Protective Effects of Ramie (Boehmeria nivea) against Oxidative Stress in C6 Glial Cells

Abstract

β amyloid protein (Aβ) plays a critical role in the pathogenesis of Alzheimer's disease (AD) and possibly in Aβ-induced mitochondrial dysfunction and oxidative stress. Aβ can directly cause reactive oxygen species (ROS) production. Overproduction of ROS is considered to be involved in the pathogenesis of neurodegeneration of AD. Here, we investigated 9 kinds of ramie (Boehmeria nivea, (L.) Gaud., BN; hereafter denoted as BN) for their protective action against oxidative stress in a cellular system using C6 glial cells. We observed loss of cell viability and high levels of ROS generation after treatment with hydrogen peroxide (H₂O₂) and Aβ_25-35. However, treatments with BN extracts led to an increase in cell viability and decrease in ROS production induced by H2O2 and Aβ25-35. In particular, the extracts of BN-01 (seobang variety from Seocheon) and BN-09 (local variety from Yeonggwang) showed excellent anti-oxidative properties. This indicates that BN extracts could prevent neurodegeneration by reducing oxidative stress in cells.

Introduction

Oxidative stress is a potential threat to most aerobic organisms and plays a crucial role in biocontrol systems. It leads to neuro-degeneration, also it has been closely implicated to neurodegenerative disorders (Di Carlo et al., 2012; Han and Wang, 2010; Lee and Cho, 2007). Oxidative stress is one of the earliest events in Alzheimer’s disease (AD) (Nunomura et al., 2001) and β amyloid protein (Aβ) is known to contribute to the oxidative damage leading to neuronal impairment (Yatin et al., 1999). In addition, oxidative stress is associated with enhanced levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Castoria et al., 2003; Macarisin et al., 2010). At low levels, ROS act as signaling molecules to regulate cellular functions. However, at high levels, they cause oxidative stress (Madeo et al., 1999; Tatone et al., 2010). In particular, the ROS containing O2―, H2O2, 1O2, HO2―, •OH, ROOH, ROO•, and RO• radicals are highly reactive and toxic. They cause damage to proteins, lipids, carbohydrates, and DNA which ultimately results in cell death (Gill and Tuteja, 2010). Neurons are most susceptible to direct oxidative injury by ROS and RNS (Juvenet, 1889; Butterfield and Lauderback, 2002). Glial cells are very important for normal brain function and oxidative damage can cause rapid changes in almost all cells including glial cell (Araujo and Cotman, 1992; Zhao et al., 2013). Therefore, one of the most promising therapeutic strategies for the treatment of neurodegenerative diseases, including AD, is the reduction of oxidative damage in cells.

Ramie (Boehmeria nivea (L.) Gaud., BN; hereafter referred to as BN) is a member of the nettle family (Urticaceae) and is mainly grown in temperate and tropical areas including China, Korea, Philippines, and India (Angelini et al., 2000; Wang et al., 2006). Popularly known as “China grass”, it is a perennial herbaceous plant and has been used as a textile fiber for centuries because of its excellent fiber quality (Wood and Angus, 1974; Liu et al., 2001). It is rich in nutrients such as vitamins, minerals, and various bioactive substances (Gupta and Wagle, 1988). Phytochemicals are bioactive substances of plants that have been associated with the protection of human health against chronic degenerative diseases (Fukumoto and Mazza, 2000). The major compounds of chemical constituents are chlorogenic acid, rutin, luteolin-7-glucoside, naringin, hesperidin, and tangeretin (Park et al., 2010). It is known to contain behenic acid, ursolic acid, β-sitosterol, cholesterol, kiwiionoside, uracil, quercetin, α-amyrin, nonacosanol, emodin, emodin-8-O-β-glucoside, physcion, polydatin, catechin, epicatechin, and epicatechin gallate (Nho et al., 2010; Shao et al., 2010). It has been used in foods such as cookies (Yoon and Jang, 2006) and Jeolpyun (traditional Korean rice cakes) (Tian et al., 2011). The edible parts of this plant - the leaves and roots - have been reported to have anti-inflammatory and anti-fungal effects (Xu et al., 2011; Sung et al., 2013), in addition to anti-hepatitis B and anti-diabetic effects (Huang et al., 2006; Kim et al., 2013). However, whether ramie has a neuroprotective effect has not been determined. In our previous study, we screened the biological activities of 90 kinds of Korean ramie (Lee et al., 2014). Among them, 9 kinds cultivated in Seocheon, Goheung, Muan, Hampyeong, and Yeonggwang showed excellent biological anti-oxidative, anti-bacterial, anti-inflammatory, and anti-cancer properties (Lee et al., 2014).

Therefore, in this study we evaluated the extracts from these 9 kinds for their potential in treating AD; for this, we measured their ability to reduce H2O2 and Aβ25-35-induced oxidative stress in C6 glial cells.

 

Materials and Methods

Plant materials and preparation of extracts

Nine kinds of ramie were collected by the staff at the Yeong-Gwang Agricultural Technology Center, Korea (Table 1). Ten grams of dried BN were extracted with MeOH (200 ml × 3) under reflux conditions and the solvent was evaporated in vacuo. Each individual MeOH extract (0.1 mg) was dissolved in dimethyl sulfoxide (DMSO) (5 μl).

Table 1.BN kinds and their collection sites

Instruments and reagents

MeOH was purchased from Sam Chun Pure Chemical Co. (Pyeongtaek, Korea). C6 glial cells were obtained from the Korea Cell Line Bank (KCLB, Korea). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin EDTA, and penicillin-streptomycin (100 unit/ml) were obtained from Welgene (Daegu, Korea). Hydrogen peroxide (H2O2) was purchased from Junsei Chemical Co. (Tokyo, Japan). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Bio Basic Canada Inc. (New York, USA). DMSO and Aβ25-35 were obtained from Sigma Chemical Co. (Louisiana, USA).

Cell culture

C6 glial cells were cultured in DMEM medium (pH 7.2) containing 100 U/ml of penicillin streptomycin and 10% FBS at 37℃ in a 5% CO2 incubator. Cells were sub-cultured 5 days with 0.05% trypsin-EDTA in phosphate buffered saline (PBS).

Cell viability assay

Once the cells reached 80-90% confluence, they were plated into 96-well plates at 5 × 104 cells/ml and cells were incubated with medium for 2 h before treatment with H2O2/A β25-35 and BN extracts. BN extracts (25 μg/ml) and H2O2 (250 μM) or Aβ25-35 (25 μM) were added, and the plates were incubated for 24 hr. Thereafter, 100 μL of MTT (5 mg/ml) solution was added to each well. After the incubation for 4 h at 37℃, the medium was removed from the plate. The resultant formazan crystals in the C6 glial cells were solubilized by adding 100 μl of DMSO. The absorbance of each well was read at 540 nm using a microplate reader (Molecular Devices, Sunnyvale, USA) (Mosmann, 1983).

ROS measurement

Cells were plated into black 96-well plates at 5 × 104 cells/ml and cells were incubated with medium for 2 h before treatment with H2O2/Aβ25-35 and BN extracts. BN extracts (25 μg/ml) were added, and the plates were incubated for 24 hr. Cells were washed twice with PBS, and incubated with dichlorodihydro-fluorescein diacetate (DCFH-DA) (Sigma Chemical Co., Louisiana, USA) 20 μM for 30 min. Thereafter, H2O2 (250 μM) or Aβ25-35 (25 μM) was treated for 24 h, and the wells were read by FLUOstar OPTIMA (BMG Labtech., Ortenberg, Germany) at an excitation wavelength of 480 nm and an emission wavelength of 535 nm (Byun et al., 2009).

Statistical analysis

Significance was verified by performing Duncan’s multiple range tests using SAS software (version 6.0, SAS Institute, Cary, USA) to analyze the differences between the control and sample treated groups. Differences between groups were considered significant when the P-value was less than 0.05. The experimental results were expressed as means ± standard deviation (SD) (n = 6).

 

Results and Discussion

Protective effects of BN extracts against H2O2-induced oxidative stress and ROS levels in C6 glial cells

ROS impairs the physiological functions of C6 glial cells and causes cell death. Free radicals, such as O2―, •OH, and •1O2, as well as non-radical species, such as H2O2, cause cell damage by increasing oxidative stress (Chen and Gibson, 2008). H2O2 is known to be a strong inducer of ROS, and if present at high levels promotes cell death (Woo et al., 2003; Miller et al., 2010). The cytotoxicity test using MTT assays is shown in Fig. 1. The treatment of BN extracts showed 77.8% to 97.5% of cell viability. Most of the BN extracts showed little or no cytotoxic activity in C6 glial cells up to 25 μg/ml, therefore we used the concentration of 25 μg/ml. The protective effect of the BN extracts against H2O2-induced oxidative stress in C6 glial cells is shown in Fig. 2. Cell viability was significantly lower (58.2%) in H2O2-treated cells than in untreated cells. Cell viability improved when the 9 kinds of BN extracts were added at concentration of 25 μg/ml. BN-04, BN-05 and BN-08 have a little cytotoxic activity in C6 glial cells, so it showed lower cell viability than control group. On the other hand, the BN-01 (64.4%), BN-02 (64.1%), BN-03 (63.5%), BN-06 (64.5%), BN-07 (78.6%), and BN-09 (70.1%) shown higher than cell viability of control group, which indicate the protective activity against oxidative stress induced by H2O2.

Fig. 1.Cytotoxicity of BN extracts at on viability of C6 glial cells.

Fig. 2.Effect of BN extracts on viability of C6 glial cells treated with H2O2.

The production of ROS was monitored using DCFH-DA, a standard compound used to detect and quantify intracellularly produced H2O2 (Myhre, 2003). The fluorescence was proportional to the amount of ROS produced by the cells (Fig. 3). ROS generation in H2O2-treated cells was markedly higher, but in cells treated with the 9 kinds of BN extracts, there was a decrease in DCFH oxidation. In particular, BN-01 (69.3%), BN-04 (70.2%), and BN-09 (67.1%) showed strong protective effect against ROS levels. These results show that BN extracts can protect against H2O2-induced oxidative stress and decrease ROS productions in C6 glial cells.

Fig. 3.Effect of BN extracts on level of reactive oxygen species in C6 glial cell treated with H2O2 (A: Time course of change in intensity of fluorescence with the BN extracts, B: The production of ROS after treatment with BN extracts).

Protective effects of BN extracts against Aβ25-35-induced oxidative stress and ROS levels in C6 glial cells

Aβ, a byproduct formed during the processing of amyloid precursor protein, is a major constituent of senile plaques, suggesting that its deposition play a role in the pathogenesis of AD (Pike et al., 1995; Xio et al., 2000). Elevation of oxidative stress and activation of the apoptotic pathway play key roles in mediating Aβ-induced toxicity and neural cell death (Behl ll., 1994; Santos et al., 2005), which is observed in the brains of patients with AD (Markesbery, 1997; Butterfield et al., 2001). In the present study, we used Aβ25-35 that was dissolved in sterile distilled water and precipitated by incubation at 37℃ for 3 days. The protective effect of the BN extracts against Aβ25-35-induced oxidative stress in C6 glial cells is shown in Fig. 4. Aβ25-35-treated cells (25 μM) showed lower viability (56.6%) than untreated cells. BN extracts were added at a concentration of 25 μg/ml. Among the 9 kinds of BN extracts, the BN-01, BN-02, BN-04, BN-05, BN-06, BN-08, and BN-09 extracts improved cell viability. In particular, BN-01 (62.3%), BN-05 (62.5%), BN-08 (64.0%), and BN-09 (61.2%) showed significant high cell viability compared to control group. It suggests that BN-01, BN-05, BN-08 and BN-09 would play the protective role against Aβ25-35-induced oxidative stress.

Fig. 4.Effect of BN extracts on viability of C6 glial cells treated with Aβ25-35.

ROS generation in Aβ25-35-treated cells was higher, but cells treated with the 9 kinds of BN extracts showed a decrease in DCFH oxidation. As shown in Fig. 5, 9 kinds of BN extracts were able to prevent the increase of ROS production induced by Aβ25-35 compared to non-treated control conditions. Therefore, BN extracts protect against Aβ25-35-induced oxidative stress and decrease ROS production in C6 glial cells. On the other hand, the protective activity from oxidative stress and ROS generation did not affect cell viability. It indicated that the other factors are related to cell survival or death. BN-04, BN-05 and BN-08 have also led to the decline of ROS generation. Combining with the cell viability and ROS generation, BN-04, BN-05 and BN-08 have cytotoxiciy at 25 μg/ml.

Fig. 5.Effect of BN extracts on level of reactive oxygen species in C6 glial cell treated with Aβ25-35 (A: Time course of change in intensity of fluorescence with the BN extracts, B: The production of ROS after treatment with BN extracts).

All of the results indicated that BN-01 and BN-09 showed excellent protective effects against oxidative stress. However, it is necessary to carry out further studies about BN’s chemical constituents to confirm the related protective mechanisms.