Noonong K et al. (2020), Neuroprotective and Neurorestorative Effects of...

ECB-ART-49386

Front Neurosci 2020 Feb 18;14:575459. doi: 10.3389/fnins.2020.575459.

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Neuroprotective and Neurorestorative Effects of Holothuria scabra Extract in the MPTP/MPP+-Induced Mouse and Cellular Models of Parkinson's Disease.

Noonong K , Sobhon P , Sroyraya M , Chaithirayanon K .

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Extracts from Holothuria scabra (HS) have been shown to possess anti-inflammation, anti-oxidant and anti-cancer activities. More recently, it was shown to have neuroprotective potential in Caenorhabditis elegans PD model. Here, we assessed whether HS has neuroprotective and neurorestorative effects on dopaminergic neurons in both mouse and cellular models of PD. We found that both pre-treatment and post-treatment with HS improved motor deficits in PD mouse model induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as determined by grid walk test. This was likely mediated by HS protective and restorative effects on maintaining the numbers of dopaminergic neurons and fibers in both substantia nigra pars compacta (SNpc) and striatum. In a cellular model of PD, HS significantly attenuated 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of DAergic-like neurons differentiated from SH-SY5Y cells by enhancing the expression of Bcl-2, suppressing the expression of cleaved Caspase 3 and preventing depolarization of mitochondrial membrane. In addition, HS could stimulate the expression of tyrosine hydroxylase (TH) and suppressed the formation of α-synuclein protein. Taken together, our in vivo and in vitro findings suggested that HS is an attractive candidate for the neuroprotection rather than neurorestoration in PD.

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	FIGURE 1. Timeline of th eexperimental procedure. Mice were randomly assigned to eight groups (n = 20 per group) consisting of two control [normal saline solution (NSS) control group and Holothuria scabra (HS) control group] and six study groups. The mice groups III to VIII were managed to subsequent behavioral tests after completed with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment at day 0 and day 3. HS extract was dissolved in NSS (0.9% NaCl). Mice were euthanized by cervical dislocation.
	FIGURE 2. The number of foot slips during grid walk on day 0 and 3 after MPTP administration is shown in histograms. Data are presented as the means ± standard error of the mean (SEM) P < 0.05, P < 0.01, and **P < 0.001, in comparison with the NSS-injected group. ###P < 0.001, in comparison with the MPTP-injected group.
	FIGURE 3. (A) Dopaminergic neurons and fibers were detected with tyrosine hydroxylase (TH) immunostaining in the substantia nigra pars compacta (SNpc) and striatum of mice brain at high magnification (20 ×) containing immunolabeled TH-positive dopamine (DA) neurons and fibers (brown). (B) Number of dopaminergic neurons with TH immunostaining in SNpc on days 0 and 3. (C) Number of dopaminergic fibers with TH immunostaining in the striatum. Data are presented as the means ± standard error of the mean (SEM) P < 0.05, P < 0.01, and **P < 0.001, in comparison with the NSS-injected group. ##P < 0.01, and ###P < 0.001, in comparison with the MPTP-injected group.
	FIGURE 4. (A) Viability of DAergic-like neurons after incubation with HS at concentrations of 0.5, 1, 5, 10, and 50 μg/ml for 24 and 48 h. (B) Effects of HS extracts on 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in DAergic-like neurons. DAergic-like neurons were pretreated with HS (0.5, 1, and 5 μg/ml) for 24 and 48 h and then exposed to 500 μM MPP+ for 24 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Data are presented as the means ± standard error of the mean (SEM) and ∗∗∗P < 0.001, in comparison with the untreated group. ##P < 0.01, and ###P < 0.001, in comparison with the MPP+-treated group.
	FIGURE 5. The mitochondria membrane potential (MMP) was assessed by JC-1 staining. Green JC-1 monomers indicate depolarized MMP, while red JC-1 dimer indicates normal MMP. Merged panel represents colocalization between red dimer and green monomer fluorescence. (A) JC-1 staining in untreated DAergic-like neurons, DAergic-like neurons treated with 500 μM MPP+, DAergic-like neurons with 1 μg/ml of HS pretreatment, followed by 500 μM MPP+. (B) Quantitative analysis of relative fluorescence between red and green in each group as measured by microplate reader. Data are presented as the means ± standard error of the mean (SEM) **P < 0.01, in comparison with the untreated group. ###P < 0.001, in comparison with the MPP+-treated group.
	FIGURE 6. (A) Effects of HS on MPP+-induced DAergic-like neuron apoptosis shown by Hoechst 33258 stain; upper panel indicates DNA condensation in the nucleus. (B) Histograms showed percentage of apoptotic cells. (C) Expression levels of Bax, Bcl-2, and cleaved caspase 3 protein as determined by Western blot (analysis; actin was used as the control and for normalizing the intensities of the target proteins in untreated, treated with 500 μM MPP+, or pretreated with 1 μg/ml of HS then followed by 500 μM MPP+. (D) Histograms showed the relative intensity of Bax, Bcl-2, and cleaved caspase 3 expressions as normalized by actin. (E) Expression levels of tyrosine hydroxylase (TH) and α-synuclein in MPP+-induced DAergic-like neurons by Western blot, comparing between the untreated control, treated with 500 μM MPP+, treated with 1 μg/ml of HS and 500 μM MPP+. (F) Histograms showed the relative intensity of TH expression as normalized by action. (G) Histograms showed the relative intensity of α-synuclein expression as normalized by action. Data are presented as the means ± standard error of the mean (SEM) ∗P < 0.05, and ∗∗∗P < 0.001, in comparison with the untreated group. ##P < 0.01, and ###P < 0.001, in comparison with the MPP+-treated group.)

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