J Vet Sci.  2014 Dec;15(4):475-483. 10.4142/jvs.2014.15.4.475.

Stanniocalcin-1 protects bovine intestinal epithelial cells from oxidative stress-induced damage

• Wu LM
• Guo R
• Hui L
• Ye YG
• Xiang JM
• Wan CY
• Zou M
• Ma R
• Sun XZ
• Yang SJ
• Guo DZ

• KMID: 2070233
• DOI: http://doi.org/10.4142/jvs.2014.15.4.475

Abstract

Chronic enteritis can produce an excess of reactive oxygen species resulting in cellular damage. Stanniocalcin-1(STC-1) reportedly possesses anti-oxidative activity, the aim of this study was to define more clearly the direct contribution of STC-1 to anti-oxidative stress in cattle. In this study, primary intestinal epithelial cells (IECs) were exposed to hydrogen peroxide (H2O2) for different time intervals to mimic chronic enteritis-induced cellular damage. Prior to treatment with 200 microM H2O2, the cells were transfected with a recombinant plasmid for 48 h to over-express STC-1. Acridine orange/ethidium bromide (AO/EB) double staining and trypan blue exclusion assays were then performed to measure cell viability and apoptosis of the cells, respectively. The expression of STC-1 and apoptosis-related proteins in the cells was monitored by real-time PCR and Western blotting. The results indicated that both STC-1 mRNA and protein expression levels positively correlated with the duration of H2O2 treatment. H2O2 damaged the bovine IECs in a time-dependent manner, and this effect was attenuated by STC-1 over-expression. Furthermore, over-expression of STC-1 up-regulated Bcl-2 protein expression and slightly down-regulated caspase-3 production in the damaged cells. Findings from this study suggested that STC-1 plays a protective role in intestinal cells through an antioxidant mechanism.

Keyword

Bcl-2; chronic enteritis; oxidative damage; stanniocalcin-1

MeSH Terms

Animals
Animals, Newborn
Blotting, Western/veterinary
Caspase 3/*genetics/metabolism
Cattle
Cattle Diseases/etiology/*genetics/metabolism
Duodenum/metabolism
Enteritis/etiology/genetics/metabolism/*veterinary
Epithelial Cells/metabolism
*Gene Expression Regulation
Glycoproteins/*genetics/metabolism
Hydrogen Peroxide/pharmacology
Male
Proto-Oncogene Proteins c-bcl-2/*genetics/metabolism
RNA, Messenger/genetics/metabolism
Real-Time Polymerase Chain Reaction/veterinary
Caspase 3
Glycoproteins
Proto-Oncogene Proteins c-bcl-2
RNA, Messenger
Hydrogen Peroxide

Figure

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  Fig. 1 Morphology of intestinal cells in culture. Phase contrast microscopy was performed to observed characteristics associated with the evolution of bovine IECs in primary cultures starting from adherent organoid structures (A) that gave rise to lager proliferating foci (B) which joined together to form a confluent monolayer. After purification, the subcultured epithelial cells grew dispersedly in the flask (C). The individual epithelial cell had a dome-like and rounded morphology when viewed with a differential interference contrast microscope (D) or scanning electron microscope (E), respectively. The nuclei and cytoplasm of the hematoxylin- and eosin-stained cells were blue and pink, respectively (F). ×40 (A), ×200 (B ~ D, and F), and ×15,000 (E). Scale bars = 200 µm (A) or 50 µm (B ~ F).

• 

  Fig. 2 Characterization of cells stained for immunocytochemistry. Panels show the primary cultures, V79, and IEC-6 cells, and indicate cells stained with antibodies against vimentin, PCK, CK18, or PBS (as the negative control), from left to right, respectively. Note that both the primary cultures (A1 and 2) and IEC-6 cells (C1 and 2) were stained yellow by the antibodies against PCK and CK18, but not by the anti-vimentin antibody (A and C). In contrast, the V79 cells displayed opposite patterns of immunoreactivity (B ~ B2). ×400 (A1 and 2), ×200 (A, A3, B ~ B3, and C ~ C3). Scale bars = 20 µm (A1 and 2) or 50 µm (A, A3, B ~ B3, and C ~ C3).

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  Fig. 3 Analysis of STC-1 mRNA and protein expression in the transfected IECs. The expression of STC-1 mNRA in cells transfected with the pcDNA/STC-1 construct was maximized approximately 48 h after transfection as measured by real-time PCR (gray column in A; *p < 0.01 or **p < 0.001 vs. the control cells). Protein expression was maximized at 72 h as measured by Western blotting (B). In contrast, no significant changes were observed in STC-1 mRNA or protein expression in cells transfected with the pcDNA3.1+ vector (black column in A and C).

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  Fig. 4 Detection of viability, apoptosis, and STC-1 expression of transfected IECs. A time-dependent decrease in cell viability (A ~ A5, cells treated with H2O2 for 0, 4, 8, 12, 16, and 24 h) and survival rate (B, *p < 0.001 vs. 0 h) was observed for cells treated with H2O2 using AO/EB double staining and a trypan blue exclusion assay, respectively. Additionally, time-dependent increases in STC-1 mRNA (C, #p < 0.01 or ##p < 0.001 vs. the control cells) and protein expression (D, C: control cells, T: H2O2-treated cells) were found in the cells exposed to 200 µM H2O2 using real-time PCR and Western blotting, respectively. ×400 (A ~ A5). Scale bars = 20 µm (A ~ A5).

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  Fig. 5 The effects of STC-1 over-expression on the viability, apoptosis rate, and apoptosis-related protein expression in IECs. (A ~ A3) Viability of cells grown normally, treated with H2O2 for 8 h, or pre-transfected with pcDNA/STC-1 or pcDNA3.1+ for 48 h before H2O2 treatment, respectively. In cells that over-overexpressed STC-1, H2O2-induced cellular injury was significantly attenuated as measured by AO/EB double staining (A3). (B) A significant decrease of apoptosis induced by H2O2 was observed in cells transfected with pcDNA/STC-1 (fifth column) as measured by a trypan blue exclusion assay (#p < 0.01 vs. the control; ap < 0.01 vs. cells treated with H2O2 alone; bp < 0.01 vs. the pcDNA3.1+-transfected cells treated with H2O2). (C) Expression of apoptosis-related factors measured by Western blotting. Lane 1, Cells grown normally as the control; Lane 2, cells treated with H2O2 alone for 8 h; Lane 3, cells treated with H2O2 alone for 8 h after transfection with pcDNA3.1+ for 48 h; Lane 4, cells treated with H2O2 alone for 8 h after transfection with pcDNA/STC-1 for 48 h. Cells transfected with pcDNA/STC-1 showed a significant up-regulation of Bcl-2 expression and a slight down-regulation of caspase 3 expression. ×400 (A1~A4). Scale bars = 20 µm (A1 ~ A4).

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