Intest Res.  2020 Oct;18(4):421-429. 10.5217/ir.2019.00120.

Pathophysiological role of Atg5 in human ulcerative colitis

• Ardali R
• Kazemipour N
• Nazifi S
• Bagheri Lankarani K
• Razeghian Jahromi I
• Sepehrimanesh M

• KMID: 2508570
• DOI: http://doi.org/10.5217/ir.2019.00120

Abstract

Background/Aims
Ulcerative colitis (UC), along with Crohn’s disease, is one of the main types of inflammatory bowel disease (IBD). On the other hand, deregulated autophagy is involved in many chronic diseases, including IBD. In this study, we aimed to investigate the role of Atg5 and microRNA-181a (miR-181a) in the pathophysiology of UC.
Methods
Colon biopsy, stool, and blood samples of 6 men and 9 women were confirmed for UC. Also, 13 men and 17 women were selected as healthy control (HC). Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used to measure the Atg-5 content of the colon biopsies. Besides, the serum and stool levels of Atg5 were measured using ELISA. Moreover, the total RNA of blood cells was extracted and evaluated for the expression of miR-181a.
Results
We found 1.2 ng/mL versus 0.46 ng/mL, 0.34 ng/mL versus 0.24 ng/mL, and 0.082 ng/mL versus 0.062 ng/mL of Atg5 in stool, intestinal tissue, and serum of UC and HCs, respectively. There was no significant difference in the expression of miR-181a in the blood samples of UC and HCs. Immunohistochemistry showed high positivity without any significant difference between the 2 groups in the quantitative analysis.
Conclusions
The significant difference observed between the stool Atg5 content of the HCs and UC patients may provide new insight into using this protein as a diagnostic biomarker, however, considering the small size of our studied population further studies are needed.

Keyword

Autophagy; Atg5; Immunohistochemistry; mi-RNA; Colitis, ulcerative

Figure

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  Fig. 1. Atg5 concentration in ulcerative colitis (UC) and healthy control (HC) groups measured using ELISA. (A) Atg5 concentration in serum samples showed no significant difference between the 2 studied groups. (B) Stool Atg5 level demonstrated a significant difference so that it was higher in UC patients. (C) Atg5 amounts in the colonic tissues of UC patients and HCs did not show a significant difference in the mentioned groups.

• 

  Fig. 2. Expression rates of microRNA-181a (miR-181a) in ulcerative colitis (UC) and healthy controls (HC) blood plasma derived from quantitative real-time polymerase chain time. Fold-change of miR-181a expression was calculated after normalized with U6 (as an internal control) employing ΔΔCT method. Mann-Whitney test was carried out to compare the studied groups. There was no statically significant difference between the 2 groups concerning the miR-181a expression.

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  Fig. 3. Tissue sections of healthy controls (HCs). A and B show the overview of tissue sections (×40) and C represents the cropped section of B (×400). (A) H&E staining of colon biopsy in HC individuals. (B, C) Immunohistochemistry staining of Atg5 in colonic tissues of the control group. Images belong to 4 HCs which are representative of 30 controls.

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  Fig. 4. Histopathology sections of colon biopsies withdrawn from ulcerative colitis (UC) patients in affected areas. A and B show the overall views of tissue sections (×40) and C is a cropped area of B (×400). (A) H&E staining of colon biopsy in UC patients. (B, C) Immunohistochemistry staining of Atg5 in the colonic tissues of UC patients. (D) The percentage of positive cells for Atg5 was measured using QuPath software, and the statistic comparison between UC and healthy controls (HCs) was done by independent t-test with P<0.05 considered as significant. No significant difference was observed between the studied groups. Images belong to colon biopsies of 4 UC patients which are representative of 15 UC patients.

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