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

Zhen-Tao Mo, Wen-Na Li, Yu-Rong Zhai, Shu-Ying Gao, Zhen-Tao Mo, Wen-Na Li, Yu-Rong Zhai, Shu-Ying Gao

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.

Keywords: Heat shock protein; cell viability; neuron-specific enolase.

Figures

Figure 1.
Figure 1.
Effect of icariin on cell viability in OGD-treated PC12 cells. Model control cells were treated with 2 h OGD. The treated cells were incubated with icariin (10−7, 10−6 or 10−5 mol/L) or nimodipine (10 μmol/L) 1 h before OGD and 2 h throughout OGD. Normal control cells were incubated in a regular cell culture incubator under normoxic conditions. After these treatments, cell viability was analyzed using MTT assay. Mean ± SD for 10 samples. ##p < 0.01 vs normal control group. **p < 0.01 vs model control group.
Figure 2.
Figure 2.
Effect of icariin on HIF-1α levels in OGD-treated PC12 cells. Model control cells were treated with 2 h OGD. The treated cells were incubated with icariin (10−7, 10−6 or 10−5 mol/L) or nimodipine (10 μmol/L) 1 h before OGD and 2 h throughout OGD. Normal control cells were incubated in a regular cell culture incubator under normoxic conditions. After these treatments, HIF-1α levels in cell supernatant fluid were analyzed using ELISA assay. Mean ± SD for six samples. ##p < 0.01 vs normal control group. **p < 0.01 vs model control group.
Figure 3.
Figure 3.
Effect of icariin on HSP-60 levels in OGD-treated PC12 cells. Model control cells were treated with 2 h OGD. The treated cells were incubated with icariin (10−7, 10−6 or 10−5 mol/L) or nimodipine (10 μmol/L) 1 h before OGD and 2 h throughout OGD. Normal control cells were incubated in a regular cell culture incubator under normoxic conditions. After these treatments, HSP60 levels in cell supernatant fluid were analyzed using ELISA assay. Mean ± SD for six samples. ##p < 0.01 vs normal control group. **p < 0.01 vs model control group.
Figure 4.
Figure 4.
Effect of icariin on HSP-70 levels in OGD-treated PC12 cells. Model control cells were treated with 2 h OGD. The treated cells were incubated with icariin(10−7, 10−6 or 10−5 mol/L) or nimodipine (10 μmol/L) 1 h before OGD and 2 h throughout OGD. Normal control cells were incubated in a regular cell culture incubator under normoxic conditions. After these treatments, HSP70 levels in cell supernatant fluid were analyzed using ELISA assay. Mean ± SD for six samples. ##p < 0.01 vs normal control group. **p < 0.01 vs model control group.
Figure 5.
Figure 5.
Effect of icariin on NSE levels in OGD-treated PC12 cells. Model control cells were treated with 2 h OGD. The treated cells were incubated with icariin(10−7, 10−6 or 10−5 mol/L) or nimodipine (10 μmol/L) 1 h before OGD and 2 h throughout OGD. Normal control cells were incubated in a regular cell culture incubator under normoxic conditions. After these treatments, NSE levels in cell supernatant fluid was analyzed using ELISA assay. Mean ± SD for six samples. ##p < 0.01 vs normal control group. **p < 0.01 vs model control group.

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