| With the global climate deterioration, livestock and poultry are facing many problems due to various stress. Stress may lead to decreasing feed intake and production performance, and also result in low reproductive effeciency, which has a negative effect on economic benefit from livestock and poultry production. Therefore, it is necessary and exigent to further study anti-stress mechanism of animals.Rats subjected to water immersion and restraint stress (WRS) are widely used in studying gastric and duodenal mucosa damage. This is an desirable experimental animal model due to characteristics of simpleness, efficiency and repeatability. IGF-1/PTEN/AKT/FOXO signaling pathway plays some important physiological roles in several animal tissues and cells. For example, IGF-1/PTEN/AKT/FOXO signaling pathway has been reported to participate in the tissue and cell demage through regulation of cell apoptosis. In stress-induced gastric ulcer, IGF-1and Akt proteins play an anti-stress role through inhibiting mucosal cell apoptosis. In addition, IGF-1, PTEN, Akt, and FoxO1proteins are also involved in spermatogenesis of animals in embryonic developmental and neonatal stage through several pathways.In current study, male rats subjected to WRS were used as the research object, investigating the effect of WRS on gastric and duodenal mucosa. We also studied the effect of WRS on male reproductive functions by morphology and radioimmunoassay. Besides, TUNEL staining and caspase-3activity assay were used to analyze the effect of WRS on cell apoptosis in gastric mucosa, duodenal mucosa and testicular tissue. Moreover, we detected the expressions and localizations of IGF-1, PTEN, total Akt and FoxO proteins in gastric mucosa, duodenal mucosa and testicular tissue in WRS and normal rats. At the same time, the related gene expressions were also investigated in gastric mucosa, duodenal mucosa, and testicular tissue by using the real time-PCR.1. Age-dependent expression of forkhead box O proteins in the duodenum of rats The O subfamily of forkhead box (FoxO) proteins is the downstream effector of the insulin like growth factor-1/phosphoinositide3-kinase/protein kinase B (IGF-1/PI3K/PKB) signal pathway. The objective of the present study was to examine the expressions of3members of FoxO proteins, FoxOl, FoxO3a and FoxO4in the duodenum of Sprague-Dawley rats at different ages. Result demonstrated that the expression of Fox04in rat duodenum showed an age-dependent manner. At Day21, there were no detectable localization and expression of FoxO4in the duodenum, while at Months2and6, localization and expression of FoxO4were distinct. In addition, FoxO4staining was primarily concentrated in the cell nuclei of the lamina propria around the intestinal gland of the duodenum in2-month-old rats, but was not detectable in the same area in6-month-old rats. Our results showed also that although FoxO3a existed in the cytoplasm of the lamina propria at a low level at the2-and6-month marks, it was still not detectable at Day21. Besides, FoxO1was not detectable in all parts and stages. Taken together, our findings suggested that the cell-specific and age-dependent expressional patterns of FoxO4and FoxO3a proteins in the duodenum, which implied some roles in the development and growth performance of the rat duodenum.2. Effect of the IGF-1/PTEN/Akt/FoxO signaling pathway on the development and healing water immersion and restraint stress-induced gastric ulcers in ratsIGF-1and Akt proteins have been reported to exhibit gastroprotective effects by reducing WRS-induced gastric mucosal cellular apoptosis. To confirm whether the IGF-1/PTEN/Akt/FoxO signaling pathway is effective in protecting against gastric ulcers, my current study was conducted to examine the expression and localization of IGF-1, PTEN, Akt, and FoxO proteins, caspase-3activity and the number of apoptotic cells in gastric mucosa of rats subjected to WRS. Our results demonstrated that WRS induced gastric ulcers by enhancing cell apoptosis in rat gastric mucosa. In addition, in normal rat gastric mucosa, PTEN, total Akt and FoxOl were stained mainly in the cell cytoplasm of fundic glands in lamina propria close to the muscularis mucosa. Also, strong staining of IGF-1, FoxO3a, and FoxO4in the gastric mucosa were primarily concentrated in the cell cytoplasm of the fundic glands in whole lamina propria. However, in rat gastric ulcer, IGF-1, total Akt, FoxO1, Fox03a, and FoxO4were localized in proximity to the base of the ulcer margin and also present in the granulation tissue of the gastric ulcer. Meanwhile, in rat gastric ulcer, mRNA transcript levels of IGF-1, PTEN, Akt-1, Akt-2, FoxOa and FoxO4were up-regulated in the gastric ulcer margin, with a peak between the4th and8th day after 7hours of WRS. In conclusion, our results imply that the IGF-1/PTEN/Akt/FoxO signaling pathway plays some role(s) in protecting against ulcer through regulation of cellular apoptosis in the development and healing of rat gastric ulcers.3. Effect of the IGF-1/PTEN/Akt/FoxO signaling pathway in duodenal mucosa of rats subjected to water immersion and restraint stressIGF-1/PTEN/Akt/FoxO signaling pathway has been reported to exhibit gastroprotective effects by reducing WRS-induced gastric mucosal cell apoptosis. To confirm whether the IGF-1/PTEN/Akt/FoxO signaling pathway plays role(s) in duodenal mucosa of rats after WRS, my current study was conducted to examine the expression and localization of IGF-1, PTEN, Akt, and FoxO proteins, caspase-3activity, and the number of apoptotic cells in duodenum mucosa of rats subjected to WRS. The results indicated that WRS enhanced cell apoptosis in duodenal mucosa. In addition, in normal rat, PTEN was stained mainly in the cellular cytoplasm of the duodenal glands and lamina propria of villi. IGF-1and total Akt were observed in the cellular cytoplasm of the duodenal glands. Also, total Akt was found in the cellular cytoplasm of the myenteric plexus. FoxO3a and FoxO4were primarily concentrated in the cellular cytoplasm of the lamina propria. Particularly, in duodenal mucosa of rat subjected to WRS, PTEN, Fox03a, and FoxO4were also localized in the cellular cytoplasm of lamina propria of restituted villi. Meanwhile, mRNA transcript levels of IGF-1, PTEN, Aktl, Akt2, FoxO3, and FoxO4were up-regulated in duodenal mucosa, with a peak between the4th and8th day after7hours of WRS. Furthermore, the results also suggested that Akt3mRNA transcript level in rats after WRS showed no significant difference in the duodenal mucosa, compared with non-WRS group. Collectively, our results implied that the IGF-1/PTEN/Akt/FoxO signaling pathway was effective in regulation of cellular apoptosis in duodenal mucosa of rats after WRS.4. Study on IGF-1/PTEN/Akt/FoxO signaling pathway in male reproduction of rats subjected to water immersion and restraint stressTo confirm whether the IGF-1/PTEN/Akt/FoxO signaling pathway plays role(s) in the male reproduction of rats after WRS, our current study was conducted to exam sperm morphology and abnormal rates, and serum testosterone concentrations after WRS. Also, we analyzed expressions and localizations of IGF-1, PTEN, Akt, and FoxO proteins, and cell apoptosis in testis of rats subjected to WRS. Our results indicated that WRS enhanced sperm abnormal rates, serum testosterone concentrations, and cell apoptosis in the testis. In addition, IGF-1and FoxO1proteins were mainly detected in sperm cytoplasm in late spermatogenesis and FoxO1protein was also localized in the leydig cytoplasm. PTEN and total Akt proteins were mainly expressed in the leydig cytoplasm and spermatogonium cytoplasm. PTEN protein was also found in vascular endothelial cells. Meanwhile, real-time PCR method was used to investigate mRNA transcripts in the testis after WRS. Results showed that IGF-1, PTEN, Aktl, Akt2, FoxO3, and FoxO4gene expressions were up-regulated, with a peak after7hours of WRS. However, in recovery stage, gene expressions of IGF-1, PTEN, Aktl, Akt2, FoxO3, and FoxO4were down-regulated gradually to normal levels. Collectively, our results demonstrated that WRS caused sperm damage and increased apoptosis in the testis. Our study also implied that the IGF-1/PTEN/Akt/FoxO signaling pathway played some roles against stress in the testis of rats after WRS.In conclusions, WRS induced different damages in gastric mucosa, duodenal mucosa, and testicular tissue in rat. Damage formation and repair was a complex physiological process and multiple factors involved. Our results showed that IGF-1/PTEN/Akt/FoxO signaling pathway played potential roles against stress through regulating the cell apoptosis in the WRS-induced tissue damage formation and repair. However, the specific regulation mechanisms still need father research. |
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