Alfalfa saponins plays protective roles in oxidative stress-induced apoptotic cells

Background: As is known, alfalfa saponin can be used as a feed additive in the pig’s diet. And the addition of alfalfa saponin to the pig’s diet could improve animal antioxidant capacity. However, the mechanism by which alfalfa saponins exerts their antioxidant effects has not been studied. To address this issue, H2O2-induced rat intestinal epithelial cell was used to explore the protective mechanism of alfalfa saponins in this study. Results: Alfalfa saponin could rescue the cell proliferation activity, elevate the amount of antioxidant enzymes and downregulate the release of MDA and LDH in H2O2-induced cells. The results indicated that the antioxidant activity of alfalfa saponin was achieved by restoring GSH homeostasis. Further results demonstrated that alfalfa saponin could inhibit cell apoptosis through activating MAPK signaling pathway. Conclusions: The mechanism by which alfalfa saponins exerts their antioxidant effects was elucidated. Therefore, alfalfa saponin could function as cellular oxidative damage inhibitor, green feed additive or potential drug candidate, providing new strategy for inhibiting cell apoptosis induced by oxidative stress in monogastric animals.


Introduction
Reactive oxygen species (ROS) are normally generated in metabolic processes in all organisms [1,2]. In general, a small amount of free radicals are constantly produced, which are also reduced or removed in time. But when this balance is disturbed by excessive ROS, oxidative stress would occur. Oxidative stress could result in cell damage, which is often re ected by decreased amount of antioxidant enzymes, increased amount of malondialdehyde (MDA) or lactate dehydrogenase (LDH), and even cell apoptosis [3][4][5]. At present, early weaning of piglets is one of the main technical means to improve the breeding environment of sows and elevate the utilization e ciency of pigsty. However, due to the physiological developmental characteristics of piglets and the combined effects of environmental, nutritional and psychological factors, oxidative stress is a common state of weaned piglets and one of the major stresses that cause economic losses in piglet production [6,7].
In recent decades, the growing evidence of the presence of compounds with antioxidant properties in the plant extracts have been studied to reduce or prevent the extent of oxidative stress in animals. Melissa o cinalis in Brazil showed effective antioxidant effects on prevention of various neurological diseases associated with oxidative stress in rat [8]. And the ower extract of Etlingera elatior has powerful antioxidant effects against lead-induced oxidative stress [9]. Furthermore, increasing attention has been paid to saponins on defending against oxidative stress. Notoginsenoside R1 could rescue cell damage in neurons by inhibiting reactive oxygen species and modulating MAPK activation [10]. Ginsenoside Rg1 plays protective roles in hydrogen peroxide-induced injury in human neuroblastoma cells [11]. Alfalfa saponins, extract of Alfalfa (Medicago sativa), are naturally bioactive compounds, which consist of a sugar moiety glycosidically linked to a hydrophobic aglycone (sapogenin), mainly pentacyclic triterpenoid [12]. Our previous study has reported that alfalfa saponins was found to be a potential source of natural antioxidants due to their marked antioxidant activity [13]. However, the mechanism of anti-oxidation of alfalfa saponins in monogastric animals, especially in the intestinal epithelial cells in vitro, has not been investigated.
To explore the antioxidant effects of alfalfa saponins on monogastric animal epithelial cells, rat intestinal epithelial cells (IEC-6 cell line) were used as experimental materials, and H 2 O 2 was exploited as a stressor to establish the oxidative stress model. In order to reveal the protective effects of alfalfa saponins on rat intestinal epithelial cells in oxidative stress, the in uence of alfalfa saponins on the cell proliferation activity and anti-oxidation of IEC-6 were explored. Furthermore, the mechanism of alfalfa saponin on IEC-6 cell apoptosis under oxidative stress was investigated, providing a new method for improving the antioxidant stress resistance of intestinal epithelial cells. Detection of cellular antioxidant enzymes, MDA and GSSG/GSH Cells (3 × 10 5 /well) were plated onto a 6-well plate. After culture for 24 h, the cells were dealt with different treatments as described above. 24 h later, the cells were collected and lysed with a sonicator to prepare a cell suspension. After centrifuge for 10 min at 1000 rpm/min, the cell supernatant were examined by T-AOC, GSH-PX, CAT, MDA and GSH/GSSG kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

LDH detection assay
Cells (1.2 × 10 4 /well) were plated onto a 96-well plate. After culture for 24 h, the cells were dealt with different treatments as described above. After cells were treated for 24 h, 120 µL cell supernatant in different groups was collected and plated onto a new 96-well plate to detect the amount of LDH using LDH Cytotoxicity Assay Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's protocol.

Annexin V assay of apoptosis
Apoptosis assay of IEC-6 cells by use of annexin V (Invitrogen) was performed according to the manufacturer's protocol. Brie y, cells (5⋅10 4 /well) were plated onto a 24-well plate. After culture for 24 h, the cells were dealt with different treatments as described above. 24 h later, cells were washed twice in cold PBS. The cells were resuspended in 195 µL Annexin V-FITC binding buffer, followed by the addition of 5 µL Annexin V-FITC and 10 µL propidium iodide (PI). The cells were incubated for 20 min in the dark, and then analyzed by uorescent microscope.

Western blot analysis
Western blot analysis was performed by SDS-PAGE. Before proteins transferred to a nitrocellulose membrane (Bio-Rad, USA), the membrane was blocked with 5% milk at 4 °C overnight. The membrane was incubated with a primary antibody for 2 h and then incubated with horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (Sigma-Aldrich, USA) for 2 h at room temperature. Proteins were detected

Statistical analysis
The mean and standard deviations for triplicate assays were calculated by one-way analysis of variance (ANOVA). The statistical signi cance between different treatments was determined using Student's t test. The effects of alfalfa saponins on proliferation rate of IEC-6 cells To explore the in uence of alfalfa saponins on proliferation rate of IEC-6 cells, the survival rate of IEC-6 cells was examined. Different concentration gradients (0, 25, 50, 100, 200, 400, 800 µg/mL) were set to treat IEC-6 cells for 24 h. The result indicated that there was no signi cant difference in cell viability when cells were treated with 0-400 µg/mL alfalfa saponins, although the cell viability decreased with increasing concentration (P 0.05, Fig. 2A). However, when IEC-6 cells were treated with 800 µg/mL alfalfa saponins, the cell activity signi cantly reduced (P 0.01, Fig. 2A). To ensure that alfalfa saponins were not toxic to IEC-6 cells, the concentration of alfalfa saponins was less than 50 µg/mL in the subsequent experiments.

Results
Thus, the low concentration of alfalfa saponins was 25 µg/mL and the high concentration of alfalfa saponins was 40 µg/mL saponins. In order to investigate whether alfalfa saponins could resist the cellular stress caused by H 2 O 2 , the cells were pre-incubated with low and high concentration (25 and 40 µg/mL) of alfalfa saponins for 24 h. The alfalfa saponins treated groups showed higher cell viability when compared with control group but without signi cant difference (P>0.05, Fig. 2B). However, 25 and 40 µg/mL alfalfa saponins could obviously rescue the cell viability in oxidative stress model. The survival rate of IEC-6 cells increased by 40% and 67%, respectively (P<0.01, Fig. 2B). Thus, the concentration of 25 and 40 µg/mL alfalfa saponins were used in subsequent experiment.
The anti-oxidant activity of alfalfa saponins in oxidative damage cells There is a stable dynamic balance of oxidation/reduction in animal. In general, a small amount of free radicals are constantly produced, which are also reduced or removed in time. But when this balance is disturbed by H 2 O 2 , a large number of free radicals accumulate in the body, exceeding the capacity of  Fig. 3A). However, the the enzyme activity of CAT gradually increased (P > 0.05) while the enzyme activity of GSH-PX could be signi cantly increased (P < 0.05) by alfalfa saponins of 25 and 40 µg/mL in cells without H 2 O 2 treatment compared with control group (cells without treatment). In oxidative stress model, the the enzyme activity of CAT and GSH-PX decreased signi cantly but could be rescued by pre-incubation of cells with alfalfa saponins of 25 and 40 µg/mL (Fig. 3A).
MDA is one kind of lipid peroxide metabolites which is produced through the non-enzymatic system when oxygen free radicals attack the membrane of polyunsaturated fatty acids (PUFA). Therefore, the amount of MDA indirectly re ects the degree of cell damage. Compared with negative control group, the amount of MDA signi cantly decreased when cells were treated with alfalfa saponin of 25 and 40 µg/mL (P < 0.05). However, the amount of MDA elevated obviously in oxidative stress model while the cells pre-incubated with alfalfa saponin of 25 and 40 µg/mL showed less amount of MDA, indicating that alfalfa saponin could protecct ells from peroxide (Fig. 3B). Large amount of free radical accumulation can cause lipid peroxidation of polyunsaturated fatty acids in intestinal epithelial cells, resulting in damage of cell membrane and increasing its permeability. LDH is a stable cytosolic enzyme that can be rapidly released into the extracellular plasm once the cell membrane is damaged. Therefore, LDH is often used as one of the indicators of degree of cell damage. In order to investigate the role of alfalfa saponins in protecting cells from damage caused by H 2 O 2 , the amount of released LDH was analyzed. Consistent with the above result (Fig. 3B), alfalfa saponins of 25 and 40 µg/mL had little effects on the release of LDH compared with that of cells without treatment (P > 0.05, Fig. 3C). However, H 2 O 2 treated cells showed much more release of LDH. The further investigation demonstrated that the pre-incubation of cells with alfalfa saponins with 25 and 40 µg/mL can signi cantly reduce the LDH release of H 2 O 2 treated cells, reducing by 45% and 56%, suggesting that alfalfa saponin reduced the LDH release dose-dependently (P < 0.01, Fig. 3C). Taken together, alfalfa saponins could protect cells from peroxide, reducing cell damage caused by H 2 O 2 .

The anti-oxidative effects of alfalfa saponins by restoring GSH homeostasis
To explore the regulatory mechanism of alfalfa saponins on oxidative damage cells, the amount of GSH and GSSG were detected. Compared with the negative control group, the intracellular GSSG concentration and GSSG/GSH ratio in H 2 O 2 group were signi cantly increased (P < 0.01, Fig. 4). After 25 and 40 µg/mL alfalfa saponins pre-treatment, the GSSG content and GSSG/GSH ratio were gradually decreased compared with that in H 2 O 2 group, indicating that the antioxidative effects of alfalfa saponins on oxidative damage cells are achieved by restoring GSH homeostasis.

The downstream pathway of H 2 O 2 -induced cell apoptosis by alfalfa saponins
To explore the mechanism of alfalfa saponins on H 2 O 2 -induced oxidative damage, the cell apoptosis rate was rstly investigated by uorescence microscope. The result demonstrated that cells treated with alfalfa saponins with 25 and 40 µg/mL showed no signi cant difference compared with negative control while cells treated with H 2 O 2 presented higher apoptosis rate ( Fig. 5A). However, the pre-incubation of cells with alfalfa saponins with 25 and 40 µg/mL could signi cantly reduce the cell apoptosis rate (  Fig. 5A). It was suggested that the antioxidant effect of alfalfa saponins on H 2 O 2 induced oxidative stress in IEC-6 cells may be achieved by inhibiting cell apoptosis. In order to further determine the regulatory mechanism of alfalfa saponins on H 2 O 2 -induced IEC-6 cell apoptosis, the expression pro le of Caspase-3, Caspase-9, Bax and Bcl-2 were determined by western blot. The results showed that there was no difference of Pro-caspase-3 expression in different groups. However, both 25 and 40 µg/mL alfalfa saponins reduced the expression of Cleaved caspase-3, caspase-9 and Bax in cells, and increased the expression of Bcl-2 gene. The addition of 40 µg/mL alfalfa saponins was more effective. After treatment with H 2 O 2 , Cleaved caspase-3, caspase-9 and Bax expression levels were signi cantly increased, and Bcl-Discussion Alfalfa saponins enhanced antioxidant system Studies have reported that H 2 O 2 can be used to induce cells to establish an oxidative stress model, which is widely applied in the modeling of oxidative stress in intestinal epithelial cells [14,15]. In general, the content of free radical maintains a dynamic balance in cells. Once this balance is destroyed, it will cause oxidative damage of cells, leading to the content changes of the substrates related to redox. T-AOC can be used to indicate the total antioxidant capacity of organism [16]. GSH-PX is one kind of important antioxidant enzyme [17]. CAT is widely distributed in living organisms, which can promote the decomposition of H 2 O 2 and protect cells from free radical damage [18]. MDA is one kind of lipid peroxide metabolites which is produced through the non-enzymatic system when oxygen free radicals attack the membrane of polyunsaturated fatty acids (PUFA) [19]. LDH is a stable cytosolic enzyme that can be rapidly released into the extracellular plasm once the cell membrane is damaged [16]. Therefore, the amount of MDA and LDH indirectly re ects the degree of cell damage [19]. At present, researchers have proved that most saponins have certain antioxidant capacity [10,11]. In this study, the survival rate of cells, the activities of T-AOC, GSH-PX and CAT were signi cantly improved by pre-incubating with alfalfa saponins. Moreover, the content of MDA and LDH were signi cantly lower in oxidative stress model preincubating with alfalfa saponins, indicating that alfalfa saponins could increase the antioxidant enzyme activity and can enhance the ability of clearing free radicals.
The antioxidative effects of alfalfa saponins are achieved by restoring GSH homeostasis oxidative stress model reduced intracellular Cleaved caspase-3, caspase-9 and Bax, and increased bcl-2 gene expression, suggesting that the mechanism of alfalfa saponins against H 2 O 2 -induced IEC-6 cell apoptosis may be achieved by up-regulating Bcl-2 and down-regulating the expression of Cleaved caspase-3, caspase-9 and Bax, thereby inhibiting mitochondrial apoptosis ( Fig. 5B).
In order to study the signaling pathway of alfalfa saponins on the anti-oxidation protection of IEC-6 cells, the expression of three signaling pathway proteins of MAPK was detected, including ERK1/2, JNK and p38. There was no signi cant difference in total protein expression of ERK1/2, JNK and p38 (P > 0.05). Compared with the negative control group, the phosphorylation levels of ERK1/2, p38 and JNK in H 2 O 2treated cells signi cantly increased (P < 0.01), while the phosphorylation levels of 3 proteins in 25 and 40 µg/mL alfalfa saponins pre-incubated groups were effectively reduced (P < 0.01, Fig. 5C). The results showed that alfalfa saponins could inhibit the activation of MAPK signaling pathway in IEC-6 cells induced by H 2 O 2 , thus protecting IEC-6 cells.
In conclusion ( Fig. 5D), alfalfa saponins pre-treatment could achieve antioxidative effects by restoring GSH homeostasis and inhibit mitochondrial apoptosis by MAPK signaling pathway, thereby further alleviating oxidative damage of cells.
The oxidative cytotoxic agent, H 2 O 2 , typically considered as a cause of oxidative stress always induces cell apoptosis [20,21]. GSH plays an important role in maintaining the redox homeostasis of organism [22]. Once the cells are damaged by oxidation, GSH participates in the antioxidant reaction to be oxidized to GSSG, resulting in an increase in the ratio of GSSG/GSH. The result of this study demonstrated that the antioxidative effects of alfalfa saponins on oxidative damage in IEC-6 cells may be achieved by increasing the amount of GSH synthesis and reducing the ratio of GSSG/GSH to restore the mechanism of GSH homeostasis.
Alfalfa saponins can inhibit cell apoptosis through MAPK signaling pathway in oxidative stress model Apoptosis is one of the important outcomes of oxidative damage in cells. Caspase-3 is an executor of cell apoptosis and is directly involved in apoptotic events. Activated Caspase-3 (Cleaved Caspase-3, 17 kD) is activated when cells undergo apoptosis [23]. In addition, anti-apoptotic genes (Bcl-2, Bcl-xL) and proapoptotic genes (Bax, Bad, Bid, Bnip3) in the Bcl-2 family are also involved in the regulation of apoptosis [24]. The results of this study showed that alfalfa saponins pre-treatment reduced expression of intracellular Cleaved caspase-3, Caspase-9 and Bax, and increased Bcl-2 expression in oxidative stress model, suggesting that the mechanism of alfalfa saponin against H 2 O 2 -induced IEC-6 cell apoptosis may be through up-regulation of Bcl-2 and down-regulation of Cleaved caspase-3, Caspase-9 and Bax gene expression. The MAPKs signal transduction pathway is involved in a series of physiological and biochemical reactions (such as cell proliferation, differentiation, metabolism, transformation and apoptosis). MAPKs are mainly composed of three subfamily proteins, ERK1/2, p38 and JNK. Zhou Y et al. reported that ERK1/2, JNK and p38 MAPK could be signi cantly activated when oxidative damage occurs in intestinal epithelial cells [25]. Moreover, the activation of these kinases will further promote the increase expression of proteoglycans, leading to the occurrence of apoptosis [26]. The results of this study found that the degree of phosphorylation of the three proteins was signi cantly reduced by the addition of alfalfa saponin in oxidative stress model. It is indicated that alfalfa saponins can prevent oxidative damage by inhibiting H 2 O 2 -induced activation of cell apoptosis, thereby protecting cells.

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