Fig 1.
Effect of the combination treatment of the LIPEF and fucoidan on the cell viability of H2O2-treated NSC-34 cells.
The cell viability was determined using MTT assay at 24 h after the treatment of 100 μM H2O2. (A) NSC-34 cells were pretreated with different intensities of LIPEF for 1 h and then exposed to 100 μM H2O2 in the continuous administration of the LIPEF for another 24 h. (n = 3, Student’s t-test; *** is used for P < 0.001). (B) Different concentrations of fucoidan were combined with the 60 V/cm LIPEF treatment on the 100 μM H2O2-treated NSC-34 cells. (n = 3, Student’s t-test; *** is used for P < 0.001). (C) Fucoidan alone at various concentrations (0 to 200 μg/ml) showed little protective effect.
Fig 2.
The protective effects on the ROS level and the GSH/GSSG ratio in the H2O2-treated NSC-34 cells.
(A) The ROS level was measured using DCFH-DA assay. (n = 3, one-way ANOVA, Turkey’s test; ** is used for P < 0.01 and *** for P < 0.001). (B) The GSH/GSSG ratio was determined using GSH colorimetric assay. (n = 3, Student’s t-test; *** is used for P < 0.001). These results showed that the protective effects of the LIPEF and fucoidan could reduce the oxidative stress in the NSC-34 cells exposed to H2O2 for 24 h.
Fig 3.
The protective effects on the MMP, the ER stress, and the nuclear condensation in the H2O2-treated NSC-34 cells.
(A) The dissipation of MMP was determined using DiOC6(3) staining. (n = 3, one-way ANOVA, Turkey’s test; *** is used for P < 0.001). (B) The ER stress was studied by ELISA method to measure the protein expression of BiP. (n = 3, one-way ANOVA, Turkey’s test; *** is used for P < 0.001). (C) The nuclei morphology was analyzed using DAPI staining under Zeiss Axio Imager A1 microscope. Scale bar = 20 μm. These results revealed that the protective effects of the LIPEF and fucoidan could maintain the mitochondrial function, reduce the ER stress, and suppress the apoptotic response in the NSC-34 cells exposed to H2O2 for 24 h.
Fig 4.
Effects of the combination treatment on the protein levels of ROCK and p-Akt.
(A) Protein expression levels of ROCK and p-Akt were measured using Western blot analysis. The expression level of ROCK and p-Akt were normalized to GAPDH and total Akt, respectively. Each relative expression level was compared with control. (B) ROCK band intensities and (C) p-Akt band intensities were quantified to understand the mechanism underlying the protective effect. (n = 3, Student’s t-test; ** is used for P < 0.01 and *** for P < 0.001).
Fig 5.
Effects of the combination treatment on the protein levels of Bcl-2 and Bax.
(A) Protein expression levels of Bcl-2 and Bax were measured using Western blot analysis. Each relative expression level was normalized to GAPDH and compared with control. (B) Band intensities were quantified to obtain the Bax/Bcl-2 ratio. (n = 3, Student’s t-test; *** is used for P < 0.001).
Fig 6.
Effect of the combination treatment on the H2O2-induced neurite retraction.
(A) The morphology of neuron cells was observed using immunofluorescence staining of beta-III tubulin under Zeiss Axio Imager A1 microscope. Scale bar = 20 μm. The average number of neurites per cell (B) and the average neurite length (C) were quantified to evaluate the neuroprotective effect. (n = 8, one-way ANOVA, Turkey’s test; *** is used for P < 0.001).