Abstract
The aim of this study was to investigate how different durations of fasting affect autophagy levels by analyzing Beclin 1 (BECN1) expression in rat small intestinal tissues using immunohistochemistry. Thirty adult male Wistar Albino rats were allocated into five groups (n=6 each). After the last feeding, the fasting groups were classified according to fasting durations of 12, 24, 36, and 48 hours, and water was provided without restriction. At the end of the experimental period, small intestine samples were collected from the rats in each group. Tissue samples fixed in Bouin’s solution were embedded in paraffin. To detect BECN1 immunolocalization, the tissues were subjected to immunohistochemical analysis using the streptavidin–biotin technique. BECN1 immunoreactivity was analyzed semi-quantitatively. At the light-microscopy level, no structural changes were detected in any region of the small intestine (duodenum, jejunum, ileum) when the fasting groups were compared with the control group. BECN1 expression was found to be localized in all groups across all small intestinal sections in villus epithelial cells, glandular epithelial cells, and the tunica muscularis layer. In all groups, BECN1 immunolocalization was observed in all regions of the small intestine, specifically in villus epithelial cells, glandular epithelial cells, and the tunica muscularis layer, with a moderate (++) staining intensity. No statistically significant difference in staining intensity was recorded between the groups (p>0.05). These results indicate that short- to moderate-term fasting (12–48 hours) does not produce a measurable change in basal BECN1-associated autophagy in the rat small intestine.
Keywords
Ethical Statement
All procedures performed on the rats were reviewed and approved by the Local Animal Experiments Ethics Committee of Burdur Mehmet Akif Ersoy. University.
Supporting Institution
Burdur Mehmet Akif Ersoy University Scientific Research Projects Coordination Unit
Project Number
0516-YL18
Thanks
This research project was supported by the Scientific Research Projects Coordination Unit of Burdur Mehmet Akif Ersoy University under project number 0516-YL18.
References
- Choi JH, Cho YS, Ko YH, Hong SU, Park JH, Lee MA. (2014). Absence of autophagy-related proteins expression is associated with poor prognosis in patients with colorectal adenocarcinoma. Gastroenterol Res Pract 2014; Article ID 179586. https://doi.org/10.1155/2014/179586
- Comincini S, Manai F, Meazza C, Pagani S, Martinelli C, Pasqua N, et al. Identification of Autophagy-Related Genes and Their Regulatory miRNAs Associated with Celiac Disease in Children. Int J Mol Sci 2017; 18: Article number 391. https://doi.org/10.3390/ijms18020391
- Foerster EG, Mukherjee T, Cabral-Fernandes L, Rocha JDB, Girardin SE, Philpott D.J. How autophagy controls the intestinal epithelial barrier. Autophagy 2022; 18: 86-103. https://doi.org/10.1080/15548627.2021.1909406
- Geng QR, Xu DZ, He LJ, Lu JB, Zhou ZW, Zhanet YQ, et al. Beclin-1 expression is a significant predictor of survival in patients with lymph nodepositive gastric cancer. PLoS One 2012; 7: e45968. https://doi.org/10.1371/journal.pone.0045968
- Giatromanolaki A, Sivridis E, Mendrinos S, Koutsopoulos AV, Koukourakis MI. Autophagy proteins in prostate cancer: relation with anaerobic metabolism and Gleason score. Urol Oncol 2014; 32: 39.e11-39.e18. https://doi.org/10.1016/j.urolonc.2013.04.003
- He C, Levine B. The Beclin 1 interactome. Curr Opin Cell Biol. 2010; 22: 140-149. https://doi.org/10.1016/j.ceb.2010.01.001
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- Kang R, Zeh H.J, Lotze M.T, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011; 18: 571–580. https://doi.org/10.1038/cdd.2010.191
- Kim HS, Lee SH, Do SI, Lim S.J, Park YK, Kim YW. Clinicopathologic correlation of beclin-1 expression in pancreatic ductal adenocarcinoma. Pathol Res Pract 2011; 207; 247-252.
- Levine B, Liu R, Dong X, Zhong Q. Beclin orthologs: integrative hubs of cell signaling, membrane trafficking, and physiology. Trends Cell Biol. 2015; 25: 533-544. https://doi.org/10.1016/j.prp.2011.02.007
- Naama M, Telpaz S, Awad A, Ben-Simon S, Harshuk-Shabso S, Modilevsky S, et al. Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress. Cell Host Microbe 2023; 31: 433-446. https://doi.org/10.1016/j.chom.2023.01.006
- Saha K, Ganapathy AS, Wang A, Morris NM, Suchanec E, Ding W, et al. Autophagy Reduces the Degradation and Promotes Membrane Localization of Occludin to Enhance the Intestinal Epithelial Tight Junction Barrier against Paracellular Macromolecule Flux. JCC 2023; 17: 433–449. https://doi.org/10.1093/ecco-jcc/jjac148
- Schläfli AM, Berezowska S, Adams O, Langer R, Tschan MP. Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry. Eur J Histochem. 2015; 59: 173-179.
- https://doi: 10.4081/ejh.2015.2481
- Song H, Xia SL, Liao C, Li YL, Wang YF , Li TP, et al. Genes encoding Pir51, Beclin 1, RbAp48 and aldolase b are up or down-regulated in human primary hepatocellular carcinoma. World J Gastroenterol 2004; 10: 509-513. https://doi: 10.3748/wjg.v10.i4.509
- Tran S, Fairlie WD, Lee EF. BECLIN1: Protein Structure, Function and Regulation. Cells 2021; 10: 1-16. https://doi.org/10.3390/cells10061522
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- Wong M, Ganapathy AS, Suchanec E, Laidler L, Ma T, Nighot P. Intestinal epithelial tight junction barrier regulation by autophagy-related protein ATG6/beclin 1. Am J Physiol Cell Physiol 2019; 316: C753–C765. https://doi.org/10.1152/ajpcell.00246.2018
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- Zhao H, Xi H, Wei B, Cai A, Wang T, Wang Y, et al. Expression of decorin in intestinal tissues of mice with inflammatory bowel disease and its correlation with autophagy. Exp Ther Med 2016; 12: 3885-3892. https://doi.org/10.3892/etm.2016.3908
Abstract
Bu çalışmanın amacı, immünohistokimyasal düzeyde sıçan ince bağırsak dokularında Beclin 1 (BECN1) ekspresyonunu analiz ederek farklı açlık sürelerinin otofaji düzeylerini nasıl etkilediğini araştırmaktı. Otuz yetişkin erkek Wistar Albino sıçan beş gruba ayrıldı (her grupta 6 sıçan). Son beslenmeden sonra, deney hayvanları 12, 24, 36 ve 48 saatlik açlık sürelerine göre sınıflandırıldı ve su kısıtlama olmaksızın verildi. Deney süresinin sonunda, her gruptaki sıçanlardan ince bağırsak örnekleri alındı. Bouin solüsyonunda tespit edilen doku örnekleri parafine gömüldü. BECN1 immünolokalizasyonunu tespit etmek için dokular, streptavidin-biotin tekniği kullanılarak immünohistokimyasal analize tabi tutuldu. BECN1 immünoreaktivitesi yarı kantitatif olarak analiz edildi. Işık mikroskobu düzeyinde, açlık grupları kontrol grubu ile karşılaştırıldığında ince bağırsağın hiçbir bölgesinde (duodenum, jejunum, ileum) yapısal değişiklik tespit edilmedi. BECN1 ekspresyonunun tüm gruplarda tüm ince bağırsak kesitlerinde villus epitel hücrelerinde, glandüler epitel hücrelerinde ve tunika muskularis tabakasında lokalize olduğu görüldü. Tüm gruplarda, BECN1 immünolokalizasyonu ince bağırsağın tüm bölgelerinde, özellikle villus epitel hücrelerinde, glandüler epitel hücrelerinde ve tunika muskularis tabakasında orta derecede (++) boyama yoğunluğu ile gözlemlenmiştir. Gruplar arasında boyama yoğunluğunda istatistiksel olarak anlamlı bir fark kaydedilmedi (p>0.05). Bu sonuçlar, kısa ila orta süreli açlığın (12-48 saat) sıçanların ince bağırsaklarında BECN1 ile ilişkili otofaji üzerinde ölçülebilir bir değişiklik yaratmadığını göstermektedir.
Keywords
Ethical Statement
Sıçanlar üzerinde gerçekleştirilen tüm prosedürler Burdur Mehmet Akif Ersoy Üniversitesi Yerel Hayvan Etik Kurulu tarafından incelenmiş ve onaylanmıştır.
Supporting Institution
Burdur Mehmet Akif Ersoy Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü
Project Number
0516-YL18
Thanks
Bu araştırma projesi Burdur Mehmet Akif Ersoy Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü tarafından 0516-YL18 proje numarası ile desteklenmiştir.
References
- Choi JH, Cho YS, Ko YH, Hong SU, Park JH, Lee MA. (2014). Absence of autophagy-related proteins expression is associated with poor prognosis in patients with colorectal adenocarcinoma. Gastroenterol Res Pract 2014; Article ID 179586. https://doi.org/10.1155/2014/179586
- Comincini S, Manai F, Meazza C, Pagani S, Martinelli C, Pasqua N, et al. Identification of Autophagy-Related Genes and Their Regulatory miRNAs Associated with Celiac Disease in Children. Int J Mol Sci 2017; 18: Article number 391. https://doi.org/10.3390/ijms18020391
- Foerster EG, Mukherjee T, Cabral-Fernandes L, Rocha JDB, Girardin SE, Philpott D.J. How autophagy controls the intestinal epithelial barrier. Autophagy 2022; 18: 86-103. https://doi.org/10.1080/15548627.2021.1909406
- Geng QR, Xu DZ, He LJ, Lu JB, Zhou ZW, Zhanet YQ, et al. Beclin-1 expression is a significant predictor of survival in patients with lymph nodepositive gastric cancer. PLoS One 2012; 7: e45968. https://doi.org/10.1371/journal.pone.0045968
- Giatromanolaki A, Sivridis E, Mendrinos S, Koutsopoulos AV, Koukourakis MI. Autophagy proteins in prostate cancer: relation with anaerobic metabolism and Gleason score. Urol Oncol 2014; 32: 39.e11-39.e18. https://doi.org/10.1016/j.urolonc.2013.04.003
- He C, Levine B. The Beclin 1 interactome. Curr Opin Cell Biol. 2010; 22: 140-149. https://doi.org/10.1016/j.ceb.2010.01.001
- Juliani J, Tran S, Harris TJ, Cruz PD, Ellis SL, Gleeson PA, et al. BECLIN-1 is essential for the maintenance of gastrointestinal epithelial integrity by regulating endocytic trafficking, F-actin organization, and lysosomal function. Autophagy Reports 2025; 4: 1-16. https://doi.org/10.1080/27694127.2025.2484494
- Kang R, Zeh H.J, Lotze M.T, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011; 18: 571–580. https://doi.org/10.1038/cdd.2010.191
- Kim HS, Lee SH, Do SI, Lim S.J, Park YK, Kim YW. Clinicopathologic correlation of beclin-1 expression in pancreatic ductal adenocarcinoma. Pathol Res Pract 2011; 207; 247-252.
- Levine B, Liu R, Dong X, Zhong Q. Beclin orthologs: integrative hubs of cell signaling, membrane trafficking, and physiology. Trends Cell Biol. 2015; 25: 533-544. https://doi.org/10.1016/j.prp.2011.02.007
- Naama M, Telpaz S, Awad A, Ben-Simon S, Harshuk-Shabso S, Modilevsky S, et al. Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress. Cell Host Microbe 2023; 31: 433-446. https://doi.org/10.1016/j.chom.2023.01.006
- Saha K, Ganapathy AS, Wang A, Morris NM, Suchanec E, Ding W, et al. Autophagy Reduces the Degradation and Promotes Membrane Localization of Occludin to Enhance the Intestinal Epithelial Tight Junction Barrier against Paracellular Macromolecule Flux. JCC 2023; 17: 433–449. https://doi.org/10.1093/ecco-jcc/jjac148
- Schläfli AM, Berezowska S, Adams O, Langer R, Tschan MP. Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry. Eur J Histochem. 2015; 59: 173-179.
- https://doi: 10.4081/ejh.2015.2481
- Song H, Xia SL, Liao C, Li YL, Wang YF , Li TP, et al. Genes encoding Pir51, Beclin 1, RbAp48 and aldolase b are up or down-regulated in human primary hepatocellular carcinoma. World J Gastroenterol 2004; 10: 509-513. https://doi: 10.3748/wjg.v10.i4.509
- Tran S, Fairlie WD, Lee EF. BECLIN1: Protein Structure, Function and Regulation. Cells 2021; 10: 1-16. https://doi.org/10.3390/cells10061522
- Tran S, Juliani J, Harris TJ, Evangelista M, Ratcliffe J, Ellis SL, et al. BECLIN1 is essential for intestinal homeostasis involving autophagy independent mechanisms through its function in endocytic trafficking. Commun. Biol. 2024; 7: Article number: 209. https://doi.org/10.1038/s42003-024-05890-7
- Wong M, Ganapathy AS, Suchanec E, Laidler L, Ma T, Nighot P. Intestinal epithelial tight junction barrier regulation by autophagy-related protein ATG6/beclin 1. Am J Physiol Cell Physiol 2019; 316: C753–C765. https://doi.org/10.1152/ajpcell.00246.2018
- Yu Y, Shiou SR, Guo Y, Lu L, Westerhoff M, Sun J, et al. Erythropoietin protects epithelial cells from excessive autophagy and apoptosis in experimental neonatal necrotizing enterocolitis. PLoS One 2013; 8: e69620. https://doi.org/10.1371/journal.pone.0069620
- Zhao H, Xi H, Wei B, Cai A, Wang T, Wang Y, et al. Expression of decorin in intestinal tissues of mice with inflammatory bowel disease and its correlation with autophagy. Exp Ther Med 2016; 12: 3885-3892. https://doi.org/10.3892/etm.2016.3908
Details
| Primary Language | English |
|---|---|
| Subjects | Health Care Administration |
| Journal Section | Research Article |
| Authors | |
| Project Number | 0516-YL18 |
| Submission Date | September 29, 2025 |
| Acceptance Date | December 16, 2025 |
| Publication Date | December 31, 2025 |
| Published in Issue | Year 2025 Volume: 13 Issue: 3 |
Cite
The Mehmet Akif Ersoy University Journal of Health Sciences Institute uses the Creative Commons Attribution License (CC BY) for all published articles.