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

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 (H 2 O 2 ), 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 H 2 O 2 . To further determine the molecular mechanism of ginsenosides, we analyzed the effects of ginsenosides on gene expression in HLE-B3 cells using microarray analysis.

H 2 O 2 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% CO 2 . Cells at 50-60% confluency were treated with H 2 O 2 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 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 × 10 5 cells), they were treated with 5, 10 or 20 μM ginsenoside for 48 h and harvested.   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 ginsenosidesinduced 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).

Ginsenoside reverses H 2 O 2 -induced growth inhibition of HLE-B3 cells
MTT assays showed that H 2 O 2 exhibited a dosedependent inhibitory effect on the viability of HLE-B3 cells. The mean inhibition rate of H 2 O 2 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 IC 50 of H 2 O 2 was 1.75 μM (ranging from 1.59 to 1.927 μM). Addition of ginsenosides significantly alleviated the growth inhibitory effect of H 2 O 2 (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).

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), 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 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). 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.

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).

Ginsenosides reduce H 2 O 2 -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 H 2 O 2 -induced apoptosis of HLE-B3 cells. Flow cytometry analysis showed that 6.16 ± 0.29% of H 2 O 2 -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     MAPK cascade, activation of MAPKK activity, protein polyubiquitination, mitotic cell cycle, stimulatory C-type lectin receptor signaling pathway, antigen processing and presentation of peptide antigen via MHC class I, antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent, DNA repair, regulation of cellular amino acid metabolic process, polyamine metabolic process, apoptotic process, cellular response to DNA damage stimulus, DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest, epidermal growth factor receptor signaling pathway, small GTPase mediated signal transduction, Ras protein signal transduction, multicellular organismal development, spermatogenesis, axon guidance, insulin receptor signaling pathway, fibroblast growth factor receptor signaling pathway, gene expression, proteasomal ubiquitin-independent protein catabolic process, positive regulation of peptidase activity, programmed cell death, viral process, cell differentiation, anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process, tumor necrosis factor-mediated signaling pathway, cellular nitrogen compound metabolic process, spermatogenesis, exchange of chromosomal proteins, NIK/NF-kappaB signaling, Fc-epsilon receptor signaling pathway, antigen processing and presentation of exogenous peptide antigen via MHC class I, regulation of apoptotic process, negative regulation of apoptotic process, regulation of mRNA stability, small molecule metabolic process, innate immune response, vascular endothelial growth factor receptor signaling pathway, neurotrophin TRK receptor signaling pathway, T  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 H 2 O 2 -induced apoptosis in HLE-B3 cells.

Discussion
In this study, we presented the first experimental evidence that ginsenosides could protect against H 2 O 2 -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 H 2 O 2 -induced injury in PC12 cells by inhibiting Akt and MAPK signaling pathways [22]. H 2 O 2 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 H 2 O 2 -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 H 2 O 2 -induced growth inhibition and inhibit H 2 O 2 -induced apoptosis in HLB cells.