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. 2018 Nov 19;2018:7383869.
doi: 10.1155/2018/7383869. eCollection 2018.

Antidepressant-Like and Neuroprotective Effects of Ethanol Extract From the Root Bark of Hibiscus syriacus L

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Antidepressant-Like and Neuroprotective Effects of Ethanol Extract From the Root Bark of Hibiscus syriacus L

Young Hwa Kim et al. Biomed Res Int. .
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Abstract

Hibiscus syriacus L. (Malvaceae) is an important ornamental shrub in horticulture and has been widely used as a medical material in Asia. The aim of this study was to assess the antidepressant and neuroprotective effects of a root bark extract of H. syriacus (HSR) and to investigate the underlying molecular mechanisms. Using an animal model of restraint stress, we investigated the effects of HSR on depressive-like behaviors and on the expression levels of serotonin, corticosterone, and neurotrophic factors in the brain. The mice were exposed to restraint stress for 2 h per day over a period of 3 weeks and orally treated with HSR (100, 200, or 400 mg/kg/day). We also examined the neuroprotective effect of HSR using corticosterone-treated human neuroblastoma SK-N-SH cells. The cells were incubated with the extract for 24 h, followed by corticosterone stimulation for 1 h, and then cell viability assay, cellular ATP assay, mitochondrial membrane potential (MMP) assay, cellular reactive oxygen species (ROS) assay, and western blotting were used to investigate the neuroprotective effects of HSR. Administration of HSR not only reduced the immobility times of the restraint-stressed mice in the forced swimming and tail suspension tests, but also significantly increased sucrose preference in the sucrose preference test. In addition, HSR significantly reduced the plasma levels of corticosterone and increased the brain levels of serotonin. The extract also increased the phosphorylation level of cyclic AMP response element-binding (CREB) protein and the expression level of brain-derived neurotrophic factor (BDNF). The in vitro assays showed that HSR pretreatment increased cell viability and ATP levels, recovered MMP, decreased ROS levels, and increased the expression of CREB and BDNF in corticosterone-induced neurotoxicity. Taken together, our data suggest that HSR may have the potential to control neuronal cell damage and depressive behaviors caused by chronic stress.

Figures

Figure 1
Figure 1
HPLC analysis of HSR. (a) HPLC chromatogram of HSR. (b) HPLC chromatogram of 11-hydroxy-9,12-octadecadienoic acid and (9Z,11E)-8,13-dihydroxy-9,11-octadecadienoic acid.
Figure 2
Figure 2
Effects of HSR on depression-related behaviors in restraint stress-induced mice. (a) Animal experimental protocol. The mice were orally administered vehicle (saline), HSR (100, 200, or 400 mg/kg), or fluoxetine (FXT; 20 mg/kg) daily for the indicated period. Restraint stress and oral administration schedules for behavioral experiments are presented. FST: forced swimming test; SPT: sucrose preference test; TST: tail suspension test. (b) Sucrose preference was measured on the indicated days. The immobility time in (c) FST and (d) TST was measured on day 23. Data are the mean ± SD (n = 6). #p < 0.05, ##p < 0.01, and ####p < 0.0001 vs. the normal group; p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 vs. the control group.
Figure 3
Figure 3
Effects of HSR on plasma levels of corticosterone (a) and brain levels of serotonin (b) in restraint stress-induced mice. Data are the means ± SD (n = 4, corticosterone; n = 3, serotonin). ####p < 0.0001 vs. the normal group; p < 0.05 and ∗∗∗∗p < 0.0001 vs. the control group.
Figure 4
Figure 4
Effects of HSR on BDNF and p-CREB/CREB expression in the brain of restraint stress-induced mice. Lysates of isolated prefrontal cortex and hippocampus were analyzed by western blotting using (a) BDNF and (b) p-CREB/CREB antibodies. β-Actin was used as the loading control. The data are representative of three independent experiments. Data are the mean ± SD (n = 3). ###p < 0.001 vs. the normal group; p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 vs. the control group.
Figure 5
Figure 5
Effects of HSR on cell viability, mitochondrial function, and mitochondrial superoxide generation in SK-N-SH cells. Cells were pretreated with 0, 10, 50, and 100 μg/mL HSF for 1 h before the addition of 0.25 mM corticosterone. (a) Cell viability. (b) ATP levels. (c) Apoptotic caspase-3/7 activity. (d) The mitochondrial membrane potential (MMP) was measured using JC-1 fluorescence. (e) Reactive oxygen species (ROS) generation was measured by H2DCFDA staining. (f) Mitochondrial superoxide production was measured by confocal microscopy (60 × 1.0). Data are expressed as a percentage of control. Data are the mean ± SD (n = 3 per group). ##p < 0.01 and ####p < 0.0001 vs. the normal group; ∗∗p < 0.01 and ∗∗∗∗p < 0.0001 vs. the control group.
Figure 6
Figure 6
Effects of HSR on corticosterone-induced proinflammatory cytokine expression. Total RNA was extracted from SK-N-SH cells, and the levels of (a) IL-1β, (b) IL-6, (c) IL-8, and (d) TNF-α mRNA were determined by qRT-PCR. Expression levels of the target genes were normalized to those of β-actin. Data are the mean ± SD (n = 3). ###p < 0.001 and ####p < 0.0001 vs. the normal group; p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 vs. the control group.
Figure 7
Figure 7
Effects of HSR on protein expression of BDNF, CREB, and MAPKs in corticosterone-treated SK-N-SH cells. (a) Western blotting analysis and (b) intensity of protein bands were quantified by densitometry. Total CREB, ERK, JNK, p38, and β-actin were used as internal standards. The data are representative of three independent experiments. Data are the mean ± SD (n = 3). ###p < 0.001 and ####p < 0.0001 vs. the normal group; p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 vs. the control group.

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