Yiqi Jiedu Formula regulates immune microenvironment of liver cancer in mice by inhibiting overactivation of NF-κB signaling pathway

doi:10.12122/j.issn.1673-4254.2026.03.16

Abstract

Abstract:

Objective To investigate the regulatory mechanism of Yiqi Jiedu Formula on immune microenvironment of hepatocellular carcinoma (HCC) in mice. Methods Forty male BALB/c mice bearing subcutaneous Hep3B cell xenografts were randomized equally into model group (with normal saline treatment), Huachansu Tablet group, and low- (0.5 g/kg), medium- (1 g/kg), and high-dose (2 g/kg) Yiqi Jiedu Formula groups. All the mice received daily gavage of the corresponding treatments for 28 consecutive days, and the changes in tumor weight and volume were recorded every 7 days, and tumor inhibition rate was calculated after the final administration. Histopathological changes in the tumor tissues were observed with HE staining, and polarization of M1/M2 macrophages was analyzed with flow cytometry. The mRNA and protein expressions of iNOS, Arg-1, p65, and p50 in the tumor tissues were detected using q-PCR and Western blotting, and CD86 and CD206 protein expressions were observed with immunofluorescence staining. Serum levels of IL-4, IL-6, IL-10, IFN‑γ, TNF‑α, and TGF‑β1 of the mice were measured using ELISA. Results The mice in the 3 Yiqi Jiedu Formula groups showed significant reductions in tumor weight and volume with decreased tumor cell count, increased tumor cell apoptosis, increased percentage of M1 macrophages, and decreased percentage of M2 macrophages. The treatment obviously upregulated CD86 and downregulated CD206 expression in the tumor tissue, lowered the protein and mRNA expressions of iNOS, Arg-1, p65, and p50, reduced serum levels of IL-4, IL-10, TNF‑α, and TGF-β1, and increased serum levels of IL-6 and IFN-γ in the tumor-bearing mice. Conclusion Yiqi Jiedu Formula effectively inhibits Hep3B cell xenograft growth in mice and induces a shift of M1/M2 ratio by promoting M1 polarization, thereby ameliorating the immunosuppressive tumor microenvironment and enhancing anti-tumor immunity possibly in association with inhibition of NF-κB signaling pathway overactivation.

Key words: primary liver cancer, Yiqi Jiedu Formula, macrophages, immune microenvironment, signaling pathway

Ze'an WANG, Lili LIU, Mei SHI, Yong HOU, Lili DONG, Guoliang ZHANG. Yiqi Jiedu Formula regulates immune microenvironment of liver cancer in mice by inhibiting overactivation of NF-κB signaling pathway[J]. Journal of Southern Medical University, 2026, 46(3): 629-637.

Figures/Tables 10

Tab.1 Primer sequences for q-PCR

mouse-β-actin F

mouse-β-actin R

mouse-iNOS F

mouse-iNOS R

mouse-Arg-1 F

mouse-Arg-1 R

mouse-P65 F

mouse-P65 R

mouse-p50 F

mouse-p50 R

CAGCCTTCCTTCTTGGGTATG

GGCATAGAGGTCTTTACGGATG

GGAGTGACGGCAAACATGACT

TCGATGCACAACTGGGTGAAC

TGTCCCTAATGACAGCTCCTT

GCATCCACCCAAATGACACAT

ACTGCCGGGATGGCTACTAT

TCTGGATTCGCTGGCTAATGG

ATGGCAGACGATGATCCCTAC

TGTTGACAGTGGTATTTCTGGTG

127

204

126

111

Tab.2 Tumor weight, tumor volume, and tumor inhibition rate in the nude mice (Mean±SD, n=5)

Group	Tumor weight (g)	P	Tumor volume (mm³)	P	Tumor inhibition rate (%)
Model	0.63±0.18	-	682.901±214.39	-	-
Low dose group of Yiqi Jiedu Formula	0.41±0.19	0.028	410.06±184.38	0.018	40.95
Medium dose group of Yiqi Jiedu Formula	0.35±0.15	0.020	375.16±128.08	0.009	43.99
High dose group of Yiqi Jiedu Formula	0.33±0.13	0.002	200.66±120.57	0.00	60.13
Cinobufagin Tablets	0.24±0.09	0.002	174.00±63.65	0.00	61.34

Fig.1 Effects of Yiqi Jiedu Formula on tumor weight and volume in Hep3B hepatoma-bearing mice. A: Model group. B: Low-dose Yiqi Jiedu Formula group. C: Medium-dose Yiqi Jiedu Formula group. D: High-dose Yiqi Jiedu Formula group. E: Cinobufagin Tablets. *P<0.05, **P<0.01 vs model group (n=5).

Fig.2 Effect of Yiqi Jiedu Formula on histopathology of tumor tissues in Hep3B hepatoma-bearing mice. A: Model group. B: Low-dose Yiqi Jiedu Formula group. C: Medium-dose Yiqi Jiedu Formula group. D: High-dose Yiqi Jiedu Formula group. E: Cinobufagin Tablets.

Fig.3 Flow cytometric analysis of macrophage polarization levels in the tumor tissues of Hep3B hepatoma-bearing mice. A: Model group. B: Low-dose Yiqi Jiedu Formula group. C: Medium-dose Yiqi Jiedu Formula group. D: High-dose Yiqi Jiedu Formula group. E: Cinobufagin Tablets. *P<0.01 vs model group.

Fig.4 Effect of Yiqi Jiedu Formula on tumor tissue assessed by immunofluorescence staining in Hep3B hepatoma-bearing mice (Original magnification: ×400). A: Model group. B: Low-dose Yiqi Jiedu Formula group. C: Medium-dose Yiqi Jiedu Formula group. D: High-dose Yiqi Jiedu Formula group. E: Cinobufagin Tablets.

Fig.5 Effect of Yiqi Jiedu Formula on immunofluorescence intensity of CD 86 and CD206 in the tumor tissues of Hep3B hepatoma-bearing mice. A: Model group. B: Low-dose Yiqi Jiedu Formula group. C: Medium-dose Yiqi Jiedu Formula group. D: High-dose Yiqi Jiedu Formula group. E: Cinobufagin Tablets. *P<0.01 vs model group (n=5).

Fig.6 Western blotting of the target proteins and their relative expression levels in tumor tissues from Hep3B hepatoma-bearing mice. A: Model group. B: Low-dose Yiqi Jiedu Formula group. C: Medium-dose Yiqi Jiedu Formula group. D: High-dose Yiqi Jiedu Formula group. E: Cinobufagin Tablets. *P<0.01 vs model group.

Fig.7 Effect of Yiqi Jiedu Formula on mRNA expression levels in tumor tissues of Hep3B hepatoma-bearing mice. A: Model group. B: Low-dose Yiqi Jiedu Formula group. C: Medium-dose Yiqi Jiedu Formula group. D: High-dose Yiqi Jiedu Formula group. E: Cinobufagin Tablets. *P<0.05, **P<0.01 vs model group.

Fig.8 Effect of Yiqi Jiedu Formula on serum levels of IL-4, IL-6, IFN-γ, TNF-α, and TGF-β1 in Hep3B hepatoma-bearing mice. A: Model group. B: Low-dose Yiqi Jiedu Formula group. C: Medium-dose Yiqi Jiedu Formula group. D: High-dose Yiqi Jiedu Formula group. E: Cinobufagin Tablets. *P<0.05, **P<0.01 vs model group.

References 30

[1]	Feng M, Pan YS, Kong RR, et al. Therapy of primary liver cancer[J]. Innov, 2020, 1(2): 100032. doi：10.1016/j.xinn.2020.100032
[2]	中华医学会肝病学分会. 原发性肝癌二级预防共识（2021年版）[J]. 中华肝脏病杂志, 2021, 29(3): 216-26. doi：10.3969/j.issn.1001-5256.2021.03.008
[3]	陈杨柳, 李媛, 官志杰. 中医药治疗原发性肝癌的研究进展[J].基层中医药, 2024, 3(12): 112-6.
[4]	Yang YM, Kim SY, Seki E. Inflammation and liver cancer: molecular mechanisms and therapeutic targets[J]. Semin Liver Dis, 2019, 39(1): 26-42. doi：10.1055/s-0038-1676806
[5]	Nault JC, Ningarhari M, Rebouissou S, et al. The role of telomeres and telomerase in cirrhosis and liver cancer[J]. Nat Rev Gastroenterol Hepatol, 2019, 16(9): 544-58. doi：10.1038/s41575-019-0165-3
[6]	Boutilier AJ, Elsawa SF. Macrophage polarization states in the tumor microenvironment[J]. Int J Mol Sci, 2021, 22(13): 6995. doi：10.3390/ijms22136995
[7]	Wang C, Ma C, Gong LH, et al. Macrophage polarization and its role in liver disease[J]. Front Immunol, 2021, 12: 803037. doi：10.3389/fimmu.2021.803037
[8]	Mantovani A, Marchesi F, Malesci A, et al. Tumour-associated macrophages as treatment targets in oncology[J]. Nat Rev Clin Oncol, 2017, 14(7): 399-416. doi：10.1038/nrclinonc.2016.217
[9]	Pittet MJ, Michielin O, Migliorini D. Clinical relevance of tumour-associated macrophages[J]. Nat Rev Clin Oncol, 2022, 19(6): 402-21. doi：10.1038/s41571-022-00620-6
[10]	中华人民共和国国家卫生健康委员会医政司. 原发性肝癌诊疗指南（2024年版）[J].协和医学杂志, 2024, 15(3): 532-59. doi：10.3877/cma.j.issn.2095-3232.2024.04.001
[11]	韩雅, 施美, 董莉莉, 等. 益气解毒方对HepG2小鼠模型免疫作用机制调控的实验研究[J]. 环球中医药, 2025, 18(1): 20-6. doi：10.3969/j.issn.1674-1749.2025.01.004
[12]	徐朦, 张鹏, 张国梁. 益气解毒方治疗原发性肝癌的作用机制:基于网络药理学及分子对接方法[J]. 南方医科大学学报, 2022, 42(6):805-14.
[13]	徐朦, 陈丽娜, 吴金玉, 等. “白花蛇舌草-半枝莲”治疗原发性肝癌的机制研究: 基于网络药理学、分子对接及体外实验验证[J]. 南方医科大学学报, 2025, 45(1): 80-9.
[14]	Cheng K, Cai N, Zhu J, et al. Tumor-associated macrophages in liver cancer: From mechanisms to therapy[J]. Cancer Commun: Lond, 2022, 42(11): 1112-40. doi：10.1002/cac2.12345
[15]	He R, He Y, Du R, et al. Revisiting of TAMs in tumor immune microenvironment: Insight from NF‑κB signaling pathway[J]. Biomed Pharmacother, 2023, 165: 115090. doi：10.1016/j.biopha.2023.115090
[16]	Dolcet X, Llobet D, Pallares J, et al. NF-kB in development and progression of human cancer[J]. Virchows Arch, 2005, 446(5): 475-82. doi：10.1007/s00428-005-1264-9
[17]	Galasso L, Cerrito L, Maccauro V, et al. Inflammatory response in the pathogenesis and treatment of hepatocellular carcinoma: a double-edged weapon[J]. Int J Mol Sci, 2024, 25(13): 7191. doi：10.3390/ijms25137191
[18]	Lawrence T. The nuclear factor NF-kappaB pathway in inflammation[J]. Cold Spring Harb Perspect Biol, 2009, 1(6): a001651. doi：10.1101/cshperspect.a001651
[19]	Farhood B, Najafi M, Mortezaee K. CD8⁺ cytotoxic T lymphocytes in cancer immunotherapy: a review[J]. J Cell Physiol, 2019, 234(6): 8509-21. doi：10.1002/jcp.27782
[20]	Shapouri-Moghaddam A, Mohammadian S, Vazini H, et al. Macrophage plasticity, polarization, and function in health and disease[J]. J Cell Physiol, 2018, 233(9): 6425-40. doi：10.1002/jcp.26429
[21]	Kashfi K, Kannikal J, Nath N. Macrophage reprogramming and cancer therapeutics: role of iNOS-derived NO[J]. Cells, 2021, 10(11): 3194. doi：10.3390/cells10113194
[22]	Bejarano L, Jordāo MJC, Joyce JA. Therapeutic targeting of the tumor microenvironment[J]. Cancer Discov, 2021, 11(4): 933-59. doi：10.1158/2159-8290.cd-20-1808
[23]	Zhang X, Lao M, Sun K, et al. Sphingolipid synthesis in tumor-associated macrophages confers immunotherapy resistance in hepatocellular carcinoma[J]. Sci Adv, 2025, 11(21): eadv0558. doi：10.1126/sciadv.adv0558
[24]	Zhou WL, Yang FT, Zhang XZ. Roles of M1 macrophages and their extracellular vesicles in cancer therapy[J]. Cells, 2024, 13(17): 1428. doi：10.3390/cells13171428
[25]	Shao N, Qiu H, Liu J, et al. Targeting lipid metabolism of macrophages: a new strategy for tumor therapy[J]. J Adv Res, 2025, 68: 99-114. doi：10.1016/j.jare.2024.02.009
[26]	Cornice J, Verzella D, Arboretto P, et al. NF‑κB: Governing macrophages in cancer[J].. Genes (Basel), 2024, 15(2):197. doi：10.3390/genes15020197
[27]	Giridharan S, Srinivasan M. Mechanisms of NF‑κB p65 and strategies for therapeutic manipulation[J]. J Inflamm Res, 2018, 11: 407-19. doi：10.2147/jir.s140188
[28]	Napetschnig J, Wu H. Molecular basis of NF-κB signaling[J]. Annu Rev Biophys, 2013, 42: 443-68. doi：10.1146/annurev-biophys-083012-130338
[29]	Yu Z, Li Y, Li Y, et al. Bufalin stimulates antitumor immune response by driving tumor-infiltrating macrophage toward M1 phenotype in hepatocellular carcinoma[J]. J Immunother Cancer, 2022, 10(5): e004297. doi：10.1136/jitc-2021-004297
[30]	Porta C, Rimoldi M, Raes G, et al. Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor kappaB[J]. Proc Natl Acad Sci USA, 2009, 106(35): 14978-83. doi：10.1073/pnas.0809784106