Elucidating the link between reactive oxygen species and p53: The pathway behind impaired diabetic wound healing
Denis Knobel, MD, James L. Crawford, BS, Parag Butala, MD, Olga Edlyn, BS, Edward H. Davidson, MD, Merideth T. Wetterau, MD, Stephen M M. Warren, MD, Pierre B. Saadeh, MD.
New York Medical Center, New York, NY, USA.
BACKGROUND: The diabetic hyperglycemic state exerts extended stress on cells, resulting in elevated p53 expression and subsequently end-organ injury. Previously, we demonstrated that topical silencing of p53 with short-interfering RNA (siRNA) improves diabetic wound healing. Here, we investigate whether the p53-reactive oxygen species (ROS)-apoptosis pathway described in cancer literature is present and active in diabetic wound healing by inhibiting the p53-upregulated modulator of apoptosis (PUMA), a pro-oxidant gene that links p53 to ROS. In addition, we utilize N-acetylcysteine (NAC), a ROS scavenger, to mitigate the effects of the ROS pathway.
METHODS: Paired 6-mm stented wounds were created on diabetic db/db mice on three treatment groups (PUMA, NAC, Control). NAC and short interfering RNA (siRNA) to PUMA were each evenly distributed in agarose-gel matrices and topically applied starting post-operative day 1. Nonsense siRNA served as control for the siRNA PUMA arm; matrix-gel alone served as control for the NAC arm. Wound closure time was photometrically assessed, and wounds were harvested on day 10 for histology, immunohistochemistry (IHC), RT-PCR, western blot and ELISA. ANOVA/t-test was used to determine statistical significance (p<=0.05).
RESULTS: Treatment with PUMA siRNA and NAC consistently accelerated wound closure (18±1.5 day, 17±1 vs. 27±1 day in control). Expression of PUMA was abrogated by respective siRNA as confirmed by western blot. Hematoxylin-eosin staining showed evenly formed new epithelium including keratinocyte coverage of the wound in treated animals, and disorganized epithelium and no keratinocytes in controls. In the PUMA siRNA and NAC-treated groups, IHC demonstrated decreased p53, caspase-3, and the oxidative DNA damage marker, 8-Hydroxy-2'-deoxyguanosine (8-OHdG) staining. DNA damage secondary to ROS (8-OHdG ELISA) decreased almost in half in each treated group (p=0.03). p53 levels decreased by 40% on ELISA (PUMA 6.1±0.12, NAC 4.38±0.08 vs. 8.8±0.42 pg/mL). VEGFα ELISA expression increased by average fold change of 2.5 in the treated groups (PUMA 3.37±0.44, NAC 4.89±0.47 vs. 1.06±0.18 pg/ml). RT-PCR confirmed near complete knockdown of pro-oxidant genes PUMA, POX and NQO-1 and increases in fold change of the anti-oxidant gene MnSOD. In, addition, RT-PCR demonstrated a 3.5-fold increase in SDF-1 expression in treated wounds.
CONCLUSIONS: In conclusion, using topical siRNA to silence PUMA resulted in decreased ROS levels and improved wound healing in our stented diabetic mouse (db-/db-) wound model. It also decreased the positive feedback from ROS and resulted in decreased levels of p53 and other pro-oxidant genes. Pharmacologic treatment of the wounds with NAC produced similar results. Our study shows that the p53-ROS-apoptosis pathway is active in diabetic wounds and that NAC holds promise for the treatment of these wounds.
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