The effects of icariin on the expression of HIF-1α, HSP-60 and HSP-70 in PC12 cells suffered from oxygen–glucose deprivation-induced injury

Abstract

Context: The effects of icariin, a chief constituent of flavonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown.Objective: To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability.Materials and methods: PC12 cells were treated with icariin (10−7, 10−6 or 10−5 mol/L) for 3 h (1 h before oxygen–glucose deprivation (OGD) plus 2 h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Results: After 2 h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly (HIF-1α: 33.3 ± 1.9 ng/L, HSP-60: 199 ± 16 ng/L, HSP-70: 195 ± 17 ng/L, NSE: 1487 ± 125 ng/L), and cell viability was significantly decreased (0.26 ± 0.03), while icariin (10−7, 10−6, or 10−5 mol/L) significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE (HIF-1α: 14.1 ± 1.4, 22.6 ± 1.8, 15.7 ± 2.1, HSP-60: 100 ± 12, 89 ± 6, 113 ± 11, HSP-70: 139 ± 9, 118 ± 7, 95 ± 9 and NSE: 1121 ± 80, 1019 ± 52, 731 ± 88), and improved cell viability (0.36 ± 0.03, 0.38 ± 0.04, 0.37 ± 0.03) in OGD-treated PC12 cells.Discussion and conclusion: These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70.

Nobiletin protects PC12 cells from ERS‑induced apoptosis in OGD/R injury via activation of the PI3K/AKT pathway

“…Cell culture. The PC12 cells were routinely cultured in DMEM/F12 medium supplemented with 10% FBS, 100 U/ml of penicillin, and 100 mg/ml of streptomycin in a humidified atmosphere of 5% CO 2 and 95% O 2 at 37˚C (16)(17)(18). The cells were fed every 2-3 days and sub-cultured once they reached 70-80% confluent.…”

“…OGD/R injury and experimental designation. To mimic the I/R conditions in vitro, the PC12 cells were subjected to OGD/R treatment according to a prior demonstrations with minor modifications (16)(17)(18). In detail, the cells were washed two times with glucose-free Earle's balanced salt solution, and were maintained in glucose-free DMEM without FBS.…”

“…Assessment of cell viability. Cell viability was determined using the CCK-8 assay as a previous method (16)(17)(18). The PC12 cells were seeded into 96-well plates at a density of 6x10 3 cells/well for 24 h of incubation.…”

“…When cells are damaged by the OGD/R injury, intracellular LDH is rapidly released into the culture supernatant. Cellular injury was measured using LDH assay (16)(17)(18). Briefly, after the indicated treatments, the supernatant of the cultured PC12 cells was harvested, centrifuged at 3,000 x g for 10 min and then subjected to a commercial kit using standard protocols according to the manufacturer's protocols.…”

“…Cell apoptosis. Apoptosis of the PC12 cell was determined using a flow cytometric assay with a commercial apoptosis detection kit (16)(17)(18). According to the manufacturer's instruction, the cells were harvested after treatment and washed twice with cold PBS.…”