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Canadian Journal of Veterinary Research logoLink to Canadian Journal of Veterinary Research
. 2020 Oct;84(4):294–301.

Effect of Achyranthes japonica Nakai extract on immunity and anti-inflammation in dogs

Gun-Hwi Lee 1, Kyung-A Hwang 1, Ji-Houn Kang 1, Kyung-Chul Choi 1,✉
PMCID: PMC7491004  PMID: 33012978

Abstract

Achyranthes japonica Nakai (A. japonica) is a medicinal herb found widely distributed throughout Korea. The biological activities of A. japonica are well-documented and include anti-fungal, anti-inflammatory, and immunity enhancement. The objective of the present study was to investigate the immune-related activities of A. japonica extract in dogs. The extract was acquired by ethanol extraction and purified by filtration. To examine the effect of A. japonica extract on immune cell viability, human lymphocytes, such as Jurkat T-cells and Ramos B-cells, were exposed to the extract. After treatment with the extract, the number of Ramos B-cells was increased, whereas Jurkat T-cells remained unaffected. Griess assay revealed decreased nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated mouse macrophage Raw 264.7 cells after exposure to A. japonica extract. To evaluate the in-vivo effect in dogs, feed containing A. japonica extract was provided to 8 dogs for 2 months. Blood samples were collected before, during, and after consumption of the feed. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples and the number of T-cells and B-cells were assessed using flow cytometry with anti-dog fluorescein isothiocyanate (FITC)-conjugated CD3 and anti-dog phycoerythrin (PE)-conjugated CD21 antibodies, respectively. We observed a significant increase in the average number of B-cells in the PBMCs during ingestion of the feed containing A. japonica. In addition, enzyme-linked immunosorbent assay (ELISA) revealed a decrease in the levels of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, in 3 out of 8 dogs and increased levels of interleukin-10 (IL-10), an anti-inflammatory cytokine, in 4 out of 8 dogs. Taken together, we believe that these changes indicate that A. japonica extract is beneficial in improving the immunity of dogs by stimulating B-cells and inducing production of anti-inflammatory responses.

Introduction

The canine immune system is a complex network consisting of specialized cells and organs designed to defend the canine body against bacteria, viruses, toxins, parasites, and any other foreign materials. Many different types of immune cells exchange information with each other, resulting in a protective system that is always ready to produce fast and effective immune responses. Immune cells originate from the lymphatic or bone marrow system (1). Lymphocytes are the main type of immune cells found in the lymph and include T-cells, B-cells, and natural killer cells (2–4).

T-cells are processed by the thymus gland and play a central role in cellular immunity (5,6). They exert their cytotoxic effect and help destroy tumor cells or virus-infected cells. Furthermore, they help the B-cells to generate antibodies and induce macrophages to develop microbicidal activity and recruit neutrophils, eosinophils, and basophils to the site of infection and inflammation. In addition, T-cells produce the cytokines and chemokines that orchestrate the immune responses; these are proteins synthesized by immune cells and aid in regulating the immune system (7,8).

Cytokines are proteins that have an effect on the cells and are important in cell signaling, as well as participating in autocrine signaling, paracrine signaling, and endocrine signaling as immune-modulating agents. Cytokines are produced by various cells, such as macrophages, B-cells, T-cells, and mast cells (9,10), and are involved in both anti-inflammatory and pro-inflammatory pathways. Proinflammatory cytokines are signaling molecules excreted by immune cells that promote inflammation and include interleukin-1 (IL-1), IL-12, IL-18, tumor necrosis factor (TNF), and interferon gamma (IFN-γ). The anti-inflammatory cytokines are immunoregulatory proteins that regulate the pro-inflammatory cytokines and include IL-4, IL-6, IL-10, IL-11, and IL-13 (11–13).

B-cells mature in the bone marrow of animals and play an essential role in humoral immunity (14,15). B-cells are responsible for generating antibodies, which are essentially proteins used to fight infections and foreign materials, and are also required to initiate the T-cell immune responses. In homeostasis and maintenance of the immune system, B-cells release immunomodulatory cytokines to regulate the lymphoid tissue organization, necrosis, and wound healing (16,17).

Achyranthes japonica Nakai (A. japonica) is a medicinal herb found widely distributed throughout Korea. It has been used in traditional medicine to treat edema and arthritis. The seed and roots contain several chemical constituents: the seed contains ecdysterone, inokosterone, and rubrosterone and the root contains triterpenoids and saponins (18–20). It is reported that A. japonica has various pharmacological effects, including anti-inflammatory, anti-oxidative, and anti-fungal activity (21,22).

In this study, we investigated the effects of prescription diets containing the extract of A. japonica on the enhancement of immunity and anti-inflammation in canines. We evaluated the number of lymphocytes (T-cells and B-cells) and cytokine levels (TNF-α and IL-10) in the blood of dogs that had consumed feed containing A. japonica for 2 mo. We found that the extract of A. japonica enhances canine immunity and we therefore suggest that the extract be included as a functional component in dog feed.

Materials and methods

Achyranthes japonica extract

Achyranthes japonica extract was provided by Regeniks (Seoul, Korea) for the in-vitro experiments. Briefly, the A. japonica extract was manufactured by immersing the leaves and roots of A. japonica in 100% ethanol for 1 to 2 d. The resultant extract was then filtered and lyophilized.

Cell culture

The human Jurkat T-cell line, human Ramos B-cell line, and the murine macrophage Raw 264.7 cell line were obtained from the Korean Cell Line Bank (KCLB) (Seoul, Korea). Jurkat T-cells and Ramos B-cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% solution of 10 U/mL penicillin and 100 μg/mL streptomycin, and 1% 10 mM HEPES. The Raw cell line was maintained in Dulbecco’s Modified Eagle medium (DMEM; HyClone Laboratories, Logan, Utah, USA) supplemented with 10% FBS, 1% solution of 10 U/mL penicillin and 100 μg/mL streptomycin, and 1% 10 mM HEPES.

WST assay

The human Jurkat T-cells and Ramos B-cells were seeded at a density of 5 × 105 cells/well in 96-well plates (SPL Life Science, Seoul, Korea) and incubated in a humidified 5% carbon dioxide (CO2) atmosphere at 37°C. After incubation for 24 h, cells were treated with varying concentrations of A. japonica extract (5 to 500 μg/mL prepared in medium) for 24 h. The cell viability was assessed by adding 20 μL of EZ-Cytox (Daeil Lab, Seoul, Korea) to each well and incubating at 37°C for 1 h. Cell viability was subsequently measured at 450 nm using a Microplate Reader (BioTek Instruments, Winooski, Vermont, USA).

Nitric oxide assay

Raw 264.7 cells were seeded at a density of 5 × 105 cells/well in 96-well plates and incubated in a humidified 5% CO2 atmosphere at 37°C for 24 h. Raw 264.7 cells were stimulated by 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, Missouri, USA) for 24 h, after which they were treated with varying concentrations of A. japonica extract (5, 50, 500 μg/mL). After incubation for 24 h, 50 μL of supernatant from each well was transferred to a new 96-well plate and 100 μL Griess solution (Promega, Madison, Wisconsin, USA) was then added to each well containing the supernatant. The nitric oxide (NO) concentration was measured at 520 nm using a Microplate Reader (BioTek Instruments). The NO concentration of each well was calculated using a nitric oxide standard reference curve. After removal of supernatant for the NO assay, EZ-Cytox solution (Daeil Lab) was added to each well containing the treated Raw 264.7 cells and incubated for 1 h at 37°C; the viability of Raw 264.7 macrophages was measured at 450 nm using a Microplate Reader (BioTek Instruments).

Animals

A total of 8 dogs, aged 1 to 8 y (4 male dogs: 3 Pomeranians and 1 poodle; 4 female dogs: 2 mixed breeds, 1 Shih Tzu, and 1 poodle) were enrolled to participate in this study after consent was obtained from their owners. All 8 dogs were housed at the Yuseong Bioscience Technology High School during the 8-week test period. The dogs were fed 90 to 100 g of the feed containing 5% A. japonica extract daily for 2 mo. In addition to the A. japonica extract, the feed contained crude protein, fat, fiber, ash, calcium, and phosphorous. The feed was manufactured and provided by Jeilfeed (Daejeon, Korea).

Evaluation of animal weight and fecal index average

The dogs were weighed a week before feeding and a week after feeding. The feces were collected and the fecal index was divided into 5 grades: grades 1 to 3 indicating diarrhea, grade 4 representing the ideal fecal state, and grade 5 indicating constipation.

Isolation of peripheral blood mononuclear cells (PBMCs)

Venous blood was collected from dogs before, during, and after feeding. The blood samples were diluted with balanced salt solution and centrifuged over Ficoll-Hypaque (GE Healthcare, Chicago, Illinois, USA) at 400 × g for 30 to 40 min, resulting in fractionation of the blood into 4 layers. The first layer was plasma and the second layer consisted of peripheral blood mononuclear cells (PBMCs). First, the plasma was transferred to a 1.5-mL tube and PBMCs were then collected using a Pasteur pipette and transferred to a 15-mL tube. Isolated PBMCs were washed 3 times and resuspended in RPMI-1640 media supplemented with 10% FBS, 1% penicillin-streptomycin solution, and 1% HEPES. The isolated PBMCs were categorized as group A (before feeding), group B (during feeding), and group C (after feeding), in accordance with the blood from which each sample was isolated. The isolated PBMCs were subsequently resuspended in freezing solution and stored in liquid nitrogen until further use.

Analysis of T- and B-lymphocyte populations in PBMCs using fluorescence-activated cell sorting (FACS)

Mouse anti-dog CD3:fluorescein isothiocyanate (FITC) antibodies (BioRad Laboratories, Hercules, California, USA) and CD21 monoclonal antibody:phycoerythrin (PE) antibodies (Thermo Scientific, Rockford, Illinois, USA) were applied to select T- and B-cells in PBMCs, respectively. The frozen PBMCs were thawed, washed, and resuspended in RPMI-1640 medium, seeded in 24 well-plate (SPL Life Science), and incubated for 4 h at 37°C in a humidified atmosphere of 5% CO2. The supernatants were collected in 15-mL tubes and centrifuged at 400 × g for 10 min. The resultant pellets were collected and resuspended in EasySep buffer (Stemcell Technologies, Vancouver, British Columbia). To assess the T- and B-cell populations in pellets, 50 μL of the suspended solution was dispended in 5-mL tubes and stained with anti-dog CD3:FITC or CD21 monoclonal antibody:PE-conjugated, for 30 min at 4°C. After adding 200 μL of EasySep buffer, the populations of T- and B-cells were analyzed by flow cytometry (SH-800; Sony Biotechnology, Japan).

Measurement of plasma TNF-α and IL-10 concentration

The plasma levels of TNF-α and IL-10 were assayed by enzyme-linked immunosorbent assay (ELISA) kits. The plasma were acquired when PBMCs were isolated from blood; 50 μL of plasma sample was seeded in each well, coated with primary antibody, and incubated for 15 min at 37°C. Secondary antibody solution conjugated with the enzyme (100 μL) was added to each well and incubated for 2 h at room temperature, followed by adding 100 μL of the substrate solution and incubating for 30 min at room temperature. The enzyme reaction was terminated by adding a stop solution. Absorbance was read at 450 nm and cytokine concentration (in pg/mL) was determined according to a standard curve prepared with the recombinant canine IL-10 and TNF-α.

Statistical analysis

Experiments were repeated 3 times and all data were statistically analyzed by GraphPad Prism (Version 5.01; GraphPad Software, San Diego, California, USA). Data are expressed as mean ± standard deviation (SD) and analyzed using 1-way analysis of variance (ANOVA), followed by post-hoc Dunnett’s multiple comparison test and Bonferroni’s multiple comparison test. A statistically significant difference was noted with an asterisk when the P-value was less than 0.05.

Results

Effect of A. japonica extract on immune cell viability

The effect of A. japonica extract on the viability of the human immune cells (Jurkat T-cells and Ramos B-cells) was measured by WST assay. No change in viability was observed after exposure of Jurkat T-cells to A. japonica extract (Figure 1A). An increase was detected in Ramos B-cell viability, however, after A. japonica extract treatments (5 to 500 μg/mL). The cell viability of B-cells was highest after exposure to the intermediate concentrations of A. japonica extract (10 and 50 μg/mL), as presented in Figure 1B.

Figure 1.

Figure 1

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Effect of A. japonica extract on the viability of human lymphocytes. A — Jurkat T-cells and B — Ramos B-cells were exposed to varying concentrations of A. japonica extract (5 to 500 μg/mL). After 24 h, the cell viability was measured by WST assay. The values are means ± SD. *P < 0.05 compared to control.

Effect of A. japonica extract on nitric oxide production by Raw 264.7 cells

To confirm the anti-inflammatory effect of A. japonica extract, an NO assay was conducted using mouse macrophage Raw 264.7 cells. Nitric oxide was produced by activated Raw 264.7 cells stimulated by lipopolysaccharide (LPS). Exposure of Raw 264.7 cells to the A. japonica extract, however, resulted in a dose-dependent decrease in NO production (Figure 2B). Furthermore, no change in viability was observed in the Raw 264.7 cells treated with A. japonica extract, as assessed in the WST assay conducted in parallel with the NO assay (Figure 2A). This indicates that the decreased production of NO in Raw 264.7 cells was not due to a cytotoxic effect of A. japonica extract on Raw 264.7 cells.

Figure 2.

Figure 2

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Effect of A. japonica extract on nitric oxide (NO) production in Raw 264.7 cells. Raw 264.7 cells were stimulated by 1 μg/mL of lipopolysaccharide (LPS) in the absence or presence of A. japonica (AJ) extract. After 24 h, (A) the cell viability was measured by WST assay and (B) the NO concentration was measured using Griess assay. The values are means ± SD. *P < 0.05 compared to control. #P < 0.05 compared to single treatment of LPS.

Effects of the feed containing A. japonica extract on weight and fecal index of dogs

Eight dogs aged 1 to 8 y old were enrolled to participate in this study after consent was obtained from their owners. The average body weight of the dogs was measured before and after consumption of the feed containing A. japonica extract. Most body weights remained unchanged: 3 dogs gained roughly 0.5 kg, but 1 dog lost 0.5 kg. The average fecal index of all 8 dogs was determined to be 4, which indicates a normal state (Table I).

Table I.

Demographics and effects of feed containing A. japonica on the body weight and fecal index of dogs.

Number Dog breeds Gender Age (y) Average fecal index Weight (kg) Remark
Before After
1 Pomeranian Male 5 4 to 5 2.7 2.7
2 Pomeranian Male 7 4 2.4 2.5
3 Pomeranian + Spitz Female — 4 5.9 5.4 −0.5 kg
4 Pomeranian Male 7 4 3.5 3.5
5 Mixed-breed dog Female 1 4 2.9 3.4 +0.5 kg
6 Poodle Male 4 4 3.4 3.7 +0.5 kg
7 Shih tzu Female 8 4 3.8 4.4 +0.5 kg
8 Poodle Female 4 4 2.8 3
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Percentage of T-cells and B-cells in canine PBMCs

To evaluate the effect of the feed containing A. japonica on the percentage of canine immune cells in the blood, flow cytometry analysis was conducted for the PBMCs isolated from the dogs before, during, and after consumption of the feed. To selectively assess B-cells and T-cells as distinguished from other immune cells, B-cells were stained with CD21+-PE and T-cells were stained with CD3+-FITC. The fluorescence-activated cell sorting (FACS) images obtained are shown in Figure 3A, where Y-axis represents the CD21+-PE B-cells and X-axis represents the CD3+-FITC T-cells. The quantitative percentage of B-cells and T-cells in the PBMCs of each dog is provided in Table II. To identify the tendency of T-cell and B-cell variations during the test period, we measured the average value for each cell number in PBMCs of all 8 dogs (Figure 3B). A decrease in the number of T-cells in the PBMCs was observed during and after consumption of the feed compared to the number before consumption, although there was no statistical significance. Conversely, it was observed that the number of B-cells in the PBMCs was highest during consumption of the feed. These results indicate that the feed containing A. japonica extract increased the proportion of B-cells in PBMCs, especially during the period of feed consumption.

Figure 3.

Figure 3

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Flow cytometry analysis of CD3+ (T-cells) and CD21+ (B-cells) in the PBMCs. The peripheral blood mononuclear cells (PBMCs) were obtained from the 8 treated dogs before, during, and after consumption of the feed containing A. japonica extract and cultured in 24-well plates containing RPMI-1640. T-cells and B-cells in PBMCs were analyzed using flow cytometry with anti-dog FITC-conjugated CD3+ and anti-dog PE-conjugated CD21+ antibodies, respectively. A — Fluorescence-activated cell sorting (FACS) images for CD3+ (T-cells) and CD21+ (B-cells) in the PBMCs of 8 dogs before (left), during (center), and after (right) feeding. B — The average number of T-cells (left panel) and B-cells (right panel) of the 8 dogs before, during, and after feeding was quantified and is represented as a graph. The values are means ± SD. *P < 0.05 compared to before feeding.

Table II.

Percentage of B-cells and T-cells in the blood of each dog consuming the food containing A. japonica.

Dog number B-cell (%) T-cell (%)
Before During After Before During After
1 0.65 17.18 4.75 81.31 61.73 72.89
2 0.74 15.57 5.36 68.12 70.76 58.89
3 0.32 4.13 0.42 84.96 72.47 82.21
4 1.17 6.79 5.47 67.76 67.91 54.89
5 1.34 7.92 1.23 81.34 56.71 74.87
6 2.13 11.99 0.96 91.69 69.21 70.51
7 0.5 1.38 0.09 91.38 85.19 90.48
8 1.76 5.12 3.41 76.96 69.24 81.41
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Plasma concentrations of cytokines TNF-α and IL-10

The plasma cytokine levels of TNF-α and IL-10 were assessed by ELISA. The plasma level of TNF-α (a pro-inflammatory cytokine) was decreased after feed consumption in 3 dogs (Dogs 4, 5, and 7), during feed consumption in 1 dog (Dog 3), and remained constant throughout the test period in 3 dogs (Dog 2, 6, and 8). The plasma level of TNF-α was increased in only 1 dog (Dog 1) during and after feed consumption (Figure 4A). The IL-10 plasma levels (an anti-inflammatory cytokine) were increased after feed consumption in 3 dogs (Dogs 4, 5, and 8), during feed consumption in 1 dog (Dog 6), but remained constant during the test period in 4 dogs (Dogs 1, 2, 3, and 7) (Figure 4B). Specifically, Dogs 5 and 8 showed a remarkable increase in plasma IL-10 level after feed consumption. Although there was a deviation among the dogs, it is likely that the feed containing A. japonica extract decreases inflammation in canines by regulating the production of inflammation-related cytokines.

Figure 4.

Figure 4

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Measurement of TNF-α and IL-10 levels in the canine plasma. The plasma levels of (A) TNF-α and (B) IL-10 in the serum samples obtained from the 8 dogs before, during, and after consumption of the feed containing A. japonica extract were measured by ELISA assay. The values are means ± SD. *P < 0.05 compared to before feeding.

Discussion

Achyranthes japonica is reported to contain diverse bioactive compounds such as saponins, caffeic acid, polysaccharide, ecdysterone, inokosterone, and rubrosterone. These chemicals exert pharmacological effects, such as anti-inflammatory and anti-arthritic outcomes. The extract of A. japonica has been reported to reduce collagen-induced arthritis in mice and inhibit LPS-induced nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) expression in mouse macrophages (23–25). Until now, most of the in-vivo effects of A. japonica ingredients have been identified in animal experiments using a rat or mouse model. The present study is the first to focus on the immune-related effects of A. japonica extract in a canine model.

We first identified and evaluated the in-vitro effect of A. japonica extract on human and mouse immune cells. In human lymphocytes, the A. japonica extract increases proliferation of B-cells, but not of T-cells. B-cells are known to play a crucial role in humoral immunity by regulating production of antibodies (26) and it has recently been reported that B-cells produce cytokines, chemokines, and growth factors (27–30), especially the pro-inflammatory cytokines IL-6 and TNF-α and anti-inflammatory cytokine IL-10 (31–33). Furthermore, A. japonica extract did not affect cell viability of mouse macrophage Raw 264.7 cells, but decreased the LPS-induced NO production in a dose-dependent manner. Nitric oxide (NO) plays a crucial role in modulating inflammatory responses by regulating nuclear factor-kappaB (NF-κB) or by acting as a mediator of pro-inflammatory cytokines such as IL-1, IL-12, and TNF-α (34,35). These results therefore imply that A. japonica extract supports humoral immunity and helps to reduce inflammation by regulating B-cell proliferation and NO production in macrophages.

In the present study, the immunity-related effects of A. japonica extract were further investigated in dogs. In the in-vivo model for 8 dogs that ingested the feed containing A. japonica extract for 2 mo, no physiological indexes such as weight and fecal index were affected. There was, however, a substantial increase in B-cell numbers, but not of T-cells, in the PBMCs of the dogs, which is similar to the results obtained in the in-vitro evaluations for human lymphocytes. In addition, in dogs that ingested the feed containing A. japonica extract, the plasma level of TNF-α was found to be mostly decreased after feeding. Conversely, the level of IL-10 was found to be significantly increased in some dogs after feeding. Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine involved in systemic inflammation by stimulating inducible NO synthase (iNOS) expression in immune cells (36,37). Interleuken-10 (IL-10), a human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine that blocks the NF-κB activity and inhibits the LPS-mediated induction of pro-inflammatory cytokines such as TNF-α and IL-1. Therefore, IL-10 inhibits the inflammation pathway and production of pro-inflammatory cytokines (38–40).

Although the effect of the feed on inflammation in dogs was assessed for only 2 kinds of cytokines, we believe that this indicates that the A. japonica extract may be beneficial in reducing inflammation in dogs. Achyranthes japonica extract also appears to enhance the immunity of dogs by activating the B-cells in blood. There is difference in the number of B-cells depending on sex and age. To compare by sex, the B-cells of the female dogs were increased more than those of the male dogs of the same age. Also compared by age, the B-cells of the young male dogs were increased more than those of older male dogs and, in the same manner, the B-cells of the young female dogs were increased more than the older female dogs. This study therefore confirmed that the young female dogs were influenced by A. japonica in their feed.

This study had a few limitations. The number of dogs that participated in the study was small and dogs enrolled were of different breeds, sex, and age. Furthermore, they were not confined to the same conditions during the experimental period, since each dog was being raised by its owner in different environments. It is therefore necessary to maintain consistent experimental conditions to achieve reliable results. In addition, the immune enhancement effect of A. japonica extract needs to be confirmed in dogs afflicted with immune-related diseases, including arthritis.

In conclusion, and despite these limitations, the results of both in-vitro and in-vivo experiments in this study demonstrate that A. japonica extract possesses immune enhancement and anti-inflammatory properties and may therefore be a useful additive in dog feeds for strengthening the canine immune system.

Acknowledgments

This work was supported by a grant from the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (no. 317021-03-1-CG000), Republic of Korea. Support was also provided by the Global Research and Development Center (GRDC) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017K1A4A3014959).

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