SBT (Composed of Panax ginseng and Aconitum carmichaeli) and Stigmasterol Enhances Nitric Oxide Production and Exerts Curative Properties as a Potential Anti-Oxidant and Immunity-Enhancing Agent

Immune dysregulation is a risk factor for several diseases, including infectious diseases. Immunostimulatory agents have been used for the treatment of immune dysregulation, but deleterious adverse effects have been reported. The present study aims to establish the anti-oxidant and immunity-enhancing effects of Sambu-Tang (SBT), composed of Panax ginseng and Aconitum carmichaeli, and stigmasterol (Stig), an active compound of SBT. Immune-related factors were analyzed in RAW264.7 macrophage cells, mouse primary splenocytes, and the serum and spleen of cyclophosphamide-induced immunosuppressed mice. Results showed that the production levels of nitric oxide (NO) and expression levels of inducible NO synthase and heme oxygenase-1 were increased following SBT or Stig treatment in RAW264.7 cells. SBT or Stig increased the production levels of G-CSF, IFN-γ, IL-12, IL-2, IL-6, and TNF-α and induced the activation of NF-κB in RAW264.7 cells. SBT or Stig promoted splenic lymphocyte proliferation and increased splenic NK cell cytotoxic activity. In addition, SBT or Stig enhanced the levels of IFN-γ, IL-12, IL-2, IL-6, or TNF-α in the serum and spleen of the immunosuppressed mice. SBT or Stig increased the superoxide dismutase activity in the spleen. Collectively, SBT and Stig possess anti-oxidant and immunomodulatory activities, so they may be considered effective natural compounds for the treatment of various symptoms caused by immune dysregulation.


Introduction
The immune system plays a critical role in controlling infections, healing injuries, and restoring homeostasis [1]. The anti-oxidant defense systems are important for the normal function of the immune system [2]. Immune dysregulation is a risk factor for several diseases, including autoimmune disease and infectious disease [3,4]. Patients with immunodeficiency disorders usually have increased frequency, severity, or persistence of infections [5]. Immunostimulatory agents have generally been used for the treatment of immune dysregulation, as well as infections or cancers [6,7]. Anti-oxidants improved immune function and decreased mortality induced by infections, such as coronavirus disease [2,8].  Cell viability was analyzed by an MTT (Sigma-Aldrich Co., St. Louis, MO, USA) assay. RAW264.7 macrophage cells and splenocytes were received with various concentrations of SBT or Stig. Then, the supernatant was discarded, and an MTT solvent (5 mg/mL) was added. The supernatant was discarded, and dimethyl sulfoxide was added to dissolve the insoluble formazan product. The absorbances at 570 nm were obtained by a microplate reader (Molecular Devices, LLC., Sunnyvale, CA, USA).

Nitrite Production
The supernatants from RAW264.7 macrophage cells were collected and reacted with Griess reagent I and Griess reagent II. A NaNO 2 standard curve was used to calculate nitrite concentrations. The nitrite concentrations were assessed at 540 nm by a microplate reader.

Measurement of Splenic Lymphocyte Proliferation
The splenocytes proliferation (1 × 10 4 ) was determined with a bromodeoxyuridine (BrdU) cell proliferation assay kit, as instructed by the manufacturer (BioVision Inc., Milpitas, CA, USA).

Measurement of NK Cell Cytotoxicity
The target cells, the YAC-1 lymphoma cells, and the effector cells, splenocytes, were seeded into 24-well plates at a ratio of effector cells to target cells of 50:1 and 200:1. The cells were treated with SBT or Stig for 20 h, followed by a mixing of the culture supernatant with a lactic acid dehydrogenase (LDH) solution (DoGenBio Co., Seoul, Korea). The NK activity of effector cells was calculated as follows: cytotoxicity (%) = (experimental release − spontaneous release/maximum release − spontaneous release) × 100.

Mice and Experimental Design
Male BALB/c mice (6 weeks of age) obtained from Dae-Han Experimental Animal Center had free access to a standard diet and water. The in vivo study was designed according to the previous reports [28,29]. After a 7-day acclimatization period, the mice were randomly divided into five groups (n = 6). From day 1 to day 3, four groups of mice were intraperitoneally injected with CTX at 100 mg/kg per day, except the normal group. The CTX-injected mice were gavaged daily with phosphate-buffered saline (PBS), SBT (100 mg/kg), Stig (1 mg/kg), and Korean Red Ginseng (RG, 400 mg/kg) for 7 consecutive days from day 4 to day 10. The body weight gains were calculated as follows: body weight gains (g) = weight on day 10 − weight on day 0. The spleen index was calculated as follows: spleen index (mg/g) = organ weight/body weight. The superoxide dismutase (SOD) activity was assayed in spleen homogenate (BioVision Inc.). The mice used in this study were handled in accordance with the guidelines for the Care and Use of Laboratory Animals (the U.S. National Institutes of Health, publication No. 85-23. Revised 1985). All experimental procedures were approved by the Animal Care Committee of Kyung Hee University (Ethics NO. KHSASP-20-472).

Statistical Analysis
Statistical significances were analyzed by one-way analysis of variance with Tukey's post hoc test (IBM SPSS software, Armonk, NY, USA). Statistical significance was considered at p < 0.05. The values show as mean ± standard error of the mean (SEM).

SBT or Stig Enhances the Secretion of NO and Expression of HO-1 in RAW264.7 Macrophage Cells
First, an MTT assay was carried out to examine the cytotoxicity of SBT and Stig. SBT at 1000 µg/mL and Stig at 10 µg/mL exhibited the cytotoxicity (p < 0.05, Figure 2A). Thus, the concentrations of SBT were determined to be 1, 10, and 100 µg/mL lower than 1000 µg/mL. The concentration of Stig was determined to be 1 µg/mL lower than 10 µg/mL, with reference to a previous report [22]. The IC50 value of Stig against RAW264.7 macrophage cells was 140 µg/mL ( Figure 2B). Activated macrophages release an effector molecule, NO, to inhibit pathogen replication [37]. In addition, NO acts as a powerful anti-oxidant [38]. Thus, we investigated whether SBT and Stig could up-regulate the NO pathways. As shown in Figure 2C, SBT or Stig significantly increased the secretion levels of NO (p < 0.05). SBT or Stig significantly increased the levels of mRNA expression of inducible NO synthase (iNOS) (p < 0.05, Figure 2D). The protein expression of iNOS was also increased by SBT or Stig ( Figure 2E). In addition, SBT or Stig significantly increased the levels of mRNA expression of a phase 2 detoxifying enzyme, HO-1, which has an important antioxidant effect [39] (p < 0.05, Figure 2F). The protein expression level of HO-1 reached the maximum value at 8 h after treatment with SBT and then decreased ( Figure 2G). Thus, the cells were treated with SBT or Stig for 8 h, and the regulatory effects on the protein expression level of HO-1 were analyzed. As expected, SBT or Stig increased the protein expression levels of HO-1 ( Figure 2H). LPS as a positive control [40] also increased the levels of NO, iNOS, and HO-1 ( Figure 2C-F,H).

SBT or Stig Enhances the Secretion of Immunostimulatory Cytokines from RAW264.7 Macrophage Cells
To evaluate whether the treatment with SBT and Stig could enhance the secretion of immunostimulatory cytokines from macrophages, SBT and Stig were treated in RAW264.7 macrophage cells. Figure 3A-F shows that the production levels of G-CSF, IFN-γ, IL-12, IL-2, IL-6, and TNF-α were increased following SBT treatment (p < 0.05). Stig significantly increased the levels of IFN-γ and IL-2 (p < 0.05, Figure 3B, D). LPS also significantly increased the production levels of G-CSF, IL-12, IL-2, IL-6 and TNF-α (p < 0.05, Figure 3A-F). Next, we observed the morphological alterations associated with macrophage activation in response to SBT and Stig. As shown in Figure 3G, most normal cells had a circular morphology, whereas the morphologies in the presence of SBT or Stig were changed irregularly with spreading and pseudopodia formation similar to LPS-treated cells ( Figure 3G), indicating the enhanced functions of RAW264.7 macrophage cells by treatment with SBT or Stig [41,42].

SBT or Stig Up-Regulates NF-κB Activation in RAW264.7 Macrophage Cells
In order to dissect the mechanisms underlying immune-stimulation following the treatment of SBT and Stig, we investigated whether SBT and Stig could increase NF-κB activation in RAW264.7 macrophage cells. Western blotting indicated that SBT or Stig remarkably increased the protein levels of nuclear NF-κB and cytoplasmic phosphorylated IκBα (pIκBα) in RAW264.7 macrophage cells ( Figure 4A). The increased expression levels of NF-κB in the nucleus were visualized by immunofluorescence microscopy ( Figure 4B).

SBT or Stig Enhances the Secretion of Immunostimulatory Cytokines from Splenocytes
We further evaluated whether SBT and Stig could enhance the secretion of immunostimulatory cytokines from splenocytes. As expected, SBT significantly increased the secretion levels of IFN-γ, IL-12, IL-2, and TNF-α (p < 0.05, Figure 5). Stig significantly increased the secretion levels of TNF-α (p < 0.05, Figure 5D). LPS also significantly increased IL-12, IL-2, and TNF-α levels.

SBT or Stig Promotes Splenic Lymphocyte Proliferation
Splenocytes proliferation elevates the expression of cytokines, potentially improving cell-mediated immune responses and resulting in immunity-enhancement [43]. The incorporation of BrdU into proliferating cells has been used as a measure of cell proliferation [44]. Thus, we evaluated whether SBT and Stig could promote splenocytes proliferation by analyzing the incorporation of BrdU. SBT or Stig significantly promoted the splenic lymphocyte proliferation (p < 0.05, Figure 6A). Interestingly, at least in this part, the splenic lymphocyte proliferation level of SBT was higher than that of LPS (p < 0.05). We further analyzed the expression levels of KI-67, which is a marker of cell proliferation [45]. The expression levels of KI-67 increased by the treatment with SBT or Stig were visualized in the nucleus by immunofluorescence microscopy ( Figure 6B). 3.6. SBT or Stig Enhances Splenic Nature Killer (NK) Cell Cytotoxic Activity NK cells as an innate immune cell have direct cytotoxic activity against tumor cells, release several cytokines, and regulate the function of innate and adaptive immune cells [46]. The cytotoxic activity of NK cells against YAC-1 targets is analyzed by measuring the LDH derived from lysed target cells in the medium following incubation of effector cells with target cells [27,47,48]. Thus, we evaluated whether SBT and Stig could increase splenic NK cell cytotoxic activity by conducting an LDH assay. We first confirmed that neither SBT nor Stig had splenocyte toxicity (Supplementary Figure S1). SBT or Stig significantly improved the splenic NK cell cytotoxic activity (p < 0.05, Figure 6C). Interestingly, at least in this part, the NK cell cytotoxic activities of SBT were higher than those of LPS (p < 0.05). NK cells are identified by the presence of NCR1, NKG2D, KLRD1, and CD49b, which are common NK cell markers [49,50]. SBT or Stig significantly increased the mRNA expression levels of NCR1, NKG2D, KLRD1, and CD49b in splenocytes, suggesting an increase in NK cell population (p < 0.05, Figure 6D-G).

SBT or Stig Enhances the Levels of Immunostimulatory Cytokines in the Serum of CTX-Induced Immunosuppressed Mice
We evaluated a regulatory effect of SBT and Stig on immunosuppression using an established murine model of CTX-induced immunosuppression ( Figure 7A). Each dose was determined by an MTT assay (Figure 2A) with reference to the previous reports [18,21,[51][52][53][54][55]. As shown in Figure 7B,C, the CTX control group showed remarkably reduced body weight gains and spleen index compared to the normal group (p < 0.05). SBT or Stig significantly increased the body weight gains and spleen index reduced by CTX (p < 0.05). In addition, the levels of IFN-γ, IL-12, IL-2, and TNF-α in the serum of the SBT-treated mice and Stig-treated mice were significantly higher than those in the CTX control group (p < 0.05, Figure 7D-G). RG was used as a positive control in the CTX-induced immunosuppressed mouse model [56].

SBT or Stig Restores the Spleen Functions in the CTX-Induced Immunosuppressed Mice
Finally, we investigated whether SBT and Stig could restore the spleen functions in the CTX-induced immunosuppressed mice. CTX exhibited a reduction in the levels of IFN-γ, IL-12, TNF-α, and IL-6 in the spleen, while the administration of SBT exhibited significant increases in the levels of IFN-γ, IL-12, TNF-α, and IL-6 (p < 0.05, Figure 8A-D). Stig significantly increased the levels of IFN-γ and IL-12 (p < 0.05, Figure 8A,B). In addition, SBT or Stig significantly increased the activity of an antioxidant enzyme, SOD, in the spleen (p < 0.05, Figure 8E). H&E stained-spleen tissues showed that the arrangement of splenocytes in the CTX control group was sparse and irregular, with necrotic areas, but the SBT or Stig-treated CTX group displayed relative tightness with fewer necrotic areas compared to the CTX control group ( Figure 8F).

Discussion
In the present study, we identified, for the first time, the potential of SBT and Stig to enhance immune functions, by studying the effects on macrophages and splenocytes under CTX-induced immunosuppressive conditions. Our results revealed that SBT or Stig increased the secretion levels of NO and immunostimulatory cytokines from macrophages or splenocytes. These effects were associated with a significant improvement of NF-κB activation. In addition, SBT or Stig increased HO-1 expression levels. SBT or Stig also enhanced splenic lymphocyte proliferation and splenic NK cell cytotoxic activity. SBT or Stig prevented immunosuppressive responses in mice.
The activated macrophages produce a variety of immunomodulators, such as NO [57]. NO, as an important component of the immune system, exhibits anti-oxidant, anti-pathogen, and anti-tumor activities [38,58]. NO is produced as a primary product of iNOS in macrophages through activation of the NF-κB pathway [38,59]. Oxidative stress was enhanced in iNOS-deficient mice after traumatic brain injury [58]. NF-κB knockout mice revealed severe defects in immune function [60]. In the present study, the enhancement of release levels of NO and expression levels of iNOS by SBT or Stig may result from the increase in NF-κB activation. An anti-oxidant, vitamin D, increased NO levels and enhanced anti-oxidant defenses [61]. Anti-oxidants, such as vitamin C and selenium, improved immune responses [62]. In addition, we found that SBT or Stig increased the HO-1 expression and SOD activity. Thus, it is conceivable that SBT or Stig as an anti-oxidant exerts immunity-enhancing effects.
In the immune response, the cytokine network is complicatedly connected between immune cells [63]. The activated macrophages release various cytokines, such as G-CSF, IFN-γ, IL-12, and TNF-α as major components of innate immune responses [64,65]. G-CSF as a hematopoietic growth factor promotes the functional maturation of neutrophils and plays an essential role in host defense against infection [65]. IFN-γ has an important role in recognizing and eliminating pathogens, and increasing the efficiency of the immune system [66]. IL-12, a well-known IFN-γ inducer, participates in host defenses against various microbial pathogens [5]. IL-2 as an immune growth factor plays a critical role in sustaining T cell responses [67]. IL-6, if not excessively synthesized, contributes to host defense through activation of immune responses when homeostasis is disrupted by infections [68]. As a proinflammatory cytokine, dysregulated excessive synthesis of IL-6 has a pathological effect on inflammatory responses. This might stem from different cellular environments, signaling pathways, and doses of IL-6 [68][69][70]. G-CSF therapy reduced the incidence of infections in patients with neutropenia [71]. When immune-supportive IFN-γ was added to antimicrobial therapy, the general condition of patient suffering from infection was improved [72]. TNF-α treatment played a protective role in the acute stage of viral myocarditis in mice [73]. The present study showed that SBT augmented the release levels of G-CSF, IFN-γ, IL-12, IL-2, IL-6, and TNF-α from macrophages, which is in concordance with studies indicating their potential as immunity-enhancing agents by increasing the levels of immunostimulatory cytokines from macrophages [29,74]. However, Stig only increased IFN-γ and IL-2 levels from macrophages. This result suggests that the effect of SBT is potentially related to the various compounds present in SBT rather Stig alone.
The activated NK cells contribute to effective immune reactions against infections and cancers via cellular cytotoxicity and cytokine production [75]. NK cells deficiency was observed in patients with recurrent viral infections and cancer [76]. Monmai et al. [27] reported that macromolecules isolated from seaweeds improve the NK cell activity, presenting it as a potent material for enhancing immunity. Becker et al. [77] reported that NK cells have been used in several clinical trials as an immunotherapy, and stimulation of NK cell activity by IL-12 and IL-2 enhances the immunotherapy of NK cells. In the present study, SBT or Stig remarkably elevated splenic NK cell cytotoxic activity, increasing the release levels of IL-12 and IL-2 from splenocytes. Thus, we speculate that SBT or Stig may have applications for NK cell-based immunotherapy.
We finally investigated whether SBT and Stig could be stable in the culture medium. The treatment with SBT or Stig did not affect the pH values of the culture medium (Supplementary Figure S2), indicating that there were no changes in conditions of the culture medium or stable conditions of the SBT or Stig-added medium. Furthermore, several reports have suggested that SBT and Stig exert beneficial effects in various long-term experiments [22,[78][79][80][81][82][83]. Thus, we presuppose that SBT or Stig may be stably maintained in the culture medium.
CTX has immunosuppressive effects and is applied as an immunosuppressant [84]. In addition, several studies reported that the injection of single doses of CTX (250-400 mg/kg) and smaller divided doses induce tolerance against ovine erythrocytes, bovine gamma globulin, and equine gamma globulin in mice [84][85][86]. To confirm the immunosuppressive properties of CTX, we assessed body weight and serum TNF-α and IL-6 levels instead of tolerance against the ovine erythrocyte test because many researchers determined the immunosuppression by measuring body weight and cytokines, such as TNF-α and IL-6 [26,[28][29][30]. We showed that the body weight and serum TNF-α and IL-6 levels were reduced on the 7th day after CTX injection, indicating immunosuppression by CTX (Supplementary Figure S3). Immunodeficiency-related diseases cause body weight loss [87]. Because immune cells in the spleen colonize and respond to immune responses, the spleen index reflects the strength of innate immune function [88]. In the present study, SBT or Stig increased the body weight gains and spleen index in the CTX-induced immunosuppressed mice and improved the immune function of mice, increasing the levels of immunostimulatory cytokines in the serum and spleen. These findings indicate that SBT or Stig counteracts the immunosuppressive effect of CTX. Despite significant advances in our understanding of the immunity-enhancing effects of SBT or Stig in the CTX-induced immunosuppressed model, further studies are needed to clarify the regulatory effects of immune responses to various antigens in the CTX-induced immunosuppressed model and the immunity-enhancing effects in various immunosuppressive models.
The beneficial effects of SBT and Stig were practically the same, with Stig having effects at a lower concentration than SBT. However, SBT enhanced the various immune-related factors, such as G-CSF, IL-12, IL-6, and TNF-α from macrophages and IFN-γ, IL-12, and IL-2 from splenocytes more so than Stig did. Ginsenoside Rg3-enriched RG or ginsenoside Re-enriched Korean Ginseng was reported to have better effects in various experimental models [89,90]. Thus, Stig-enriched SBT could be expected to have a better effect on immunity enhancement.

Conclusions
In summary, the results indicate that SBT or Stig-mediated immune-enhancement, characterized by increased levels of immunostimulatory molecules in macrophages and splenocytes, may restore immunosuppressive responses in mice. The HO-1 expression levels, splenic lymphocyte proliferation, and splenic NK cell cytotoxic activity were elevated by SBT or Stig. Therefore, we speculate that SBT or Stig may exert curative properties as a potential immunity-enhancing agent.