AZATHIOPRINE LINKED WITH IMPAIRED INTESTINAL EPITHELIAL REGENERATION IN CROHN’S DISEASE
Katarzyna Borycka-Kiciak2, Anna Pietrzak*1,2, Karolina Ferenc3, Piotr Pietrzak3,4, Lukasz Janaszek2, Wieslaw Tarnowski2
1Gastroenterology, Institute of Oncology, Warsaw, Poland; 2Medical Centre of Postgraduate Education , Warsaw, Poland; 3Warsaw University of Life Sciences, Warsaw, Poland; 4Warsaw Agricultural University,, Warsaw, Poland
Background
In patients operated due to active Crohn’s disease (CD), the negative effect of steroids and biologics on intraabdominal and wound septic complications is known. Until now, azathioprine (AZA) is considered to be safe. The aim of our study was to assess the impact of AZA on intestinal epithelial cells damage as well as restoration and regeneration in patients with active CD as a surrogate marker of healing.
Methods
Intestinal specimens taken from macroscopically healthy surgical margins of all consecutive CD patients operated due to active isolated ileocecal disease during the study period (2014-2016) were evaluated. Expression of caspase-3, p-53, and Ki-67 as markers of cell apoptosis, DNA damage, and proliferation respectively, were immunohistochemically tested and assessed using a confocal microscope and Microimage for in-tissue-cytometry analysis. Western-blot analysis was performed for the evaluation of cellular integrity using ZO-1 and E-cadherin as a markers. 30-day clinical outcomes were assessed. The study was approved by the institutional Ethics Committee.
Results
From 61 operated due to active CD patients, 35 met the inclusion criteria. Patients were divided accordingly to preoperative treatment: treated with no immunomodulators (N-9 pts), on steroids (S-14 pts), on AZA(A-6 pts), and on combination therapy, AZA + steroids (AS-6 pts). There were no substantial differences between groups. We found statistically significant increase of apoptosis in A group compared to N (5.33±1.05 vs 1.29±0.51, p=0.011), but also S group (1.58±0.68, p=0.014) and increase of DNA damage in A, AS and S groups compared with N group (p =0,01; p=0,032; p=0,035 respectively) (Figure 1a-c). P53-mediated cell cycle arrest and apoptosis through a caspase-3-dependent pathway in response to DNA damage was the most intensive in A group (Figure 2a). Reduction of cell proliferative activity in group A did not reach statistical significance (p=0,057). A reduction of ZO-1 in A group and increased level of E-cadherin in S group were noticed. The effect of the decreased number of tight junctions and disintegration of the mucosa layer was observed in A group (Figure 2b). Clinically, in 30-day postoperative follow-up, six wound healing complications and one anastomotic leak were found, all in patients treated with immunomodulators.
Conclusion
We found that in epithelial cells of the small and large intestine of patients treated with AZA, apoptotic activity, and DNA damage processes are increased when regeneration processes and mucosal integrity are significantly disturbed. These abnormalities of intestinal epithelial regeneration may be a surrogate marker of impaired mucosal healing.
Figure 1 Quantification (%) of apoptosis (caspase-3), DNA damage (p53) and mitosis (Ki-67) of epithelial cells depending on type of treatment. Statistically significant results (p<0,05) are marked with lines with an asterisk
Figure 2 (a) Microphotographs showing co-expression of caspase-3 (green fluorescence) and -p53 (red fluorescence). Severity of the changes corresponds to the severity of apoptotic processes. (b) Light microscope visualization of differences in epithelial cells integrity (arrows) due to the changed level of peripheral plasma-membrane proteins like ZO-1 and cadherin in all groups
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