Zebrafish glafenine-intestinal injury is ameliorated by mu-opioid signaling via enhancement of Atf6-dependent cellular stress responses Short Title: Glafenine intestinal injury in zebrafish

Summary Beside their analgesic properties, opiates exert beneficial effects on the intestinal wound healing response. In this study, we investigated the role of mu-opioid receptor (MOR) signaling on the unfolded protein response (UPR) using a novel zebrafish model of NSAID-induced intestinal injury. The NSAID glafenine was administered to 5 days-post-fertilization (dpf) zebrafish larvae for up to 24h in the presence or absence of the MOR-specific agonist DALDA. By analysis with histology, transmission electron microscopy, and vital dye staining, glafenine-treated zebrafish showed evidence of endoplasmic reticulum (ER) and mitochondrial stress with disrupted intestinal architecture and halted cell stress responses, alongside accumulation of apoptotic intestinal epithelial cells in the lumen. While the early UPR marker BiP was induced with glafenine-injury, downstream atf6 and s-xbp1 expression were paradoxically not increased, explaining the halted cell stress responses. The mu-opioid agonist DALDA protected against glafenine-induced injury through induction of atf6-dependent UPR. Our findings show that DALDA prevents glafenine-induced epithelial damage through induction of effective UPR.