The protective effect of non-invasive low intensity pulsed electric field and fucoidan in preventing oxidative stress-induced motor neuron death via ROCK/Akt pathway

Abstract

With the expansion of the aged population, it is predicted that neurodegenerative diseases (NDDs) will become a major threat to public health worldwide. However, existing therapies can control the symptoms of the diseases at best, rather than offering a fundamental cure. As for the complex pathogenesis, clinical and preclinical researches have indicated that oxidative stress, a central role in neuronal degeneration, is a possible therapeutic target in the development of novel remedies. In this study, the motor neuron-like cell line NSC-34 was employed as an experimental model in probing the effects induced by the combination of non-invasive low intensity pulsed electric field (LIPEF) and fucoidan on the H2O2-induced neuron damage. It was found that single treatment of the LIPEF could protect the NSC-34 cells from oxidative stress, and the protective effect was enhanced by combining the LIPEF and fucoidan. Notably, it was observed that single treatment of the LIPEF obviously suppressed the H2O2-enhanced expression of ROCK protein and increased the phosphorylation of Akt in the H2O2-treated NSC-34 cells. Moreover, the LIPEF can be easily modified to concentrate on a specific area. Accordingly, this technique can be used as an advanced remedy for ROCK inhibition without the drawback of drug metabolism. Therefore, we suggest the LIPEF would be a promising strategy as a treatment for motor neurodegeneration and warrant further probe into its potential in treating other neuronal degenerations.

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).