Reversal of stress- or chemotherapy-induced immunosuppression by socheongryong-tang aqueous extract

Reversal of stress- or chemotherapy-induced immunosuppression by socheongryong-tang aqueous extract | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Reversal of stress- or chemotherapy-induced immunosuppression by socheongryong-tang aqueous extract Youngsic Jeon, Hyeonseok Ko, Dong-Young Woo, Taejung Kim, Ki Sung Kang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4096694/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Socheongryong-tang (SCRT) has been recognized as a traditional medication for managing chills and fever in East Asian countries, including Korea, China, and Japan. This study aimed to elucidate the novel biological activity and mode of action underlying the immunity-boosting effects of SCRT in murine macrophages. Our findings demonstrate that SCRT significantly enhances phagocytic activity, productions of nitric oxide (NO) and prostaglandin E 2 (PGE 2 ), and mRNA expression of cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). These effects are attributed to the activation of the reactive oxygen species (ROS)/mitogen activated protein kinases (MAPKs)/nuclear factor-κB (NF-κB) signaling axis. Importantly, SCRT maintains its immunomodulatory effects even under stressful conditions induced by hydrocortisone (HCOR) treatment or chemotherapy with 5-fluorouracil (5-FU). This resilience against stress or chemotherapy-induced immunosuppression underscores the potential of SCRT aqueous extract as a promising therapeutic agent for mitigating immunosuppression associated with stress or chemotherapy. Biological sciences/Cell biology Biological sciences/Immunology Socheongryong-tang Immunity-boosting Reactive oxygen species Mitogen activated protein kinases Nuclear factor-κB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The immune response is a multifaceted process involving both innate and adaptive components. The innate immune response encompasses the initial defense mechanisms mediated by macrophages and natural killer cells, while the adaptive immune response involves T and B lymphocytes 1 . Macrophages play a crucial role in tissue immune responses through phagocytosis and non-specific protection against bacterial infections 2 . Activation of phagocytic cells is essential for initiating inflammation, which involves the release of cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), along with other inflammatory mediators like nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) 3 , 4 . Inducible nitric oxide synthase (iNOS) is responsible for generating NO during the immune response, while cyclooxygenases (COX-1 and − 2) produce PGE 2 by converting arachidonic acid. Given the role of NO and PGE 2 as key pro-inflammatory mediators in macrophages, enhancing their production could be a beneficial strategy for boosting the immune system 5 , 6 . Lipopolysaccharide (LPS) is a potent inflammatory stimulant commonly used in screening for anti-inflammatory and immune-enhancing compounds. Previous studies have utilized high doses of LPS (e.g., 0.1-1 µg/ml) to induce acute inflammation in RAW264.7 macrophage cells for screening anti-inflammatory agents 7 – 9 . Conversely, low doses of LPS (e.g., 1–10 ng/ml) are used as a positive control to stimulate murine macrophages in screening for immune-boosting agents 10 . Stress-induced immunosuppression refers to the suppression or weakening of the body's immune response as a result of exposure to stressors, which can be psychological, physiological, or environmental. One of the mechanisms through which stress induces immunosuppression is by activating the hypothalamic-pituitary-adrenal axis, leading to the release of stress hormones such as cortisol 11 . Cortisol, also known as hydrocortisone (HCOR) when supplied as a drug, is a steroid hormone produced by the adrenal glands in response to stress 12 . It plays a pivotal role in regulating various physiological processes, including metabolism, inflammation, and immune function 13 , 14 . Especially, when released into the bloodstream, cortisol binds to glucocorticoid receptors on immune cells, thereby suppressing immune responses 15 . Chemotherapy is a fundamental component of cancer treatment, yet it carries the risk of inducing immunosuppression, which can potentially impact patient outcomes. While chemotherapy effectively targets cancer cells, it can also affect rapidly dividing cells in the immune system, including leukocytes and bone marrow precursors, leading to compromised immune function 16 , 17 . Consequently, patients may experience heightened susceptibility to infections, delayed wound healing, and other immune-related complications. Managing and monitoring immune function during chemotherapy are crucial to mitigate the risk of infections and other adverse effects, underscoring the importance of comprehensive patient care. Socheongryong-tang (SCRT) has traditionally been used as an herbal medicine formula for managing bronchitis, asthma, rhinitis, and cold-related symptoms across East Asia, including Korea, China, and Japan 17 , 18 . SCRT composes 8 herbal components: Pinellia ternata Rhizoma, Paeonia lactiflora Pall. Radix, Ephedra sinica Stadf. Herba, Zingiber officinale Rosc. Rhizoma, Glycyrrhiza glabra L. Radix, Cinnamomum cassia Blume. Ramulus, Asarum sieboldii F. Maekawa Radix, and Schizandra chinensis Fructus 18 . The major components of SCRT include 6-gingerol, liquiritigenin, and glycyrrhizin, as well as phenylpropanoids such as cinnamic acid, cinnamaldehyde, and coumarin; monoterpene glycosides such as paeoniflorin and albiflorin; lignans including schizandrin, gomisin A, and gomisin N; and alkaloids including ephedrine and pseudoephedrine 19 – 21 . In this study, we aimed to investigate the immunomodulatory properties of SCRT aqueous extract and elucidate its underlying mode of action in macrophages. Our findings provide a molecular trait for the reversal of stress- or chemotherapy-induced immunosuppression by SCRT for the first time, and could be used to help in the development of pharmaceuticals aimed at manipulating immunomodulation. 2. Results 2.1. Effects of SCRT on phagocytic capacity on RAW264.7 macrophages To determine the optimal concentration of SCRT without cytotoxicity, we first evaluated the cell viability on RAW264.7 cells. SCRT did not have cytotoxic effect for 24 h up to a concentration of 50 µg/mL (Fig. 1 A). Based on this result, SCRT was used at a concentration of 12.5–50 µg/mL in further experiments. Next, we confirmed the phagocytic activity of macrophagocytes. We observed a dose-dependent increase in phagocytic activity upon treatment with SCRT (Fig. 1 B). Specifically, we observed irregular polygons and an elevated number of engulfed particles surrounding the cells in response to SCRT treatment (25–50 µg/mL), mirroring the effects observed with LPS treatment (Fig. 1 C). These findings suggest that SCRT has the potential to enhance phagocytic activity in a dose-dependent manner compared to the normal condition. 2.2. SCRT enhances NO and PGE2 production on RAW264.7 macrophages To investigate the potential immunomodulatory effects of SCRT on RAW264.7 macrophages, we assessed the production of NO and PGE 2 by using a griess assay and PGE 2 enzyme-linked immunosorbent assay (ELISA). Our results revealed that SCRT significantly increased the concentration of NO and PGE 2 compared to the normal condition (Fig. 2 A and 2 B). Next, we investigated whether SCRT can enhance the expression of Inos and Ptgs2 . The treatment of SCRT dose-dependently increased the levels of Inos and Ptgs2 compared to the normal condition (Fig. 2 C and 2 D). Additionally, the protein expression of iNOS and COX-2 were significantly enhanced by SCRT (Fig. 2 E). To verify whether SCRT treatment affect the expression of immunomodulatory-related pro-inflammatory cytokines, such as Tnf , Il1b , and Il6 in RAW264.7 cells, we examined the expression levels of these cytokines induced by SCRT (Fig. 2 F). Indeed, these cytokines were known to induce M1 polarized macrophage associating with pro-inflammatory phenotype 22 , 23 . 2.3. SCRT is associated with activation of ROS/MAPK/NF-κB signaling axis Given that p65, the subunit of NF-κB, has been implicated in the regulation of pro-inflammation and expression of cytokines, such as TNF-α, IL-1β, and IL-6 24,25 , we further evaluated that the SCRT could modulate immunomodulatory effects through the activation of p65 associated with Tnf , Il1b , and Il6 . SCRT-treated macrophages displayed significantly increased the p65 phosphorylation compared to those in non-treated macrophages (Fig. 3 A). Next, to monitor p65 trans-activity, we assessed the NF-κB reporter assay and observed that SCRT treatment increased the trans-activity of NF-κB compared to the normal condition (Fig. 3 B). As NO and PGE 2 production were previously found to be activated by MAPK pathway, core regulatory molecules 26 , we further evaluated the phosphorylation level of ERK1/2, p38, and JNK in RAW264.7 macrophages and found that SCRT-treated macrophages exhibited the elevation of ERK1/2, p38, and JNK phosphorylation (Fig. 3 C). In addition, based on the previously determined the association between reactive oxygen species (ROS) generation and various pathological states, including the regulation of inflammation by M1 polarized macrophages 27 , our investigation attempted to determine whether SCTR treatment was associated with elevated ROS release. We found that ROS production was significantly increased by SCRT treatment in a dose-dependet manner (Fig. 3 D). Taken together, the immunomodulatory properties of SCRT are associated with p65 and MAPK activation caused by ROS production. 2.4. SCRT recovers the immunomodulatory properties during stress-induced immunosuppression To verify whether SCRT treatment can improve its immunomodulatory properties even under stressful condition, we examined the immunity-boosting effects in HCOR-treated RAW264.7 cells. HCOR reduced LPS-stimulated production of NO and PGE 2 , as well as the mRNA expression of Inos and Ptgs2 ; however, SCRT treatment ameliorated the situation in a dose-dependent manner (Fig. 4 A- 4 D)). In addition, the downregulation of mRNA expression of cytokines (e.g., Tnf , Il1b , and Il6 ) induced by HCOR was also reversed by SCRT treatment (Fig. 4 E- 4 G). Taken together, our findings indicate that SCRT treatment restores immunomodulatory properties during chemotherapy-induced immunosuppression. These results demonstrate that SCRT treatment restores immunomodulatory properties during stressful-induced immunosuppression. 2.5. SCRT recovers the immunomodulatory properties during chemotherapy-induced immunosuppression Subsequently, we examined the effect of various anti-cancer drugs [e.g., sorafenib, etoposide, tamoxifen, and 5-fluorouracil (5-FU)] on chemotherapy-induced immunosuppression in LPS-stimulated RAW264.7 cells. Among them, only 5-FU induced immunosuppression, a phenomenon improved by SCRT treatment ( Supplementary Fig. 1A-1C ). The decreased LPS-stimulated production of NO and PGE 2 induced by 5-FU, as well as the mRNA expression of Inos and Ptgs2 , were dose-dependently reversed by SCRT (Fig. 5 A − 5 D ). Furthermore, similar results were observed regarding the modulation of cytokines mRNA expression (e.g., Tnf , Il1b , and Il6 ) by SCRT (Fig. 5 E- 5 G). Taken together, our findings suggest that SCRT treatment restores immunomodulatory properties during chemotherapy-induced immunosuppression. 3. Discussion The therapeutic potential of SCRT in managing seasonal colds and inflammatory disorders has long been recognized in oriental traditional medicine. However, despite some evidence of its anti-inflammatory activity, the precise mechanistic significance of SCRT in immunomodulation remains uncertain 17 , 18 , 28 . In this study, we aimed to elucidate the immunomodulatory effects of SCRT on RAW264.7 macrophages. Our findings demonstrated that SCRT treatment enhanced phagocytic activity and induced the release of NO and PGE 2 . These findings are consistent with previous reports linking NO and PGE 2 production to immunomodulation, particularly in M1 polarized macrophages, which play crucial roles in pro-inflammatory responses and cytokine production 23 , 29 . Furthermore, SCRT treatment activated the ROS/MAPKs/NF-κB signaling pathway, leading to the production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. These responses indicate fundamental immunostimulatory effects, which contradict its traditional use in managing inflammatory conditions. In previous studies where anti-inflammatory effects were observed, SCRT was administered at high concentrations (up to 1000 µg/ml). In our study, demonstrating immune-boosting effects, SCRT was administered at low concentrations (up to 50 µg/ml) 18 . We speculate that these differences in SCRT treatment concentrations may have contributed to the distinct effects observed between anti-inflammation and immune enhancement, although the underlying mechanisms governing SCRT's dual activities remain unclear. Especially, it is conceivable that SCRT contains bioactive compounds capable of modulating key signaling pathways involved in immunomodulation. Among the various ingredients of SCRT, Paeonia lactiflora Pall. Radix has been known for its vasodilatory effects, while Zingiber officinale Rosc. Rhizoma and Schizandra chinensis Fructus have been recognized for their immune-enhancing properties 30 – 33 . Inactivation or activation of the ROS/MAPKs/NF-κB signaling pathway by SCRT may contribute to controlling its mode of action, reflecting opposite mechanisms for anti-inflammation and immune enhancement. Nonetheless, comprehensive studies are required to thoroughly elucidate these processes. Interestingly, SCRT also demonstrated resilience against stress- or chemotherapy-induced immunosuppression, highlighting its potential as a therapeutic agent for mitigating immune dysfunction associated with these conditions. The ability of SCRT to restore immunomodulatory effects in combination with HCOR and 5-FU, respectively, as evidenced by the reversal of NO, PGE 2 , and cytokine downregulation induced by stressors or chemotherapy agents, underscores its versatility and therapeutic potential in managing immune-related disorders under stress- and chemotherapy-induced immunosuppressive conditions. In conclusion, the contradictory activities of SCRT, exhibiting both anti-inflammatory and immune-enhancing effects, provide novel insights on its therapeutic potential. Particularly, the ability of SCRT to ameliorate stress- or chemotherapy-induced immunosuppression is crucial. This study will contribute to our understanding of immunomodulatory properties and aid in identifying the specific physiologically active components responsible for SCRT's pharmacological effects. Materials And Methods 4.1. Preparation of socheongryong-tang water extract SCRT, known as Hanpoong Socheongryongtang Ext. Granule (ATC code: R05F), is a product sold by Hanpoong Pharmaceutical Co., Ltd. under strict quality control (Jeonju, Republic Korea) ( Supplementary Table 1 ). The ingredient was added to 90 mL of distilled water and boiled at 90–100°C for 3 hours. The resulting decoction was then filtered through filter paper with a 5-micrometer pore size, and the filtrate was concentrated using a rotary evaporator. The remaining concentrate was vacuum-dried to obtain a powder. The SCRT powder was dissolved in dimethyl sulfoxide for in vitro experiments. 4.2. HPLC (High-Performance Liquid Chromatography) analysis A chromatogram of SCRT was obtained using an HPLC-PDA-MS. Resultantly, Seven constituents (ephedrine, albiflorin, paeoniflorin, liquiritin apioside, liquiritin, isoliquiritin apioside, and glycyrrhizin) were confirmed within 30 min using mobile phases comprising solvent A (0.1%, v/v formic acid in water) and solvent B (0.1%, formic acid in acetonitrile) under optimized chromatography conditions: An analytical reversed phase Shimadzu Nexera X2 system comprising of a solvent degassing unit (DGU − 20A), binary pump (LC − 30AD), auto sampler (SIL − 30AC), system controller unit (CBM − 20A), photodiode array detector (SPD − M20A), and column oven unit (CTO − 20AC) was used for qualitative and/or quantitative analysis. Electrospray ionization (ESI)-mass spectrometry (MS) was performed using a Shimadzu LCMS-2020 system for qualitative analysis. A Phenomenex Luna Omega polar C18 column (150×2.1 mm, 1.6 µm) was used. The flow rate was set to 0.2 mL/min and the gradient flow program was as follows – initial to 1 min: 15% B, 40.0 min: 70% B, 45.0 min: 100% B, 48.0 min: 100% B, and 53.0 min: 15% B. The detection wavelengths for analytes were set at 230, 254, and 280 nm, and the injection volume of 1,000 ppm concentration sample was 3 µL ( Supplementary Fig. 2 ). 4.3. Cell culture The murine macrophage RAW264.7 cell line was procured from the American Type Culture Collection (ATCC, Manassas, VA). These murine cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Corning, NY) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA) and 1% penicillin and streptomycin. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . 4.4. Cell viability RAW264.7 cells were treated with 12.5–50 µg/mL of SCRT for 24 h, and cell proliferation was assessed using a Quanti-Max™ WST-8 Cell Viability Assay kit (Biomax QM3000, Seoul, Republic of Korea) following the manufacturer’s instructions. 4.5. Phagocytic activity RAW264.7 cells were seeded in 96-well plates at a density of 5 × 10 4 cells/well and incubated overnight until they adhered to the plates. The adherent cells were then treated with the specified concentrations of SCRT or 1 ng/mL of LPS (055:B5, Sigma Aldrich, St. Louis, MO) for 24 h. Following treatment, the phagocytic capacity of murine macrophages was assessed using the Phagocytosis Assay Zymosan Substrate Kit (Abcam, MA, USA) according to the manufacturer’s instructions. 4.6. Griess assay RAW264.7 cells were incubated with phenol red-free medium containing the SCRT, HCOR, anti-cancer drugs or LPS for 24 h. Then, the supernatant was collected, mixed with an equal volume of Griess reagent (1% sulfanilamide, 5% phosphoric acid, and 0.1% N- (1-naphthyl)-ethylenediamine), and incubated at room temperature for 5–10 min. Absorbance was determined using a microplate reader at a wavelength of 540 nm. The experiments were performed in triplicate. 4.7. PGE 2 ELISA RAW264.7 cells were incubated with fresh medium containing the SCRT, HCOR, anti-cancer drugs or LPS for 24 h. Then, the production of PGE 2 was measured using the PGE 2 Parameter Assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocols. 4.8. DCF-DA assay Intracellular ROS levels were assessed using the DCF-DA staining method. RAW264.7 cells were seeded in 96-well plates at a density of 1 × 10 5 cells/well and treated with SCRT or LPS for 24 h. After treatment, the medium was replaced with fresh medium containing 20 µM 2’,7’-dichlorofluorescin diacetate (DCF-DA, Sigma-Aldrich) and incubated at 37°C with 5% CO 2 for 20 min. Fluorescence intensity was measured at excitation wavelength of 485 nm and emission wavelength of 530 nm using an Infinite PRO microreader (TECAN, Männedorf, Switzerland). 4.9. Real-Time PCR Analysis Total RNA was extracted using the RNeasy Mini kit (QIAgen, MD, USA), and cDNA was synthesized using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). Quantitative real-time PCR was performed using QuantStudio 6 Pro real-time PCR System (Applied Biosystems, Foster City, CA), TaqMan® Fast Advanced Master Mix (Applied Biosystems), or AccuPower® 2xGreenStarTM qPCR Master Mix (BIONEER, Daejeon, Republic of Korea). The primer sequences and information for qRT-PCR are provided in Supplementary Tables 2 and 3 . 4.10. Western blotting Protein samples were subjected to western blot analysis following previously described methods 34 . Monoclonal antibodies against iNOS, COX-2, phospho-NF-κB/p65 (Ser536), NF-κB/p65, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-JNK (Thr183/Tyr185), JNK, and GAPDH were obtained from Cell Signaling Technology Inc (Boston, MA, USA). 4.11. Dual-luciferase reporter assay RAW264.7 cells seeded in 96-well white plates were co-transfected with pNF-κB-Luc and pNL-Luc vectors using FuGENE HD Transfection Reagent (Promega, Madison, WI) following the manufacturer’s instructions. After co-transfection for 24 h, the cells were treated with 50 µg/mL SCRT or 1 ng/mL LPS for 6 h. Subsequently, the cells were lysed, and luciferase activity was measured using a dual-luciferase reporter assay system (Promega) and GloMax® Navigator Microplate Luminometer (Promega) according to the manufacturer’s instructions. The relative firefly luciferase activity was normalized to NanoLuc® luciferase expression to adjust for variations in transfection efficiency. 4.12. Statistical analysis Data are presented as the mean ± standard deviation (S.D.) of at least three independent readings for each experiment. Statistical analysis was performed using Student’s t-tests and one-way ANOVA to evaluate deviations between the means. Abbreviations TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; NO, nitric oxide; PGE 2 , prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenases 2; LPS, lipopolysaccharide; HCOR, hydrocortisone; SCRT, socheongryong-tang; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; 5-FU, 5-fluorouracil Declarations Conflicts of interests : The authors declare that they have no conflicts of interest. Funding: This work was supported by the Korea Institute of Science and Technology (KIST) institutional program (Project No. 2E33301 and 2Z07011). Author Contribution Y.J. and H.K. conducted experiments, interpreted data, and prepared the manuscript. D.W. also conducted experiments. T.K., K.S.K., and S.N.K provided valuable contributions to result interpretation. Y.K. collaborated in manuscript writing, supervised, and provided financial support for the study. Data availability: The data generated and analyzed during the current study are available from the corresponding author upon reasonable request. References Abbas, A. K. & Janeway, C. A., Jr. Immunology: improving on nature in the twenty-first century. Cell 100, 129–138, doi: 10.1016/s0092-8674(00)81689-x (2000). Tosi, M. F. Innate immune responses to infection. J Allergy Clin Immunol 116, 241–249; quiz 250, doi: 10.1016/j.jaci.2005.05.036 (2005). Fujiwara, N. & Kobayashi, K. Macrophages in inflammation. 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P. et al. Ginger polysaccharides enhance intestinal immunity by modulating gut microbiota in cyclophosphamide-induced immunosuppressed mice. Int J Biol Macromol 223, 1308–1319, doi: 10.1016/j.ijbiomac.2022.11.104 (2022). Yu, J. et al. Immunomodulatory effect of Schisandra polysaccharides in cyclophosphamide-induced immunocompromised mice. Exp Ther Med 15, 4755–4762, doi: 10.3892/etm.2018.6073 (2018). Jeon, Y. et al. YAP inactivation in estrogen receptor alpha-positive hepatocellular carcinoma with less aggressive behavior. Exp Mol Med 53, 1055–1067, doi: 10.1038/s12276-021-00639-2 (2021). Additional Declarations No competing interests reported. Supplementary Files Supplementarydata2nd.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4096694","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":284776218,"identity":"7a7dc278-7a3c-4d0a-bd53-e795adbbed42","order_by":0,"name":"Youngsic Jeon","email":"","orcid":"","institution":"Korea Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Youngsic","middleName":"","lastName":"Jeon","suffix":""},{"id":284776220,"identity":"f7673a69-0204-49c6-be04-a42408fc9321","order_by":1,"name":"Hyeonseok Ko","email":"","orcid":"","institution":"HLB Life Science R\u0026D","correspondingAuthor":false,"prefix":"","firstName":"Hyeonseok","middleName":"","lastName":"Ko","suffix":""},{"id":284776222,"identity":"9055eff5-7a63-4d7c-af85-279e3c330593","order_by":2,"name":"Dong-Young Woo","email":"","orcid":"","institution":"Korea Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Dong-Young","middleName":"","lastName":"Woo","suffix":""},{"id":284776224,"identity":"e890ee90-8bba-47e0-8c6c-db3599b75c6b","order_by":3,"name":"Taejung Kim","email":"","orcid":"","institution":"Korea Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Taejung","middleName":"","lastName":"Kim","suffix":""},{"id":284776225,"identity":"a0a7f261-7aeb-4298-9842-03ef158f8b1b","order_by":4,"name":"Ki Sung Kang","email":"","orcid":"","institution":"Gachon University","correspondingAuthor":false,"prefix":"","firstName":"Ki","middleName":"Sung","lastName":"Kang","suffix":""},{"id":284776226,"identity":"915506e4-5892-406b-8686-1cd9333dd35e","order_by":5,"name":"Su Nam Kim","email":"","orcid":"","institution":"Korea Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Su","middleName":"Nam","lastName":"Kim","suffix":""},{"id":284776227,"identity":"f894290a-2c1f-4aed-b4fc-9c5e8b6bf84e","order_by":6,"name":"Young-Joo Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie2QsYoCMRCG5xDGJrhtRFh9hECqg4CvkuUgNoqvEBDW0tZ7m1kH4ivsE1x9YrPXHGa1solbWuSrJmE+/p8ByGTekgD08W8Ejmugxw8OUABdORH9YAcp/QKyLqWLwxBFNaE6tcJV9fSnav462BYx8zepUCBeS1PVM0csLHx+e+RjWjl7XquYMlt5jsWUovEuXeyuWI7Fzr7porJ8rfTFiDVKJBJ9SjxGUplSsLzxrkThLAsnlWT8SiqTNujrxhsx3wd96YxRxb7WSWXR0tNbAoySAsD84F9sZDKZTOYGMuVPds16pvQAAAAASUVORK5CYII=","orcid":"","institution":"Korea Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Young-Joo","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-03-14 02:29:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4096694/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4096694/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53882356,"identity":"f85fdf82-9097-4a07-9d01-084342cd0aea","added_by":"auto","created_at":"2024-04-01 18:03:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":365659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSCRT treatment enhances phagocytic activity\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) RAW264.7 cells were treated with various concentrations of SCRT for 24 h and subjected to WST-8 assay. (\u003cstrong\u003eB\u003c/strong\u003e,\u003cstrong\u003eC\u003c/strong\u003e) RAW264.7 cells were incubated with 1 ng/mL LPS and 12.5 – 50 μg/mL SCRT for 24 h. The levels of phagocytic capacity by SCRT were assessed using Phagocytosis Assay Kit. The data are shown as mean ± S.D. of triplicate experiment. Statistical significance is indicated (* p \u0026lt; 0.05, ** p \u0026lt; 0.01, and *** p \u0026lt; 0.001; Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4096694/v1/894709e7ca3547644b42ba31.jpg"},{"id":53882911,"identity":"80848e65-a102-415d-86d3-48021ee3cb9c","added_by":"auto","created_at":"2024-04-01 18:11:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":363240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSCRT treatment enhanced \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eInos\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eptg2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTnf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIl1b\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIl6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression. \u003c/strong\u003eRAW264.7 cells were treated with 1 ng/mL LPS and the indicated concentrations of SCRT for 24 h. (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) The supernatant was used for detecting NO and PGE\u003csub\u003e2\u003c/sub\u003e concentration using the griess assay and PGE\u003csub\u003e2\u003c/sub\u003e Parameter Assay, respectively. (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e) Bar plots show mRNA expression of \u003cem\u003eInos\u003c/em\u003e and \u003cem\u003ePtgs2\u003c/em\u003e using qRT-PCR. (\u003cstrong\u003eE\u003c/strong\u003e) Protein expression of iNOS and COX-2 were determined by western blotting. (\u003cstrong\u003eF\u003c/strong\u003e) Bar plots show mRNA expression of cytokines, \u003cem\u003eTnf, Il1b,\u003c/em\u003e and \u003cem\u003eIl6,\u003c/em\u003e using qRT-PCR. Expression of each gene was normalized to that of \u003cem\u003eGapdh\u003c/em\u003e. The data are shown as mean ± S.D. of triplicate experiment. Statistical significance is indicated (*p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001, Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4096694/v1/54df570738830f27075d4e77.jpg"},{"id":53882357,"identity":"9d204d37-7019-49af-b101-df73808fbd27","added_by":"auto","created_at":"2024-04-01 18:03:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":298889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSCRT treatment activates ROS/MAPKs/NF-κB signaling axis. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Protein levels of p-p65 and p65 were determined by western blotting. (\u003cstrong\u003eB\u003c/strong\u003e) NF-κB transactivity was evaluated using the dual-luciferase assay in RAW264.7 cells. (\u003cstrong\u003eC\u003c/strong\u003e) Protein levels of p-ERK, ERK, p-JNK, JNK, p-p38, and p38 were determined by western blotting. (\u003cstrong\u003eD\u003c/strong\u003e) Bar plots show the production of ROS using DCF-DA assay. GAPDH was used as control for protein expression analysis. The data are shown as mean ± S.D. of triplicate experiments. Statistical significance is indicated (*p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001, Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4096694/v1/38e17cc1111ef4bde9a53eae.jpg"},{"id":53882359,"identity":"0cb74d1f-30aa-4324-a840-62453e6e4b0b","added_by":"auto","created_at":"2024-04-01 18:03:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":329090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSCRT recovers the immunomodulatory properties under stress-induced condition. \u003c/strong\u003eRAW264.7 cells were pretreated with the indicated concentrations of SCRT or 1 ng/mL LPS for 2 h, and then exposed to 100 ng/mL HCOR for 24 h. (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) The supernatant was used for detecting NO and PGE\u003csub\u003e2\u003c/sub\u003e concentration using the griess assay and ELISA, respectively. (\u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e) Bar plots show the mRNA expression of \u003cem\u003eInos\u003c/em\u003e, \u003cem\u003ePtgs2\u003c/em\u003e, \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e using qRT-PCR. Expression of each gene was normalized to that of \u003cem\u003eGapdh\u003c/em\u003e. The data are shown as mean ± S.D. of triplicate experiments. Statistical significance is indicated (vs. with LPS; \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001, vs. with LPS and HCOR; *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001, one-way ANOVA).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4096694/v1/2d4094472ba8e4041ee9b86c.jpg"},{"id":53882360,"identity":"fd53f036-c243-4e58-b7b7-dcee8c11a7e9","added_by":"auto","created_at":"2024-04-01 18:03:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":332655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSCRT recovers the immunomodulatory properties under chemotherapy-induced immunosuppression. \u003c/strong\u003eRAW264.7 cells were pretreated with the indicated concentrations of SCRT or 1 ng/mL LPS for 2 h, and then exposed to 50 nM 5-FU for 24 h. (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) The supernatant was used for detecting NO and PGE\u003csub\u003e2\u003c/sub\u003e concentration using the griess assay and ELISA. (\u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e) Bar plots show the mRNA expression of \u003cem\u003eInos\u003c/em\u003e, \u003cem\u003ePtgs2\u003c/em\u003e, \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e using qRT-PCR. Expression of each gene was normalized to that of \u003cem\u003eGapdh\u003c/em\u003e. The data are shown as mean ± S.D. of triplicate experiments. Statistical significance is indicated (vs. with LPS; \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001, vs. with LPS and 5-FU; *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001, one-way ANOVA).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4096694/v1/a1c43180661000212a5195d7.jpg"},{"id":57668848,"identity":"021e2579-a159-435c-98dc-8aa5d57532bf","added_by":"auto","created_at":"2024-06-04 05:53:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2383527,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4096694/v1/5f983c9e-2332-45c6-8448-b00a71920857.pdf"},{"id":53882361,"identity":"dd76f01b-a6dd-4c7e-a435-ca23fb5518ae","added_by":"auto","created_at":"2024-04-01 18:03:30","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":665476,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata2nd.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4096694/v1/c5158188a082dd27897ca169.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reversal of stress- or chemotherapy-induced immunosuppression by socheongryong-tang aqueous extract","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe immune response is a multifaceted process involving both innate and adaptive components. The innate immune response encompasses the initial defense mechanisms mediated by macrophages and natural killer cells, while the adaptive immune response involves T and B lymphocytes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Macrophages play a crucial role in tissue immune responses through phagocytosis and non-specific protection against bacterial infections\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Activation of phagocytic cells is essential for initiating inflammation, which involves the release of cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), along with other inflammatory mediators like nitric oxide (NO) and prostaglandin E\u003csub\u003e2\u003c/sub\u003e (PGE\u003csub\u003e2\u003c/sub\u003e)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Inducible nitric oxide synthase (iNOS) is responsible for generating NO during the immune response, while cyclooxygenases (COX-1 and \u0026minus;\u0026thinsp;2) produce PGE\u003csub\u003e2\u003c/sub\u003e by converting arachidonic acid. Given the role of NO and PGE\u003csub\u003e2\u003c/sub\u003e as key pro-inflammatory mediators in macrophages, enhancing their production could be a beneficial strategy for boosting the immune system\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Lipopolysaccharide (LPS) is a potent inflammatory stimulant commonly used in screening for anti-inflammatory and immune-enhancing compounds. Previous studies have utilized high doses of LPS (e.g., 0.1-1 \u0026micro;g/ml) to induce acute inflammation in RAW264.7 macrophage cells for screening anti-inflammatory agents\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Conversely, low doses of LPS (e.g., 1\u0026ndash;10 ng/ml) are used as a positive control to stimulate murine macrophages in screening for immune-boosting agents\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStress-induced immunosuppression refers to the suppression or weakening of the body's immune response as a result of exposure to stressors, which can be psychological, physiological, or environmental. One of the mechanisms through which stress induces immunosuppression is by activating the hypothalamic-pituitary-adrenal axis, leading to the release of stress hormones such as cortisol\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Cortisol, also known as hydrocortisone (HCOR) when supplied as a drug, is a steroid hormone produced by the adrenal glands in response to stress\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. It plays a pivotal role in regulating various physiological processes, including metabolism, inflammation, and immune function\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Especially, when released into the bloodstream, cortisol binds to glucocorticoid receptors on immune cells, thereby suppressing immune responses\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChemotherapy is a fundamental component of cancer treatment, yet it carries the risk of inducing immunosuppression, which can potentially impact patient outcomes. While chemotherapy effectively targets cancer cells, it can also affect rapidly dividing cells in the immune system, including leukocytes and bone marrow precursors, leading to compromised immune function\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Consequently, patients may experience heightened susceptibility to infections, delayed wound healing, and other immune-related complications. Managing and monitoring immune function during chemotherapy are crucial to mitigate the risk of infections and other adverse effects, underscoring the importance of comprehensive patient care.\u003c/p\u003e \u003cp\u003eSocheongryong-tang (SCRT) has traditionally been used as an herbal medicine formula for managing bronchitis, asthma, rhinitis, and cold-related symptoms across East Asia, including Korea, China, and Japan\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. SCRT composes 8 herbal components: Pinellia ternata Rhizoma, Paeonia lactiflora Pall. Radix, Ephedra sinica Stadf. Herba, Zingiber officinale Rosc. Rhizoma, Glycyrrhiza glabra L. Radix, Cinnamomum cassia Blume. Ramulus, Asarum sieboldii F. Maekawa Radix, and Schizandra chinensis Fructus\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The major components of SCRT include 6-gingerol, liquiritigenin, and glycyrrhizin, as well as phenylpropanoids such as cinnamic acid, cinnamaldehyde, and coumarin; monoterpene glycosides such as paeoniflorin and albiflorin; lignans including schizandrin, gomisin A, and gomisin N; and alkaloids including ephedrine and pseudoephedrine\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate the immunomodulatory properties of SCRT aqueous extract and elucidate its underlying mode of action in macrophages. Our findings provide a molecular trait for the reversal of stress- or chemotherapy-induced immunosuppression by SCRT for the first time, and could be used to help in the development of pharmaceuticals aimed at manipulating immunomodulation.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Effects of SCRT on phagocytic capacity on RAW264.7 macrophages\u003c/h2\u003e \u003cp\u003eTo determine the optimal concentration of SCRT without cytotoxicity, we first evaluated the cell viability on RAW264.7 cells. SCRT did not have cytotoxic effect for 24 h up to a concentration of 50 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Based on this result, SCRT was used at a concentration of 12.5\u0026ndash;50 \u0026micro;g/mL in further experiments. Next, we confirmed the phagocytic activity of macrophagocytes. We observed a dose-dependent increase in phagocytic activity upon treatment with SCRT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Specifically, we observed irregular polygons and an elevated number of engulfed particles surrounding the cells in response to SCRT treatment (25\u0026ndash;50 \u0026micro;g/mL), mirroring the effects observed with LPS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These findings suggest that SCRT has the potential to enhance phagocytic activity in a dose-dependent manner compared to the normal condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. SCRT enhances NO and PGE2 production on RAW264.7 macrophages\u003c/h2\u003e \u003cp\u003eTo investigate the potential immunomodulatory effects of SCRT on RAW264.7 macrophages, we assessed the production of NO and PGE\u003csub\u003e2\u003c/sub\u003e by using a griess assay and PGE\u003csub\u003e2\u003c/sub\u003e enzyme-linked immunosorbent assay (ELISA). Our results revealed that SCRT significantly increased the concentration of NO and PGE\u003csub\u003e2\u003c/sub\u003e compared to the normal condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, we investigated whether SCRT can enhance the expression of \u003cem\u003eInos\u003c/em\u003e and \u003cem\u003ePtgs2\u003c/em\u003e. The treatment of SCRT dose-dependently increased the levels of \u003cem\u003eInos\u003c/em\u003e and \u003cem\u003ePtgs2\u003c/em\u003e compared to the normal condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Additionally, the protein expression of iNOS and COX-2 were significantly enhanced by SCRT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). To verify whether SCRT treatment affect the expression of immunomodulatory-related pro-inflammatory cytokines, such as \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e in RAW264.7 cells, we examined the expression levels of these cytokines induced by SCRT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Indeed, these cytokines were known to induce M1 polarized macrophage associating with pro-inflammatory phenotype\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. SCRT is associated with activation of ROS/MAPK/NF-κB signaling axis\u003c/h2\u003e \u003cp\u003eGiven that p65, the subunit of NF-κB, has been implicated in the regulation of pro-inflammation and expression of cytokines, such as TNF-α, IL-1β, and IL-6\u003csup\u003e24,25\u003c/sup\u003e, we further evaluated that the SCRT could modulate immunomodulatory effects through the activation of p65 associated with \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e. SCRT-treated macrophages displayed significantly increased the p65 phosphorylation compared to those in non-treated macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Next, to monitor p65 trans-activity, we assessed the NF-κB reporter assay and observed that SCRT treatment increased the trans-activity of NF-κB compared to the normal condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). As NO and PGE\u003csub\u003e2\u003c/sub\u003e production were previously found to be activated by MAPK pathway, core regulatory molecules\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, we further evaluated the phosphorylation level of ERK1/2, p38, and JNK in RAW264.7 macrophages and found that SCRT-treated macrophages exhibited the elevation of ERK1/2, p38, and JNK phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, based on the previously determined the association between reactive oxygen species (ROS) generation and various pathological states, including the regulation of inflammation by M1 polarized macrophages\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, our investigation attempted to determine whether SCTR treatment was associated with elevated ROS release. We found that ROS production was significantly increased by SCRT treatment in a dose-dependet manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Taken together, the immunomodulatory properties of SCRT are associated with p65 and MAPK activation caused by ROS production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. SCRT recovers the immunomodulatory properties during stress-induced immunosuppression\u003c/h2\u003e \u003cp\u003eTo verify whether SCRT treatment can improve its immunomodulatory properties even under stressful condition, we examined the immunity-boosting effects in HCOR-treated RAW264.7 cells. HCOR reduced LPS-stimulated production of NO and PGE\u003csub\u003e2\u003c/sub\u003e, as well as the mRNA expression of \u003cem\u003eInos\u003c/em\u003e and \u003cem\u003ePtgs2\u003c/em\u003e; however, SCRT treatment ameliorated the situation in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD)). In addition, the downregulation of mRNA expression of cytokines (e.g., \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e) induced by HCOR was also reversed by SCRT treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Taken together, our findings indicate that SCRT treatment restores immunomodulatory properties during chemotherapy-induced immunosuppression. These results demonstrate that SCRT treatment restores immunomodulatory properties during stressful-induced immunosuppression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. SCRT recovers the immunomodulatory properties during chemotherapy-induced immunosuppression\u003c/h2\u003e \u003cp\u003eSubsequently, we examined the effect of various anti-cancer drugs [e.g., sorafenib, etoposide, tamoxifen, and 5-fluorouracil (5-FU)] on chemotherapy-induced immunosuppression in LPS-stimulated RAW264.7 cells. Among them, only 5-FU induced immunosuppression, a phenomenon improved by SCRT treatment (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A-1C\u003c/b\u003e). The decreased LPS-stimulated production of NO and PGE\u003csub\u003e2\u003c/sub\u003e induced by 5-FU, as well as the mRNA expression of \u003cem\u003eInos\u003c/em\u003e and \u003cem\u003ePtgs2\u003c/em\u003e, were dose-dependently reversed by SCRT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u0026minus;\u0026thinsp;5\u003cb\u003eD\u003c/b\u003e). Furthermore, similar results were observed regarding the modulation of cytokines mRNA expression (e.g., \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e) by SCRT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Taken together, our findings suggest that SCRT treatment restores immunomodulatory properties during chemotherapy-induced immunosuppression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe therapeutic potential of SCRT in managing seasonal colds and inflammatory disorders has long been recognized in oriental traditional medicine. However, despite some evidence of its anti-inflammatory activity, the precise mechanistic significance of SCRT in immunomodulation remains uncertain\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In this study, we aimed to elucidate the immunomodulatory effects of SCRT on RAW264.7 macrophages. Our findings demonstrated that SCRT treatment enhanced phagocytic activity and induced the release of NO and PGE\u003csub\u003e2\u003c/sub\u003e. These findings are consistent with previous reports linking NO and PGE\u003csub\u003e2\u003c/sub\u003e production to immunomodulation, particularly in M1 polarized macrophages, which play crucial roles in pro-inflammatory responses and cytokine production\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Furthermore, SCRT treatment activated the ROS/MAPKs/NF-κB signaling pathway, leading to the production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. These responses indicate fundamental immunostimulatory effects, which contradict its traditional use in managing inflammatory conditions.\u003c/p\u003e \u003cp\u003eIn previous studies where anti-inflammatory effects were observed, SCRT was administered at high concentrations (up to 1000 \u0026micro;g/ml). In our study, demonstrating immune-boosting effects, SCRT was administered at low concentrations (up to 50 \u0026micro;g/ml)\u003csup\u003e18\u003c/sup\u003e. We speculate that these differences in SCRT treatment concentrations may have contributed to the distinct effects observed between anti-inflammation and immune enhancement, although the underlying mechanisms governing SCRT's dual activities remain unclear. Especially, it is conceivable that SCRT contains bioactive compounds capable of modulating key signaling pathways involved in immunomodulation. Among the various ingredients of SCRT, Paeonia lactiflora Pall. Radix has been known for its vasodilatory effects, while Zingiber officinale Rosc. Rhizoma and Schizandra chinensis Fructus have been recognized for their immune-enhancing properties\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Inactivation or activation of the ROS/MAPKs/NF-κB signaling pathway by SCRT may contribute to controlling its mode of action, reflecting opposite mechanisms for anti-inflammation and immune enhancement. Nonetheless, comprehensive studies are required to thoroughly elucidate these processes.\u003c/p\u003e \u003cp\u003eInterestingly, SCRT also demonstrated resilience against stress- or chemotherapy-induced immunosuppression, highlighting its potential as a therapeutic agent for mitigating immune dysfunction associated with these conditions. The ability of SCRT to restore immunomodulatory effects in combination with HCOR and 5-FU, respectively, as evidenced by the reversal of NO, PGE\u003csub\u003e2\u003c/sub\u003e, and cytokine downregulation induced by stressors or chemotherapy agents, underscores its versatility and therapeutic potential in managing immune-related disorders under stress- and chemotherapy-induced immunosuppressive conditions.\u003c/p\u003e \u003cp\u003eIn conclusion, the contradictory activities of SCRT, exhibiting both anti-inflammatory and immune-enhancing effects, provide novel insights on its therapeutic potential. Particularly, the ability of SCRT to ameliorate stress- or chemotherapy-induced immunosuppression is crucial. This study will contribute to our understanding of immunomodulatory properties and aid in identifying the specific physiologically active components responsible for SCRT's pharmacological effects.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Preparation of socheongryong-tang water extract\u003c/h2\u003e \u003cp\u003eSCRT, known as Hanpoong Socheongryongtang Ext. Granule (ATC code: R05F), is a product sold by Hanpoong Pharmaceutical Co., Ltd. under strict quality control (Jeonju, Republic Korea) (\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e). The ingredient was added to 90 mL of distilled water and boiled at 90\u0026ndash;100\u0026deg;C for 3 hours. The resulting decoction was then filtered through filter paper with a 5-micrometer pore size, and the filtrate was concentrated using a rotary evaporator. The remaining concentrate was vacuum-dried to obtain a powder. The SCRT powder was dissolved in dimethyl sulfoxide for in vitro experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.2. HPLC (High-Performance Liquid Chromatography) analysis\u003c/h2\u003e \u003cp\u003eA chromatogram of SCRT was obtained using an HPLC-PDA-MS. Resultantly, Seven constituents (ephedrine, albiflorin, paeoniflorin, liquiritin apioside, liquiritin, isoliquiritin apioside, and glycyrrhizin) were confirmed within 30 min using mobile phases comprising solvent A (0.1%, v/v formic acid in water) and solvent B (0.1%, formic acid in acetonitrile) under optimized chromatography conditions: An analytical reversed phase Shimadzu Nexera X2 system comprising of a solvent degassing unit (DGU\u0026thinsp;\u0026minus;\u0026thinsp;20A), binary pump (LC\u0026thinsp;\u0026minus;\u0026thinsp;30AD), auto sampler (SIL\u0026thinsp;\u0026minus;\u0026thinsp;30AC), system controller unit (CBM\u0026thinsp;\u0026minus;\u0026thinsp;20A), photodiode array detector (SPD\u0026thinsp;\u0026minus;\u0026thinsp;M20A), and column oven unit (CTO\u0026thinsp;\u0026minus;\u0026thinsp;20AC) was used for qualitative and/or quantitative analysis. Electrospray ionization (ESI)-mass spectrometry (MS) was performed using a Shimadzu LCMS-2020 system for qualitative analysis. A Phenomenex Luna Omega polar C18 column (150\u0026times;2.1 mm, 1.6 \u0026micro;m) was used. The flow rate was set to 0.2 mL/min and the gradient flow program was as follows \u0026ndash; initial to 1 min: 15% B, 40.0 min: 70% B, 45.0 min: 100% B, 48.0 min: 100% B, and 53.0 min: 15% B. The detection wavelengths for analytes were set at 230, 254, and 280 nm, and the injection volume of 1,000 ppm concentration sample was 3 \u0026micro;L (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Cell culture\u003c/h2\u003e \u003cp\u003eThe murine macrophage RAW264.7 cell line was procured from the American Type Culture Collection (ATCC, Manassas, VA). These murine cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s medium (DMEM, Corning, NY) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA) and 1% penicillin and streptomycin. The cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Cell viability\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were treated with 12.5\u0026ndash;50 \u0026micro;g/mL of SCRT for 24 h, and cell proliferation was assessed using a Quanti-Max\u0026trade; WST-8 Cell Viability Assay kit (Biomax QM3000, Seoul, Republic of Korea) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Phagocytic activity\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were seeded in 96-well plates at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well and incubated overnight until they adhered to the plates. The adherent cells were then treated with the specified concentrations of SCRT or 1 ng/mL of LPS (055:B5, Sigma Aldrich, St. Louis, MO) for 24 h. Following treatment, the phagocytic capacity of murine macrophages was assessed using the Phagocytosis Assay Zymosan Substrate Kit (Abcam, MA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Griess assay\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were incubated with phenol red-free medium containing the SCRT, HCOR, anti-cancer drugs or LPS for 24 h. Then, the supernatant was collected, mixed with an equal volume of Griess reagent (1% sulfanilamide, 5% phosphoric acid, and 0.1% N- (1-naphthyl)-ethylenediamine), and incubated at room temperature for 5\u0026ndash;10 min. Absorbance was determined using a microplate reader at a wavelength of 540 nm. The experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.7. PGE\u003csub\u003e2\u003c/sub\u003e ELISA\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were incubated with fresh medium containing the SCRT, HCOR, anti-cancer drugs or LPS for 24 h. Then, the production of PGE\u003csub\u003e2\u003c/sub\u003e was measured using the PGE\u003csub\u003e2\u003c/sub\u003e Parameter Assay kit (R\u0026amp;D Systems, Minneapolis, MN) according to the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.8. DCF-DA assay\u003c/h2\u003e \u003cp\u003eIntracellular ROS levels were assessed using the DCF-DA staining method. RAW264.7 cells were seeded in 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and treated with SCRT or LPS for 24 h. After treatment, the medium was replaced with fresh medium containing 20 \u0026micro;M 2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescin diacetate (DCF-DA, Sigma-Aldrich) and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 20 min. Fluorescence intensity was measured at excitation wavelength of 485 nm and emission wavelength of 530 nm using an Infinite PRO microreader (TECAN, M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.9. Real-Time PCR Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the RNeasy Mini kit (QIAgen, MD, USA), and cDNA was synthesized using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). Quantitative real-time PCR was performed using QuantStudio 6 Pro real-time PCR System (Applied Biosystems, Foster City, CA), TaqMan\u0026reg; Fast Advanced Master Mix (Applied Biosystems), or AccuPower\u0026reg; 2xGreenStarTM qPCR Master Mix (BIONEER, Daejeon, Republic of Korea). The primer sequences and information for qRT-PCR are provided in \u003cb\u003eSupplementary Tables\u0026nbsp;2 and 3\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.10. Western blotting\u003c/h2\u003e \u003cp\u003eProtein samples were subjected to western blot analysis following previously described methods\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Monoclonal antibodies against iNOS, COX-2, phospho-NF-κB/p65 (Ser536), NF-κB/p65, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-JNK (Thr183/Tyr185), JNK, and GAPDH were obtained from Cell Signaling Technology Inc (Boston, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.11. Dual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003eRAW264.7 cells seeded in 96-well white plates were co-transfected with pNF-κB-Luc and pNL-Luc vectors using FuGENE HD Transfection Reagent (Promega, Madison, WI) following the manufacturer\u0026rsquo;s instructions. After co-transfection for 24 h, the cells were treated with 50 \u0026micro;g/mL SCRT or 1 ng/mL LPS for 6 h. Subsequently, the cells were lysed, and luciferase activity was measured using a dual-luciferase reporter assay system (Promega) and GloMax\u0026reg; Navigator Microplate Luminometer (Promega) according to the manufacturer\u0026rsquo;s instructions. The relative firefly luciferase activity was normalized to NanoLuc\u0026reg; luciferase expression to adjust for variations in transfection efficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.12. Statistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (S.D.) of at least three independent readings for each experiment. Statistical analysis was performed using Student\u0026rsquo;s t-tests and one-way ANOVA to evaluate deviations between the means.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTNF-\u0026alpha;, tumor necrosis factor-alpha; IL-1\u0026beta;, interleukin-1 beta; IL-6,\u0026nbsp;interleukin-6; NO, nitric oxide; PGE\u003csub\u003e2\u003c/sub\u003e, prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenases 2; LPS, lipopolysaccharide; HCOR, hydrocortisone; SCRT, socheongryong-tang; ELISA, enzyme-linked immunosorbent assay; ROS, reactive oxygen species; 5-FU, 5-fluorouracil\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interests\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by the Korea Institute of Science and Technology (KIST) institutional program (Project No. 2E33301 and 2Z07011).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.J. and H.K. conducted experiments, interpreted data, and prepared the manuscript. D.W. also conducted experiments. T.K., K.S.K., and S.N.K provided valuable contributions to result interpretation. Y.K. collaborated in manuscript writing, supervised, and provided financial support for the study.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e \u003cp\u003eThe data generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbas, A. K. \u0026amp; Janeway, C. A., Jr. Immunology: improving on nature in the twenty-first century. Cell 100, 129\u0026ndash;138, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0092-8674(00)81689-x\u003c/span\u003e\u003cspan address=\"10.1016/s0092-8674(00)81689-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTosi, M. F. 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This study aimed to elucidate the novel biological activity and mode of action underlying the immunity-boosting effects of SCRT in murine macrophages. Our findings demonstrate that SCRT significantly enhances phagocytic activity, productions of nitric oxide (NO) and prostaglandin E\u003csub\u003e2\u003c/sub\u003e (PGE\u003csub\u003e2\u003c/sub\u003e), and mRNA expression of cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). These effects are attributed to the activation of the reactive oxygen species (ROS)/mitogen activated protein kinases (MAPKs)/nuclear factor-κB (NF-κB) signaling axis. Importantly, SCRT maintains its immunomodulatory effects even under stressful conditions induced by hydrocortisone (HCOR) treatment or chemotherapy with 5-fluorouracil (5-FU). This resilience against stress or chemotherapy-induced immunosuppression underscores the potential of SCRT aqueous extract as a promising therapeutic agent for mitigating immunosuppression associated with stress or chemotherapy.\u003c/p\u003e","manuscriptTitle":"Reversal of stress- or chemotherapy-induced immunosuppression by socheongryong-tang aqueous extract","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 18:03:24","doi":"10.21203/rs.3.rs-4096694/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bfa7d97b-8918-4ffc-8a97-6cbc34665c07","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29979155,"name":"Biological sciences/Cell biology"},{"id":29979156,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2024-06-04T05:45:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-01 18:03:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4096694","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4096694","identity":"rs-4096694","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}