Ethanol extract of Oenanthe javanica increases cell proliferation and neuroblast differentiation in the adolescent rat dentate gyrus.

Oenanthe javanica is an aquatic perennial herb that belongs to the Oenanthe genus in Apiaceae family, and it displays well-known medicinal properties such as protective effects against glutamate-induced neurotoxicity. However, few studies regarding effects of Oenanthe javanica on neurogenesis in the brain have been reported. In this study, we examined the effects of a normal diet and a diet containing ethanol extract of Oenanthe javanica on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus of adolescent rats using Ki-67 (an endogenous marker for cell proliferation) and doublecortin (a marker for neuroblast). Our results showed that Oenanthe javanica extract significantly increased the number of Ki-67-immunoreactive cells and doublecortin-immunoreactive neuroblasts in the subgranular zone of the dentate gyrus in the adolescent rats. In addition, the immunoreactivity of brain-derived neurotrophic factor was significantly increased in the dentate gyrus of the Oenanthe javanica extract-treated group compared with the control group. However, we did not find that vascular endothelial growth factor expression was increased in the Oenanthe javanica extract-treated group compared with the control group. These results indicate that Oenanthe javanica extract improves cell proliferation and neuroblast differentiation by increasing brain-derived neurotrophic factor immunoreactivity in the rat dentate gyrus.

Introduction

The hippocampus is involved in cognitive processes such as declarative memory, context-dependent and spatial learning processes (Wang et al., 2011; Gil-Mohapel et al., 2013; Jelenkovic et al., 2014). It has been reported that two regions in the brain, the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricle, can improve self-renewal activity throughout the animal's life (Yamaguchi et al., 2000; Yoo et al., 2012; Yang et al., 2014). In the SGZ of the hippocampal DG, neural precursor cells undergo cell proliferation, maturation, migrate into the granule cell layer, and integrate into pre-existing neural circuitry (Kempermann, 2008; Chen et al., 2013).

It has been reported that neurogenesis in the DG can be altered by various factors, such as pharmacological drugs, aging and neurodegenerative diseases (Choi et al., 2012). Many studies have shown that brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) are important neurotrophic factors (Pencea et al., 2001; Li et al., 2012; Kim et al., 2014), and they are widely studied in various research areas, such as neurogenesis in the hippocampal DG (Ji et al., 2014; Zeng et al., 2014). In particular, some researchers have reported that chronic stress in animal models inhibits neurogenesis, suggesting a lower expression of BDNF in the DG (Roceri et al., 2002; Garza et al., 2012). In addition, VEGF is an angiogenic and vascular permeability factor, and blood vessels influence neurogenesis in the SGZ of the DG in the adult rat (Palmer et al., 2000). Jin et al. ( 2002) have reported that VEGF can stimulate neurogenesis in major neuroproliferative zones (SGZ and SVZ) of the adult rat brain.

Oenanthe javanica is an aquatic perennial herb that belongs to the Oenanthe genus in Apiaceae family, and it has well-known medicinal properties (Jiang et al., 2014). The fresh stems and leaves are widely used as a seasoning in diet in Korea (Seo and Baek, 2005), and they are used in traditional Chinese medicine for the treatments of fever, leucorrhea, mumps, difficult urination and hypertension (Kim et al., 2013). Previous studies indicate that Oenanthe javanica has effects of anti-inflammatory (Yang et al., 2013), antioxidant and antigenotoxic activities (Kwon et al., 2006). In clinical applications, it may have some efficacy on acute liver diseases (Yang et al., 2014), sepsis, and septic shock (Kim et al., 2013). It has been also reported that persicarin, a main constituent of Oenanthe javanica, significantly protects primary cultured rat cortical cells from glutamate-induced toxicity (Ma et al., 2010). However, few studies regarding effects of Oenanthe javanica on neurogenesis in the brain have been reported.

Therefore, in the present study, we investigated the effects of Oenanthe javanica extract (OJE) on cell proliferation and neuroblast differentiation in the DG of the adolescent rat using Ki-67 (an endogenous marker for cell proliferation) and doublecortin (DCX, a marker for neuroblast). In addition, we examined the effects of OJE on immunoreactivities of BDNF and VEGF in the DG of the adolescent rats.

Materials and Methods

Experimental animals

Fourteen male Wistar rats, weighing 110–130 g, aged 5 weeks, were purchased from Orient Bio Inc. (Seongnam, South Korea). They were housed in a conventional state under adequate temperature (23°C) and humidity (60%) control with a 12-hour light/dark cycle, and free access to food and water. The procedures for handling and caring for the animals adhered to the guidelines that are in compliance with the current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985, revised 1996), and were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University. All of the experiments were conducted with an effort to minimize the number of animals used and the suffering caused by the procedures used in the present study.

Preparation of OJE

Oenanthe javanica was collected by Professor Jong Dai Kim in Kangwon province (South Korea), in March 2013 and kept in a deep freezer (−70°C). Oenanthe javanica was extracted with 10 vol(v/w) of 70% ethanol at 70°C for 4 hours, and extraction was repeated three times. The extracts were filtered through Whatman filter paper (No.2), concentrated with a vacuum evaporator, and completely dried with a freeze-drier and then ground into fine powder. The extraction yield was 14.5%. OJE was mixed by 0.5% in normal composition diet. Compositions of experimental diets are represented in .

Table 1

Composition of experimental diets

Treatment of OJE

The rats were divided into two experimental groups: (1) vehicle-treated group as the control (n = 7) and (2) OJE-treated group (n = 7). As previously described (Tae et al., 2014), rats of the control group were fed with a normal diet, and rats of the OJE-treated group were fed with a diet containing OJE () for 4 weeks, because, in traditional Chinese medicine, extracts from plants have been taken orally and there are no data about the absorption and metabolism of OJE. Each rat was housed and fed individually in plastic cage (40 cm × 25 cm× 15 cm) during experiment. Each rat was given experimental diets (approximately 50 g in total) every other day. The body weight was measured before diet or after diet for 4 weeks, and food intake was recorded at regular time as 2 days interval for 4 weeks. There were no significant differences in body weight and food intake between the control and OJE-treated groups ().

Table 2

The effects of Oenanthe javanica extract (OJE) diet on body weight and food intake

Tissue processing for histology

For histological analysis, the rats (n = 7 in each group) were anesthetized with 30 mg/kg Zoletil 50 (Virbac, Carros, France) and perfused transcardially with 0.1 M phosphate-buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The brains were removed and postfixed in the same fixative for 4 hours. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter, frozen tissues were serially sectioned on a cryostat (Leica, Wetzlar, Germany) into 30-μm coronal sections, and they were then collected into 6-well plates containing PBS for further process.

Immunohistochemistry

To obtain accurate data for immunohistochemistry, the sections were carefully processed under the same conditions. The tissue sections were selected between −3.00 and −4.08 mm posterior to the bregma in reference to the rat. The sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 minutes and 10% normal goat serum in 0.05 M PBS for 30 minutes. The sections were next incubated with diluted rabbit anti-Ki-67 (1:100; Abcam, Cambridge, UK), goat anti-DCX (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-BDNF (1:400; Abcam), rabbit anti-VEGF (1:200; Santa Cruz Biotechnology) overnight at 4°C. Thereafter, the sections were exposed to biotinylated goat anti-rabbit or rabbit anti-goat IgG (1:200; Vector, Burlingame, CA, USA) for 2 hours at room temperature and streptavidin peroxidase complex (1:200; Vector) for 1 hour at room temperature. They were visualized with 3,3′-diaminobenzidine tetrahydrochloride in 0.1 M Tris-HCl buffer and mounted on the gelatin-coated slides. After dehydration, the sections were mounted in canada balsam (Kanto Chemical, Japan).

Images of Ki-67, DCX, BDNF and VEGF-immunoreactive structures were taken from the dentate gyrus through AxioM1 light microscope (BX53, Olympus, Germany) equipped with digital camera (DP72, Olympus) connected to a PC monitor. Total number of Ki-67 or DCX positive cells in all the groups was counted in 15 sections/rat using an image analyzing system equipped with a computer-based CCD camera (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ, USA). Cell counts were obtained by averaging the counts from the sections taken from each rat. In addition, the staining intensity of BDNF and VEGF-immunoreactive structures was evaluated on the basis of an optical density (OD), which was obtained after the transformation of the mean gray level using the formula: OD = log (256/mean gray level). The OD of background was taken from areas adjacent to the measured area. After the background density was subtracted, a ratio of the OD of image file was calibrated as % (relative OD, ROD) using Adobe Photoshop version 8.0 and then analyzed using NIH Image 1.59 software. All measurements were performed in order to ensure objectivity under blind conditions, by two observers for each experiment, and experimental samples were measured under the same conditions.

Statistical analysis

The data shown here are expressed as the mean ± SEM. Differences of the means among the groups were statistically analyzed by one-way analysis of variance with Duncan's post hoc test. Statistical significance was considered at P < 0.05.

Results

Effects of OJE on cell proliferation

Ki-67-immunoreactive cells were found in the SGZ of the DG in all the experimental groups (). In the control group, Ki-67-immunoreactive cells were easily observed in the SGZ, and the mean number of them was 5.7 ± 0.41 per section (Figure and ). In the OJE-treated group, the number of Ki-67-immunoreactive cells was significantly increased in the DG compared with the control group, and the mean number of them was 16.2 ± 0.12 per section (about 284% of the control group) (P < 0.05; Figure and ).

Figure 1

Ki-67 immunohistochemistry in the dentate gyrus of rats in the control and Oenanthe javanica extract (OJE)-treated groups.

Immunohistochemistry for Ki-67 in the DG of the control (A and a) and OJE- (B and b) treated groups. Ki-67-immunoreactive cells are significantly increased in the OJE-treated group compared with the control group. ML: Molecular layer; GCL: granule cell layer; PL: polymorphic layer. Scale bars: 200 μm (A and B), 40 μm (a and b). Insets show higher magnification to point out Ki-67-immunoreactive cells. (C) Analysis of Ki-67-immunoreactive cells per section of each group (n = 7 per group; *P < 0.05, significantly different from the control group; one-way analysis of variance with Duncan's post hoc test). The bars indicate the mean ± SEM.

Effects of OJE on neuronal differentiation

In all the experimental groups, DCX-immunoreactive neuroblasts were mainly detected in the SGZ of the DG (). In the control group, DCX-immunoreactive neuroblasts were easily observed in the SGZ of the DG, and some of them had poorly developed processes (Figure and ). The mean number of DCX-immunoreactive neuroblasts was 13.7 ± 2.24 per section (Figure and ). In the OJE-treated group, the number of DCX-immunoreactive neuroblasts was significantly increased in the SGZ of the DG, and their processes were very long and thick compared with those in the control group (Figure and ). In addition, the mean number of DCX-immunoreactive neuroblasts was 33.4 ± 3.19 per section and significantly higher (about 244 % of the control group) compared with the control group (P < 0.05; (Figure and ).

Figure 2

Doublecortin (DCX) immunohistochemistry in the dentate gyrus of the control and Oenanthe javanica extract (OJE)-treated groups.

DCX immunohistochemistry in the DG of the control (A and a) and OJE- (B and b) treated groups. In the OJE-treated group, the number of DCX-immunoreactive neuroblasts is significantly increased, and their processes are much longer and thicker than those in the control group. ML: Molecular layer; GCL: granule cell layer; PL: polymorphic layer. Scale bar: 200 μm (A and B), 40 μm (a and b). Insets show higher magnification to point out DCX-immunoreactive neuroblasts. (C) Analysis of DCX-immunoreactive neuroblasts per section of the control and OJE-treated groups (n = 7 per group; *P < 0.05, significantly different from the control group; one-way analysis of variance with Duncan's post hoc test). The bars indicate the mean ± SEM.

Effects of OJE on immunoreactivities of BDNF and VEGF

In the control group, weak BDNF immunoreactivity was observed in the granule cell layer of the DG (). In the OJE-treated group, BDNF immunoreactivity was significantly increased in the granule cell layer of the DG compared with the control group (Figure and ).

Brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) immunohistochemistry in the dentate gyrus of the control and Oenanthe javanica extract (OJE)-treated groups.

BDNF (A and B) and VEGF (C and D) immunohistochemistry in the dentate gyrus of the control and OJE-treated groups. BDNF immunoreactivity (asterisk) is significantly increased in the OJE-treated group compared with the control group. However, VEGF immunoreactivity in the OJE-treated group is not changed. ML: Molecular layer; GCL: granule cell layer; PL: polymorphic layer. Scale bar: 200 μm. (E) Relative optical density (ROD) as percentage of BDNF- and VEGF-immunoreactive structures in the control and OJE-treated groups (n = 7 per group; *P < 0.05, significantly different from the control group; one-way analysis of variance (ANOVA) with Duncan's post hoc test). The bars indicate the mean ± SEM.

On the other hand, weak VEGF immunoreactivity was detected in the granule cell layer of the DG in the control group, and the VEGF immunoreactivity in the OJE-treated group was similar to that in the control group (Figure and ).

Discussion

In the present study, we examined the effects of OJE on cell proliferation and neuroblast differentiation in the SGZ of the DG of the adolescent rats. Our results showed markedly increased numbers of Ki-67-immunoreactive cells and DCX-immunoreactive neuroblasts in the OJE-treated group compared with those in the control group. Furthermore, in the OJE-treated group, the neuroblasts displayed well-developed processes.

Recently, some researchers demonstrated that dendrite maturation was closely related to the survival of newborn neurons (Plumpe et al., 2006). In addition, Snyder et al. (2005) reported that newly generated cells might be associated with spatial learning and memory of normal hippocampal function in the adult hippocampus. It is well known that DCX is a specific marker for neuronal precursors and neurogenesis in the development of the adult CNS (Bonfanti and Theodosis, 1994; Brown et al., 2003; Jin et al., 2004). It was reported that the increase of DCX immunoreactivity could result in increasing branch points and complexity of the dendrites of DCX-immunoreactive neuroblasts (Cohen et al., 2008; Yoo et al., 2011). Therefore, our present finding in the OJE-treated-group indicates that OJE may be involved in neuroblast maturation.

Some plants have ability to create hippocampal neurogenesis. For example, Rosa damascene enhances adult neurogenesis, hippocampal volume and synaptic plasticity in a rat model of Alzheimer's disease (Esfandiary et al., 2014). In this study, we used Oenanthe javanica, which is an aquatic perennial herb, and our results showed that OJE improved cell proliferation and neuroblast differentiation in the SGZ of the DG.

We also detected the immunoreactivities of BDNF and VEGF in the DG of the OJE-treated group, and we found that the immunoreactivity of BDNF, not VEGF, was significantly increased after OJE treatment. It was reported that the expression of BDNF was increased in the DG of the hippocampus, and the elevation might enhance neurogenesis in the brain (Kaloustian et al., 2008; Hwang et al., 2009). In addition, Lee et al. (2002) showed that neurogenesis was significantly increased after the administration of BDNF, and, recently, Dong et al. (2014) reported that the induction of hippocampal neurogenesis was linked with the activation of ERK1/CREB signaling pathway by upregulating the level of BDNF.

It is well known that VEGF is a signal protein linked with the regulation of physiological and pathological angiogenesis (Ferrara and Davis-Smyth, 1997; Nowacka and Obuchowicz, 2013). Administration of VEGF could markedly reduce neuronal death (Qiu et al., 2003) and increase neurogenesis and angiogenesis in the ischemic brain in vitro and in vivo (Jin et al., 2002). However, in this study, we did not find that the immunoreactivity of VEGF was increased in the DG after OJE treatment. This finding indicates that OJE does not increase the expression of endogenous VEGF, although we cannot exactly explain why VEGF immunoreactivity is increased in the rat DG after OJE treatment.

In brief, in this study, we showed that cell proliferation and neuroblast differentiation were markedly increased in the DG of the OJE-treated adolescent rats. In addition, BDNF immunoreactivity was significantly increased after OJE administration. These results suggest that OJE has a potential to significantly increase cell proliferation and neuroblast differentiation. These effects of OJE might be associated with the increase of BDNF immunoreactivity in the rat DG.