The protective effect of soybean protein‐derived peptides on apoptosis via the activation of PI3K‐AKT and inhibition on apoptosis pathway

Abstract Soybean protein‐derived peptides (SBP) are a rich source of various bioactive peptides with multiple health benefits. However, the prospective effects of SBP on human cells are still unclear. Therefore, this article investigated the effects of small molecular weight SBP on MG132‐induced apoptosis in RAW264.7 cells. SBP inhibited MG132‐induced apoptosis of RAW264.7 cells in a dose‐dependent manner by flow cytometry. To further study its molecular mechanisms, Western blot analysis demonstrated that SBP could activate the PI3K‐AKT pathway by increasing the phosphorylation of PI3K and AKT and inhibiting apoptosis pathway by downregulating the expressions of pro‐apoptotic proteins of Bim, Bax, Fas, and Fasl and promoting the expressions of anti‐apoptotic proteins of Bcl‐xL and Bcl‐2. These results indicated the protective effect of SBP on MG132‐induced apoptosis in RAW264.7 cells.

pathway (Fritsch et al., 2019). However, cell survival also requires to actively control apoptosis by inhibiting the expression of pro-apoptotic factors and promoting the expression of anti-apoptotic factors (Altomare & Testa, 2005;Yap et al., 2011). The PI3K-AKT pathway is an intracellular signaling pathway regulates many normal cellular processes including cell proliferation, survival, growth, motility, and longevity. Multiple survival factors activate the PI3K pathway, leading to activation of AKT, which plays an important role in cell survival signaling (Nitulescu et al., 2016). PTEN can negatively regulate the PI3K-AKT pathway (Seo, Lee, Sung, et al., 2016). Activated AKT inhibits the pro-apoptotic Bcl-2 family members Bad and Bax (Luo, Budihardjo, Zou, Slaughter, & Wang, 1998;Palmer, 2003).
Abnormal apoptosis can affect the balance of cell growth and death, leading to organ dysfunction. The cell initiates an apoptotic program in the presence of environmental pollution (China-Smog), radiation (Fukushima Daiichi disaster), genetic diseases, malnutrition, or other types of cell damage caused by severe stress cause premature cell apoptosis and damage to health. Inhibiting cell apoptosis can delay cell aging, which is beneficial to health and longevity (Hoda & Hoda, 2005). It has been reported that the active peptides of natural products can inhibit apoptosis. BH4-domain peptide from Bcl-xL exerted anti-apoptotic activity in vivo (Sugioka et al., 2003). Selenoprotein hydrolysate (SPH) significantly reduced Pb 2+ -induced caspase-3 activation, reversed Pb 2+ -induced Bax upregulation and cytochrome corelease, and downregulated intracellular Bcl-2 expression (Fang et al., 2017). Nicolai Gronne Jorgensen et al have used a peptide derived from an anti-apoptotic protein to perform a Phase I test of peptide vaccination on multiple myeloma with no adverse effects (Jørgensen et al., 2016). Liu et al. (2014) reported that Trp-Asn-Trp-Ala-Asp, a pentapeptide derived from egg white ovomucin pepsin hydrolysates, upregulated the level of anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic protein level. Soybean protein-derived peptides (SBP) are a rich source of various bioactive peptides and associated with many potential health benefits, including reducing the risk of obesity, high cholesterol levels, cardiovascular disease, insulin-resistance/type II diabetes, certain types of cancers, and immune disorders (Kim et al., 2013(Kim et al., , 2018Li, Luo, & Zhang, 2014;Rayaprolu, Hettiarachchy, Horax, Kumar-Phillips, et al., 2017). SBP like lunasin and soymorphins possess more than one of these properties and play a role in the prevention of multiple chronic diseases (Chatterjee, Gleddie, & Xiao, 2018). SBP also induce stem cell proliferation by mediating signal transduction, including activation of ERK and TGF-β1 pathways (Lee, Roh, Kim, Lee, & Park, 2012). The external interventions, including environmental pollution, food safety, drug side effects, etc., can lead to accelerated cell death, resulting in various diseases that cause harm to human body. Proteins extracted from soy can be hydrolyzed by proteases to produce SBP with biological functions. SBP have been identified in different experimental systems to have biological properties .
In this study, SBP were obtained by enzymatic reaction of soybean protein isolate (SPI). The obtained SBP having a molecular weight of 189-1,000 Da was then evaluated for its preventive effect on MG-132-induced apoptosis of RAW264.7 cells. Which cell signaling pathway from SBP used to inhibit RAW264.7 cell apoptosis or damage were determined by Western Blot. It provides a theoretical basis for SBP to inhibit premature cell apoptosis caused by external factors.

| Materials
The soybean protein isolate (SPI) used in this study were provided by Nutrily Biotechnology, Ltd. (Patent No.: CN107674900A) and SBP were prepared according to the method described (Liu & Scotland, 2017;Zhao et al., 2018). As given in Figure

| Determination of SBP molecular weight distribution
The measurement was performed on the LC20A by gel filtration chromatography. The column used was a TSK-GEL G2000SWXL column (5 μm, 7.8 mm × 300 mm). The mass concentration of the F I G U R E 1 Preparation of SBP from SPI prepared SBP is 1 mg/ml, and the preparation concentration of all molecular weight standards is 1 mg/ml. Before injection, samples and standards (Gly-Gly-Gly, Gly-Tyr-Arg, bacitracin, and insulin) were filtered through a 0.22 μm filter. The injection volume was 20 µl, the mobile phase was V(water)/V(acetonitrile)/V(trifluoroace tic acid) = 80:20:0.1, the flow rate was 0.5 ml/min, and the detection wavelength was 220 nm.

| MG-132 and SBP treatment modeling and cell viability tests
To establish cellular apoptosis model, RAW264.7 cells were seeded into 6-well plate at a density of 4 × 10 5 cells/ml. MG132 was dissolved in the medium with the concentration (0, 3, 5, 10, 20, 30, 40, 50 and 60 μM) for 4 hr. Then, cells were collected for viability detection. Cell viability was determined by MTT assay.
To assess the effect of SBP on cellular apoptosis, RAW264.7 cells were pretreated with 10 ml of fresh DMEM containing different concentrations of SBP substrates (0.00, 0.06, 0.13, 0.25, 0.50, 1.00 and 2.00 mg/ml) followed by MG-132 treatment. Then, cells were collected for viability detection. Cell viability was determined by MTT assay. Briefly, 4 × 10 5 cells/ml were plated in 96-well plates a day before SBP or MG132 treatment. After pretreatment by SBP (24 hr) or MG132 (4 hr), MTT solution (20 μl, 5 mg/ml) was added to each well to dissolve formazan crystals.
Absorbance was measured at 450 nm by using a plate reader (Bio-Rad Laboratories Ltd.).

| Apoptosis detection of RAW264.7 cells by flow cytometry
RAW264.7 cells were placed in 6-well plates at a density of 4 × 10 5 cells/ml and treated with different concentrations of SBP substrates (0.13, 0.25, and 0.50 mg/ml). After incubation of 24 hr, cells were treated with MG-132 at the concentration of 1 mg/ml for 4 hr. The cells were harvested by trypsinization, washed twice with precooled PBS, and centrifuged at 1,000 g for 5 min to collect cell pellets. They were resuspended by adding 150 μl 2 × binding buffer. Then 5 μl of annexin V-FITC was added and incubated with the cells at room temperature for 20 min. Add 5 μl PI to the cells for 5-min incubation, and then 100 μl 2 × sample buffer before transferring to the machine.
Fluorescence intensity reflecting apoptosis rate was measured using a CytoFLEX FCM (Beckman).

| Western Blot analysis
RAW 264.7 cells were collected and lysed by ice-cold RIPA and incubated on ice for 30 min. Cell proteins were extracted, and total protein concentration was measured by BCA method and adjusted to a protein concentration of 2 μg/μl. Each protein sample was subjected to SDS-PAGE electrophoresis in a volume of 5 μl and transferred to the membrane. After that, it was blocked with 5% BSA for 1-2 hr at room temperature, and then washed with TBST for three times, each time for 5 min. Then, each primary antibody was added, and incubation of the primary antibody was at 4°C overnight. The incubation of HRP-conjugated secondary antibody was for 1.5 hr at room temperature. At last, the ECL substrate was applied to the blot for image capture. GAPDH was used as a loading control.

| Data analysis
All experiments were repeated three times and the results were expressed as mean and standard deviation (SD). One-way analysis of variance (ANOVA) was performed on all data using SPSS 16 software. A level of p value < .05 was considered statistically significant.

| Characteristics of SBP
The Shimadzu LC20A instrument was operated by gel filtration chromatography to determine the molecular weight distribution of SBP.
As shown in Table 1, the ratio of molecular weight 189-1,000 Da was observed to be 84.01%, the average molecular weight was about 723.12 Da, and consisted of about 3-6 amino acids.  The MTT assay was performed to rule out the potential cytotoxic effects caused by the SBP. As shown in Figure 2b, the cell viability decreased in a concentration-dependent manner with the increase of sample concentration. Cell viability decreased significantly (p < .05) when the SBP concentrations were 1.00 and 2.00 mg/ml, indicating severe RAW264.7 cell damage.

| Effect of MG-132 and SBP on cell viability
Therefore, the appropriate concentration of the SBP was 0.50 mg/ ml. In addition, two experimental samples were established using F I G U R E 2 Effects of MG-132 on the viability of RAW264.7 cells (a), and the effects of SBP on the viability of RAW264.7 cells (b). All results were expressed as the means ± SD (n = 3). There was no significant difference in the results of the same letters (p > .05) F I G U R E 3 Apoptosis was detected by annexin V-FITC staining using by flow cytometry (a). The histogram summarizes the characteristics of apoptosis changes (b). All results were expressed as the means ± SD (n = 3). There was no significant difference in the results of the same letters (p > .05) concentrations of 0.25 and 0.13 mg/ml to further investigate the protective effects of SBP on cells.

| Effect of SBP on cellular apoptosis rate
The effect of SBP on RAW264.7 cell apoptosis treated with MG-132 at different concentrations (0.13, 0.25, and 0.5 mg/ml) for 24 hr were evaluated. Apoptosis data obtained by flow cytometry, as shown in Figure 3a. The apoptosis rate of the control group was 1.67 ± 0.13%, the control + MG-132 group was 18.04 ± 0.63%, the SBP 0.13 mg/ ml + MG-132 group was 9.15 ± 0.48%, the SBP 0.25 mg/ml + MG-132 was 7.71 ± 0.14%, and the SBP 0.50 mg/ml + MG-132 group was 4.62 ± 0.24%. The results as shown in Figure 3b, the apoptosis rate of the control + MG-132 group was almost ten times higher than that in the control group. The apoptosis rate of control + MG-

| Effects of SBP on PI3K-AKT pathway
To understand the molecular mechanism of SBP on MG132-induced apoptosis, we focused on the PI3K-AKT pathway first. As shown in Figure 4a, compared with the blank group, MG-132 significantly

| Effects of SBP on exogenous apoptosis pathway
Next, the influence of SBP and MG132 on exogenous apoptosis was investigated by detection the expression levels of related proteins.
As shown in Figure

| Effects of SBP on intrinsic apoptosis pathway
To fully understand the apoptosis pathway, protein levels of the in-

| D ISCUSS I ON
To explore the molecular mechanism of SBP on MG-132-induced apoptosis, we investigated the influence of SBP on the PI3K-AKT and apoptosis pathways in cells, and further studied expression levels of important proteins in these pathways. Some cytotoxic drugs, like cisplatin, gemcitabine, can trigger apoptosis by inducing G2/M arrest and p53 expression or preventing Myc protein expression (Hung et al., 2010). In our study, MG-132 induced the apoptosis by regulating Fas-mediated and mitochondria-mediated protein expression. ti-angiogenesis (Li, Dong, et al., 2014). Lunasin is another chemo preventive SBP that is closely associated with the BBI. Lunasin binds to nonacetylated H3 and H4 histones to prevent their acetylation, exerting anti-carcinogenic activity (Davis & Inaba, 2016). In human breast cancer cell line MCF-7, lunasin induced apoptosis in MCF-7 cells by upregulation of tumor suppressor PTEN. Whereas, lunasin is also able to inactivate the tumor suppressor proteins, Rb, p53, and p32, and compete with the histone acetyltransferases to bind to the core deacetylated histones H3 and H4, and stop the transcription, leading to arrest of the G1/S phase and apoptosis (Jiang et al., 2016).
Based on these reports, the influence of SBP has multiple levels of regulation effects on apoptosis.
Upon the SBP treatments, the protein alterations of Bad, Bim, BCL-XL, Fas, and FasL were significantly dose-dependent, while the alterations of other proteins were not dose dependence. The effect of SBP on apoptosis may also be an indirect influence of AKT pathway. AKT, accompanied with PI3K to block cell apoptosis by inhibiting pre-apoptosis Bcl-2 family members (Radogna & Diederich, 2018).
Similar to Zhang's result, glucagon-like peptide-1 (GLP-1) protects cardiomyocytes from AOPP-induced apoptosis, mainly by increasing the expression of PI3K and AKT, and inhibiting the protein expression of Bad (Zhang et al., 2016). Generally, the activation of PI3K-AKT path- of <20 amino acids can use endocytosis and translocation to internalize into cells, however, the internalization mechanism of these SBP in cells still remains largely controversial (Barone & Zimmer, 2016;Jiao et al., 2009). So, we also propose the hypothesis that SBP may utilize endocytosis or other internalization mechanism to permeate into RAW264.7 cell, which further bind to adaptor protein mTORC1 of PI3K-AKT pathway, or apoptosis pathway regulator proteins to perform the protective effect on apoptosis induced by MG-132.
Using different approaches, many soybean bioactive peptides have a variety of physiological functions. However, the exact components of SBP which possess the biological function were not isolated in our current study. It is a bit one-sided to separate the mixed peptides to study their activity, which cannot fully represent peptides as functional foods. The efficacy of peptide mixtures is higher than that of a single peptide, nutrition is more comprehensive, and amino acids are more balanced. Especially as a functional food, it has a wide range of applications in assisting certain medical diseases. Among them, unhealthy diet nutrition is related to the development and deterioration of many disease states, thus explaining the important role of diet in nutrition balance (Kaminski et al., 2019). Therefore, this study may help to better understand the role of bioactive peptides in preventing injury. However, more studies are required to further identify their target organs and elucidate their biological mechanisms of action in order to be potentially used as functional food or even therapeutics for prevention or treatment of chronic diseases (Li-Chan, 2015).
The present study evaluated anti-apoptosis activity induced by MG-132 of SBP on RAW264.7 cell. The supplement of SBP did not show cytotoxic activity, while MG-132 stimulated strong apoptosis effect on RAW264.7 cell. SBP mediated anti-apoptosis effects through the activation of PI3K-AKT pathway, whereas inhibited the exogenous apoptosis pathways by down-regulating Fas and FasL expressions. Furthermore, SBP also reduced the mitochondria-mediated apoptosis pathway through regulating Bax and Bcl-2 expressions. Results of this study suggested SBP has a novel anti-apoptosis effect, which may broaden its application as functional food ingredients.

| CON CLUS IONS
Soybean is a promising source of peptides with a wide range of biological activities. In the present study, soybean protein was hydrolyzed by mix proteases to produce SBP which display anti-apoptosis effect in RAW264.7 cells through the activation of PI3K-AKT pathway or indirect binding to Fas and FasL receptor. Further studies are needed for better understanding of the active ingredients of SBP, and their absorption, metabolism, and target tissues in animal models, as well as clarification of their mechanism of actions in other cellular signaling pathways.

ACK N OWLED G M ENTS
This research was funded by the National Key Research and Development Program of China, grant Number: 2016YFD0400401

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.