Immune-Enhancing Activity of Vitis coignetiae Extract via Increasing Cytokine and Natural Killer Cell Activity in Splenocytes and Cyclophosphamide-Induced Immunosuppressed Rats

Plant and fruit extracts exhibit fewer side efects than pharmaceuticals and can display therapeutic qualities. Consequently, they have attracted increased attention among health-conscious individuals


Introduction
Te immune system plays an important role in the pathophysiology of diseases, including immune disorders (psoriasis and arthritis) and cancer, in animals and humans [1,2].Disruption of the immune system can therefore be a starting point for disease development.Recently, several factors (excess alcohol and tobacco consumption, exposure to particulate matter 10 and 2.5, stress, and drug therapy) have been shown to reduce human and animal immunity [3][4][5][6].Terefore, individuals are increasingly turning towards immuneboosting nutritional supplements or foods.Functional foods and botanical drug products, including natural extracts, are known to display few side efects and confer various benefcial efects (such as immunomodulatory, anti-infammatory, and anticancer efects) [7,8].Natural extracts from roots (ginseng, red ginseng, and Heracleum moellendorfi root), fowers (Paulownia tomentosa), and fruits (blueberry and black chokeberry) have shown evidence of antioxidant (reactive oxygen species chelation) and anti-infammatory efects (decreased cytokine release and infammatory pathway inhibition) [9][10][11][12][13][14][15].Accordingly, research is being actively conducted on natural extracts, and products (foods and drugs) using natural extracts are being increasingly produced.
Cyclophosphamide (Cy) is an alkylating agent with a broad spectrum of activity in various diseases; it is widely used as an anticancer agent, especially in chemotherapy [16].Cy is converted to 4-hydroxycylophosphamide/aldophosphamide in the liver, difuses into cells in the body, and is fnally converted to the active compound phosphoramide mustard.Finally, it passes through the nuclear membrane and induces apoptosis [17].Terefore, Cy kills both the cancer and normal cells, including immune cells.In addition, it displays side efects such as hepatotoxicity, nausea, vomiting, hemorrhagic bladder infammation, and alopecia, and long-term therapy results in the loss of body weight (BW), thymus weight, white blood cells (WBCs), bone marrow cells, and natural T cells [18,19].Previous studies have utilized Cy-induced immunosuppression as a tool with which to study the immune-enhancing efects of drugs or natural products; one example is that regarding HemoHIM (Atomy Co., Korea), of which the herbal preparation showed evidence of anticancer efects, memory improvement, and immune enhancement [20,21].
In this study, we hypothesized that Vitis coignetiae extract (VCE) would confer an immune-enhancing efect and aimed to verify whether the administration would increase immune-related activity in Wistar rat splenocytes and in Cyinduced immunosuppressed rats.

Preparation of VCE.
Vitis coignetiae fruit (washed with distilled water) was ground using a blender for 20 s.VCE was extracted using 20% EtOH (ground fruit, 100 g with 20% EtOH, 900 g).Ten, VCE was fltered using a qualitative flter paper (11 μm, GE Healthcare, UK).Te fltered solution was evaporated in a vacuum rotary evaporator.Ten, the concentrated extract was lyophilized in a freeze dryer at −50 °C for 6 d.

Animals and Oral Administration of VCE.
All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Animals.Tis study was approved by the Institutional Animal Care and Use Committee of INVIVO Co., Ltd.(IV-RB- 17-2206-20).Specifc pathogen-free Wistar rats (fve weeks, male) were purchased from Orient Bio Co. (Seongnam, Korea).After one week, the rats were divided into six groups (normal; control; VCE 50 mg/kg; VCE 250 mg/kg; VCE 500 mg/kg; and positive, HemoHIM 1,000 mg/kg).All rats, except those from the normal group, were orally administered with Cy (5 mg/kg) for immunosuppression.All animals' BWs were monitored weekly during the experimental period.Wistar rats were kept in a temperature-controlled room with a 12 h light and 12 h dark cycle.

Cell Viability of Splenocytes.
In brief, primary splenocytes (5 × 10 5 cells/well) were seeded in 96-well plates and incubated at 37 °C for 24 h.Ten, the stabilized splenocytes were treated with VCE (indicated doses in Figure 1) and VCE with Cy 0.8 mg/mL, and incubated for 24 h in a 5% CO 2 incubator.Cell viability rate was measured using the WST-1 assay kit (ITSBio, Seoul, Korea) and the Sunrise ™ enzyme- linked immunosorbent assay (ELISA) plate reader (Tecan, Männedorf, Switzerland).

Nitric Oxide (NO) Release in Splenocytes.
NO production by Cy-treated splenocytes was evaluated using a Griess assay [10].Splenocytes (5 × 10 5 cells/well) were seeded in a 96-well plate.Te cells were treated with Cy (0.8 mg/mL) and VCE (0-300 μg/mL) for 24 h, where the control group contained only RPMI-1640.Ten, the supernatant (80 μL/well) was collected and added to a new 96-well plate and mixed with the same amount of Griess reagent.Before detection of the absorbance, the mixture was shaken in the dark for 10 min.Te absorbance was measured at 540 nm.

Natural Killer (NK) Cell
Activity.NK cell activity was investigated as previously described [26].In brief, AR42J (Cat.CRL-1492, ATCC, VA, USA) cells were used as target cells for the NK cell activity, and primary splenocytes (from all experimental groups) were used as efector cells.Splenocytes were cocultured with AR42J cells in 96-well plates at a ratio of 25 : 1 in a 5% CO 2 incubator at 37 °C for 24 h.AR42J viability rate was measured by the WST-1 assay kit.

Complete Blood Cell (CBC) Count Analysis.
At the time of autopsy, whole blood was collected from the abdominal vena cava of experimental groups of Wistar rats.Ten, it was kept in a ethylene-diamine-tetraacetic acid (EDTA)-coated tubes (BD Caribe, Ltd., USA).Te total numbers of white blood cells (WBCs), lymphocytes, granulocytes, and midsized cells in EDTA-coated tubes were analyzed using a Mindray BC-2800 hematological analyzer (Mindray, Bath, UK).

Histological Analysis.
Histochemical analyses were performed as previously described [26].In brief, after the animals were euthanized, their spleens were removed, weighed, and fxed in 10% neutral-bufered formalin.Te organs were then embedded in parafn and sectioned into 4μm-thick slices using a microtome (Termo Scientifc, Waltham, MA, U.S.A.).Sectioned tissues were stained with hematoxylin and eosin (H&E).Spleen images were scanned using a MoticEasyScan One (Hong Kong, China) and detected using Motic DSAssistant (X86) software.
2.12.Statistical Analysis.All data are expressed as the mean ± standard error of the mean (SEM), and diferences between groups were analyzed using one-way ANOVA (Duncan's multiple-range test).All analyses were performed using SPSS (version 23.0; SPSS, Inc. USA).Each value represents the mean of at least three independent experiments for each group.Statistical signifcance was set at p < 0.05.

NO Production in Splenocytes.
Te production of NO was investigated for anti-infammatory as well as immuneenhancing efects.Appropriate immune stimulation boosts immunity [9].Terefore, we investigated NO production in the splenocytes of Wistar rats (Figures 1(c) and 1(d)).Te production of NO was increased by VCE (3.43 ± 0.17 μM at 300 μg/mL) and VCE with Cy (at 4.87 ± 0.21 μM at 300 μg/mL).

Journal of Food Biochemistry
Journal of Food Biochemistry

Infammation Pathways in Splenocytes.
A previous study showed that nonexcessive stimulation of immune pathways (such as the mitogen-activated protein kinase (MAPK) and NFκB pathways) helps to increase immunity [9].To determine immune-related protein expression, western blot analysis was performed for the expression of inducible nitric oxide synthase (iNOS) and phosphorylation of MAPKs (Erk, JNK, and p38) and NFκB.As shown in Figure 4, immunosuppression by Cy (0.8 mg/mL) did not afect the expression of iNOS and the phosphorylation of MAPKs but suppressed the phosphorylation of NFκB.In the presence of VCE (30, 100, and 300 μg/mL), the expression of iNOS signifcantly increased with VCE 100 μg/mL, and concentration-dependent increases of MAPKs were observed, with the exception of p38 (Figures 4(b)-4(e)).NFκB phosphorylation increased in a concentration-dependent manner (Figure 4(f )).Tese results show that VCE stimulates immune-related pathways in a concentrationdependent manner.also unchanged, while the spleen weight increased compared tocontrol rats.Tese results suggest that VCE enhances immunity by activating the immune system in the spleen.

CBC and Cytokines in Cy-Induced Immunosuppressed
Rats.A previous study showed reductions in WBC, granulocyte, lymphocyte, midsize cell, and infammatory cytokine (such as IL-2, IL-12, IFN-c, and TNF-α) levels in Cytreated splenocytes and Cy-induced immunosuppressed rats [19,28].Terefore, we investigated whole blood and serum cytokine levels (Figures 6 and 7).After respiratory anesthesia, whole blood was collected immediately from the vena cava, followed by an analysis of the CBC (WBC, granulocytes, lymphocytes, and midsized cells), and serum was separated.As shown in Figure 6, WBCs, granulocytes, lymphocytes, and midsized cells signifcantly decreased following Cy intake (5 mg/kg) and increased following VCE administration (250 mg/kg).CBC levels in VCE 50 rats were similar to those in control rats.As shown in Figure 3, infammatory cytokine levels decreased in Cy-treated splenocytes.Terefore, we investigated the levels of infammatory cytokines in the serum of Cy-induced immunosuppressed rats (Figure 7).Following oral administration of Cy for four weeks, IL-2, IL-12, IFN-c, and TNF-α levels decreased.VCE administration (500 mg/kg) increased cytokine levels to levels similar to those of normal rats.In addition, IgG and IgM levels were investigated to determine immunity (Figures 7(e) and 7(f )).Levels of IgG and IgM were decreased in control rats, while IgG increased after VCE 250 treatment and IgM increased after VCE 500 treatment.In the control group, IgG and IgM levels decreased, while these were signifcantly increased in the VCE 250 and positive control groups (HemoHIM).Tese results show that VCE can recover CBC, cytokine, IgM, and IgG levels to normal levels in immunosuppressed rats.Terefore, VCE is efective in increasing immunity.[29].To determine the efect of VCE on NK cell activity, we analyzed lactate dehydrogenase (LDH) production in splenocytes (Figure 8(a)), Cy-induced immunosuppressed rats (Figure 8(b)), and AR42J cells.In Figure 8(a), NK cell activity (%) in splenocytes increased in a concentration-dependent manner with VCE.In addition, oral administration of VCE increased NK cell activity in Cyinduced immunosuppressed rats.Tese results indicate that VCE enhances immune function by increasing NK cell activity in splenocytes.

NK Cell Activity. Enhancing NK cell activity protects against viral infection
3.9.Histological Assay.We observed morphological changes in the spleen and evaluated the efects of VCE (Figure 9).In the control group, white pulp (WP) and lymph nodule (LN) depletion were observed using H&E staining in the spleen, Various natural products afect the immune system [30].Recently, natural products (favonoids and phenols) have been shown to have various efects (such as antioxidant, anti-infammatory, anticancer, and immune-regulating effects) and increase immunity through various actions such as cellular and nonspecifc immunity as a stimulus to immune organs.Various efects of natural products defend against external harm [9].A previous study reported that the intake of a natural extract (Platycodon grandiforum) helps to improve immunity by recovering IL levels and the collapse of spleen tissue [19].VCE has been reported to have strong antiinfammatory efects on macrophages and anticancer efects (proliferation, migration, and invasion) in cancer cells [22,27].Te spleen is essential for immune system function; it is important for fltering blood, removing bacteria and infecting organisms in the bloodstream, and destroying and producing Immunoglobulins.In a previous study, Cyinduced immunosuppression induced decreases in CBC and IL levels and led to the collapse of the spleen tissue.To date, there have been no reports on the results of studying the efcacy of VCE in Cy-induced immunosuppression.

Discussion
Our results show that VCE is a natural product containing phenols (4-hydratebenzoic acid, gallic acid, and syringic acid) and the favonoid quercetin (Figure 2).It has been suggested that VCE afects the immune regulation and could be efective in enhancing immunity.Terefore, the immuneenhancing efects of VCE were evaluated.We performed a cytotoxicity evaluation in Wistar rat splenocytes to ensure the safety of VCE.Te results show that there is no cytotoxicity up to a concentration of 300 μg/mL.Moreover, cytokine (TNF-α, INF-c, IL-2, and IL-12) levels were decreased in Cy-treated splenocytes and were recovered following VCE administration.Previous studies have shown the stimulation of infammatory pathways (MAPK and NFκB pathways) and related protein expression (iNOS and cyclooxygenase-2 (COX-2)) in immune cells by various natural extracts [31,32].Furthermore, infammatory pathway activation and related protein expression increased by infammatory mediators (such as lipopolysaccharide, IL-1β, and TNF-α) have been shown to be reduced by natural extracts [31,33].However, this is increased by natural extracts due to their immune-enhancing efect [9].Our results showed that VCE increased the expression of iNOS and phosphorylation of MAPKs (Erk and JNK) and NFκB.
Tese results suggest that VCE shows immune-enhancing efects at nontoxic concentrations in vitro.
A decrease in immunity indicates a condition in which immune system disruption may fail to prevent harmful external intrusion.Tis decreases the weight of immunerelated tissues (spleen and thymus) and WBC count in vivo [34,35].Our results showed that CBC and cytokine levels, and thymus and spleen weights, were reduced in Cy-induced immunosuppressed rats.Elevated cytokine regulation indicates that infammation has occurred, while downregulation indicates that immunity has decreased [36].In our results, Cy-induced cytokine reduction was recovered by VCE to normal levels.In addition, WBC, lymphocyte, granulocyte, and midsized cell levels were recovered in the whole blood of Cy-induced immunosuppressed rats.Finally, we observed morphological changes in the spleen.In our results, the WP and LN collapsed in the Cy group and recovered in the VCE (500 mg/kg) and positive control groups (HemoHIM).Our fndings suggest that VCEinduced cytokine (TNF-α, IFN-c, IL-2, IL-12, IgG, and   Figure 8: Efect of Vitis coignetiae extract (VCE) on natural killer (NK) cell activity in cyclophosphamide (Cy)-treated Wistar rat splenocytes and Cy-induced immunosuppressed rats.Wistar rat splenocytes were treated with VCE (0-300 μg/mL) for 24 h and Cy-induced immunosuppressed rat splenocytes were treated with Cy (5 mg/kg/d), oral VCE (0, 50, 250, and 500 mg/kg/d), and HemoHIM (positive control; 1,000 mg/kg/d) once daily for four weeks, after which spleens were collected for analysis.Splenocytes were cocultured with target cells (AR42J) in 96-well plates.Ten, the cells were incubated for 24 h in a 5% CO 2 incubator with a ratio of efector to target cells of 20 : 1. NK cell activity was calculated as the survival rate of AR42J cells compared to those of the control group.Bars labeled with diferent superscripts have signifcantly diferent values (p < 0.05).
IgM), CBC (WBC, granulocyte, lymphocyte, and midsize cell), and NK cell activity increases in Cy-treated splenocytes and Cy-induced immunosuppressed rats, as well as the stimulation of infammatory pathways, constitutes immune enhancement.

Conclusions
In conclusion, our fndings demonstrate that VCE mitigated the decrease in cytokine levels in Cy-treated splenocytes, CBC levels in whole blood, and cytokine levels in serum, and diminished the increased thymus and spleen weight increases in Cyc-induced immunosuppressed rats.Tese results suggest that VCE is a healthy functional food that can boost immunity during chemical immunosuppression.
Terefore, VCE may have applications in the development of immune-enhancing functional foods and medicines.In this study, VCE was nontoxic in vivo; however, further toxicity assessments are needed before its use in humans [37][38][39][40].

Figure 6 :
Figure6: Efects of Vitis coignetiae extract (VCE) on (a) white blood cell, (b) granulocyte, (c) lymphocyte, and (d) midsize cell absolute counts in the whole blood of cyclophosphamide (Cy)-induced immunosuppressed rats.Wistar rats were treated with saline, Cy (5 mg/kg/d), oral VCE (0, 50, 250, and 500 mg/kg/d), and HemoHIM (positive control; 1,000 mg/kg/d) once daily for four weeks, after which whole blood samples were collected for analysis.Te levels of white blood cells, granulocytes, lymphocytes, and midsize cells in the blood.Bars labeled with diferent superscripts have signifcantly diferent values (p < 0.05).