益气解毒方通过抑制NF-κB通路的过度激活改善小鼠肝癌免疫微环境

doi:10.12122/j.issn.1673-4254.2026.03.16

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (3): 629-637.doi: 10.12122/j.issn.1673-4254.2026.03.16

• 基础研究 • 上一篇

益气解毒方通过抑制NF-κB通路的过度激活改善小鼠肝癌免疫微环境

王泽安¹(), 刘丽丽², 施美², 侯勇², 董莉莉³, 张国梁²()

^1.安徽中医药大学第一临床医学院，安徽合肥 230038
^2.安徽中医药大学第一附属医院感染科，安徽合肥 230031
^3.上海曙光医院安徽医院肝病科，安徽合肥 230031

收稿日期:2025-08-05 出版日期:2026-03-20 发布日期:2026-03-26
通讯作者: 张国梁 E-mail:zf_wangzean@126.com;zhangguoliang61@sina.com
作者简介:王泽安，在读硕士研究生，E-mail: zf_wangzean@126.com
基金资助:
全国名中医肝胆系疾病学术思想传承及特色方药的临床开发研究(202204295107020040);国家中医药管理局全国名老中医药专家传承工作室建设项目（国中医药人教函(2022175号);徐经世国医大师工作室创新团队项目(皖秘2023-11)

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

Ze'an WANG¹(), Lili LIU², Mei SHI², Yong HOU², Lili DONG³, Guoliang ZHANG²()

^1.First Clinical Medical College, Anhui University of Chinese Medicine, Hefei 230038, China
^2.Department of Infectious Diseases, First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230031, China
^3.Department of Liver Diseases, Anhui Hospital of Shanghai Shuguang Hospital, Hefei 230031, China

Received:2025-08-05 Online:2026-03-20 Published:2026-03-26
Contact: Guoliang ZHANG E-mail:zf_wangzean@126.com;zhangguoliang61@sina.com

摘要/Abstract

摘要：

目的探究益气解毒方对Hep3B肝癌小鼠模型中免疫微环境的调控机制。方法选取40只SPF级雄性小鼠，小鼠腋下注射Hep3B肿瘤细胞，建立异位小鼠模型，将小鼠随机分为模型组（生理盐水0.2 g/kg），华蟾素片组（0.22 g/kg），益气解毒方低（0.5 g/kg）、中（1 g/kg）、高（2 g/kg）剂量组，8只/组。连续灌胃给药28 d，每7 d记录各组小鼠瘤重、体积。给药结束后计算抑瘤率，通过HE染色观察小鼠肿瘤组织病理变化，流式细胞术检测肿瘤组织中M1/M2型巨噬细胞极化比例，免疫荧光术检测CD86、CD206蛋白表达，Western blotting检测小鼠瘤体组织中iNOS、Arg-1、p65、p50蛋白表达，q-PCR检测iNOS、Arg-1、p65、p50 mRNA表达，ELISA法检测血清IL-4、IL-6、IL-10、INF-γ、TNF-α、TGF-β1水平。结果与模型组比较，益气解毒方各组小鼠肿瘤质量、体积减小；肿瘤细胞数量减少，细胞核缩小，坏死区域增大；M1型巨噬细胞比例升高，M2型巨噬细胞比例降低；肿瘤组织中CD86表达上升，CD206表达下降；iNOS、Arg-1、p65、p50蛋白及mRNA表达降低；血清IL-4、IL-10、TNF-α、TGF-β1水平降低，IL-6、INF-γ水平升高（P<0.05）。结论益气解毒方能有效抑制Hep3B肝癌荷瘤小鼠肿瘤生长，逆转肿瘤相关巨噬细胞的极化状态，将M1/M2比例从M2主导调整至M1主导，从而改善肿瘤免疫抑制微环境，增强抗肿瘤效应，其作用机制可能与抑制NF-κB信号通路的过度激活有关。

关键词: 原发性肝癌, 益气解毒方, 巨噬细胞, 免疫微环境, 信号通路

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

王泽安, 刘丽丽, 施美, 侯勇, 董莉莉, 张国梁. 益气解毒方通过抑制NF-κB通路的过度激活改善小鼠肝癌免疫微环境[J]. 南方医科大学学报, 2026, 46(3): 629-637.

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.

图/表 10

表1 各基因引物序列

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

表2 裸鼠体质量、瘤质量、肿瘤体积与抑瘤率

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

图1 益气解毒方对各组Hep3B肝癌小鼠肿瘤质量、体积的影响

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).

图2 益气解毒方对各组Hep3B肝癌小鼠肿瘤组织病理的影响

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.

图3 流式检测各组Hep3B肝癌小鼠中巨噬细胞极化水平

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.

图4 益气解毒方对各组Hep3B肝癌小鼠组织免疫荧光染色观察

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.

图5 益气解毒方对各组Hep3B肝癌小鼠组织免疫荧光相对表达

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).

图6 各组Hep3B肝癌小鼠组织中目标蛋白免疫印迹分析及相对蛋白表达量

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.

图7 各组Hep3B肝癌小鼠组织中相关mRNA的影响

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.

图8 各组Hep3B肝癌小鼠血清IL-4, IL-6, IL-10, INF-γ, TNF-α, TGF-β1水平的影响

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.

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益气解毒方通过抑制NF-κB通路的过度激活改善小鼠肝癌免疫微环境

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

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