IGF-1/PTEN/Akt/FoxO信号通路在大鼠水浸束缚应激中作用研究
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摘要
随着全球气候变化,各种应激给畜禽生产带来了极大的威胁,导致畜禽采食量减少,产量下降,繁殖性能降低,严重影响畜禽生产的经济效益,因此,深入研究动物机体的抗应激机制具有重要意义。
水浸束缚应激(WRS)大鼠是一个广泛应用于研究胃和十二指肠黏膜损伤机制的动物模型,具有简单、高效和重复性高等特点,是一个理想的实验动物模型。建模之前,饥饿处理24h,然后对大鼠采取束缚处理,并浸泡于温度20士2℃的水里,水位达到大鼠剑突部。IGF-1/PTEN/Akt/FoxO信号通路在许多动物组织和细胞内发挥重要生理效应,如通过调控细胞凋亡参与组织和细胞损伤机制。在应激性胃溃疡中,IGF-1和Akt蛋白通过抑制黏膜组织细胞凋亡发挥抗应激作用。同时,IGF-1, PTEN, Akt和FoxO1通过各种途径参与了雄性动物胚胎发育和新生阶段睾丸精子发生过程。
本论文以雄性WRS大鼠为研究对象,重点研究了WRS处理对大鼠胃和十二指肠黏膜结构的影响；通过形态学和放射免疫测定研究了WRS处理对雄性大鼠生殖机能的影响；通过TUNEL和caspase-3活性检测了WRS处理对大鼠胃、十二指肠黏膜以及睾丸组织细胞凋亡的影响；以免疫组织化学和免疫印迹法分析了IGF-1、PTEN、总Akt和FoxO蛋白在正常和WRS大鼠胃、十二指肠黏膜以及睾丸组织表达和定位情况；同时我们还通过实时荧光定量PCR研究了WRS大鼠胃、十二指肠黏膜以及睾丸组织IGF-1/PTEN/Akt/FoxO信号通路相关基因的表达水平。
1.FoxO蛋白在大鼠十二指肠表达定位研究
FoxO蛋白是IGF-1/PI3K/Akt信号通路下游重要的靶蛋白。本文旨在揭示FoxO家族中的FoxO1、FoxO3a和FoxO4三种蛋白在不同年龄Sprague-Dawley大鼠十二指肠中的表达情况。结果显示,Fox04在大鼠十二指肠中呈年龄依赖性表达。21日龄时FoxO4在大鼠十二指肠中没有检测到表达,2月龄和6月龄时FoxO4有不同的定位和表达。Foxo4在2月龄大鼠十二指肠肠腺附近的固有层细胞核和细胞质中表达,但6月龄时只在固有层细胞质中表达,并且2月龄的表达量显著高于6月龄。另外,FoxO3a在2月龄和6月龄的固有层细胞质都有表达,表达量差异不显著。类似地,FoxO3a在21日龄大鼠十二指肠也没有检测到表达。此外,在各阶段大鼠十二指肠都没有检测到FoxO1的表达。综上所述,我们认为大鼠十二指肠中FoxO4和Fox03a的表达具有细胞特异性和年龄依赖性特征,提示其在大鼠十二指肠的生长过程中发挥作用。
2. IGF-1/PTEN/Akt/FoxO信号通路在大鼠应激性胃溃疡发生和愈合中的作用研究
在应激性胃溃疡中,IGF-1和Akt蛋白通过抑制黏膜组织细胞凋亡发挥抗应激作用。为了探明IGF-1/PTEN/Akt/FoxO信号通路是否在应激性胃溃疡中发挥抗应激作用,本试验研究了IGF-1、PTEN、Akt和FoxO蛋白在胃黏膜组织的表达与定位,检测了胃溃疡各时期胃黏膜组织的细胞凋亡水平。结果表明：WRS处理诱使大鼠胃黏膜发生溃疡,并导致胃黏膜细胞凋亡水平升高。另外,通过免疫组织化学方法检测发现,正常大鼠胃黏膜组织中,PTEN、总Akt和FoxO1蛋白主要定位于靠近黏膜肌层的胃底腺细胞质中,IGF-1、FoxO3a和FoxO4蛋白广泛定位于黏膜固有层内腺体细胞质中；在胃溃疡组织中,IGF-1、总Akt、FoxO3a和FoxO4蛋白在溃疡基部和肉芽组织都有表达,而PTEN和FoxO1蛋白在WRS前后定位模式没有变化。通过实时荧光定量PCR研究发现,WRS导致IGF-1、PTEN、Aktl、Akt2、FoxO3和FoxO4基因表达水平升高,并且在WRS结束后4-8d达到峰值,而Akt3和FoxO1基因表达水平在WRS前后没有变化。综上所述,结果表明IGF-1/PTEN/Akt/FoxO信号通路可能通过参与调控黏膜组织细胞凋亡,在应激性胃溃疡发生和愈合中发挥重要作用。
3. IGF-1/PTEN/Akt/FoxO信号通路在水浸束缚应激大鼠十二指肠中的作用研究
IGF-1/PTEN/Akt/FoxO信号通路可以通过抑制WRS处理诱导的胃黏膜细胞凋亡,从而达到胃黏膜保护效应。为了确认IGF-1/PTEN/Akt/FoxO信号通路的相关效应是否适用于十二指肠,在本试验中,我们分析了IGF-1、PTEN、Akt和FoxO蛋白在WRS大鼠十二指肠黏膜的表达与定位；检测了WRS处理各时期十二指肠黏膜的细胞凋亡水平。结果表明WRS可促进十二指肠黏膜细胞凋亡。另外,在正常大鼠十二指肠黏膜中,PTEN主要定位于十二指肠腺和绒毛固有层细胞胞质中；IGF-1和Akt主要定位于肠腺细胞质中,Akt在肌间神经丛细胞质也有表达；FoxO3a和FoxO4主要定位于绒毛固有层细胞质中。在WRS大鼠模型的十二指肠黏膜中,PTEN、FoxO3a和FoxO4在新生绒毛固有层细胞质中有表达。此外,WRS处理导致大鼠十二指肠黏膜中IGF-1、 PTEN、Akt1、Akt2、FoxO3和FoxO4基因表达水平升高,并且在WRS处理后的第4和8d分别达到峰值。不过,WRS处理没有引起大鼠十二指肠黏膜Akt3基因表达水平发生变化。综上所述,我们认为在WRS大鼠十二指肠中,IGF-1/PTEN/Akt/FoxO信号通路可能参与了调控黏膜细胞凋亡。
4.水浸束缚应激对雄性大鼠生殖机能的影响及IGF-1/PTEN/Akt/FoxO信号通路在其中的作用
本试验目的是研究WRS处理对雄性大鼠生殖机能的影响及IGF-1/PTEN/Akt/FoxO信号通路在其中的作用。我们研究了WRS处理对大鼠附睾精子形态、畸形率以及血清睾酮浓度的影响；分析了IGF-1、PTEN、Akt和FoxO蛋白在大鼠睾丸组织的表达与定位；检测了WRS处理各时期睾丸组织的细胞凋亡水平。结果表明：WRS处理导致了大鼠附睾精子畸形率、血清睾酮浓度和睾丸组织细胞凋亡水平升高。另外,通过免疫组织化学方法检测发现,IGF-1和FoxO1蛋白广泛表达于精子发生后期的精子细胞质中,并且FoxO1蛋白在睾丸间质细胞质中也有表达；PTEN和总Akt蛋白主要定位于睾丸间质细胞和精原细胞胞质中,其中PTEN在血管内皮细胞也有表达。通过实时荧光定量PCR研究发现,WRS处理导致了大鼠睾丸组织IGF-1, PTEN, FoxO1和Akt2基因表达量升高,在WRS处理7h后达到峰值；在恢复阶段,这些基因表达量逐渐降到正常水平。综上所述,WRS处理造成了大鼠睾丸精子的损伤,并导致睾丸组织细胞凋亡水平升高,IGF-1/PTEN/Akt/FoxO信号通路在WRS大鼠睾丸组织中可能发挥了抗应激相关作用。
综上所述,WRS处理对大鼠胃、十二指肠黏膜以及睾丸组织都造成了不同程度的损伤。不过,组织损伤的发生和修复是一个复杂的生理过程,多个因素在该过程中起作用。本研究结果表明IGF-1/PTEN/Akt/FoxO信号通路可能通过调控细胞凋亡参与组织损伤的发生和修复,从而发挥抗应激相关的作用机制。然而,IGF-1/PTEN/Akt/FoxO信号通路在其中的具体调控机制还需要进一步研究。
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 rats
IGF-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 stress
IGF-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 stress
To 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.
水浸束缚应激(WRS)大鼠是一个广泛应用于研究胃和十二指肠黏膜损伤机制的动物模型,具有简单、高效和重复性高等特点,是一个理想的实验动物模型。建模之前,饥饿处理24h,然后对大鼠采取束缚处理,并浸泡于温度20士2℃的水里,水位达到大鼠剑突部。IGF-1/PTEN/Akt/FoxO信号通路在许多动物组织和细胞内发挥重要生理效应,如通过调控细胞凋亡参与组织和细胞损伤机制。在应激性胃溃疡中,IGF-1和Akt蛋白通过抑制黏膜组织细胞凋亡发挥抗应激作用。同时,IGF-1, PTEN, Akt和FoxO1通过各种途径参与了雄性动物胚胎发育和新生阶段睾丸精子发生过程。
本论文以雄性WRS大鼠为研究对象,重点研究了WRS处理对大鼠胃和十二指肠黏膜结构的影响；通过形态学和放射免疫测定研究了WRS处理对雄性大鼠生殖机能的影响；通过TUNEL和caspase-3活性检测了WRS处理对大鼠胃、十二指肠黏膜以及睾丸组织细胞凋亡的影响；以免疫组织化学和免疫印迹法分析了IGF-1、PTEN、总Akt和FoxO蛋白在正常和WRS大鼠胃、十二指肠黏膜以及睾丸组织表达和定位情况；同时我们还通过实时荧光定量PCR研究了WRS大鼠胃、十二指肠黏膜以及睾丸组织IGF-1/PTEN/Akt/FoxO信号通路相关基因的表达水平。
1.FoxO蛋白在大鼠十二指肠表达定位研究
FoxO蛋白是IGF-1/PI3K/Akt信号通路下游重要的靶蛋白。本文旨在揭示FoxO家族中的FoxO1、FoxO3a和FoxO4三种蛋白在不同年龄Sprague-Dawley大鼠十二指肠中的表达情况。结果显示,Fox04在大鼠十二指肠中呈年龄依赖性表达。21日龄时FoxO4在大鼠十二指肠中没有检测到表达,2月龄和6月龄时FoxO4有不同的定位和表达。Foxo4在2月龄大鼠十二指肠肠腺附近的固有层细胞核和细胞质中表达,但6月龄时只在固有层细胞质中表达,并且2月龄的表达量显著高于6月龄。另外,FoxO3a在2月龄和6月龄的固有层细胞质都有表达,表达量差异不显著。类似地,FoxO3a在21日龄大鼠十二指肠也没有检测到表达。此外,在各阶段大鼠十二指肠都没有检测到FoxO1的表达。综上所述,我们认为大鼠十二指肠中FoxO4和Fox03a的表达具有细胞特异性和年龄依赖性特征,提示其在大鼠十二指肠的生长过程中发挥作用。
2. IGF-1/PTEN/Akt/FoxO信号通路在大鼠应激性胃溃疡发生和愈合中的作用研究
在应激性胃溃疡中,IGF-1和Akt蛋白通过抑制黏膜组织细胞凋亡发挥抗应激作用。为了探明IGF-1/PTEN/Akt/FoxO信号通路是否在应激性胃溃疡中发挥抗应激作用,本试验研究了IGF-1、PTEN、Akt和FoxO蛋白在胃黏膜组织的表达与定位,检测了胃溃疡各时期胃黏膜组织的细胞凋亡水平。结果表明：WRS处理诱使大鼠胃黏膜发生溃疡,并导致胃黏膜细胞凋亡水平升高。另外,通过免疫组织化学方法检测发现,正常大鼠胃黏膜组织中,PTEN、总Akt和FoxO1蛋白主要定位于靠近黏膜肌层的胃底腺细胞质中,IGF-1、FoxO3a和FoxO4蛋白广泛定位于黏膜固有层内腺体细胞质中；在胃溃疡组织中,IGF-1、总Akt、FoxO3a和FoxO4蛋白在溃疡基部和肉芽组织都有表达,而PTEN和FoxO1蛋白在WRS前后定位模式没有变化。通过实时荧光定量PCR研究发现,WRS导致IGF-1、PTEN、Aktl、Akt2、FoxO3和FoxO4基因表达水平升高,并且在WRS结束后4-8d达到峰值,而Akt3和FoxO1基因表达水平在WRS前后没有变化。综上所述,结果表明IGF-1/PTEN/Akt/FoxO信号通路可能通过参与调控黏膜组织细胞凋亡,在应激性胃溃疡发生和愈合中发挥重要作用。
3. IGF-1/PTEN/Akt/FoxO信号通路在水浸束缚应激大鼠十二指肠中的作用研究
IGF-1/PTEN/Akt/FoxO信号通路可以通过抑制WRS处理诱导的胃黏膜细胞凋亡,从而达到胃黏膜保护效应。为了确认IGF-1/PTEN/Akt/FoxO信号通路的相关效应是否适用于十二指肠,在本试验中,我们分析了IGF-1、PTEN、Akt和FoxO蛋白在WRS大鼠十二指肠黏膜的表达与定位；检测了WRS处理各时期十二指肠黏膜的细胞凋亡水平。结果表明WRS可促进十二指肠黏膜细胞凋亡。另外,在正常大鼠十二指肠黏膜中,PTEN主要定位于十二指肠腺和绒毛固有层细胞胞质中；IGF-1和Akt主要定位于肠腺细胞质中,Akt在肌间神经丛细胞质也有表达；FoxO3a和FoxO4主要定位于绒毛固有层细胞质中。在WRS大鼠模型的十二指肠黏膜中,PTEN、FoxO3a和FoxO4在新生绒毛固有层细胞质中有表达。此外,WRS处理导致大鼠十二指肠黏膜中IGF-1、 PTEN、Akt1、Akt2、FoxO3和FoxO4基因表达水平升高,并且在WRS处理后的第4和8d分别达到峰值。不过,WRS处理没有引起大鼠十二指肠黏膜Akt3基因表达水平发生变化。综上所述,我们认为在WRS大鼠十二指肠中,IGF-1/PTEN/Akt/FoxO信号通路可能参与了调控黏膜细胞凋亡。
4.水浸束缚应激对雄性大鼠生殖机能的影响及IGF-1/PTEN/Akt/FoxO信号通路在其中的作用
本试验目的是研究WRS处理对雄性大鼠生殖机能的影响及IGF-1/PTEN/Akt/FoxO信号通路在其中的作用。我们研究了WRS处理对大鼠附睾精子形态、畸形率以及血清睾酮浓度的影响；分析了IGF-1、PTEN、Akt和FoxO蛋白在大鼠睾丸组织的表达与定位；检测了WRS处理各时期睾丸组织的细胞凋亡水平。结果表明：WRS处理导致了大鼠附睾精子畸形率、血清睾酮浓度和睾丸组织细胞凋亡水平升高。另外,通过免疫组织化学方法检测发现,IGF-1和FoxO1蛋白广泛表达于精子发生后期的精子细胞质中,并且FoxO1蛋白在睾丸间质细胞质中也有表达；PTEN和总Akt蛋白主要定位于睾丸间质细胞和精原细胞胞质中,其中PTEN在血管内皮细胞也有表达。通过实时荧光定量PCR研究发现,WRS处理导致了大鼠睾丸组织IGF-1, PTEN, FoxO1和Akt2基因表达量升高,在WRS处理7h后达到峰值；在恢复阶段,这些基因表达量逐渐降到正常水平。综上所述,WRS处理造成了大鼠睾丸精子的损伤,并导致睾丸组织细胞凋亡水平升高,IGF-1/PTEN/Akt/FoxO信号通路在WRS大鼠睾丸组织中可能发挥了抗应激相关作用。
综上所述,WRS处理对大鼠胃、十二指肠黏膜以及睾丸组织都造成了不同程度的损伤。不过,组织损伤的发生和修复是一个复杂的生理过程,多个因素在该过程中起作用。本研究结果表明IGF-1/PTEN/Akt/FoxO信号通路可能通过调控细胞凋亡参与组织损伤的发生和修复,从而发挥抗应激相关的作用机制。然而,IGF-1/PTEN/Akt/FoxO信号通路在其中的具体调控机制还需要进一步研究。
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 rats
IGF-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 stress
IGF-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 stress
To 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.
引文
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