Ginsenosides induce extensive changes in gene expression and inhibit oxidative stress-induced apoptosis in human lens epithelial cells

Abstract

Background

The effect of ginsenosides on the growth and apoptosis of human lens epithelial (HLE) B3 cells exposed to H₂O₂ was investigated. In addition, the effect of ginsenosides on gene expression in HLE-B3 cells was analyzed using microarray assays to determine its molecular mechanism.

Methods

HLE-B3 cells were treated with 1.75 M H₂O₂ in the presence or absence of 5, 10 or 20 μM ginsenosides. Cell viability and apoptosis were examined by MTT assays and flow cytometry, respectively, at 24 to 120 h after the treatment. Furthermore, HLE-B3 cells were treated with 20 μM ginsenosides for 8 days and total RNA was isolated and analyzed using the Affymetrix GeneChip Array. Principal component analysis was performed to visualize the microarray data.

Results

Addition of ginsenosides significantly alleviated the growth inhibitory effect of H₂O₂ on HLE-B3 cells and the percentage of viable cells was increased by more than 3 folds. Flow cytometric analysis showed that 6.16 ± 0.29% of H₂O₂-treated HLE-B3 cells were early apoptotic cells, and the percentage was reduced to 4.78 ± 0.16% (P < 0.05) in the presence of 20 μM ginsenosides. Principal component analysis revealed that ginsenoside caused extensive changes in gene expression in HLE-B3 cells. A total of 6219 genes showed significant differential expression in HLE-B3 cells treated with ginsenoside; among them, 2552 (41.0%) genes were significantly upregulated, whereas 3667 (59.0%) genes were significantly downregulated. FOXN2, APP and RAD23B were the top three upregulated genes while WSB1, PSME4 and DCAF7 were the top three downregulated genes in HLE-B3 cells treated with ginsenosides.

Conclusion

Ginsenosides induce extensive changes in the expression of genes involved in multiple signaling pathways, including apoptotic signaling pathway and DNA damage response signaling pathway. Ginsenosides alleviate H₂O₂-induced suppression of the growth of HLB cells and inhibit H₂O₂-induced apoptosis of HLB cells.

Background

It has been estimated that 95 million people worldwide are affected by cataracts [1]. Age-related cataracts remain highly prevalent in the elderly population, and elderly patients with cataracts account for a significant proportion of visually impaired elderly people globally [2]. Surgery has been shown to be effective for cataract correction, but is not without risks and problems [3]. Currently, there is a lack of effective alternative treatment modalities for cataract surgery. Although many risk factors for cataractogenesis have been identified, such as long-term corticosteroid use [4], smoking, excessive UV-B exposure, and diabetes [5], other than a healthy lifestyle such as smoking cessation, there are no effective preventive measures, including pharmacological treatment of cataract formation.

Cataract is a multifactorial eye disease. Although the exact molecular mechanism of cataractogenesis remains elusive, oxidative stress (OS) has been implicated as the main culprit of cataract lens opacity [6]. Hydrogen peroxide (H₂O₂), a non-free radical member of the active oxygen family, is the major intracellular reactive oxygen species (ROS) in the aqueous humor, which generates hydroxyl radicals that irreversibly damage the lens epithelium. It can activate multiple signaling events such as the activation of apoptosis-associated molecules or pathways, including caspases, the Bcl-2 family, the mitogen-activated protein kinases (MAPKs), and NF-кB pathways, which lead to apoptosis of lens epithelial cells (HLE), ultimately resulting in lens opacification and subsequent cataract development [7, 8]. A variety of antioxidant nutrients, such as flavonoids, phenolic acids, carotenoids, and vitamins, have been tested for their ability to prevent or delay cataract development in animal studies, but their protective effects have not been demonstrated unequivocally [9].

Ginsenosides, also known as ginseng saponins, are isolated from the total saponins of Panax notoginseng and have been tested against various diseases including ischemic stroke [10]. Ginsenosides have antioxidant and antioxidant-related properties in a variety of cell types. Ginsenosides were shown to significantly inhibited UV-B-induced ROS elevation in HaCaT keratinocytes [11]. Ginsenoside Rg1 mediated by ultrasound-targeted microbubble destruction can reduce the level of OS, relieve intraocular pressure and reduce ganglion cell damage in glaucomatous optic nerve of rabbits. Ginsenosides Rb1 and Rd. were also shown to protect the retina from intense light-induced degeneration in BALB/c mice exposed to intense light [12]. However, the possible effect of ginsenosides on cataracts has not been examined. Given that ginsenosides have been shown to protect against UV-B exposure in keratinocytes [13, 14], we hypothesized that ginsenosides may also exert protective effects against OS-induced lens epithelial damage. In the current study, we investigated the effect of ginsenosides on the growth and apoptosis of human lens epithelial (HLE-B3) cells exposed to H₂O₂. To further determine the molecular mechanism of ginsenosides, we analyzed the effects of ginsenosides on gene expression in HLE-B3 cells using microarray analysis.

Methods

H₂O₂ treatment

Human lens epithelial (HLE-B3) cells purchased from American Type Culture Collection were cultured in minimal essential medium (MEM) containing 20% of fetal bovine serum in a humidified incubator at 37 °C with 5% CO₂. Cells at 50–60% confluency were treated with H₂O₂ at concentrations from 0.039 to 2.5 μM for 24 h, and the concentration was incremented by a factor of two. Cell viability was examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays using Cell Proliferation Reagent Kit I (MTT; Roche Applied Science) following the manufacturer’s protocol and as previously described [15]. Each experiment was repeated at least three times independently in quintuplicate. Optical density (OD) was measured at 490 nm using a microplate reader (iMark, USA). A standard curve was drawn to determine IC₅₀ of H₂O₂. All cells were treated with H₂O₂ at IC₅₀ for 24 h in all subsequent experiments.

Ginsenoside treatment

HLE-B3 cells (passage 6) were plated at 6 × 10³ cells per well in 96-well plates. After the medium was changed, the cells at 30–40% confluency were treated with H₂O₂ at IC₅₀ in the absence or presence of 5, 10 or 20 μM ginsenoside (Fleton Natural Products, Chengdu, China; HPLC grade pure 99.8%). Ten microliter of MTT (at a final concentration of 5 mg/ml) was added to each well and the cells were incubated for another 4 h, and the supernatant was discarded. DMSO (150 μL) was added to each well to dissolve the precipitate. Cell viability was examined by MTT assays after 24 to 120 h of drug treatment following the manufacturer’s instructions (Sigma, St. Louis, MO, USA). Each experiment was repeated at least three times independently in quintuplicate.

Flow cytometry

Flow cytometry was performed as previously described [16]. Briefly, HLE-B3 cells were seeded in 6-well plates. When the cells were 70% confluent (5 × 10⁵ cells), they were treated with 5, 10 or 20 μM ginsenoside for 48 h and harvested. The treated cells were washed once with phosphate-buffered saline (PBS), trypsinized, and washed again in PBS containing 2% fetal bovine serum and fixed in ice-cold ethanol for at least 1 h at − 20 °C. The cells were washed, and stained with FITC-annexin V (Beyotime Biotechnology Research Institute, China) and propidium iodide (30 μg/mL) and treated with RNase (0.6 mg/mL) in PBS containing 0.5% (v/v) Tween 20 and 2% fetal bovine serum. Fluorescence-activated cell sorting analysis was performed on a FACS Calibur flow cytometer (BD Biosciences) using Cellquest software, and the Mod-Fit program (Verity Software House Inc., Topsham, ME) was used to analyze the percentage of apoptotic cells.

Microarray

HLE-B3 cells were treated with 20 μM ginsenoside for 8 days. Total RNA was extracted using the Recover All™ Total Nucleic Acid Isolation Kit (Ambion, AM1975) following the manufacturer’s protocol. RNA was biotin-labeled using the FlashTag™ Biotin HSR RNA Labeling Kit (Affymetrix). An input of 400 nanograms of total RNA was used for each reaction. Hybridization, washing and staining were performed using the commercially available Affymetrix GeneChip Hybridization, Wash and Stain Kit. All samples were hybridized to the Affymetrix GeneChip Array. Expression data were normalized using the robust multi-array average (just RMA) method where the raw intensity values were background-corrected, log₂-transformed and then quartile-normalized. A linear model was fitted to the normalized data to obtain a measure of expression for each probe set on each array.

Gene analysis

Principal component analysis was carried out to visualize the microarray data. All genes were plotted on the first and second principal components. The first principal component (PC1) measured the grand mean expression and the second (PC2) measured the ginsenosides-induced expression changes. In addition, a scatter plot of principal component analysis of differential gene expression patterns in ginsenosides-treated cells and control cells was drawn. The Pearson correlation of gene expression patterns in HLE-B3 cells treated with ginsenoside was calculated between the vectors pointing from the overall mean of the entire dataset and the respective group mean. For hierarchical clustering, genes in the dataset were subjected to complete-linkage hierarchical clustering using a Euclidean distance metric. The pathway and function analyses were performed using KEGG and Gene Ontology (GO).

Results

Ginsenoside reverses H₂O₂-induced growth inhibition of HLE-B3 cells

MTT assays showed that H₂O₂ exhibited a dose-dependent inhibitory effect on the viability of HLE-B3 cells. The mean inhibition rate of H₂O₂ steadily increased from 6.6% at a concentration of 0.039 μM to 15.3% at a concentration of 1.25 μM and rapidly reached to 85.4% at the final concentration of 2.5 μM (Fig. 1). The IC₅₀ of H₂O₂ was 1.75 μM (ranging from 1.59 to 1.927 μM). Addition of ginsenosides significantly alleviated the growth inhibitory effect of H₂O₂ (1.75 μM) on HLE-B3 cells and the percentage of viable cells was increased by more than three folds (Fig. 1). There was no statistical difference in the percentage of viable HLE-B3 cells treated with low (5 μM), mid (10 μM) and high (20 μM) dose of ginsenosides (P > 0.05).

Fig. 1

Ginsenoside reverses H₂O₂-induced growth inhibition of HLE-B3 cells. HLE-B3 cells were treated with 1.75 μM H₂O₂ and low (5 μM), mid (10 μM) and high dose (20 μM) of ginsenoside. Viabilities of HLE-B3 cells were examined by MTT assays as detailed in Methods. Ginsenoside significantly alleviated the growth inhibitory effect of H₂O₂ on HLE-B3 cells with more than 3-fold increase in the percentage of viable HLE-B3 cells. No statistical difference was observed in the percentage of viable HLE-B3 cells treated with low (5 μM), mid (10 μM) and high dose (20 μM) of ginsenoside (P > 0.05)

Ginsenosides induce extensive changes in gene expression

Principal component analysis revealed that ginsenosides caused extensive changes in gene expression in HLE-B3 cells (Fig. 2). The differential gene expression patterns in ginsenoside-treated and control cells were further shown in a principal component analysis scatter plot (Fig. 3), indicating a linear relationship in gene expression patterns. The Pearson correlation matrix of gene expression patterns in HLE-B3 cells treated with ginsenosides is shown in Fig. 4. In addition, the volcano plot showed that more genes were downregulated than upregulated in ginsenosides-treated HLE-B3 cells (Fig. 5). Hierarchical clustering of gene expression patterns in HLE-B3 cells treated with ginsenosides further detailed the upregulated and downregulated genes (Fig. 6).

Fig. 2

Principal component analysis for visualization of microarray data. All genes are plotted on to the first and second principal components. The first principal component (PC1) measures total average expression, and the second (PC2) measures changes in expression induced by ginsenoside. The ellipse at the center contains 95% of the genes. Pink color represents genes from ginsenoside-treated cells and green color indicates genes from control cells

Fig. 3

A principal component analysis scatter plot of differential gene expression patterns in ginsenoside-treated cells and control cells. Genes with equal expression values line up on the diagonal identity line and higher expression values are further away from the origin. Points below the diagonal represent genes with higher expression in ginsenoside-treated cells plotted on the x-axis. Points above the diagonal represent genes with higher expression values in control cells plotted on the y-axis. The further away a point-of-interest is from the diagonal line the larger is the difference in expression in ginsenoside-treated cells compared with the control cells

Fig. 4

Correlation matrix of gene expression patterns in HLE-B3 cells treated with ginsenoside. The Pearson correlation was calculated between the vectors pointing from the overall mean of the entire dataset to the respective group mean. The values at each position in the matrix characterize the expression level of a particular gene under a particular experimental condition. Each box is a log ratio of gene expression of HLE-B3 cells treated with ginsenoside/the control cells. Rows represent genes and columns represent measurements from individual arrays

Fig. 5

The volcano plot shows ginsenoside-induced changes of gene expression in HLE-B3 cells. The x axis shows fold changes in gene expression while the y axis shows statistical significance (−log10 of P values). The horizontal green line shows where P = 0.05 with points above the line having P < 0.05 and points below the line having P > 0.05. The volcano plot is color coded; those points having a fold-change less than 2 (log2 = 1) are shown in gray. Red indicates points-of-interest that display statistically significant differences. Points-of-interest left to the vertical green line represent downregulated genes and those that lie right to the vertical green line are upregulated genes

Fig. 6

Hierarchical clustering. Genes in the data set were subjected to complete-linkage hierarchical clustering using a Euclidean distance metric. Genes that are upregulated appear in red, and those that are downregulated appear in green, with the relative log2 (ratio) reflected by the intensity of the color

A total of 6219 genes showed significant differential expression in ginsenosides-treated and control HLE-B3 cells. Among them, 2552 (41.0%) genes were significantly upregulated while 3667 (59.0%) genes were significantly downregulated.

The 10 most upregulated and downregulated genes are shown in Tables 1 and 2, respectively. FOXN2, APP and RAD23B are the top three upregulated genes while WSB1, PSME4 and DCAF7 are the top three downregulated genes in HLE-B3 cells treated with ginsenosides. Ginsenoside caused one to two-fold increase in the ten most upregulated genes and one to two-fold decrease in the ten most downregulated genes in HLE-B3 cells.

Table 1 Ten most upregulated genes in HLE-B3 cells treated by ginsenoside

Table 2 Ten most downregulated genes in HLE-B3 cells treated by ginsenoside

Ginsenosides upregulate the expression of genes involved in apoptosis and DNA damage response

Gene ontology analysis showed that three of the top ten upregulated genes were related to apoptosis, including APP, LMNB1 and MAPK8 (Table 3). Besides, three genes were involved in DNA damage response, including RAD23B, MAPK8 and TLK2. Specifically, MAPK8 is involved in the cellular response to hydrogen peroxide and APP to OS. Furthermore, PSME4, which is one of the top ten downregulated genes, is involved in the negative regulation of apoptosis (Table 4).

Table 3 Annotated functions of ten most upregulated genes by ginsenoside

Table 4 Annotated functions of ten most downregulated genes by ginsenoside

Ginsenosides reduce H₂O₂-induced apoptosis of HLE-B3 cells

Since gene ontology analysis revealed that ginsenosides modulated the expression of apoptosis-related genes, such as LMNB1 and PSME4, we examined the effect of ginsenosides on H₂O₂-induced apoptosis of HLE-B3 cells. Flow cytometry analysis showed that 6.16 ± 0.29% of H₂O₂-treated HLE-B3 cells were early apoptotic cells (Fig. 7a), and the percentage was significantly reduced to 5.22 ± 0.59%, 4.98 ± 0.29% and 4.78 ± 0.16% by the presence of low (5 μM), mid (10 μM) and high (20 μM) dose of ginsenosides (P < 0.05), respectively (Fig. 7b to e), suggesting that ginsenosides reduce H₂O₂-induced apoptosis in HLE-B3 cells.

Fig. 7

Ginsenoside reduces H₂O₂-induced apoptosis of HLE-B3 cells. HLE-B3 cells were treated with 1.75 μM H₂O₂ (a) and low (5 μM) (b), mid (10 μM) (c) and high dose (20 μM) (d) of ginsenoside. The percentage of apoptosis cells was examined by flow cytometry. e Data are expressed as mean ± SD of at least three independent experiments. P < 0.05 of the treatment groups versus 1.75 μM H₂O₂. Representative histograms are shown in (a) to (d)

Discussion

In this study, we presented the first experimental evidence that ginsenosides could protect against H₂O₂-induced growth inhibition and apoptosis in HLE-B3 cells. Furthermore, ginsenosides caused widespread changes in gene expression, including changes in genes involved in DNA damage response and apoptosis, suggesting that ginsenosides act through multiple molecular mechanisms.

Microarray data showed that FOXN2, APP and RAD23B were the top three upregulated genes while WSB1, PSME4 and DCAF7 were the top three downregulated genes by ginsenosides in HLE-B3 cells. FOXN2 is a member of the Forkhead box transcription factors. Its role in cataractogenesis has not been confirmed. A recent study showed that FOXN2 could suppress the proliferation of lung cancer cells [17]. RAD23B, MAPK8 and TLK2 have been shown to be involved in DNA damage response [18]. Effect of ginsenosides on MAPK has been well documented [19,20,21]. A Chinese herbal medicine containing ginsenosides was found to attenuate H₂O₂-induced injury in PC12 cells by inhibiting Akt and MAPK signaling pathways [22]. H₂O₂ in the aqueous humor can activate MAPK signaling in HLE cells [7, 23], but the exact effect of ginsenosides on MAPK in HLB cells remains to be elucidated.

We also showed that ginsenosides reduced the percentage of early apoptotic HLB cells. This is consistent with our finding that ginsenosides modulate the expression of apoptosis-related genes such as LMNB1 and PSME4. In fact, PSME4 is among the top ten downregulated genes and is involved in negative regulation of apoptosis. In addition, three of the top ten upregulated genes are also related to apoptosis, including APP, LMNB1 and MAPK8. Apoptosis of HLB cells is increased during cataractogenesis [24], while ginsenosides may attenuate cataractogenesis by inhibiting H₂O₂–induced expression of apoptosis-related genes in HLB cells.

Conclusion

Ginsenosides can induce widespread changes in the expression of genes involved in multiple signaling pathways, including apoptotic signaling and DNA damage response signaling. Ginsenosides can alleviate H₂O₂-induced growth inhibition and inhibit H₂O₂-induced apoptosis in HLB cells.