基于程序性死亡受体1/程序性死亡配体1信号通路的益气解毒方对肝癌裸鼠恒定自然杀伤T细胞功能的影响OA
Effect of Yiqi Jiedu Formula on the expression of PD-1 and PD-L1 in iNKT cells and tumor tissues of hepatocellular carcinoma nude mice
目的 探究基于程序性死亡受体 1(programmed cell death protein 1,PD-1)/程序性死亡配体 1(programmed cell death ligand 1,PD-L1)信号通路的益气解毒方对肝癌裸鼠恒定自然杀伤 T细胞(invariant natural killer T cells,iNKT)功能的影响,探究益气解毒方对肝癌裸鼠抗肿瘤的作用机制.方法 选取 50 只裸鼠,雌雄各半,随机分为 10 只正常组和 40 只肝癌模型诱导组.采用人肝细胞癌 Hep3B 细胞构建裸鼠皮下肝癌移植瘤模型,将造模成功的裸鼠随机分为中药组低、中、高剂量组、模型组、华蟾素片组,共五组,每组 8 只.正常组和模型组每天以生理盐水 0.2g/kg 灌胃;华蟾素片组将华蟾素片规格为每片250 mg、每天以 0.22 g/kg 剂量给药;中药高、中、低剂量组分别每天以益气解毒方0.4 g/kg、0.2 g/kg、0.1 g/kg 剂量给药,连续给药28 天,观察裸鼠每天的生长情况,记录裸鼠的肿瘤重量、体积,计算肿瘤生长抑制率.麻醉处死后,通过苏木精—伊红(hematoxylin and eosin,HE)染色观察各组裸鼠肿瘤组织病理变化;流式细胞实验检测 iNKT 细胞活性;酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测血清白细胞介素-2(interleukin-2,IL-2)、白细胞介素-4(interleukin-4,IL-4)、白细胞介素-10(interleukin-10,IL-10)及 γ-干扰素(interferon-γ,IFN-γ)水平;蛋白质免疫印迹试验(western blot,WB)、实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)和免疫荧光单染检测肿瘤组织 PD-1、PD-L1 表达.结果 与模型组对比,中药组和华蟾素片组裸鼠的瘤重、肿瘤体积显著降低(P<0.01,P<0.05)且呈剂量依赖性,中药组、华蟾素片组在不同程度上改善了肿瘤的形状和炎症浸润情况;中药组和华蟾素片组分化簇3 阳性(cluster of differentiation 3 positive,CD3+)、IL-4、IL-10 水平显著降低(P<0.01,P<0.05);IL-2、IFN-γ、分化簇 69 阳性/分化簇 1d 阳性(CD69+/CD1d+)与模型组水平显著升高(P<0.01,P<0.05);中药高剂量组和华蟾素片组 PD-1、PD-L1 蛋白的表达、PD-1、PD-L1 mRNA 表达显著降低(P<0.05,P<0.01),益气解毒方低、中剂量组无明显变化,差异无统计学意义.中药组和华蟾素片组肿瘤生长速度较缓慢,对比模型组体积较小,抑瘤率效果显著.结论 高剂量益气解毒方可以通过增强 iNKT 细胞活性和抗肿瘤免疫应答,抑制 PD-1/PD-L1 信号通路的表达,改善肝癌裸鼠的免疫微环境并抑制肿瘤生长.
Objective To explore the effects of Yiqi Jiedu Formula,a traditional Chinese medicine formula based on the PD-1/PD-L1 signaling pathway,on the function of iNKT cells in hepatocellular carcinome nude mice,and to investigate the mechanism of its anti-tumor effects.Methods Fifty mice,half male and half female,were selected and randomly divided into 10 normal mice and 40 liver cancer model mice.A subcutaneous liver cancer xenograft model in mice was established using Hep3B cells.After successful modeling,the mice were randomly assigned into five groups:traditional Chinese medicine(TCM)groups(low,medium and high doses of the Yiqi Jiedu Formula),model group and Huachansu Tablet group,with 8 mice in each group.The normal and model groups were orally administered 0.2 g/kg normal saline daily;the Huachansu Tablet group received 0.22 g/kg per day,with each tablet containing 250 mg;the high,medium,and low dose TCM groups received daily doses of 0.4 g/kg,0.2 g/kg,and 0.1 g/kg,respectively,for 28 consecutive days.Mice were monitored daily for growth,and tumor weight and volume were recorded to calculate tumor growth inhibition rates.After euthanasia under anesthesia,HE staining was used to observe pathological changes in tumor tissues across groups;flow cytometry was used to evaluate iNKT cell activity;ELISA was employed to detect serum levels of IL-2,IL-4,IL-10,and IFN-γ;Western blot,qRT-PCR,and single immunofluorescence staining were used to detect PD-1 and PD-L1 expression in tumor tissues.Results Compared with the model group,tumor weight and volume were significantly reduced in the TCM and Huachansu Tablet groups(P<0.01,P<0.05)in a dose-dependent manner.Tumor morphology and inflammatory infiltration were improved to varying degrees in the TCM and Huachansu Tablet groups;CD3+,IL-4,and IL-10 levels were significantly decreased in these groups(P<0.01,P<0.05);IL-2,IFN-γ,CD69+/CD 1d+levels were significantly increased compared with the model group(P<0.01,P<0.05);PD-1 and PD-L1 protein and mRNA expression were significantly reduced in the high-dose TCM group and Huachansu Tablet group(P<0.05,P<0.01),while the low-and medium-dose TCM groups showed no significant changes.Tumor growth in the TCM and Huachansu Tablet groups was slower,with smaller volumes and significant tumor inhibition rates compared with the model group.Conclusion The traditional Chinese medicine Yiqi Jiedu Formula can inhibit PD-1/PD-L1 signaling,enhance iNKT cell activity and antitumor immune response,improve the immune microenvironment in hepatocellular carcinoma nude mice and suppress tumor growth.
王震元;张国梁;刘丽丽;韩雅;董丽丽;侯勇;施美
230031 合肥,安徽中医药大学第一附属医院感染科||安徽中医药大学研究生院230031 合肥,安徽中医药大学第一附属医院感染科||国家中医药管理局徐经世国医大师传承工作室||张国梁全国名医工作室230031 合肥,安徽中医药大学第一附属医院感染科||国家中医药管理局徐经世国医大师传承工作室||张国梁全国名医工作室安徽中医药大学研究生院安徽中医药大学研究生院230031 合肥,安徽中医药大学第一附属医院感染科||国家中医药管理局徐经世国医大师传承工作室||张国梁全国名医工作室230031 合肥,安徽中医药大学第一附属医院感染科||国家中医药管理局徐经世国医大师传承工作室||张国梁全国名医工作室
医药卫生
益气解毒方肝癌裸鼠恒定自然杀伤T细胞程序性死亡受体1/程序性死亡配体1信号通路作用机制
Yiqi Jiedu Formulahepatocellular carcinoma nude miceiNKT cellsPD-1/PD-L1 signaling pathwaymechanism of action
《环球中医药》 2026 (5)
982-990,9
国家自然科学基金(81874451)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[2022]175号)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)国家中医药管理局徐经世国医大师传承工作室建设项目(2015072)徐经世国医大师工作室创新团队项目(皖秘2023-11)
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