Radioprotective effect of R-spondin1 against radiation induced gastrointestinal syndrome

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Radiation-induced gastrointestinal syndrome (RIGS) results from cytocidal effect of irradiation on intestinal crypt cells, resulting in villous depletion, malabsorption, watery stool, sepsis and death. Human R-spondin1 is an intestinal stem cell growth factor with mitogenic effect on intestinal crypt cells. We, therefore, attempted to discern whether R-spondin has any protective role on RIGS. Male C57Bl6 mice received 5 × 109 particles of recombinant adenovirus expressing human R-spondin1 (AdRspo1) or E. coli Lacz (AdLacz) via tail-vein injection, 3 and 1 day before whole body (WBI) and abdominal irradiation. Post-irradiation survival was analyzed by Kaplan Meier analysis. Paraffin sections of jejunum were stained with hematoxylin and eosin and TUNEL and BrdU immunohistochemistry were performed for determining apoptotic and proliferative responses. Xylose absorption test was performed to assess the functional integrity of intestinal mucosal barrier post-radiation. Systemic administration of AdRspo1 did not cause any adverse toxicities. All mice in the AdLacz control groups were dead within 12 days after 10.4 Gy WBI. In contrast, 80% of mice from the AdRspo1 cohort survived for > 25 days (p<0.003, log-rank test). Although there was improvement in survival following 12 Gy (45%) and 14 Gy (40%) of abdominal irradiation (p 16Gy. Compared to AdLacZ cohort, there was a significant increase in proliferation of crypt cells (P<0.05), number of intestinal crypt regenerative foci (p<0.001) and an increase in the crypt depth (p<0.001) in AdRspo-1-treated animals on 3.5 days after radiation exposure (10.4-14 Gy). There was a higher xylose absorption in AdRspo1-treated animals indicating a rapid intestinal regeneration following WBI. Since, R-spondin1 stimulates the Wnt-1 pathway, there was increased expression of beta-catenin protein in both the cytosolic and nuclear fractions of intestinal epithelial cells in AdRspo1-treated jejunum. Quantitative PCR demonstrated an upregulation of beta-catenin target genes (Ephb2, EphB3, TCF4, Lef1) in Ad-Rspo1-treated animals. In conclusion, systemic administration of AdRspo1 provided intestinal radioproptection in C57Bl/6 mice. Rspo1 induced an increase in intestinal regeneration and induction of beta-catenin and its target genes suggesting possible involvement of Wnt/beta-catenin pathway in the R-spondin1 mediated radioprotection of RIGS.