ENHANCED ANTIOXIDANT SIGNALING BY MITOCHONDRIAL-TARGETED DRUG AUPHOS DRIVES ATTENUATION OF COLITIS

Uncontrolled oxidative stress due to reactive oxygen species (ROS) overproduction and impaired antioxidant defense system are central to IBD pathogenesis. Damaging effect of excessive ROS activates pro-inflammatory signaling pathways, compromising intestinal epithelial barrier function and exacerbates existing inflammation. In the present study, we investigate the underlying antioxidant defense mechanism of our previously tested mitochondria-targeted drug, AuPhos (CCC2022-24). We postulate that AuPhos-induced mitochondrial function enhances anti-oxidant levels and improves tissue inflammation in IBD.

Effects of AuPhos on cellular antioxidant defense system were evaluated in normal human colonic epithelial cells (NCM460), piroxicam (Px)-accelerated Il10^-/- colitis mice and human ulcerative colitis (UC) biopsies. Briefly, NCM460 cells were treated overnight with AuPhos (0.5uM) +/- low-dose TNF(1ng/ml) and processed for protein analysis by western blot (WB). The Px-Il10^-/-mice were given Px in the chow for 2 weeks (Brown et al.; Inflamm Bowel Dis. 2008) then treated with AuPhos (25mg/kg) or vehicle by oral gavage every 3d (n=15/group) starting at D28. Colonic tissues were collected at D56 in AllProtect® and subjected for RNA analysis. Biopsy samples from consented normal and UC patients (Mayo 1-3) were collected, and treated ex-vivo for 3h with vehicle or AuPhos (0.5uM) at 4^oC. Transcript and protein levels of antioxidant enzymes were analyzed using RT-qPCR and WB. Effect of AuPhos on cytosolic ROS (H₂O₂) production was determined by Amplex red assay. AuPhos effect on oxidative injury was assessed by IHC staining of 4-HNE and 8-hydroxydeoxyguanosine (8-OHdG).

AuPhos increases expression of NRF2, a master regulator of antioxidant signaling, in human intestinal epithelial cells (IECs). Protein analysis in NCM460 cells show that AuPhos increases downstream antioxidant markers such as manganese superoxide dismutase (MnSOD), glutathione peroxidase-4 (GPX4), thioredoxin reductase-2 (Trx2), peroxiredoxin-3 (Prdx3) and catalase expression when compared to the TNF or control samples. AuPhos-fed Px-Il10^-/- mice and ex vivo treated colonic biopsies also show significant increase in antioxidant enzymes (MnSOD, CuZnSOD, Gpx, Prdx) at mRNA and protein levels. Furthermore, in human UC biopsies AuPhos treatment reduced cytosolic ROS (H₂O₂) production as well as markers of oxidative stress (4-HNE and 8-OHdG).

Increased AuPhos-induced Nrf2 signaling enhances anti-oxidant enzymes and attenuates oxidant injury in UC tissue biopsies raising the possibility that enhanced mitochondrial function limits oxidant injury, a major mechanism for mucosal repair. This suggests the potential of AuPhos in treating IBD by attenuating oxidative injury.