Immuno-enhancement effects of Platycodon grandiflorum extracts in splenocytes and a cyclophosphamide-induced immunosuppressed rat model
Platycodon grandiflorum is a flowering plant that is used in traditional medicine for treating pulmonary and respiratory disorders. It exerts various pharmacological effects, including immunomodulatory and anti-cancer activities. The purpose of this study was to confirm the in vitro and in vivo immune-enhancing effects of P. grandiflorum extract (PGE) on splenocytes isolated from cyclophosphamide (CP)-induced immunosuppressed rats.
For in vitro analysis, splenocytes were treated with PGE at various doses along with CP. Cell viability was measured by a WST-1 assay, and NK cell activity and cytotoxic T lymphocyte (CTL) activity was also examined. In addition, immunoglobulin A (IgA), IgG, and cytokine levels were measured. For in vivo analysis, Sprague Dawley rats were treated with various doses of PGE along with CP. Complete blood count (CBC) was performed, and plasma levels of IgA, IgG, TNF-α, IFN-γ, IL-2, and IL-12 were quantified. Additionally, tissue damage was assessed through histological analyses of the thymus and spleen.
PGE treatment enhanced cell viability and natural killer cell and cytotoxic T lymphocyte activity, and increased the production of CP-induced inflammatory cytokines (TNF-α, IFN-γ, IL-2, and IL-12) and immunoglobulins (IgG and IgA) in splenocytes. In addition, in CP-treated rats, PGE treatment induced the recovery of white blood cell, neutrophil, and lymphocyte counts, along with mid-range absolute counts, and increased the serum levels of inflammatory cytokines (TNF-α, IFN-γ, IL-2, and IL-12) and immunoglobulins (IgG and IgA). Moreover, PGE attenuated CP-induced spleen and thymic damage.
Our results confirmed that PGE exerts an immune-enhancing effect both in vitro and in vivo, suggesting that PGE may have applications as a component of immunostimulatory agents or as an ingredient in functional foods.
KeywordsPlatycodon grandiflorum Immunostimulation Cyclophosphamide Cytokine Immune cells
Complete blood count
Cytotoxic T lymphocyte
Mid-range absolute counts
P. grandiflorum extract
Platycodon grandiflorum (PG) roots have traditionally been used as a herbal medicine and food for the treatment of various pulmonary diseases and respiratory disorders in China, Japan, and Korea . PG has been found to contain numerous chemical components, including saponins, polysaccharides, flavonoids, phenolic acids, fatty acids, and sterols . In addition, PG performs various pharmacological activities that have anti-diabetic , anti-cancer , anti-inflammatory , immunomodulatory , and anti-allergic  effects.
Organs of the immune system, such as the bone marrow, thymus, lymph nodes, and spleen, play a major role in mediating the immune response and to ensure that a protective response to negate the effects of harmful stimuli is established . These highly sophisticated and complex biological responses involve macrophages, dendritic cells, and various immune cells, including lymphocytes . Lymphocytes, which comprise white blood cells, can be divided into T cells, B cells, and natural killer cells (NK). NK cells belong to the innate immune system and play a major role in defending the host from cancer cells, bacteria, and virus-infected cells . T cells and secreted cytokines are associated with adaptive or cell-mediated immune responses, whereas B cells and antibodies are key factors in the humoral immune response . Further, the imbalances in the immune system are associated with autoimmune diseases and disorders caused by inflammatory responses in various organs .
Cyclophosphamide (CP), a commonly used alkylating agent for chemotherapy, exerts myelosuppressive, immunosuppressive, and cytotoxic effects [13, 14]. Administration of CP can lead to a sudden change in Th1/Th2 bias, resulting in immunosuppression. In previous studies, the immunological effects of CP were found to decrease the proliferation of T cells along with the levels of secreted Th1 cytokines (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, interleukin [IL]-2, and IL-12) and Th2 cells (IL-4, IL-6, and IL-10) [15, 16].
In order to develop potential immune-enhancing agents that can be used as components of functional foods, increasing attention is being paid to the immunomodulatory properties of plants with a broad spectrum of therapeutic properties, as plant-based therapeutic agents are associated with relatively low toxicities [17, 18]. Although a recent report indicated that PG exerts an anti-tumor effect by improving the immune function , to our knowledge, no data are available regarding its immune-enhancing effects in a CP-induced immunosuppression model. Therefore, in the present study, we confirmed the in vitro and in vivo immune-enhancing effects of PG extract (PGE) on splenocytes isolated from immunosuppressed rats generated by CP administration.
Preparation of PGE
PG was manufactured by JIRISANDONGUICHON Co. Ltd. (Sancheong-Gun, Gyeongnam, Korea). It was formally identified by the Rural Development Administration of the Korean Government. The dried roots of PG were purchased from JIRISANDONGUICHON, and a voucher specimen (no. 180426) was deposited at INVIVO Co. Ltd., Iksan, Korea. The extract was prepared as follows: briefly, the dried roots (100 g) were extracted in an electric boiling pot for 3 h with 1000 mL of distilled water at 100 °C, and filtered using a 0.45-μm syringe filter. The extract solution was evaporated under 40 mmHg using a rotary evaporator (N-1110S-W; Eyela, Tokyo, Japan), and was then used for each experiment.
The main components present in PGE were analyzed using an HPLC system (Shimadzu LC-30 Series, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) with an SHISEIDO Capcell Pak C18 column (3.0 mm i.d. × 50 mm, 3 μm). Signals were detected using a MS system (Shimadzu LCMS-8030, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). The levels of Platycodin D in PGE were quantitated using a calibration curve established by injecting dilutions of each Platycodin D standard (1–10 μg/mL) into the HPLC system (correlation coefficient ≥ 0.996). Standards (Platycodin D) for HPLC analysis were obtained from Sigma-Aldrich (St Louis, MO, USA).
Animal experiments were performed as previously described . Sprague Dawley (SD) rats were purchased from Samtaco Inc. (Osan, Gyeonggi-do, Korea), and acclimated to the following environmental conditions for 7 days: 12-h light/12-h dark cycle; temperature, 23 ± 1 °C; humidity, 50 ± 5%; and illumination, 150–300 lx. Rats were randomly assigned to five groups (10 rats per group). The protocols used for these animal studies were approved by the Committee on Care and Use of Laboratory Animals of Wonkwang University (Iksan, Jeollabuk-do, Korea; approval no. WKU18–17).
Cells were cultured as previously described . Briefly, the spleen of an 8-week-old SD rat was aseptically dissected to obtain splenocytes. The spleen was gently pressed with forceps and then forced through a 70-μm cell strainer (SPL Life Sciences, Pocheon-si, Gyeonggi-do, Korea). Cells were treated with red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, U.S.A.). Isolated splenocytes were incubated in RPMI-1640 containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin, streptomycin) (Invitrogen) in a 5% CO2 incubator.
Cell viability were performed as previously described , using a WST-1 Assay Kit (ITSBio, Seoul, Gyeonggi-do, Korea) according to the manufacturer’s instructions. Briefly, splenocytes (2 × 105 cells/well) were seeded into 96-well plates and then treated with PGE at various doses along with CP (1600 μg/mL), and incubated for 24 h in an incubator with 5% CO2 atmosphere. Cell viability was assessed using a WST-1 Assay Kit and a microplate plate reader (Tecan, Männedorf, Switzerland).
NK cell activity assay
NK cell activity was measured as previously described . Briefly, AR42J rat pancreas tumor cells obtained from the American Type Culture Collection (ATCC, Manassas, Va., U.S.A.) AR42J cells were used as target cells for the NK cell activity assay, and splenocytes were used as effector cells. Splenocytes were co-cultured with AR42J cells in 96-well plates at a 25:1 ratio of effector cells to target cells. The viability of AR42J cells was assessed using a CytoTox detection kit (TaKaRa, Shiga, Japan) and a microplate plate reader. NK cell activity was calculated as the viability of AR42J cells compared to control cells.
Cytotoxic T lymphocyte (CTL) activity assay
CTL activity was determined as previously described . Briefly, CTL activity was analyzed using the CytoTox detection kit (TaKaRa) according to the manufacturer’s protocol. Tumor cells (HL-60) obtained from ATCC were used as target cells for the CTL assay, and an effector cell to target cell ratio of 25:1 was used. CTL activity was calculated as the survival rate of HL-60 cells compared to that of control cells.
Measurement of immunoglobulin a (IgA), IgG, and cytokine levels in splenocytes
Immunoglobulin (Ig) and cytokine levels were measured as previously described . Briefly, splenocytes (2 × 105 cells/well) were seeded into 96-well plates, after which cells were treated with various concentrations of PGE (0, 10, 30, 50, 100, 300, or 500 μg/mL) and CP (1600 μg/mL), and incubated for 24 h. The levels of IgA and IgG were measured using an immunoglobulin ELISA kit (AFFymetrix, Santa Clara, Ca., U.S.A and Abcam, Cambridge, U.K.) according to the manufacturer’s instructions. The levels of TNF-α, IFN-γ, IL-2, and IL-12 in the culture medium measured using Cytokine Activation Analysis Kits (R&D Systems, Minneapolis, MN, U.S.A.), according to the manufacturer’s instructions. The immunoglobulin and cytokine levels were measured using an ELISA reader.
Complete blood count (CBC) and IgA, IgG, and cytokine analyses
These analyses were conducted as previously described . Briefly, SD rats were orally administered PGE (0, 10, 30, or 100 mg/kg/day) and CP (5 mg/kg, once per day) for 28 days. After the final drug administration, rats were weighed and anesthetized with 5% isoflurane. Whole blood was collected. Next, the rats were euthanized by exsanguination. The numbers of white blood cells (WBCs), lymphocytes, neutrophils, and mid-range absolute counts (MID) were measured using a Hemavet 950 system (Drew Scientific Group, Dallas, Tx., U.S.A.). The levels of IgA, IgG, TNF-α, IFN-γ, IL-2, and IL-12 in the plasma were quantified using ELISA kits, according to the manufacturer’s instructions.
Thymus and spleen histochemical analyses
Histochemical analyses of the thymus and spleen were performed as previously described . Thymus and spleen tissues were fixed in 10% neutral buffered formalin. The organs were then processed for dehydrating with alcohol and embedding in paraffin. Samples were cut to 4-μm-thick section and stained with hematoxylin and eosin. Tissue damage was evaluated under a microscope.
All experimental data are presented as mean ± standard error (SE), calculated using the statistical program SPSS version 12.0 (SPSS Inc., Chicago, IL, U.S.A). Results were analyzed by a one-way analysis of variance (ANOVA) and Duncan’s multiple range tests using SAS software (version 9.3; SAS Institute Inc., Cary, NC., U.S.A.). P-values < 0.05 were considered statistically significant.
HPLC analysis of PGE
Effect of PGE on CP-induced splenocyte viability
Effect of PGE on cytokine expression in CP-treated splenocytes
Effects of PGE on IgG and IgA levels in CP-treated splenocytes
Effects of PGE on NK and CTL cytotoxicity in splenocytes
Effect of PGE on immune cell number in rats
Effect of PGE on serum levels of immune-related cytokines in CP-induced immunosuppressed rats
Effects of PGE on serum levels of IgG and IgA in CP-induced immunosuppressed rats
Effect of PGE on spleen and thymus damage in CP-induced immunosuppressed rats
The study of immune-enhancing effects of natural products represents an active area of current research [18, 25, 26]. Previous studies have reported immune enhancement following administration of PG [27, 28]. However, these reports have only determined the effect of PG on immune enhancement of lymphocytes and macrophages. In this study, we first showed that PG could revert the immunosuppression caused by CP. Specifically, PGE-treatment alleviated CP-induced reduction in cell proliferation, cytokine and immunoglobulin levels, and NK cell and CTL activity in splenocytes. Additionally, PGE increased immune cell numbers and serum cytokines and immunoglobulin levels, and stimulated the recovery of the spleen and thymus in immunosuppressed rats. These findings indicate that PGE improves the weakened immune response brought about by CP treatment.
CP is an important chemotherapeutic drug in tumor therapy, but it is not beneficial for healthy cells and side effects such as bone marrow suppression, immunosuppression, oxidative stress, and occasionally life-threatening effects, are often observed . In previous studies, CP-treated rats have been used as immunosuppression models in animals. CP-induced immunosuppression significantly reduces red blood cell, WBC, and platelet counts, and additionally inhibits spleen NK cell and CTL activity, as well as decreasing CD4 T lymphocyte count and the CD4/CD8 ratio [30, 31]. In addition, the levels of cytokines (IL-2, IFN, and IL-10) and immunoglobulins (IgA, IgM, and IgG), along with serum hemoglobin, were reduced by CP . Moreover, administration of CP has been shown to cause damage to the spleen and thymus, which are important organs in the immune response . Thus, we investigated the immune-enhancing effects of PGE under conditions of CP-induced immunosuppression in this study.
Proliferation of macrophages and lymphocytes is important in the early activation stage of cellular and humoral immune responses. Splenocytes are composed of various cells, such as T and B cells, macrophages, and dendritic cells with different immune functions . Stimulation of splenocytes proliferation ultimately increases the expression of cytokines, potentially improving cell-mediated immune responses and leading to immune-enhancement . We found that PGE reversed the effect of reduced immune cell viability caused by CP (Fig. 2b and Fig. 6).
Cytokines expressed by various immune cells play an important role in lymphocyte differentiation, inflammation control, host defenses against bacterial infection, cell survival, apoptosis, and the immune response [36, 37]. Th cells activated after antigen recognition are classified into Th1 and Th2 cells based on their function and differences in cytokines secreted [38, 39] . The different types of cytokines secreted by Th1 and Th2 cells are important factors in determining cell function. Th1 cells secrete IL-2, TNF-α, and IFN-γ, which are involved in cell-mediated immune responses [40, 41], whereas Th2 cells mainly secrete IL-4, IL-6, and IL-10 through the humoral immune response . In particular, TNF-α is produced by T and B cells, NK cells, and macrophages, and regulates inflammation and the host defense through inhibiting bacterial infections and suppressing acute stress [43, 44]. IFN-γ and IL-2 are produced by T-helper cells . IFN-γ is a natural immune mediator that promotes the expression of major histocompatibility complex molecules through activation of monocytes/macrophages . IL-2, produced by activated T lymphocytes, induces growth, proliferation, and differentiation of immature T cells into effector T cells . Finally, IL-12 is known to be produced by a variety of immune cells, including dendritic cells, macrophages, neutrophils, and human B-lymphocytes. It is responsible for differentiating native T lymphocytes into Th1 lymphocytes , and promotes the production of TNF-α and IFN-γ in T lymphocytes and NK cells, respectively [49, 50]. Therefore, we confirmed the effect of PGE on the expression of Th1 cytokines, and hypothesized that PGE would promote the Th1 immune response under conditions of CP-induced immunosuppression (Fig. 3 and Fig. 7).
IgG, IgA, and IgM comprise major immunoglobulins that play a role in the neutralization of toxins, bacteria, or viruses, along with opsonization and complement activation . Recently, numerous studies have reported that natural products can enhance the humoral immune response by promoting the production of IgA, IgM, and IgG . Our results demonstrate that PGE can increase levels of IgG and IgA in CP-immunosuppressed rats. This result suggests that PGE plays a role in enhancing the humoral immune response.
NK cells and CTLs comprise two major populations that can target and kill foreign and abnormal cells. NK cells and CTLs play an important role in the early immune response . NK cells and CTLs are activated by stimulation of cytokines and chemokines and play a central role in the regulation of tumor growth and metastasis, as well as the removal of viruses . Thus, measurement of NK cell and CTL activity represents a useful method for assessing the cellular immune response of the host . Our results showed that spleen NK cell cytotoxic activity was significantly increased after treatment with PGE (Fig. 5). Additionally, we assessed the NK cell and CTL activity of Red ginseng extract (RGE) as a positive control, and found that, although the RGE extracts were highly cytotoxic, NK cell activity was greater in the RGE group than in the PGE group at concentrations below 50 μg/mL. However, CTL activity was higher in the PGE group compared to the RGE group, and in particular, PGE had a pronounced effect on CTL activity, which was higher than for the RGE group (Additional file 1: Figure S1). These results suggest that PGE enhances the cell-mediated immune response through modulating NK cell activity.
Immunosuppression is a condition in which the immune system is not sufficiently activated to defend the host from infections. The immune system can be weakened by various factors, such as birth and aging, which may lead to shrinkage of immune tissues and a decrease in the number of WBCs . Mature blood cells such as WBCs, red blood cells, and platelets are produced by hematopoietic stem cells, which are multipotent and self-renewable . We found that, although CP treatment decreased the WBC, lymphocyte, MID, and neutrophil populations, consistent with prior reports , these cell counts were significantly restored by PGE administration in a dose-dependent manner. These results suggest that PGE enhances immunity by protecting against CP-induced myelosuppression.
In addition, we confirmed the effect of PGE on CP-induced immune organ damage. Histopathological examination of the spleen and thymus of immunosuppressed rats (CP-treated) revealed CP-induced damage. Notably, PGE-treatment significantly reduced CP-induced pathological changes in the spleen and thymus (Fig. 9), suggesting that PGE may in fact reverse the CP-induced atrophy in immune organs.
In summary, we demonstrated the immune-enhancing effects of PGE in CP-treated splenocytes using an immunosuppressed rat model. Our results confirmed that PGE stimulates innate and adaptive immune responses under immunosuppressive conditions, and increases cytokine, antibody, and immune cell production and NK cell and CTL activity, thereby enhancing the immune response and host defense. These findings suggest that PG may have applications in the development of functional foods and medicines that can be used for immune-enhancement.
YRL and HYL designed the study. EMN, JMK and HKS performed the experiments. MJK, HJY and KSK analyzed the data. S. Jung provided the roots of Platycodon grandiflorum and provided comments and editorial review of the manuscript. YRL drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by “Food Functionality Evaluation program” under the Ministry of Agriculture (no. 31607203), Food and Rural Affairs and National Research Council of Science & Technology (NST) grant by the Korea Government (MSIP) (no. CAP-16-07-KIOM) and Korea Food Research Institute (E015302–05). The funding bodies had no involvement in the concept, design, analysis or writing the manuscript.
Ethics approval and consent to participate
This animal study was approved by the Committee on Care and Use of Laboratory Animals of Wonkwang University.
Consent for publication
The authors declare that they have no competing of interests.
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