Introduction

Influenza viruses, especially influenza A viruses, have caused several human epidemics and pandemics, inflicting enormous suffering and economic losses [1]. Influenza virus infections can have added complications such as pneumonia and ischemic heart disease that considerably increase the rate of hospitalization and mortality, especially in young children and elderly individuals [2,3]. M2 ion channel inhibitors such as amantadine and rimantadine and neuraminidase inhibitors such as zanamivir and oseltamivir are used to treat influenza virus infections. Recently, there have been reports of neuraminidase inhibitor-resistant influenza A viruses [4-7]; hence, a new drug, favipiravir (FRV), for influenza virus infections was developed [8,9]. Furthermore, baloxavir marboxil, an endonuclease inhibitor, has also been developed against influenza A virus [10]. However, the adverse effects and disadvantages of these drugs have also been reported. The risk of teratogenesis with FPV use might be higher than that with other drugs in the same class in clinical practice [11]. The emergence of polymerase acidic protein variants with I38T/M/F substitutions, leading to reduced susceptibility to baloxavir, occurred in 2.2% and 9.7% of baloxavir recipients in phase 2 trial and phase 3 trials, respectively [12].

Since prehistoric times, humans have applied poultices and imbibed infusions of numerous indigenous plants. Several of these infusions include food extracts that contain biologically active substances that possess antimicrobial properties [13]. Moerman [14] compared the medicinal and food floras of the native peoples of North America and discovered an overlap of these floras by both family and taxon. That is, many medical plants also double as food and add secondary herbal compounds to diet, providing medicinal benefits with low or no adverse effects. For example, herbal teas are convenient and safer alternatives to pharmaceuticals to improve sleep quality [15]. Therefore, a library of food and foodrelated substance extracts would be a good source for discovering novel antimicrobial agents. The anti-influenza virus effects of food extracts have been reported both in vivo and in vitro [16-23]. Hot water extracts of some of the most popular crops worldwide, such as soybeans, adlay seeds, and naked barley seeds, inhibit the growth of influenza viruses [24]. Previously, we reported the antiinfluenza effects of soybeans, and fount that one of the components of soybean extracts might suppress the virus growth by enhancing the defense ability of infected cells rather than directly inhibiting the virus [25]. At low concentrations, soybean extracts have a broad-spectrum anti-influenza virus activity without cytotoxicity in vitro. However, to the best of our knowledge, except one reports on tofu (fermented soybean food) extract [26], there are no reports on the effects of soybean extracts in in vivo models. Furthermore, although several studies have examined the antiviral effects of soybean- [24-26] and daidzein-containing [27] diets, studies focusing on the antiviral activity of the soybean component are needed.

As studies on the in vivo effects of soybean extracts on influenza viruses are lacking, we aimed to demonstrate the inhibitory effect and mechanism of action of soybean extract on influenza virus in mice in this study.

Materials and Methods

Cells and viruses

Madin–Darby Canine Kidney cells were grown in Eagle’s minimum essential medium (MEM; Sigma-Aldrich, Inc., St. Louis, MO, USA) containing 7% fetal bovine serum (FBS; Biowest Biotechnology Company, Nuaillé, Pays De La Loire, France). The influenza A virus H1N1 (A/PR/8/34) was used in the study. For cell infection, the virus was diluted in serum-free MEM supplemented with 0.4% bovine serum albumin (BSA, fraction V; Sigma-Aldrich, and added to the cells at a multiplicity of infection (MOI) of 0.001 for 1 h at 37 °C. The medium was then replaced with FBS-free Dulbecco’s modified Eagle’s medium (SigmaAldrich) supplemented with 10% BSA and acetyl-trypsin (1 µg/ ml; Sigma-Aldrich) for the remaining infection period.

A mouse-adapted strain of influenza virus A/PR/8/34(H1N1) was derived by serial passages of pulmonary homogenates from the infected mice in naive mice. The viral stocks were stored at −80 °C.

Hot water extracts of soybeans

Roasted soybean flour was used in the study. To prepare hot water extracts of the flour, 2.2 g (soybeans) of flour was extracted with 50 ml of hot water (80 °C) for 50 min using a hot water bath. The extract was filtered through a No. 2 filter paper and then a Millex GV membrane (pore size 0.22 mm; Millipore, Billerica, MA, USA) and stored at -30 °C before use. The antiviral activities of the extracts were stable for at least 2 months under these conditions.

Focus-forming assay to evaluate virucidal activities

Focus formation was achieved using the method of Morimoto et al [28]. Each viral dilution was selected to obtain a final count of approximately 30 Focus-Forming Units (FFU) per well. The antiviral activity is expressed as the reciprocal of the highest dilution that reduced the number of foci to 50% or less of the control value.

Approval for animal testing

This study was performed with the approval of the Animal Experiment Committee of Hyogo College of Medicine, Nishinomiya, Hyogo, Japan (approval number: 15–007) and complied with the ordinance of the Regulation for Enforcement of the Act on Welfare and Management of Animals related to the care and management of experimental animals.

Mice feeding and infection with the virus

Five-week-old female BALB/c mice (20–22 g) were obtained from Charles River Laboratories International, Inc., USA. The mice were broadly divided into two groups and were maintained in a laminar flow hood at the Animal Experiment Facility of the Hyogo College of Medicine under suitable conditions (12 h light/ dark cycle, 25 °C, relative humidity of 50%). One group was fed an AIN-76 purified mouse diet (containing no soybean component; Table I) (CLEA Japan, Inc., Japan), whereas the other group was fed a CE-2 diet (a general feed containing soybean components; Table 1 and Table 2 for 1 week. The major difference between AIN-76 and CE-2 is the nitrogen-free extract contained in CE-2. In addition, secondary metabolites such as soybean polyphenols are contained in the nitrogen-free extract. The mice were housed in groups of four or five in plastic cages with paper bedding (Paper Clean, SLC, Hamamatsu, Japan). After 1 week, the 60 six-weekold BALB/c female mice in the AIN-76 fed group were further divided into 6 groups of 10 mice, and the A/PR/8/34 influenza virus (20 µl each: 10, 25, 50, 75, 100 and 800 PFU/mouse), a mouse-adapted strain, was administered through one nostril of the mice. Similarly, the 40 CE-2 fed mice were further divided into 4 groups of 10 mice, and the influenza virus (20 µl each: 75, 800, 1000, and 1200 PFU/mouse) was administered.

 

AIN-76			CE-2
Component	content rate (%)	Source	Component
Casein	20	Protein	Soybean meal, fishmeal, yeast
DL-Methionine	0.3
Cornstarch	15	Carbohydrate	Wheat, corn, milo
Sucrose	50
Corn oil	5	Lipid	Germ, soybean oil
Cellulose powder	5	Fiber	Wheat bran, rice bran, alfalfa meal
Mineral mixture	3.5	Mineral
Vitamin mixture	1	Vitamin
Choline bitartrate	0.2

Table 1 : Components of mouse diet.

Nutrient components	Nutrient components (%)
Moisture	8.7
Crude protein	26.46
Crude fat	5
Crude fiber	4.04
Crude ash	8.6
Nitrogen free extract	46.2

Table 2 : Mean values from analyses in fiscal 2021 of CE-2 diet. This data is modified the data in the catalog of CLEA Japan, Inc.

Evaluating the effect of soybean extracts on influenza virus in BALB/c mouse

Five-week-old BALB/c female mice were maintained in a laminar flow hood at the Animal Experiment Facility and acclimatized to feed AIN-76 for 1 week. Body weight was measured every morning during the study. The mice were randomly divided into two groups (10 per group) on day 1. On day 6, the mice were anesthetized via an intraperitoneal injection of triple anesthesia (medetomidine hydrochloride, midazolam, and butorphanol tartrate) (0.2 ml/mouse), and one group (test group) was inoculated with 20 μl of soybean extract (SE: 20 mg/kg/d) into one nostril. The mock-treated group was administered an equal volume of phosphate-buffered saline (PBS). The MLD₅₀ of the first virus lot shown in Table 3 was 10^1.5 PFU in the mice fed AIN-67 and 10^2.4 PFU in the mice fed CE-2. Second virus lot shown in Figure 1 was less pathogenic than those shown in Table 3, and the MLD₅₀ was 10^2.0 PFU in the mice fed AIN-67. Therefore, in the subsequent experiments, 10^2.0 PFU was inoculated. On day 7, the mice were anesthetized by intraperitoneal injection of the triple anesthesia and infected with 100 PFU influenza virus by inoculating 20 μl of virus solution into one nostril. After 1 h, SE was inoculated into the test group mice and PBS was inoculated into the mock-treated group mice. The SE-administered and mocktreated mice were inoculated with SE and PBS, respectively, for 3 d. Survival was monitored for 14 d after virus infection. The changes in body weight were monitored for 14 d from the day of infection. The mice were maintained in accordance with the ordinance of the Regulation for Enforcement of the Act on Welfare and Management of Animals related to the care and management of experimental animals (Ordinance, 2016).

 

Figure 1: Effect of nasal administration of SE on the prevention of death in influenza virus-infected mice.

Survival rate of mice treated with SE via the nasal cavity and mock-treated mice. Twenty microliters of SE or PBS was inoculated, via the nasal cavity, for 5 d from 1 d before virus infection to 3 d after virus infection. All mice were inoculated with 100 PFU of the second lot influenza virus in Table 3. A: Body weights of the SE-administered and control mice. B: Survival rates of mice in the SEadministered group and the mock-treated group. Both groups contained 10 mice each. *: p<0.05

		Feed
	CE-2	AIN-67
MLD50 (PFU/mouse)	102.4	101.5

Table 3 : Mouse lethal dose 50 (MLD50) of mouse-adapted influenza virus A/PR/8/34 in the mice fed AIN-67 and CE-2.

These MLD50 values were obtained by the first viral lot, and the pathogenicity of the second lot was weaker, with an MLD50 was of 102.0 in mice fed AIN-67.

Sampling of plasma, nasal cavity lavage fluid, and lung

To prevent blood coagulation, 0.2 ml of 1000 U/ml heparin solution was intraperitoneally administered per mice and left for 30 min. After 30 min, 0.3 ml of triple anesthesia (medetomidine, midazolam, and butorphanol) was intraperitoneally administered. First, the femoral vein was cut, blood was collected, and plasma was collected by centrifuging the blood at 3000 rpm for 15 min at 4 °C. The lungs were removed, weighed, and then 5 ml of MEM (-) was added for homogenization. After centrifugation at 2500 rpm for 20 min at 4 °C, the supernatant was collected and used as a sample. In addition, the lungs were removed from the trachea and fixed with neutral buffered formalin solution to prepare a lung specimen. PBS (-) (500 µl) was injected using a syringe, and the nasal cavity lavage fluid was collected, centrifuged at 3000 rpm for 15 min at 4 °C, and the lavage supernatant (LS) was used as a sample.

Histopathological examination of the lung

The mice lungs were removed from the trachea, and a neutral buffered formalin solution was injected through the trachea to inflate the alveoli. The lungs soaked in neutral buffered formalin solution were washed with water, immersed in 70% ethanol solution for 30 min, set in an automatic embedding device, and then dehydrated with ethanol and replaced in xylene. After replacement, the lungs were hardened with paraffin to prepare paraffin blocks. The paraffin blocks were sliced to a thickness of 2 µm using a microtome, attached to a glass slide, and dried. Deparaffinization was performed with xylene and 100% alcohol. Subsequently, the nucleus was stained with hematoxylin solution, and the cytoplasm was stained with eosin solution (H&E slides). A mounting medium was dropped on the edge of the slide, and the slide was covered with a cover glass and dried at room temperature for a day. The H&E slides were scanned and converted into whole slide images using a Nanozoomer (Hamamatsu Photonics, Japan), and the images were observed using NDP.view2 image viewing software. The histopathological terminology for non-neoplastic lung lesions is in accordance with the International Harmonization Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice Project [23]. The severity of infiltration of mononuclear cells and neutrophils, necrosis of alveoli, and necrosis of bronchiolar epithelial cells were graded on a four-point scale of 1. minimal, 2. mild, 3. moderate, and 4. marked. Histopathological examinations were conducted by pathologists certified by the International Academy of Toxicologic Pathology.

Measurement of blood IgG and nasal cavity lavage fluid IgA

The level of immunoglobulin A (IgA) and IgG was measured using the Mouse IgA ELISA Quantitation Set (Bethyl Laboratories, Inc., Montgomery, TX, USA) and Mouse IgG ELIZA Quantitation Set (Bethyl Laboratories, Inc.) following the manufacturerrecommended protocols.

Measurement of blood cytokines

The level of cytokines in the blood was measured using the tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), IL-4, IL-12, and interferon-γ (IFN-γ) measurement kit (R&D Systems, Minneapolis, Minnesota, USA) following the manufacturer’s instructions.

Lung virus titer measurement

The LSs were serially diluted four-fold in 10 diluted steps in 1.5 ml Eppendorf centrifuge tubes. Thereafter, focus formation by each LS dilution was evaluated following the method described by Morimoto et al [28]. The antiviral activity is expressed as the reciprocal of the highest dilution that reduced the number of foci to 50% or less of the control value.

Statistical analyses

Statistical analyses were performed using the unpaired t-test and analysis of variance with the Tamhane test using SPSS (version 24.0; SPSS, Inc., Chicago, IL, USA). The amount of virus in the time-of-addition assay and the antiviral assay were analyzed using Student’s t-test in Excel Toukei (version 6.0; Esumi, Tokyo, Japan). The survival rate of the mock- and virus-infected mice was analyzed using the Kaplan–Meier method and log-rank test in Excel Toukei Statcel 3 (OMS, Saitama, Japan). Values are presented as mean ± standard deviation. Statistical significance was set at p < 0.05.

Results

Survival rate of CE-2- and AIN-76-fed mice upon influenza virus infection

The survival rate of CE-2-fed mice after influenza infection, and the viral load indicating that the MLD₅₀ was approximately 10^2.4 plaque-forming units (PFU)/animal (Table 3 ). The survival rate of AIN-76-fed mice after influenza infection, and the viral load indicating that the MLD ₅₀ was approximately 10^1.5 PFU/ animal (Table 3). The MLD₅₀ value of the virus in the CE-2-fed mice was approximately 8 times higher compared to that in the AIN-76-fed mice.

Effect of SE administration on the survival rate of virusinfected mice

We treated the mice with approximately 20 mg/kg/d SE (SEadministered group) or PBS (mock-treated group) and evaluated their survival rates. The body weight of the SE-administered and mock-treated mice was not significantly different (Figure 1A). Both SE-administered and mock-treated groups started dying simultaneously after influenza virus infection, but the SEadministered group had a significantly longer survival time than the mock-treated group (Figure 1B).

Effect of SE administration on the lungs of the virus-infected mice

(Figure 2) shows an image of the lungs removed from the SE-administered and mock-treated (PBS-treated) mice. The site and extent of inflammation are indicated using arrows. On days 2 and 3 post-infection (p.i.), inflammation was observed in certain places in both SE-administered and mock-treated groups, but no significant difference was observed. However, on day 4 p.i., there was increased inflammation and overall lung enlargement in the mock-treated group than in the SE-administered group.

 

Figure 2: Effect of nasal administration of SE on the lung in influenza virus-infected mice.

Lung samples were obtained from days 2, 3, and 4 p.i. A: Photo of the lungs of SE-treated mice. B: mock-treated (PBStreated) mice. C: Control (mock-infected) mice on day 4 p.i. The red arrows and circled areas show the inflammation in the lungs. Control mice were mock infected, which is the same as being PBStreated, and mock-treated (PBS-treated). A photograph of the lung used in the experiment in Figure 3 was taken.

(Figure 3A) demonstrates the weight of the lungs. There was no difference between the groups on days 2 and 3 p.i., but the lung weight of the mock-treated (PBS-treated) group significantly increased compared with that of the SE-administered group on day 4 p.i. (p<0.05).

 

Figure 3: Effect of nasal administration of SE on lung against lung weight and virus titer in influenza virus-infected mice.

A: Lung weight. Lung samples from SE-administered or mock-treated (PBS-treated) mice were obtained from days 2, 3, and 4 p.i. B: virus titer. The lungs were homogenized using a Teflon homogenizer with 5 mL of MEM. The homogenates were centrifuged, and the supernatants were used as the virus suspension of the lungs. The details are shown in the Materials and Methods. Both treatment groups used 5 mice each on days 2, 3 and 4 p.i., respectively. *: p<0.05, **: p<0.01

The virus titer per 1 g of the lung is shown in (Figure 3B). On day 3 p.i., the SE-administered group showed significantly lower viral titers than the mock-treated group (p<0.01). In addition, the virus titer of the mock-treated group increased from day 2 to day 3, whereas there was no considerable change in the SE-administered group.

The H&E staining of the lungs is shown in (Figure 4). In the lungs of influenza virus-infected mice, the infiltration of mononuclear cells and neutrophils and shedding and necrosis of bronchiolar epithelial cells, which cause apoptotic bronchopneumonia, were observed. In the mock-treated group, the severity of infiltration of mononuclear cells and neutrophils, necrosis of alveoli, and necrosis of bronchiolar epithelial cells on Day 3 was 3, 3, and 4 on average. The influx of neutrophils into the bronchiolar lumen (black arrow), bleeding into the alveoli (white arrow), and cell death due to apoptosis (red arrow) were prominent, and the alveolar structure completely collapsed. Although similar findings were observed in the SE-administered group, the severity of infiltration of mononuclear cells and neutrophils, necrosis of alveoli, and necrosis of bronchiolar epithelial cells on Day 3 was 1, 1, and 1 on average. The degree of damage to the bronchiolar epithelium and alveoli was low.