Protective effect of Lepidium sativum seed extract against hydrogen peroxide-induced cytotoxicity and oxidative stress in human liver cells (HepG2)

Abstract

Context: Garden cress [Lepidium sativum (Brassicaceae)] has been widely used to treat a number of ailments in traditional medicine. The pharmacological and preventive potential of Lepidium sativum, such as anti-inflammatory, antipyretic, antihypertensive, anti-ashthamatic, anticancer, and anti-oxidant, are well known.

Objective: The present investigation was designed to study the protective effects of chloroform extract of Lepidium sativum seed (LSE) against oxidative stress and cytotoxicity induced by hydrogen peroxide (H₂O₂) in human liver cells (HepG2).

Materials and methods: Cytotoxicity of LSE and H₂O₂ was identified by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red uptake (NRU) assays, and morphological changes in HepG2. The cells were pre-exposed to biologically safe concentrations (5–25 μg/ml) of LSE for 24 h, and then cytotoxic (0.25 mM) concentration of H₂O₂ was added. After 24 h of the exposures, cell viability by MTT, NRU assays, and morphological changes in HepG2 were evaluated. Further, protective effects of LSE on reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), and reduced glutathione (GSH) levels induced by H₂O₂ were studied.

Results: Pre-exposure of LSE significantly attenuated the loss of cell viability up to 48% at 25 µg/ml concentration against H₂O₂ (LD₅₀ value = 2.5 mM). Results also showed that LSE at 25 µg/ml concentration significantly inhibited the induction of ROS generation (45%) and LPO (56%), and increases the MMP (55%) and GSH levels (46%).

Discussion and conclusion: The study suggests the cytoprotective effects of LSE against H₂O₂-induced toxicity in HepG2. The results also demonstrate the anti-oxidative nature of LSE.

• Glutathione
• lipid peroxidation
• MMP
• morphological changes
• ROS generation

Introduction

Oxidative stress plays a significant role in the etiology of variety of human diseases (Dhalla et al., 2000; Rahman, 2005). The role of oxidative stress in liver cells induced by various toxins is also well known (Rodeiro et al., 2008). Reports showed that overproduction of reactive oxygen species (ROS) plays a major role in the hepatocarcinoma (Lima et al., 2006; Zhang et al., 2011), which leads to cellular damage (Lin et al., 2007; Zhang et al., 2012). A number of in vitro studies have demonstrated that oxidative stress induced by chemical oxidants, such as hydrogen peroxide (H₂O₂), leads to cell death (Cai et al., 2008; Hwang & Yen, 2008; Kim et al., 2009; Siddiqui et al., 2011). H₂O₂ has also been reported to induce apoptotic changes, which subsequently lead to death in a variety of cell systems (Jung et al., 2006; Kanno et al., 2003; Sattayasai et al., 2013), including human liver cells (HepG2) (Alia et al., 2005; Chen et al., 2011). Therefore, we have selected H₂O₂ to induce the oxidative stress-mediated cytotoxicity in the HepG2.

Garden cress [Lepidium sativum (Brassicaceae)] has been widely used to treat a number of ailments in traditional system of medicine. Lepidium sativum is a fast-growing edible herb belonging to the family Brassicaceae (Cruciferae) or mustard family, and is being cultivated as culinary vegetable in North America, Europe, and all over Asia including India (Al-Sheddi et al., 2013; Nadkarni, 1976). The pharmacological and preventive properties of L. sativum, such as anti-oxidant (Yadav et al., 2010), anti-inflammatory, anticoagulant (Al-Yahya et al., 1994), antidiabetic (Eddoaks et al, 2005), antidiarrheal (Manohar et al., 2009), antihypertensive, diuretic (Mohamed et al., 2003; Umang et al., 2009), antirheumatic (Ahsan et al., 1989), anti-asthmatic (Paranjape & Mehta, 2006), chemoprotective (Fekadu et al, 2002), hypercholesterolemic (Al Hamedan, 2010), laxative (Najeeb et al., 2011), and fracture healing (Abdullah & Abdullah, 2007) are well documented. The seeds of L. sativum are also known to be useful in leprosy, skin, dysentery, diarrhea, splenomegal, and asthma diseases (Kirtikar & Basu, 2006). The phytoconstituent of L. sativum include carbohydrate, protein, fatty acid, riboflavin, niacin, flavonoids, isothiocynates glycoside, essential aromatic oils, and fatty oils (Al-Sheddi et al., 2013).

In view of the significant lack of the mechanism(s) defining the protective effects of L. sativum on oxidative stress and ROS generation in HepG2, the present study was designed to develop a model system to investigate the protective effects of chloroform extract of L. sativum seed (LSE) against H₂O₂-induced cytotoxicity in HepG2. The HepG2 are involved in the metabolism (Kim et al., 2011), and compose a good model to assess the toxicity or detoxification of various compounds against oxidative stress inducers (Farshori et al., 2013; Lima et al., 2006; Zhang et al., 2012).

Materials and methods

Chemicals and consumables

DMEM culture medium, antibiotics-atimycotic solution, fetal bovine serum (FBS), and trypsin were purchased from Invitrogen, Life Sciences, Grand Island, NY. Consumables and culture wares used in the study were procured from Nunc, Roskilde, Denmark. H₂O₂ and all other specified chemicals and reagents were purchased from Sigma Chemical Company Pvt. Ltd. St. Louis, MO.

Preparation of extract

The L. sativum seeds used in this study were obtained from the local market of Riyadh, Saudi Arabia in October 2013. The seeds were identified by Dr. Mohammad Atiqur Rahman, taxonomist of Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, Saudi Arabia and a specimen (#15967) was submitted in the herbarium of the King Saud University. The seeds were screened manually to remove bad ones. They were then dried to constant weight in an oven at 70°C, ground using mechanical grinder, put in air-tight containers, and stored in a desiccator. For the preparation of chloroform extract, the seeds were macerated in chloroform and then filtered. The procedure was repeated several times. The solvent was then evaporated using a rotary evaporator and the residue so obtained was formed as the chloroform extract.

Cell culture

HepG2, sourced from the American Type Culture Collection (Accession HB-8065, Rockville, MD) were cultured in DMEM, supplemented with 10% fetal bovine serum, 0.2% sodium bicarbonate, and antibiotic/antimycotic solution (100×, 1 ml/100 ml of medium). Cells were grown in 5% CO₂ at 37°C in high humid atmosphere. Before the experiments, the viability of cells was assessed following the protocol of Siddiqui et al. (2008). HepG2 showing more than 98% cell viability and passage number between 20 and 22 were used in this study.

Experimental design

To determine the biologically safe concentrations of LSE, HepG2 were exposed to 5–500 μg/ml of LSE and to determine the LD₅₀ value of H₂O₂, the cells were exposed to 0.01–2 mM of H₂O₂ for 24 h. Further, to evaluate the protective effects of LSE on HepG2, the cells were pre-exposed to biologically safe concentrations (5, 10, and 25 μg/ml) of LSE. Then H₂O₂ (0.25 mM) was added and cells were incubated at 37°C for 24 h. After the exposures, percentage cell viability, morphological changes, ROS generation, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), and reduced glutathione (GSH) were evaluated.

Drug solutions

The LSE was not completely soluble in culture medium; therefore, the stock solutions of extract were prepared in dimethylsulphoxide (DMSO) and diluted in culture medium to reach the desired concentrations. The concentration of DMSO in culture medium was not more than 0.1% and this medium was used as control. Hydrogen peroxide (H₂O₂) was freshly diluted in culture medium before adding to the cells.

Cytotoxicity screening of LSE extract and H₂O₂

The biologically safe or non-cytotoxic concentration of LSE and cytotoxicity of H₂O₂ was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red uptake (NRU), and cellular morphology assays. For the cytotoxicity assessment, HepG2 were exposed to various concentrations (5–500 μg/ml) of LSE and H₂O₂ (0.01–2 mM) for 24 h. The cellular morphological analysis was performed by phase contrast-inverted microscope.

MTT assay

Percentage cell viability was assessed using the MTT assay as described (Siddiqui et al., 2008). Briefly, HepG2 (1 × 10⁴) were allowed to adhere for 24 h CO₂ incubator at 37°C in 96-well culture plates. After 24 h exposure of HepG2 with increasing concentrations (5–500 μg/ml) of LSE and H₂O₂ (0.01–2 mM) for 24 h, MTT (5 mg/ml of stock in PBS) was added (10 μl/well in 100 μl of cell suspension), and plates were incubated for 4 h. The supernatant was discarded and 200 μl of DMSO was added to each well and mixed gently. The developed color was read at 550 nm using multiwell microplate reader (Thermo Scientific, Waltham, MA). Untreated sets were also run under identical conditions and served as control.

NRU assay

NRU assay was carried out following the protocol described by Siddiqui et al. (2010). Briefly, after 24 h exposure of HepG2 with increasing concentrations (5–500 μg/ml) of LSE and H₂O₂ (0.01–2 mM) for 24 h, the medium was aspirated and cells were washed twice with PBS, and incubated for 3 h in a medium supplemented with neutral red (50 μg/ml). The medium was washed off rapidly with a solution containing 0.5% formaldehyde and 1% calcium chloride. Cells were subjected to further incubation of 20 min at 37°C in a mixture of acetic acid (1%) and ethanol (50%) to extract the dye. The plates were read at 540 nm using multiwell microplate reader (Thermo Scientific, Waltham, MA). The values were compared with the control sets run under identical conditions.

Morphological analysis

Morphological changes in HepG2 exposed to increasing concentrations of LSE (5–500 μg/ml) and H₂O₂ (0.01–2 mM) for 24 h were observed by phase contrast inverted microscope (Olympus, Center Valley, PA) attached with automatic image analysis software. Further, to observe the protective effects of LSE on cellular morphology, HepG2 were treated with biologically safe concentrations (5, 10, and 25 μg/ml) of LSE before exposure to cytotoxic concentration of H₂O₂ (0.25 mM) for 24 h.

ROS generation

The production of intracellular ROS was measured using 2,7-dichlorofluorescin diacetate (DCFH-DA) as described by Wang and Joseph (1999) with slight modifications. In brief, after the exposure of HepG2 with LSE and H₂O₂, the cells were washed twice with PBS. The cells were then exposed to 20 μM of DCFH-DA fluorescent dye for 1 h at 37°C in dark. The fluorescence intensity of specific dye was measured at 485 nm excitation and 530 nm emission wavelengths using spectrofluorometer (Fluoroskan, Thermo Scientific, Waltham, MA).

Mitochondrial membrane potential

MMP was measured following the protocol of Zhang et al. (2011). In brief, control and treated cells were washed twice with PBS. The cells were further treated with 10 µg/ml of Rhodamine-123 fluorescent dye for 1 h at 37°C in dark. The fluorescence intensity of Rhodamine-123 was measured at 485 nm excitation and 530 nm emission wavelengths using spectrofluorometer.

Lipid peroxidation

LPO was performed using thiobarbituric acid-reactive substances (TBARS) protocol (Buege & Aust, 1978). Briefly, after the exposure of HepG2 with LSE and H₂O₂, HepG2 were collected by centrifugation and sonicated in ice cold potassium chloride (1.15%) and centrifuged again for 10 min at 3000 × g. The resulting supernatant (1 ml) was collected and 2 ml of thiobarbituric acid (TBA) reagent (15% TCA, 0.7% TBA, and 0.25N HCl) was added. The solution was heated at a temperature of 100°C for 15 min in a boiling bath. The sample was then placed at a cold temperature and centrifuged at 1000 × g for 10 min. The absorbance of the supernatant was measured at 535 nm.

GSH level

Intracellular reduced GSH level was estimated as described by Chandra et al. (2002) with some modifications. Briefly, HepG2 exposed to LSE and H₂O₂ were collected by centrifugation and the cellular proteins were precipitated by incubating 1 ml sonicated cell suspension with 10% TCA (1 ml) on ice for 1 h followed by centrifugation at 3000 rpm for 10 min. The supernatant was then added to 2 ml buffer (0.4 M Tris and 0.02 M EDTA; pH 8.9) and 0.01 M of 5,5′-dithionitrobenzoic acid (DTNB) to reach a final volume of 3 ml. The tubes were incubated for 10 min at 37°C in a shaking water bath. The absorbance of yellow color developed was read at 412 nm.

Statistical analysis

Results were expressed as mean ± SE of at least three independent experiments (each in triplicate). Statistical analysis was performed using one-way ANOVA using Dunnett’s post hoc test employed to compare the values between control and treated groups. The values showing p < 0.05 were considered as statistically significant.

Results

Cytotoxicity assessments of LSE and H₂O₂

The cytotoxicity of LSE was assessed using MTT assay, NRU assay, and cellular morphology, after exposing the HepG2 to various concentrations of LSE and H₂O₂. Our results showed that LSE at 5, 10, and 25 μg/ml concentrations did not cause any significant effect on cell viability of HepG2 (Figures 1(A, B) and 2). Therefore, the concentrations 5, 10, and 25 μg/ml of LSE were chosen to study the protective effects against H₂O₂-induced toxicity in HepG2. Further, based on the LD₅₀ value obtained, 0.25 mM of H₂O₂ was used to induce the toxicity in further experiments (Figure 3A and B).

Figure 1. Cytotoxicity assessment by (A) MTT assay and (B) NRU assay in HepG2 following the exposure of various concentrations of LSE for 24 h. Values are mean ± SE of three independent experiments. *p < 0.05, **p < 0.01 versus control.

Figure 2. Alterations in the morphology of HepG2 exposed for 24 h. Various concentrations of LSE for 24 h. Image were taken using phase contrast-inverted microscope at 20× magnification.

Figure 3. Cytotoxicity assessments by (A) MTT and (B) NRU assays in HepG2 following the exposure of various concentrations of hydrogen peroxide (H₂O₂) for 24 h. Values are mean ± SE of three independent experiments. *p < 0.05, **p < 0.01 versus control.

Protective effects of LSE against H₂O₂-induced cell death

MTT and NRU assays

The protective potential of LSE in HepG2 observed by MTT and NRU assays is presented in Figure 4(A) and (B). A significant (p < 0.01) reduction in percentage cell viability was observed in HepG2 following the exposure of H₂O₂ (0.25 mM) for 24 h by MTT assay (Figure 4A), and NRU assay (Figure 4B). HepG2 pre-treated with LSE at 5, 10, and 25 μg/ml for 24 h significantly attenuated the H₂O₂ induced loss of cell viability in a concentration-dependent manner. An increase of 45% in the cell viability of HepG2 was recorded at 25 μg/ml of LSE (Figure 4A). A similar kind of concentration-dependent increase in the cell viability was also observed by NRU assay in LSE pre-exposed HepG2. An increase of 48% in cell viability of HepG2 was recorded at 25 μg/ml of LSE (Figure 4B).

Figure 4. Percent cell viability by (A) MTT and (B) NRU assay in HepG2. HepG2 were exposed to increasing concentrations of LSE for 24 h. Then, cells were exposed to H₂O₂ (0.25 mM) for 24 h. Values are mean ± SE of three independent experiments. *p < 0.05, **p < 0.01 versus H₂O₂ exposure.

Morphological changes

The alterations in the morphology of HepG2 on exposure of LSE and H₂O₂ are shown in Figure 5(A–E). Cells exposed to 0.25 mM of H₂O₂ reduced the normal morphology of HepG2, and cell adhesion capacity as compared with control. Most of the cells exposed to H₂O₂ lost their typical morphology and appeared smaller in size (Figure 5B). HepG2 exposed to increasing concentrations of LSE for 24 h prior to H₂O₂ exposure significantly restore their original morphology in a concentration-dependent manner (Figure 5C–E).

Figure 5. Alterations in the morphology of HepG2. Cells were pre-exposed with LSE for 24 h, then hydrogen peroxide (H₂O₂) for 24 h. Image were taken using phase contrast-inverted microscope at 20× magnification. (A) Control, (B) H₂O₂ (0.25 mM), (C) LSE (5 μg/ml) + H₂O₂ (0.25 mM), (D) LSE (10 μg/ml) + H₂O₂ (0.25 mM), and (E) LSE (25 μg/ml) + H₂O₂ (0.25 mM).

Protective effects of LSE on ROS generation induced by H₂O₂

The results of ROS generation in HepG2 exposed to H₂O₂ and various concentrations of LSE are presented in Figure 6(A). Exposure of HepG2 with H₂O₂ at 0.25 mM for 24 h resulted in an increase of 154 ± 5.6% of increase (p < 0.01) in the production of ROS. Pre-treatment of cells with LSE at 5, 10, and 25 μg/ml concentration significantly reduced the levels of H₂O₂-induced ROS production in HepG2 (Figure 6A).

Figure 6. Protective potential of LSE on (A) reactive oxygen species (ROS) generation; (B) mitochondrial membrane potential (MMP); (C) lipid peroxidation (LPO); (D) glutathione (GSH) depletion in HepG2 following the exposure of different concentrations of LSE for 24 h prior to the addition of H₂O₂ for 24 h. All values represent the mean ± SE. *p < 0.05, **p < 0.01 versus H₂O₂ exposure.

Protective effects of LSE on MMP

The intensity of mitochondrial membrane potential (MMP) in HepG2 exposed to H₂O₂ and various concentrations of LSE is presented in Figure 6(B). Results showed that HepG2 exposed to H₂O₂ (0.25 mM) for 24 h resulted in 46 ± 2.6% reduction (p < 0.01) in the level of MMP. Pre-treatment of cells with LSE at 5, 10, and 25 μg/ml concentrations prior to H₂O₂ treatment significantly increased the intensity of MMP in HepG2 (Figure 6B).

Protective effects of LSE on LPO

Protective potential of various concentrations of LSE on H₂O₂ induced lipid peroxidation in HepG2 is summarized in Figure 6(C). As shown in figure, exposure of H₂O₂ resulted in a significant increase of 61% (p < 0.01) in LPO as compared with control cells. HepG2 pre-treated with LSE at 5, 10, and 25 μg/ml concentration for 24 h prior to H₂O₂ treatment significantly (p < 0.01) reduced the LPO (Figure 6C).

Protective effects of LSE on GSH level

Protective potential of LSE on H₂O₂-induced depletion in GSH level is summarized in Figure 6(D). As shown in figure, HepG2 exposed with 0.25 mM of H₂O₂ significantly reduced the GSH level by 48% (p < 0.01) as compared with control. Results also showed that LSE at 5, 10, and 25 μg/ml significantly prevented (p < 0.01) the depletion of GSH levels caused H₂O₂ by in HepG2 (Figure 6D).

Discussion

Oxidative stress has been proven to play an important role in the progression of a number of human diseases (Dhalla et al., 2000; Rahman, 2005). H₂O₂ is known to induce oxidative stress in a variety of cells (Kumar et al., 2010; Siddiqui et al., 2011; Sattayasai et al., 2013). There is a plethora of evidence documenting the essential involvement of oxidative stress-induced damages and increased levels of H₂O₂ in a variety of diseases (Spencer et al., 2009; Yamashita et al., 2008). Since oxidative stress seems to be involved in many diseases, the administration of anti-oxidants may be useful in the prevention and treatment of these diseases (Sekler et al., 2008). Currently, there is an increasing interest in naturally derived bioactive compounds with potential cytoprotective effects against oxidative stress induced cell death (Ali et al., 2014; Jaydeokar et al., 2014). Lepidium sativum has been widely used to treat a number of diseases in conventional medical system. It has been reported that seed extracts of L. sativum possess anti-asthmatic (Paranjape & Mehta, 2006) and bronchodilatory (Mali et al., 2008) activities, and are also being used in the treatment of bronchial asthma (Archana & Mehta, 2006). However, protective effects of L. sativum on ROS generation and oxidative stress induced by H₂O₂ in HepG2 have not been explored so far. Therefore, in order to explore the protective potential of LSE against H₂O₂-induced oxidative stress and ROS generation, the present study was designed. Our results demonstrated that LSE exhibited significant cytoprotective effects against H₂O₂-induced cell death in HepG2. Based on the LD₅₀ value obtained by MTT and NRU assays, 0.25 mM of H₂O₂ was selected to induce oxidative stress-mediated cytotoxicity in HepG2. The results of this study are consistent with the previous reports of concentration-dependent cytotoxicity of H₂O₂ (Kumar et al., 2010; Okello et al., 2011; Siddiqui et al., 2011). Response of H₂O₂ in various cells has been reported in similar fashion in the literature and we believe that the same kind of oxidative stress-related events have also been induced in HepG2. Our results showed that pre-exposure of LSE significantly increased the cell viability of H₂O₂-exposed HepG2. These data are comparable with previous studies reporting that crude extracts from natural products exhibited significant cyto-protection against the H₂O₂ in PC12 and SK-N-SH cells (Kumar et al., 2010; Sattayasai et al., 2013) and in liver cells against other toxicants (Alia et al., 2006; Goya et al., 2007; Martin et al., 2008; Verma et al., 2013).

The induction of intracellular ROS generation in HepG2 indicates that H₂O₂ can cause oxidative stress. In this study, the restoration of the intra-cellular ROS level by pre-treatment with LSE showed the protective effects of various concentrations of LSE against H₂O₂-induced ROS generation in HepG2. Our results are in agreement with the previous reports showing that the administration of natural products suppress the increased level of intra-cellular ROS generation (Farshori et al., 2013; Molina et al., 2003; Sattayasai et al., 2013; You et al., 2010). It is known that apoptosis-signaling cascades can be initiated by mitochondria impairment (Taveira et al., 2014). It has also been suggested by some authors that the mitochondria plays an important role in cell protection and could be used as an essential therapeutic target (Swerdlow et al., 2010). In this study, we found that the administration of LSE can protect mitochondrial membrane dysfunction in H₂O₂ exposed HepG2. As shown in Figure 6(B), the LSE revealed potential capacity by preserving the mitochondrial membrane potential (MMP).

Lipid peroxidation is one of the mechanisms of oxidative stress and cell death (Schmidtmann et al., 1991; Tribble et al., 1897). The increase in the lipid peroxidation caused by H₂O₂ has been reported (Siddique et al., 2012) and our results are in well concurrence with these findings. This increase in lipid peroxidation in HepG2 by H₂O₂ may be due to an enhancement in the hepatic MDA from the peroxidation of polyunsaturated fatty acids. Our results also showed that the H₂O₂-induced increase in the level of lipid peroxidation was significantly decreased when cells were pre-exposed to various concentrations of LSE. Glutathione (GSH) is a main non-enzymatic anti-oxidant defense system within the cells and plays an important role in the protection against oxidative stress (Scharf et al., 2003). As shown in Figure 6(D), H₂O₂ reduced the GSH level to 48% in HepG2 as compared with untreated control. However, the pre-treatment of HepG2 with LSE (5–25 μg/ml) significantly restored the decrease in the GSH level caused by H₂O₂. It is also known that the glutathione peroxidase catalyses GSH oxidation to GSSG at the expense of H₂O₂ and glutathione reductase recycles oxidized GSH back to reduced GSH (Goya et al., 2007; Ursini et al., 1995). Therefore, it could be inferred that the LSE pre-exposed HepG2 quenches the intracellular damaging peroxide and recovers the concentration of reduced GSH. The significant revival in the level of GSH clearly indicates a positive response of LSE in cell defense system against the H₂O₂ oxidative insult.

Conclusion

Our results demonstrate that LSE has the ability to protect HepG2 against the H₂O₂-induced cytotoxicity by modulating ROS generation and lipid peroxidation, as well as reducing MMP and GSH levels. The results from this study produced more insight on the potential biological activities of LSE and suggested that LSE could be a source of an anti-oxidant. However, detailed future studies on the mechanism(s) of detoxification of H₂O₂ by which LSE exert cytoprotective effects in HepG2 are needed.

Declaration of interest

The authors report that they have no conflict of interest. This work was supported by NSTIP Strategic Technologies Program number (12-MED2491-02) in the Kingdom of Saudi Arabia.