RESUMEN
BACKGROUND: Growing evidence suggests that the Wnt/ß-catenin signaling cascade is implicated in the control of stem cell activity, cell proliferation, lineage commitment, and cell survival during normal development and tissue regeneration of the gastrointestinal epithelium. The roles of this signaling cascade in stimulation of cell proliferation after massive small bowel resection are unknown. The purpose of this study was to evaluate the role of Wnt/ß-catenin signaling during late stages of intestinal adaptation in a rat model of short bowel syndrome (SBS). METHODS: Male rats were divided into two groups: sham rats underwent bowel transection and SBS rats underwent a 75 % bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's digital gene expression analysis was used to determine Wnt/ß-catenin signaling gene expression profiling. Twelve Wnt/ß-catenin-related genes and ß-catenin protein expression were determined using real-time PCR, western blotting and immunohistochemistry. RESULTS: From the total number of 20,000 probes, 20 genes related to Wnt/ß-catenin signaling were investigated. From these genes, seven genes were found to be up-regulated and eight genes to be down-regulated in SBS vs. sham animals with a relative change in gene expression level of 20 % or more. From 12 genes determined by real-time PCR, nine genes were down-regulated in SBS rats compared to control animals including target gene c-Myc. SBS rats also showed a significant decrease in ß-catenin protein compared to control animals. CONCLUSION: Two weeks following massive bowel resection in rats, Wnt/ß-catenin signaling pathway is inhibited. In addition, it appears that cell differentiation rather than proliferation is most important in the late stages of intestinal adaptation.
Asunto(s)
Regulación hacia Abajo/genética , Intestino Delgado/cirugía , Síndrome del Intestino Corto/cirugía , Transducción de Señal/genética , Proteínas Wnt/genética , beta Catenina/genética , Adaptación Fisiológica/genética , Análisis de Varianza , Animales , Apoptosis/genética , Western Blotting/métodos , Proliferación Celular , Modelos Animales de Enfermedad , Expresión Génica/genética , Masculino , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Síndrome del Intestino Corto/genéticaRESUMEN
PURPOSE: The primary toxic effects of methotrexate (MTX) are myelosuppression and/or intestinal mucositis. The objective of the present study is to investigate the effect of MTX on germ cell apoptosis and spermatogenesis in a rat. METHODS: Male Sprague-Dawley rats were divided into three experimental groups: control rats treated with vehicle; MTX-2 rats treated with one dose (20 µg/kg) of MTX given IP and killed on the second day; and MTX rats treated with IP MTX (20 µg/kg) and killed on day 4. Johnsen's criteria and the number of germinal cell layers in the testes were used to categorize the spermatogenesis. TUNEL assay was used to determine germ cell apoptosis. Western blotting was used to determine Bax and Bcl-2 protein levels. Statistical analysis was performed using the non-parametric Kruskal-Wallis ANOVA test, with p less than 0.05 considered statistically significant. RESULTS: On day 2, MTX-treated animals demonstrated minimal changes in the histological parameters of spermatogenesis, but germ cell apoptosis increased significantly (threefold increase, p = 0.002) compared to control rats. On day 4, MTX-treated rats demonstrated a trend toward a decrease in germ cell apoptosis, compared to day 2, and showed histological signs of impaired spermatogenesis (decreased number of germ cell layers and Johnsen's criteria). A significant increase in cell apoptosis in MTX-treated rats was correlated with higher Bax/Bcl-2 protein levels. CONCLUSIONS: MTX induced germ cell apoptosis and impaired spermatogenesis in rat testes.