breast cancer agent.

Edaravone inhibits apoptosis caused by ischemia/reperfusion injury in a porcine hepatectomy model

AIM To investigate the effect of E3-methyl-1-phenyl-2-pyrazolin-5-one (Edr) on hepatic ischemia-reperfusion (I/R) injury and liver regeneration in a porcine hepatectomy model. METHODS One hour ischemia was induced by occluding the vessels and the bile duct of the right and median lobes. A 40% left hepatectomy was performed after reperfusion. Six animals received Edr (3 mg/kg per hour) intravenously and six control animals received saline just before reperfusion. Remnant liver volume, hemodynamics, aspartate aminotransferase (AST), alanine aminotransferase, lactate dehydrogenase and lactic acid, were compared between the groups. The expression of transforming growth factor-β (TGF-β1) and toll-like receptor (TRL) mRNA in hepatic tissues was examined using reverse transcription polymerase chain reaction. Apoptosis was demonstrated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. RESULTS Serum AST (P = 0.029), and toll like receptor 4 level (P = 0.043) were significantly lower after 3 h in animals receiving Edr. In addition, TUNEL staining in Edr-treated pigs showed significantly fewer hepatocytes undergoing apoptosis compared with control pigs. After 1 mo, all factors were non-significantly different between the two groups. CONCLUSION Edr is considered to reduce hepatic injury in the early stage of I/R injury in a porcine model.

Total Colectomy Improves Altered Distribution of Regulatory T Cells in Patients with Ulcerative Colitis

BackgroundIt is now generally believed that regulatory T cells (Tregs) play an important role in peripheral tolerance, and a defect in Tregs is considered one of the most important factors in the induction of various kinds of autoimmune disease including ulcerative colitis (UC). Here, we examined the change in frequency of Tregs phenotype in five patients with UC whose condition had not been controllable by conventional conservative therapy and who were scheduled for total colectomy.AimsThe aim of this study was to elucidate the role of Tregs in the pathogenesis of UC in a clinical setting.MethodsPeripheral blood mononuclear cells (PBMCs) were obtained from five patients with UC, and the change in frequency of Tregs was analyzed by flow cytometry before total colectomy and on postoperative days 1, 7, and 20. Tregs were defined as CD4+ CD25+ CD45RA+ T cells, and data (%) were expressed as frequency of Tregs/CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) from healthy blood donors (n = 5) and from patients undergoing other types of major surgery (n = 5) were used as controls.ResultsComparison with normal subjects showed that generation of Tregs was suppressed in UC patients before they underwent total colectomy (0.95 versus 5.06; P = 0.009). The frequency of Tregs increased shortly after total colectomy. The frequency at postoperative days 1, 7, and 20 was 3.81%, 8.13%, and 3.76%, respectively. There were significant differences in the change in frequency in the period before surgery and postoperative day 1, between postoperative days 1 and 7, and between days 7 and 20.ConclusionsElimination of targeted antigens residing in the colorectal mucosa by total colectomy improved the suppressed distribution of Tregs in UC patients. The present study provides the first direct clinical evidence that Tregs play a pivotal role in the pathogenesis of UC.

Influence of Tumor Peroxisome Proliferator-Activated Receptor γ and δ Expression on Postoperative Mortality of Patients Undergoing Colorectal Cancer Surgery

Purpose: To evaluate the influence of peroxisome proliferator-activated receptor γ (PPAR γ) and δ (PPAR δ) expression on postoperative mortality of patients with colorectal cancer (CRC). Methods: Optimal cutoff values were determined for each relative expression ratio (RER) (RER = PPAR expression of tumor/PPAR expression of normal mucosa) of PPAR, and patients were divided into two groups as follows (PPAR staging): patients with elevated RERs of PPAR γ (> 2.0) or PPAR δ (> 1.0) were termed Group H, and patients showing none of these elevated RERs of PPARs were termed Group L. Prognostic significance was analyzed by univariate and Kaplan–Meier analyses. Results: In total, 26 CRC patients were studied. Univariate analysis revealed that PPAR γ (> 2.0/ ≤ 2.0) (odds ratio, 11.43; 95% C.I., 1.154–113.1; p =. 0373), PPAR δ (> 1.0/ ≤ 1.0) (odds ratio, 15.00; 95% C.I., 1.503–149.7; p =. 0210) and PPAR staging (H/L) (odds ratio, 63.00; 95% C.I., 4.956–800.8; p =. 0014) were significant predictors of postoperative mortality. Kaplan–Meier analysis revealed that the survival curve of patients with CRC was clearly divided by PPAR staging (log rank test, p <.0001). Conclusions: Evaluation of PPAR γ and δ expression is useful for predicting postoperative mortality in patients undergoing CRC surgery.