A multiomics study uncovers the immune-enhancing effects and mechanisms of Zhenqi granules

Abstract

Many nations have prohibited the use of antibiotic growth promoters because of concerns regarding the increased development of antimicrobial resistance and the risks associated with drug residues. Traditional Chinese medicines (TCMs) are valuable alternatives to antibiotics. Our previous research demonstrated that Zhenqi granules (ZQ), a formula containing Huangqi and Nvzhenzi, significantly promoted the growth of pigs. This study further investigated the fundamental mechanism from the perspective of immune enhancement by combining transcriptomics, network pharmacology, and cell experiments. Piglet experiments revealed that ZQ administration significantly increased the production of antibodies against CSFV following vaccination. Moreover, compared with control piglets, ZQ-treated piglets presented significantly increased serum cytokine levels and peripheral blood lymphocytes proliferation, indicating the immune-enhancing effect of ZQ. Comparative transcriptomic analysis of spleen tissues revealed that ZQ administration altered the expression of numerous immunity-related genes, with significant enrichment in the calcium signaling pathway. Network pharmacology analysis demonstrated that multiple active components of ZQ interact with immune-related targets, particularly those in the calcium signaling pathway. In vitro cell experiments confirmed that ZQ treatment activated key proteins and downstream transcription factors in this pathway. These findings provide mechanistic insights into the immune-enhancing effect of ZQ, in which the calcium signaling pathway plays a key role.

Keywords

Immunomodulation / Zhenqi granules / Growth promoter / Transcriptomics / Network pharmacology / Calcium signaling pathway

References

[1] Amadori M, Zanotti C. Immunoprophylaxis in intensive farming systems: The way forward. Veterinary Immunology and Immunopathology, 2016, 181: 2-9

[2] Besedovsky HO, Del Rey A. Introduction: Immune-neuroendocrine network. Frontiers of Hormone Research, 2002, 29: 1-14

[3] Boyman O, Kolios AGA, Raeber ME. Modulation of T-cell responses by IL-2 and IL-2 complexes. Clinical and Experimental Rheumatology, 2015, 33: S54-S57

[4] Bui JD, Calbo S, Hayden-Martinez K, Kane LP, Gardner P, Hedrick SM. A role for CaMKII in T-cell memory. Cell, 2000, 100: 457-467

[5] Cheng Y, Chen Y, Li J, Qu H, Zhao Y, Wen C, Zhou Y. Dietary β-sitosterol regulates serum lipid level and improves immune function, antioxidant status, and intestinal morphology in broilers. Poultry Science, 2020, 99: 1400-1408

[6] Dong Z, Jia X. Feeding and management factors and prevention measures that cause low immunity in pigs. World Tropical Agriculture Information, 2023, 11: 69-70

[7] Feske S. Calcium signaling in lymphocyte activation and disease. Nature Reviews Immunology, 2007, 7: 690-702

[8] Feske S, Wulff H, Skolnik EY. Ion channels in innate and adaptive immunity. Annual Review of Immunology, 2015, 33: 291-353

[9] Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K. Development of monocytes, macrophages, and dendritic cells. Science, 2010, 327: 656-661

[10] Hao S-H, Ma X-D, Xu L, Xie J-D, Feng Z-H, Chen J-W, Chen R-X, Wang F-W, Tang Y-H, Xie D, Cai M-Y. Dual specific phosphatase 4 suppresses ferroptosis and enhances sorafenib resistance in hepatocellular carcinoma. Drug Resistance Updates, 2024, 73 101052

[11] Huang, Y., Luo, W., Yao, Z., Qiu, X., Zhuo, W., Zhang, L., Liu, J., Huang, Q., Zhou, R. 2024. Effect of Zhenqi granules on weaning stress in piglets. Journal of Traditional Chinese Veterinary Medicine 43: 1–7+105.

[12] Hynes NE, Lane HA. ERBB receptors and cancer: The complexity of targeted inhibitors. Nature Reviews Cancer, 2005, 5: 341-354

[13] Jukes TH, Stokstad ELR, Tayloe RR, Cunha TJ, Edwards HM, Meadows GB. Growth-promoting effect of aureomycin on pigs. Archives of Biochemistry, 1950, 26: 324-325

[14] Kong X, Zhang Y, Yin Y, Wu G, Zhou H, Tan Z, Yang F, Bo M-J, Huang R, Li T, Geng M. Chinese yam polysaccharide enhances growth performance and cellular immune response in weanling rats. Journal of the Science of Food and Agriculture, 2009, 89: 2039-2044

[15] Lagattuta KA, Kang JB, Nathan A, Pauken KE, Jonsson AH, Rao DA, Sharpe AH, Ishigaki K, Raychaudhuri S. Repertoire analyses reveal T-cell antigen receptor sequence features that influence T-cell fate. Nature Immunology, 2022, 23: 446-457

[16] Lee T-I, Yang C-S, Fang K-M, Tzeng S-F. Role of ciliary neurotrophic factor in microglial phagocytosis. Neurochemical Research, 2009, 34: 109-117

[17] León-Ponte M, Ahern GP, O'connell PJ. Serotonin provides an accessory signal to enhance T-cell activation by signaling through the 5-HT7 receptor. Blood, 2007, 109: 3139-3146

[18] Li LL, Fugui Y, Zhang B, Hanhui P, Li FN, Nong Z, De-Xing H, Yulong Y, Jie L, Tang ZR, Guilin L. Dietary supplementation with Atractylodes macrophala Koidz polysaccharides ameliorate metabolic status and improve immune function in early-weaned pigs. Livestock Science, 2011, 142: 33-41

[19] Li, Q., T. Zhao, G. Mao, W. Feng, Y. Chen, T. Zou, L. Yang, and J.Y. Qian. 2023. A Se-enriched Grifola frondosa polysaccharide induces macrophage activation by TLR4-mediated MAPK signaling pathway. International Journal of Biological Macromolecules 238:124108. https://doi.org/10.1016/j.ijbiomac.2023.124108.

[20] Li T, Qin W, Wu B, Jin X, Zhang R, Zhang J, Du L. Effects of glycyrrhiza polysaccharides on growth performance, meat quality, serum parameters and growth/meat quality-related gene expression in broilers. Frontiers in Veterinary Science, 2024, 11: 1357491

[21] Lillehoj H, Liu Y, Calsamiglia S, Fernandez-Miyakawa ME, Chi F, Cravens RL, Oh S, Gay CG. Phytochemicals as antibiotic alternatives to promote growth and enhance host health. Veterinary Research, 2018, 49: 76

[22] Lin S-S, Tzeng B-H, Lee K-R, Smith RJH, Campbell KP, Chen C-C. Cav3.2 T-type calcium channel is required for the NFAT-dependent Sox9 expression in tracheal cartilage. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111: E1990-E1998

[23] Liu J, Nile SH, Xu G, Wang Y, Kai G. Systematic exploration of Astragalus membranaceus and Panax ginseng as immune regulators: Insights from the comparative biological and computational analysis. Phytomedicine, 2021, 86 153077

[24] Liu S, Geng R, Lin E, Zhao P, Chen Y. ERBB1/2/3 expression, prognosis, and immune infiltration in cutaneous melanoma. Frontiers in Genetics, 2021, 12 602160

[25] Liu L, Jiao Y, Yang M, Wu L, Long G, Hu W. Network pharmacology, molecular docking and molecular dynamics to explore the potential immunomodulatory mechanisms of deer antler. International Journal of Molecular Sciences, 2023

[26] Long LN, Kang BJ, Jiang Q, Chen JS. Effects of dietary Lycium barbarum polysaccharides on growth performance, digestive enzyme activities, antioxidant status, and immunity of broiler chickens. Poultry Science, 2020, 99: 744-751

[27] Luan S, Wang C. Calcium signaling mechanisms across kingdoms. Annual Review of Cell and Developmental Biology, 2021, 37: 311-340

[28] Luo W, Huang Y, Qiu X, Zhuo W, Tao Y, Wang S, Li H, Shen J, Zhao L, Zhang L, Li S, Liu J, Huang Q, Zhou R. Growth-promoting effects of Zhenqi granules on finishing pigs. Animals, 2022, 12 3521

[29] Menshova RV, Anastyuk SD, Ermakova SP, Shevchenko NM, Isakov VI, Zvyagintseva TN. Structure and anticancer activity in vitro of sulfated galactofucan from brown alga alaria angusta. Carbohydrate Polymers, 2015, 132: 118-125

[30] Moore PR, Evenson A. Use of sulfasuxidine, streptothricin, and streptomycin in nutritional studies with the chick. Journal of Biological Chemistry, 1946, 165: 437-441

[31] Mössner R, Lesch KP. Role of serotonin in the immune system and in neuroimmune interactions. Brain, Behavior, and Immunity, 1998, 12: 249-271

[32] Poggi A, Carosio R, Spaggiari GM, Fortis C, Tambussi G, Dell'antonio G, Dal Cin E, Rubartelli A, Zocchi MR. NK cell activation by dendritic cells is dependent on LFA-1-mediated induction of calcium-calmodulin kinase II: Inhibition by HIV-1 Tat C-terminal domain. The Journal of Immunology, 2002, 168: 95-101

[33] Prakriya M, Lewis RS. Store-operated calcium channels. Physiological Reviews, 2015, 95: 1383-1436

[34] Qin L, Chen S, Xie L, Yu Q, Chen Y, Shen M, Xie J. Mechanisms of RAW264.7 macrophages immunomodulation mediated by polysaccharide from mung bean skin based on RNA-seq analysis. Food Research International, 2022, 154 111017

[35] Qiu S, Zhang B, Chen X, Liu Y, Tan J, Wu C. Effects of traditional medicine combined with CSF vaccine on the levels of antibody against CSFV and blood routine indexes in pigs. Animal Husbandry & Veterinary Medicine, 2016, 48: 85-89

[36] Rahman MRT, Fliss I, Biron E. Insights in the development and uses of alternatives to antibiotic growth promoters in poultry and swine production. Antibiotics, 2022

[37] Renda G, Gökkaya İ, Şöhretoğlu D. Immunomodulatory properties of triterpenes. Phytochemistry Reviews, 2022, 21: 537-563

[38] Schreck R, Albermann K, Baeuerle PA. Nuclear factor kappa B: An oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radical Research Communications, 1992, 17: 221-237

[39] Shao B-M, Xu W, Dai H, Tu P, Li Z, Gao X-M. A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb. Biochemical and Biophysical Research Communications, 2004, 320: 1103-1111

[40] Shi H. Effect of Fuzheng Jiedu granule on the immune response to swine fever vaccine, 2016, Master, South China Agricultural University

[41] Sim GC, Radvanyi L. The IL-2 cytokine family in cancer immunotherapy. Cytokine & Growth Factor Reviews, 2014, 25: 377-390

[42] Song B, Guan S, Lu J, Chen Z, Huang G, Li G, Xiong Y, Zhang S, Yue Z, Deng X. Suppressive effects of fisetin on mice T lymphocytes in vitro and in vivo. Journal of Surgical Research, 2013, 185: 399-409

[43] Song, K. 2023. Preliminary study on the effect and mechanism of Qilian Powder on PRRS vaccine immunity. Doctor, JIlin Universuty.

[44] Trebak M, Kinet J-P. Calcium signaling in T cells. Nature Reviews Immunology, 2019, 19: 154-169

[45] Vaeth M, Kahlfuss S, Feske S. CRAC channels and calcium signaling in T-cell-mediated immunity. Trends in Immunology, 2020, 41: 878-901

[46] Van Boeckel TP, Pires J, Silvester R, Zhao C, Song J, Criscuolo NG, Gilbert M, Bonhoeffer S, Laxminarayan R. Global trends in antimicrobial resistance in animals in low- and middle-income countries. Science, 2019

[47] Wang X, Hu L-P, Qin W-T, Yang Q, Chen D-Y, Li Q, Zhou K-X, Huang P-Q, Xu C-J, Li J, Yao L-L, Wang Y-H, Tian G-A, Yang J-Y, Yang M-W, Liu D-J, Sun Y-W, Jiang S-H, Zhang X-L, Zhang Z-G. Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis. Nature Communications, 2021, 12: 174

[48] Wang T, Jiang G, Lv S, Xiao Y, Fan C, Zou M, Wang Y, Guo Q, Ahsanul Kabir M, Peng X. Avian safety guardian: Luteolin restores Mycoplasma gallisepticum-induced immunocompromise to improve production performance by inhibiting the IL-17/NF-kB pathway. International Immunopharmacology, 2023, 124 110946

[49] Wang H, Xu S, Mao H, Wang B, Feng Y, Ihsan A, Li S, Wang X. Pharmacodynamic evaluation and network pharmacology analysis of a novel anti-heat stress Chinese herbal formula. Animal Diseases, 2024, 4: 42

[50] Wang H, Zheng X, Zheng D, Wang X, Zhao Z, Zhao M, Guo Q, Mu Y. Monoclonal antibody against porcine LAG3 inhibits porcine reproductive and respiratory syndrome virus infection. Veterinary Sciences, 2024

[51] Wang H, Zhu W, Hong Y, Wei W, Zheng N, He X, Bao Y, Gao X, Huang W, Sheng L, Li M, Li H. Astragalus polysaccharides attenuate chemotherapy-induced immune injury by modulating gut microbiota and polyunsaturated fatty acid metabolism. Phytomedicine, 2024, 128 155492

[52] Wu Y, Li N, Zhang T, Che Y, Duan K, Wang Y, Zhou H, Wan X, Lei H, Nguyễn AD, De Souza C, Li K, Wu Y, Liu J, Wang D. Glycyrrhiza polysaccharides can improve and prolong the response of chickens to the newcastle disease vaccine. Poultry Science, 2022, 101 101549

[53] Xiao C, Li K, Teng C, Wei Z, Li J, Zhang S, Liu L, Lv H, Zhong R. Dietary Qi-Weng-Huangbo powder enhances growth performance, diarrhea and immune function of weaned piglets by modulating gut health and microbial profiles. Frontiers in Immunology, 2023, 14: 1342852

[54] Xiong Y, Zhang S, Lu J, Sun S, Song B, Xu L, Yang Z, Guan S. Investigation of effects of farrerol on suppression of murine T lymphocyte activation in vitro and in vivo. International Immunopharmacology, 2013, 16: 313-321

[55] Yang JX, Maria TC, Zhou B, Xiao FL, Wang M, Mao YJ, Li Y. Quercetin improves immune function in arbor acre broilers through activation of NF-κB signaling pathway. Poultry Science, 2020, 99: 906-913

[56] Yang M, Yang T, Chen F, Ma J, Chen Q. Effect of achyranthes bidentata polysaccharides on antibody titer and blood routine indexs of swine fever vaccine in vaccine-vaccinated piglets. Chinese Journal of Veterinary Science, 2020, 40: 2293-2297

[57] Yu Q, Nie S-P, Wang J-Q, Huang D-F, Li W-J, Xie M-Y. Signaling pathway involved in the immunomodulatory effect of Ganoderma atrum polysaccharide in spleen lymphocytes. Journal of Agricultural and Food Chemistry, 2015, 63: 2734-2740

[58] Zeng P, Li J, Chen Y, Zhang L. The structures and biological functions of polysaccharides from traditional Chinese herbs. Progress in Molecular Biology and Translational Science, 2019, 163: 423-444

[59] Zhang X. Eukaryotic expression of E2 protein and preliminary evaluation of immune efficacy in classical swine fever, 2024, Master, Huazhong Agricultural University

[60] Zhao Y, Yan B, Wang Z, Li M, Zhao W. Natural polysaccharides with immunomodulatory activities. Mini Reviews in Medicinal Chemistry., 2020, 20: 96-106

[61] Zhao L, Zhang H, Li N, Chen J, Xu H, Wang Y, Liang Q. Network pharmacology, a promising approach to reveal the pharmacology mechanism of Chinese medicine formula. Journal of Ethnopharmacology, 2023, 309 116306

[62] Zhou H, Dai C, Cui X, Zhang T, Che Y, Duan K, Yi L, Nguyen AD, Li N, De Souza C, Wan X, Wu Y, Li K, Liu Y, Wu Y. Immunomodulatory and antioxidant effects of glycyrrhiza uralensis polysaccharide in lohmann brown chickens. Frontiers in Veterinary Science, 2022, 9 959449

Funding

the National Key R&D Plans of China(No. 2024YFE0104800)

Natural Science Foundation of Hubei Province(No. 2024AFB734)

RIGHTS & PERMISSIONS

The Author(s)