| PartⅠObjective: To investigate the protective effect and molecular mechanism of astaxanthin against OGD-induced damage.Methods: SH-SY5Y cells were cultured. SH-SY5Y cells were incubated with different astaxanthin concentrations (5, 10, or 20 μM) for 24 h before incubation with OGD for 6 h. Cell viability was measured by the MTT assay. ROS production was detected by using the DCFH2-DA probe. Rhodamine123 is used in mitochondrial membrane potential. Cell apoptosis rate was measured by Annexin V-FITC/PI double-staining assay kit. SOD activity and MDA contents were measured according to the manufacture’s protocol. The protein expression of AKT, P-AKT, GSK3β, p-GSK3β,Nrf2, HO-1, Bax, Bcl-2, Caspase-3 were tested by Western blot. Cells were incubated with a PI3K inhibitor LY294002 (10 μM) or the GSK3β inhibitor LiCl (10 μM) for 60 min, and then were incubated with astaxanthin for 24 h before incubation with OGD for 6 h, the potein expression of AKT, P-AKT, GSK3β,p-GSK3β, Nrf2, HO-1 were tested.Results: Pre-treatment with astaxanthinin (5-20 μM) for 24 h significantly decreased the oxygen and glucose deprivation (OGD)-induced viability loss, decreased apoptotic rate, MDA levels and attenuated OGD-mediated reactive oxygen species (ROS)production in a concentration-dependent manner (p < 0.05). In addition, astaxanthin inhibited OGD-induced mitochondrial membrane potential and increased the SOD levels and B-cell lymphoma 2 (Bcl-2)/Bax ratio (p < 0.05). Phosphorylated AKT(p-AKT), p-GSK3p, nucleus Nrf2 and heme oxygenase 1 protein levels increased in the presence of astaxanthin (20 μM) and subsequently decreased in the presence of LY294002 and increased in the presence of LiCl in SH-SY5Y cells.Conclusions: Astaxanthinin may confer neuroprotection against OGD-induced apoptosis via the PI3K/AKT/GSK3p/Nrf2 signaling pathway.Part IIObjective: To investigate the effect of astaxanthin on angiogenesis and its molecular mechanism.Methods: Human brain micro vascular endothelial cell (HBMEC) culture and rat aortic smooth muscle cell (RASMC) culture. HBMEC were incubated with different astaxanthin concentrations (3-30 μM) for 24 h before incubation with OGD for 6 h. The effects of astaxanthin on HBMEC proliferation, invasion and tube formation were measured by flow cytometry instrument, Scratch adhesion test, Matrigel tube formation assay and observe the effects of astaxanthin on cell viability and LDH leakage in vitro.The protein expression of wnt7a、GSK3β、β-catenin、cyclin D1 and Caspase-3 were tested by Western blot. HBMEC were incubated with a inhibitor IWR-1-endo (5μM)for 12 h, and then were incubated with astaxanthin (10 μM ) for 24 h before incubation with OGD for 6 h, the potein expression of wnt7a、GSK3β、β-catenin、cyclin D1 and tube formation were tested, respectively. RASMC were incubated with different astaxanthin concentrations (1-10 μM) for 48 h before incubation with OGD for 6 h. The effects of astaxanthin on RASMC proliferation, invasion and tube formation were measured by flow cytometry instrument, Scratch adhesion test, Matrigel tube formation assay and observe the effects of astaxanthin on cell viability and LDH leakage in vitro.The protein expression of wnt5a、GSK3β、β-catenin、cyclin D1 were tested by Western blot. RASMC were incubated with a inhibitor IWR-1-endo (5 μM) for 12 h, and then were incubated with astaxanthin (3 μM ) for 24 h before incubation with OGD for 6 h,the potein expression of wnt5a、 GSK3β、 β-catenin、 cyclin D1 and tube formation were tested, respectively.Results: Our data suggested that astaxanthin induces angiogenesis by increasing proliferation, invasion and tube formation in a concentration-dependent mannerin vitro(p < 0.05). Wnt and β-catenin expression were increasing by astaxanthin and counteracted by IWR-1-endo in HMBECs and RASMCs. Tube formation was increased by astaxanthin and counteracted by IWR-1-endo.Conclusions: It may be suggested that astaxanthin induces angiogenesis in vitro via a programmed Wnt/β-catenin signaling pathway. |
PDF Full Text Request