Protective effect of luteolin on skin ischemia-reperfusion injury through an AKT-dependent mechanism

Abstract

Cutaneous ischemia‑reperfusion (I/R) injury is one of the most crucial problems in flap surgery, which affects the survival of the skin flap and patient prognosis, luteolin, a plant derived flavonoid, has previously been shown to exert a variety of beneficial effects for reducing I/R injury in several organs. The aim of the present study was to evaluate the anti‑inflammatory and anti‑oxidative stress effects of luteolin on cutaneous I/R injury. The in vitro study were performed using a permanent human immortalized epidermal keratinocyte cell line (HaCaT), cells were cultured in the presence of luteolin and were then treated with hydrogen peroxide, the cell viability, mitochondrial membrane potential and the cell survival/apoptosis related signaling pathway activation were assessed to investigate the cytoprotective effects of luteolin. For in vivo experiments, skin flap I/R injury animal model was established in Sprague‑Dawley rats, by measuring the area of flap survival, analyzing the expression of pro‑inflammatory cytokine and evaluation of the histological changes in the skin tissue, the protective effects of luteolin on skin I/R injury were investigated. The function of protein kinase B (AKT) and heme oxygenase‑1 (HO‑1) activation on luteolin mediated I/R injury protection was assessed by administration of phosphoinositide‑3‑kinase/AKT inhibitor LY294002 and HO‑1 inhibitor ZNPP. The results showed that luteolin treatment significantly increased the viability of HaCaT cells upon exposure to hydrogen peroxide, and the administration of luteolin in vivo significantly improved skin flap survival in the I/R injury rat model. The mechanisms underlying these beneficial effects included increased phosphoinositide‑3‑kinase/protein kinase B activation, improved expression of antioxidant enzyme, and scavenging the cytotoxic effects of reactive oxygen species (ROS). Taken together, the results suggested that luteolin preconditioning yielded significant protection against cutaneous I/R injury by protecting skin keratinocytes from ROS‑induced damage.

Figure 1

Chemical structure of the luteolin and the surgical procedure. (A) Cell viability of HaCaT cells were detected using an MTS assay. (B) Diagram showing the surgical procedure. Briefly, the rat was anesthetized and an island skin flap measuring 3×6 cm over the lower chest and abdomen was raised; the flaps were transected proximally, leaving the superficial epigastric vessels as the only connection, and epigastric vessels close to the femoral artery and vein were occluded with a 2-V microvascular clamp during the ischemic period. The clamps were removed following 4 h of ischemia.

Figure 2

Luteolin preconditioning decreases the cytotoxic effect of hydrogen peroxide in HaCaT keratinocyte cells. (A) Cell viability of HaCaT cells detected using a MTT assay in the presence of hydrogen peroxide and luteolin pretreatment; (B) Percentage of apoptotic cells was assessed by FACS with Annexin-V and PI staining. (C) Mitochondrial membrane potential assay was performed using 6,6′-tetrachloro-1,1′,3,3′-tetraethylimidacarbocyanineiodide staining (magnification, ×100). Results are expressed as the mean ± standard deviation (^*P<0.05, ^**P<0.01 compared with the H₂O₂-treated group). Ctl, control; Luo, luteolin; PI, propidium iodide.

Figure 3

Effect of luteolin on hydrogen peroxide-induced keratinocyte apoptosis. The protein levels of markers of apoptosis, BAX, caspase 3 and BCL-2, were evaluated using a western blot assay. The HaCaT cells were exposed to 100 µM of hydrogen peroxide in the presence or absence of increased concentration of luteolin. +, 3 µg/ml; ++, 6 µg/ml; +++, 12 µg/ml; AKT, protein kinase B; P-AKT, phosphorylated AKT; HO-1, heme oxygenase-1; BCL-2, B-cell lymphoma 2; BAX, BCL-2-assocated X protein; I/R, Luo, luteolin. ^*P<0.05, vs H₂O₂ treatment.

Figure 4

Luteolin administrations ameliorates I/R injury-induced skin tissue damage by inhibition of acute inflammation and oxidative stress levels. (A) Pro-survival effects of luteolin on I/R-injured skin tissue in rats undergoing skin flap surgery; flap survival area was significantly improved in rats subjected to I/R injury with luteolin treatment. (B) Relative expression of pro-inflammatory cytokines in the skin tissue biopsy was detected by reverse transcription-quantitative polymerase chain reaction analysis. (C) I/R-injured skin tissues were stained with hematoxylin and eosin and the histopathologic changes were visualized under a light microscope at different magnifications (×40 and ×100). (D) Effect of luteolin administration on I/R-induced oxidative damage in skin tissue; the relevant biomarkers were assessed using commercial kits. Values are expressed as the mean ± standard deviation (n=8). ^*P<0.05, vs. Ctl. I/R, ischemia-reperfusion; Luo, luteolin; IL, interleukin; TNF-α, tumor necrosis factor; Ctl, control; MPO, myeloperoxidase; MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 5

Protective effect of luteolin against I/R injury is mediated via the PI3K/AKT pathway. Immunofluresence staining of the skin tissue sections isolated (A) 1 day and (B) 7 days post-I/R injury. (C) Flap survival area in rats subjected to I/R injury and luteolin treatment were reduced in the rats that received intraperitoneal injection of PI3K/AKT inhibitor LY294002, but not by HO-1 inhibitor ZnPP. ^*P<0.05, vs. Ctl. I/R, ischemia-reperfusion; PI3K, phosphoinositide-3-kinase; AKT, protein kinase B; HO-1, heme oxygenase-1; Luo, luteolin; LY, LY294002; CTL, control.