Rosmarinic Acid Alleviates Ischemia-Reperfusion-Induced Flap Injury by Regulating Autophagy and Apoptosis through Nrf2/HO-1 and MAPK Pathway

Abstract

Background: Rosmarinic acid (RA) exerts an antioxidant property and alleviates some tissues’ ischemia/reperfusion (I/R) injury. However, its role in I/R-induced flap injury is still unknown. Therefore, this study investigates RA’s feasibility in treating flap injury caused by I/R. Methods: Rats were given the same dose of normal saline or RA for 7 days, and the flap operation was performed on day 8. The survival of the flap was observed after 7 days of operation, and the flap tissues were taken for hematoxylin-eosin (H&E) staining. Besides, human keratinocytes (HaCaT) were cultured under an anoxic environment for 12 h and reoxygenated for 1 h to induce I/R model in vitro. RA pre-treatment and small interfering (si) for nuclear factor erythroid 2-related factor 2 (Nrf2) transfection were conducted before I/R treatment. Western blot was used to detect Nrf2, heme oxygenase-1 (HO-1), PTEN-induced kinase 1 (PINK1), Parkin, light chain 3 (LC3)II/LC3I, p62, p38, p-p38, B cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) expression levels in flap tissues or HaCaT cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry were employed to measure cell viability and apoptosis, respectively. Results: RA alleviated I/R-induced flap injury in rats and apoptosis of HaCaT cells and increased cell viability. Besides, I/R inhibited Nrf2, HO-1, Bcl-2, PINK1, Parkin and LC3II/LC3I levels, but enhanced p62, p-p38 and Bax levels. RA could partially reverse the effect of I/R. siNrf2 counteracted the effects of RA on the viability, apoptosis and expression levels of HO-1, Bcl-2, PINK1, Parkin, LC3II/LC3I, p-p38 and Bax in HaCaT cells. Conclusions: RA alleviated I/R-induced flap injury by regulating Nrf2/HO-1 and p38 MAPK (mitogen-activated protein kinase) signaling pathways.