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. 2022 May 30;31(7):849–856. doi: 10.1007/s10068-022-01104-0

Anti-inflammatory effects of mung bean protein hydrolysate on the lipopolysaccharide- induced RAW264.7 macrophages

Jingjing Diao 1,2,✉, Xue Miao 3, Hongsheng Chen 3
PMCID: PMC9203638  PMID: 35720459

Abstract

The anti-inflammatory effects of mung bean protein hydrolysate (MBPH) on the lipopolysaccharide (LPS)-induced macrophages were investigated herein. MBPH was shown to affect the cell morphology, proliferation, cell cycle, cytokine levels at different culture times, and the expression level of nuclear factor-kappa B (NF-κB). The obtained results revealed that different fractions of MBPH promote cell proliferation, alter the cell cycle by decreasing the proportion of cells in the S stage and increasing the proportion of cells in the G2 stage, increase the expression of cytokines, included IL-6, IL-1β, and TNF-α, and negatively affect the LPS-induced inflammatory cytokines. Based on the analysis of cytokine expression at different points in time, it is concluded that cytokine secretion of MBPH-treated group reaches a peak at 24 h, the result was significantly different compared to other treatment groups (P < 0.05). It can be observed that the inflammatory response induced by LPS in the MBPH-III treatment group is reduced compared with other fractions (P < 0.05). In addition, MBPH inhibits the activation of NF-κB signaling pathway by inhibiting the nuclear transcription of p65 and phosphorylation of IκBα in macrophages induced by LPS. Our results demonstrated that lower molecular weight MBPH exerted stronger anti-inflammatory effects than other molecular fractions. Thus, MBPH could be utilized as a functional food ingredient to prevent inflammation in chronic diseases.

Keyword: Mung bean protein hydrolysate, Cytokines, NF-κB signaling pathway, Immune regulation

Practical applications

MBPH has good immune effect and other functional characteristics, so MBPH can be used as a raw material for good health products. At the same time, with the in-depth study of MBPH, it is expected to be applied in immune-modulatory drugs, so as to achieve the purpose of prevention and treatment of diseases.

Introduction

Mung bean (Vigna radiata (L.) Wilczek) is a traditional Chinese legume crop used in both medicine and food. It contains a variety of nutrients, such as proteins, starch, minerals, and trace elements (Keong et al., 2015; Liyanage et al., 2017; Nair et al., 2015). Mung bean contains approximately 19.5–33.1% of protein, which is 2–3 times higher than the protein content of corn, wheat flour, or millet (Mendoza et al., 2001). Moreover, this crop contains 2 times more essential amino acids than the cereal grains (Chen et al., 2014; Zhu et al., 2018). At present, processing mung bean is predominantly done in China to produce starch and mung bean vermicelli. In addition, by-products from mung bean processing are mainly used as low-value feed or industrial waste. This causes waste of high-quality protein resources while causing environmental pollution (Mendoza et al., 2001). In recent years, numerous studies have shown that enzymatic hydrolysis of proteins is the most effective way to use them. Protein hydrolysate has been demonstrated to exhibit various biological activities, such as anti-oxidant, antibacterial, immunomodulatory, and angiotensin I-converting enzyme (ACE) inhibitory effects (Lu, 2018; Sonklin et al., 2018; Wang et al., 2018; Yathisha et al., 2018). Notably, there is increasingly evidence that some protein hydrolysates prepared from common food proteins display significant biological activities, including anti-cancer properties. Administering hydrolysates is also associated with increased immunity and reduced incidence of chronic diseases (Chalamaiah et al., 2018; Cian et al., 2012; Hou et al., 2012; Petrov et al., 2015; Zheng et al., 2015). Our previous study found that mung bean protein hydrolysate (MBPH) inhibited the inflammatory response of macrophages induced by LPS (Diao et al., 2019). Furthermore, other studies have shown that protein hydrolysates exhibit anti-inflammatory effects by inhibiting the activation of nuclear factor-kappa B (NF-κB) signaling pathway. It is noteworthy that activated NF-κB can promote the secretion of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and other pro-inflammatory cytokines. Moreover, our previous studies demonstrated that MBPH inhibits the secretion of IL-1β, IL-6, and other pro-inflammatory cytokines in LPS-induced macrophages and stimulates the secretion of anti-inflammatory cytokines (e.g., IL-10) (Diao et al., 2019). Nonetheless, the effects of different fractions of MBPH on the proliferation of RAW264.7 cells and the cycle progression as well as the anti-inflammatory mechanism are not fully understood. Thus, the aim of the present study was to evaluate the effects of different fractions of MBPH on the proliferation of the RAW264.7 cells and the cell cycle progression as well as to investigate the anti-inflammatory effects in vitro.

Materials and methods

Materials

Mung bean protein (80.3%, g/g) was obtained from Zhaoyuan Biological Protein Co. (Yantai, China). Alcalase was purchased from Novo Co. (Novozyme Nordisk, Bagsvaerd, Denmark). The Sephadex G15 column was provided by Baoman Co. (Shanghai, China). The neutral red cell proliferation and cytotoxicity assay kit and ELISA kit were obtained from Beyotime Co. (Shanghai, China). The polyacrylamide difluoride (PVDF) membrane, antibody (β-actin, p65, s536, IκBα), ECL Plus, and the cell cycle and apoptosis analysis kit were also purchased from Beyotime Co. (Shanghai, China). Inactivated fetal bovine serum, Dulbecco’s Modified Eagle’s Medium (DMEM), bicinchoninic acid (BCA) kit, and the penicillin–streptomycin liquid were obtained from Solarbio Co. (Beijing, China).

Preparation of MBPH

Mung bean protein (7%, w/w) was hydrolyzed by Alcalase 2.4 L. The ratio of the enzyme to the protein substrate was 1:100. The hydrolysis conditions were pH 8.5 at 55 °C. The hydrolysis was terminated when the degree of hydrolysis (DH) reached 25%. The hydrolysates were spray-dried and stored at room temperature until required.

MBPH fractionation

MBPH was fractionated utilizing Sephadex G15 chromatography according to the method previously described by Diao and Zhang (2014). MBPH was diluted in distilled water to a concentration of 100 mg/mL. The resulting solution (2 mL) was pumped on a Sephadex G15 column (1 × 40 cm) and eluted with distilled water at a flow rate of 1 mL/min. The elution was monitored by ultraviolet (UV) absorbance at 220 nm. The three peak areas of MBPH were observed and designated as MBPH-I, MBPH-II, MBPH-III. The molecular weight (MW) standards were also eluted under the same conditions. These included aprotinin (6500 Da), bacitracin (1450 Da), and glycyl-glycyl-tyrosyl-arginine (450 Da). Subsequently, the peak areas of MBPH were calculated employing the Collection Microsoft software (Jingke Co., Shanghai, China). The MW of the three fractions was determined at 2400.58, 1900.41, and 1165.28 Da, respectively.

Morphology of macrophages

The RAW264.7 macrophages were inoculated into 6-well plates at a concentration of 1 × 105 /mL for 2 h at 37 °C and 5% CO2. To observe the morphology, the macrophages were incubated with 200 mg/mL of MBPH (MBPH-I, MBPH-II, or MBPH-III) or 20 μL of LPS (200 μg/mL) for 12 h. The control group was incubated with phosphate buffer solution (PBS) equal to the amount of the MBPH fraction. Following incubation, the cell morphology was evaluated and recorded with biological microscope (CX23, Shenzhen, China).

Cell proliferation assay

The RAW264.7 cells (1 × 105 cells/well) macrophages were seeded in 96-well plates maintained at 37 °C in a 5% CO2 incubator for 12 h. The cells were treated with PBS (200 mg/mL), MBPH (200 mg/mL, MBPH-I, MBPH-II, or MBPH-III), or 20 μL of LPS (200 μg/mL) for 4 h, removed the medium. Thereafter, add 200 μL of sterile filtered MTT solution (1 mg/mL) to each well and culturing cells under the same conditions for 4 h. After supernatant is discarded, 150 μL DMSO was added to each well and shake for 10 min. Absorbance was measured at 490 nm using the microplate reader.

Cell cycle distribution

The RAW264.7 macrophages were inoculated into 6-well plates at a concentration of 1 × 105 /mL for 24 h at 37 °C and 5% CO2. After the macrophages were incubated with 200 mg/mL of PBS, 200 mg/mL of MBPH (MBPH-I, MBPH-II, or MBPH-III), or 20 μL of LPS (200 μg/mL) for 12 h. Subsequently, the cells were treated according to the instructions of the cell cycle detection kit and analyzed using CyFlow space flow cytometry (CytoFLEX, Beckman Coulter, USA). Using of trypsin digested adherent cell, centrifuged at 3000 g/min for 10 min, then add 1 mL pre-cooled PBS, centrifuged for 10 min again, add 1 mL 70% of the ethanol solution and refrigerate at 4 °C for 12 h for later use. After centrifugation to remove ethanol solution, PBS cleaning, centrifugation to obtain cell precipitation, add propylene iodide dyeing solution, staining 30 min at 37℃, the flow cytometer detects red fluorescence at excitation wavelengths of 488 nm wavelengths, light scattering is also detected.

Determination of cytokines in macrophages

Macrophages were incubated under different culture medium conditions (LPS, MBPH-I, MBPH-II, MBPH-III, or MBPH-I + LPS, MBPH-II + LPS, MBPH-III + LPS) at 37 °C in 5% CO2 for 2, 4, 6, 8, 12, and 24 h. The secretion amounts of IL-6, IL-1β, and TNF-α were detected using specific cytokine ELISA kits (Beyotime Co, Shanghai, China) according to the manufacturer’s instructions.

Western blot

The cells were incubated under different medium conditions (PBS, LPS, MBPH-I, MBPH-II, MBPH-III, or MBPH-I + LPS, MBPH-II + LPS, MBPH-III + LPS) at 37 °C and 5% CO2 for 12 h. The cell protein was extracted and its concentration was determined utilizing the BCA protein quantification kit. The cell protein was separated by electrophoresis using a 5–10% polyacrylamide gel. The gel was transferred onto a PVDF membrane, which was subsequently immersed in 5% dried skim milk for 1 h. The membranes were incubated with primary antibodies against p65, s536, and β-actin (dilution ratio 1:500) at 4 °C overnight prior to washing 5 times with TBST. A secondary antibody (1:10,000 dilution) was added and the membranes were further incubated at room temperature for 2 h. The blots were evaluated using the ECL assay kit. The Western blot data were analyzed utilizing the Image J2x software.

Statistical analysis

The data were analyzed employing the General Linear Models procedure in the Statistix 8.0 software package (Tallahassee FL, USA). Significant differences (P < 0.05) between the means were identified using the Tukey’s post hoc tests.

Results and discussion

Morphology of the macrophages

To determine whether MBPH could over-activate the RAW264.7 macrophages, i.e., initiate the production of inflammatory responses, the morphology of the macrophages was evaluated using an inverted microscope. As illustrated in Fig. 1A, the macrophages in the control group were small and spherical, and a small portion appeared to exhibit a fusiform morphology. In contrast, the presence of LPS stimulated the macrophages to display a dendritic form (cell body hypertrophy and irregularity) and to produce numerous pseudopodia and cytoplasmic particles (Fig. 1B), indicating excessive activation of cells. The morphology of MBPH-treated cells were slightly larger than that of control group, but there was almost no pseudopodia (Fig. 1C, 1D, and 1E). Overall, the morphology was not significantly different from that of the control group. These results demonstrate that MBPH exhibited no adverse effects on the macrophages.

Fig. 1.

Fig. 1

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Morphological changes of different fractions of MBPH in the RAW264.7 macrophages (× 100) (A) Untreated RAW264.7 cells (control); (B) LPS-treated RAW264.7 cells; (C) MBPH-I-treated RAW264.7 cells; (D) MBPH-II-treated RAW264.7 cells; (E) MBPH-III-treated RAW264.7 cells

Proliferation activity

The proliferation ability of macrophages is an important indicator of macrophage activation as well as improvement of the immune ability of the body (Ye et al., 2019). The cell proliferation rate of different fractions of MBPH was shown in Fig. 2. The proliferation rate was significantly increased in the LPS-treated group and the MBPH-treated group of different fractions compared with the control group. The proliferative effects on RAW 264.7 cells in the MBPH-I, MBPH-II and MBPH-III treatment groups were significantly different (P < 0.05). Among them, the MBPH-III treatment group was significantly lower than the LPS-treated group, but stronger than other groups, indicating that MBPH-III had a certain promoting effect on RAW264.7 cell proliferation.

Fig. 2.

Fig. 2

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Proliferation activity of the RAW264.7 macrophages following treatment with different fractions of MBPH. The results are presented as mean ± SD (n = 3 for each group at every time point). Significant differences between the treated groups and the control at every time point are indicated as (a)–(c)

Changes in the cell cycle distribution

To investigate the effects of different fractions of MBPH on the differentiation of the RAW264.7 cells, we performed the cell cycle analysis on the macrophages. The cell cycle is the whole process that cells go through from the completion of one division to the end of the next, which can be divided into interphase and mitotic phase. The interphase can be divided into three phases: DNA presynthetic phase (G1 phase), DNA synthetic phase (S phase) and DNA postsynthetic phase (G2 phase). The G1 phase is the synthesis of RNA and ribosomes that cells need for their own growth. This phase is characterized by a metabolic activity and significant increase in cell volume. DNA replication is performed in phase S and large amounts of RNA and protein are produced in phase G2. Figure 3 shows the population percentages at G1, S, and G2 phases of variably treated cells. The G1, S, and G2 phases of the untreated cells were determined at 56.36 ± 1.15%, 2.69 ± 0.05%, and 26.59 ± 0.51%, respectively. Following the LPS treatment, the percentages of G1 and S phases significantly increased to 62.99 ± 1.25% and 8.19 ± 0.15%, respectively. On the other hand, the G2 percentage decreased to 14.91 ± 0.21%. Compared with the LPS-treated group, the G1 and S cell percentages of different fractions of the MBPH-treated group decreased to 60.23 ± 1.13%, 52.65 ± 0.98%, 52.60 ± 0.84%, 7.24 ± 0.14%, 3.33 ± 0.05%, and 3.52 ± 0.07%. Conversely, the percentage of G2 increased to 22.13 ± 0.39%, and 24.32 ± 0.37% (Table 1). The results for the MBPH-III group was similar to that of the untreated group. Based on the above results, low molecular weight MBPH-treated group could decrease the proportion of G1 phase cells, but increase the proportion of S phase cells, indicating that MBPH promoted DNA synthesis and promoted cell cycle differentiation from G1 phase to S phase. Notably, the percentage of S phase and G2 phase cells in MBPH-III-treated group was significantly higher than that of other MBPH-treated groups. This is consistent with the results that MBPH-III promoting the proliferation of RAW264.7 cells. Conversely, LPS cause an increase in the proportion of cells in G1 phase and S phase, and a decrease the proportion of cells in G2 phase. The results of Fig. 2 showed that LPS could induce a large number of cell proliferation, combined with the cell cycle result, it could be concluded that LPS induced cell arrest, lengthened the preparation period of cell replication, and reduced the cell growth activity (Olszewski et al., 2015; Wang, 2016). Moreover, this result was consistent with numerous studies have shown that LPS induces apoptosis (Wu et al., 2015).

Fig. 3.

Fig. 3

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Cell cycle distribution of the RAW264.7 macrophages following treatment with different fractions of MBPH. (A) Untreated RAW264.7 cells (control); (B) LPS-treated RAW264.7 cells; (C) MBPH-I-treated RAW264.7 cells; (D) MBPH-II-treated RAW264.7 cells; (E) MBPH-III-treated RAW264.7 cells

Table 1.

Cell cycle distribution ratio of the RAW264.7 macrophages following treatment with different fractions of MBPH

Group G1 phase/% S phase/% G2 phase/%
Control 56.36 ± 1.15c 2.69 ± 0.05e 26.59 ± 0.51a
LPS 62.99 ± 1.25a 8.19 ± 0.15a 14.91 ± 0.21e
MBPH-I 60.23 ± 1.13b 7.24 ± 0.14b 16.33 ± 0.31d
MBPH-II 52.65 ± 0.98d 3.33 ± 0.05d 22.13 ± 0.39c
MBPH-III 52.60 ± 0.84d 3.52 ± 0.07c 24.32 ± 0.37b
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The results are presented as mean ± SD (n = 3 for each group at every time point). Significant differences between the treated groups and the control at every time point are indicated as a–e

MBPH regulates the release of cytokines in the RAW264.7 macrophages

As demonstrated in Fig. 4, different fractions of MBPH and LPS led to the production of IL-6, IL-1β, and TNF-α in the RAW264.7 macrophages at 2, 4, 6, 8, 12, and 24 h. We found that different fractions of MBPH exhibited weaker effects on the cytokines in normal macrophages, and significantly lower than that of LPS-induced group (P < 0.05). The levels of cytokines upon exposure to MBPH and LPS increased with the prolongation of culture time. It is noteworthy that the expression levels of the cytokines were high at 12 h, and no significant change was noted at 24 h. In addition, our results demonstrate that the secretion levels of IL-6, IL-1β, and TNF-α for MBPH-III were higher than those for the other fractions. Some studies have shown that moderate expression of pro-inflammatory cytokines allows macrophages to be activated and the body to enter a state of cellular immunity (Zeng et al., 2004). In contrast, overexpression can cause an inflammatory response. In addition, our results also found that the expression level of IL-6 in MBPH-treated gourp was higher than these of IL-1β and TNF-α, the reason was that macrophages have the functions of capturing, processing and presenting antigen, secreting active substances and regulating mimunity. Some studies have demonstrated that protein hydrolysates can activate macrophages, and secrete cytokines, including TNF-α, IL-1β, IL-6. Furthermore, the secreted TNF-α and IL-1β could induce the activation of NF-κB pathway, resulting in the production of more IL-6 (Nakayama et al., 2016).

Fig. 4.

Fig. 4

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Effects of different fractions of MBPH on the production 53 of cytokines in the RAW264.7 macrophages. The levels of IL-6 (A), IL-1β (B), and TNF-α (C) in the culture media were measured at 2, 4, 6, 8, 12, and 24 h following administration of LPS or MBPH. The results are presented as mean ± SD (n = 3 for each group at every time point). Significant differences between the treated groups and the control at every time point are indicated as (a)–(c)

MBPH regulates the release of cytokines in LPS-stimulated RAW264.7 macrophages

The effects of different fractions of MBPH on the cytokine secretion levels (including IL-6, IL-1β, and TNF-α) were examined in LPS-stimulated RAW264.7 macrophages (Fig. 5). It was determined that LPS caused a significant production of IL-6, IL-1β, and TNF-α in macrophages at 2, 4, 6, 8, 12, and 24 h (P < 0.05). Compared with the LPS-treated group, different fractions of the MBPH treatment groups attenuated the excessive release of IL-6, IL-1β, and TNF-α in the LPS-stimulated RAW264.7 macrophages, and the ability to suppress inflammation was strongest at 12 h. In addition, the obtained results revealed that the MBPH-III treatment group exhibited stronger inhibitory activity than the other MBPH fractions. These results indicate that different fractions MBPH improved the vitality of cells and exerted anti-inflammatory activity in LPS-stimulated macrophages.

Fig. 5.

Fig. 5

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Effects of different fractions of MBPH on the production 61 of cytokines in LPS-stimulated RAW264.7 macrophages. The levels of IL-6 (A), IL-62 1β (B), TNF-α (C) in the culture media were measured at 2, 4, 6, 8, 12, and 24 h following LPS or MBPH administration. The results are presented as mean ± SD (n = 3 in each group at every time point). Significant differences between the treated groups and the control at every time point are indicated as (a)–(d)

NF-κB signaling pathway

The effects of protein hydrolysates on the immunity has been confirmed. Specifically, hydrolysates were shown to modulate inflammation related to immune response e.g., through inhibition of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) (Li et al., 2012). Our above results revealed that different fractions of MBPH enhanced the expression of IL-6, IL-1β, and TNF-α in macrophages. Moreover, it was shown that MBPH inhibits the production of the pro-inflammatory cytokines in LPS-stimulated macrophages. Previous studies found that the immune-modulatory effects of immunopotentiators are related to the NF-κB signaling pathway. Ren (2010) also reported that polysaccharo-peptides significantly activated the NF-κB signaling pathway in normal cells and blocked the nuclear translocation of NF-κB in LPS-stimulated cells. To investigate the anti-inflammatory potential of the different fractions of MBPH through the NF-κB pathway, the expression levels of protein factors related to inflammation and immunity in macrophages were determined by western blot analysis (Fig. 6). Phosphorylation IκBα and nuclear p65 are signs of an activated NF-κB pathway. The activated NF-κB then would stimulate target genes in the nucleus, leading to the release of proinflammatory cytokines (Diao et al., 2019). Different fractions of the MBPH-treated groups promoted the expression of p65, phosphorylated p65 (S536) and IκBα, and the expression levels were marginally higher than that of the untreated group (P > 0.05). The protein expression levels of nuclear p65 and IκBα were considerably lower than those of the LPS-treatment group, indicating that different fractions of MBPH activated the NF-κB signaling pathway and stimulated the cell cytokine expression; however, the inflammatory response was significantly lower than that of the LPS-treatment group (P < 0.05). Moreover, the obtained outcomes suggested that different fractions of MBPH reduced the expression of p65 and IκBα in LPS-stimulated macrophages. The protein expression level of MBPH-III was higher than those of the remaining MBPH treatment groups, suggesting that different fractions of MBPH exhibited inhibitory effects on the IκBα phosphorylation, leading to inactivation of NF-κB. Based on the experimental results, we speculated that the anti-inflammatory effects of MBPH were a consequence of the NF-κB pathway inhibition.

Fig. 6.

Fig. 6

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Effects of MBPH on the regulation of the NF-κB pathway 79 in the untreated and LPS-stimulated RAW264.7 macrophages evaluated by western blotting analyses. (A) p65 and IκBα phosphorylation as assessed by western blotting analyses; (B) Relative protein levels of p65 and IκBα are shown in (A). Significant differences of p65 expression level between the treated groups and the control group are indicated by a-e; significant differences of IκBα expression level between the treated groups and the control group are indicated by A–D

Previous studies demonstrated that immune-modulatory properties of food-derived protein hydrolysates are related to the length, sequence, and amino acid composition (Chalamaiah et al., 2018). According to the above results, MBPH-III (MW < 1165 Da) had strong immune regulation and anti-inflammatory effects, due to the small molecular weight of MBPH-III, which can propitiously penetrate the cell membrane and be transferred to the target more effectively to play its role (Liu et al., 2014). Moreover, numerous studies have confirmed that molecular weight of protein hydrolysate is correlated with its immune activity. Kong et al. (2008) reported that lower molecular weight soy protein hydrolysate was effective in stimulating lymphocyte proliferation. Wu and Pan (1999) found that bovine casein contains two kinds of immunoactive peptides, Leu-Leu-Try, Thr-Thr-Met-Pro-Leu-Tyr. Zeng et al. (2004) confirmed that hexapeptides obtained from soybean protein hydrolysate have the ability to stimulate macrophage phagocytosis. These immunoactive peptides are characterized by low molecular weight.

In the present study, MBPH enhanced the vitality of macrophages and activated the expression of cytokines, including IL-1β, IL-6, and TNF-α in normal macrophages. The secretion of the cytokines reaches a peak at 12–24 h. MBPH was also shown to inhibit the secretion of pro-inflammatory cytokines in LPS-induced cells, which exhibited potent inhibitory effects at 12 h. The obtained results demonstrated that MBPH displayed anti-inflammatory effects on LPS-induced macrophages by inhibiting the nuclear translocation of the p65 protein, phosphorylation of IκBα, and inhibition of the over-activation of the NF-κB signaling pathway.

Acknowledgements

This work was supported by the Heilongjiang Major Science and Technology Projects (2021ZX12B06), National Program on Key Research Project (2018YFE0206300), and Heilongjiang Postdoctoral Fund (LBH-Z20206).

Abbreviations

LPS

Lipopolysaccharide

NF-κB

Nuclear factor kappa-B

MBPH

Mung bean protein hydrolysate

IL-6

Interleukin-6

IL-1β

Interleukin-1β

TNF-α

Tumor necrosis factor-α

Author contributions

JJD and XM designed the study, interpreted the results and drafted the manuscript. HSCh collected the test data and modified the manuscript.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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