余甘子叶总黄酮对H22荷瘤小鼠免疫功能调节的影响
Effect of Phyllanthus emblica L.total flavonoids on immunomodulatory function in H22 tumor-bearing mice
陈文雅 1李璐 2郭尔楚 3容少玲 1陈奔2
作者信息
- 1. 广西中医药大学药学院,广西南宁 530001
- 2. 广西中医药大学广西中医药科学实验中心,广西南宁 530001
- 3. 广西中医药大学教学实训中心,广西南宁 530001
- 折叠
摘要
目的:探讨余甘子叶总黄酮对H22荷瘤小鼠免疫调节的影响与肿瘤微环境的变化及其相互关系.方法:昆明小鼠50只腋下H22细胞荷瘤造模后,根据瘤体大小采用随机区组法分为对照(Control)组,5-氟尿嘧啶(5-FU)组,余甘子叶总黄酮低(YGZY-L:0.496g·kg-1)、中(YGZY-M:0.991 g·kg-、高(YGZY-H:1.982g·kg1)剂量组,连续给药 14d.观察瘤质量及抑瘤率;ELISA法检测肿瘤组织中IL-2、IL-6、IL-10、IFN-γ、TNF-α含量水平;HE染色观察瘤组织形态;免疫组化法检测瘤组织CD4+T、CD8+T细胞比例;制备瘤组织单细胞悬浮液,分离淋巴细胞,并用流式细胞术观察肿瘤组织中浸润的淋巴细胞比例;Western blot 观察 NF-κB1、FLT3、PD-1、BTLA、CTLA-4 表达情况.结果:抑瘤率为 5-FU 组 62.2％,YGZY-L 组 13.3％,YGZY-M组40.7％,YGZY-H组53.5％;HE染色显示余甘子叶总黄酮使实体瘤细胞坏死、核固缩;免疫组化显示余甘子叶总黄酮和5-FU可以激活肿瘤微环境CD4+T细胞和CD8+T细胞数量(P＜0.01);ELISA检测显示余甘子叶总黄酮显著上调肿瘤微环境中IL-2、IL-6、IFN-γ、TNF-α,下调IL-10(P＜0.01);瘤组织浸润淋巴细胞流式分析发现,与Control组相比,余甘子叶总黄酮降低CD4+CD25+Foxp3+Treg、CD4+PD-1+TIL、CD8+PD-1+TIL 比例,升高 CD8+CD28+CTL比例(P＜0.05或P＜0.01);Western blot分析显示,与Control组相比,余甘子叶总黄酮显著降低肿瘤微环境中NF-κB 1、FLT3表达量(P＜0.01),及PD-1、CTLA-4、BTLA表达量(P＜0.05或P＜0.01).结论:余甘子叶总黄酮可以通过改善肿瘤免疫微环境,调节荷瘤小鼠免疫功能来抵抗肝癌肿瘤.
Abstract
OBJECTIVE To explore the effect of Phyllanthus emblica L.(PEL)total flavonoids on immunomodulatory function in H22 tumor-bearing mice and examine its relationship with changes in tumor microenvironment.METHODS After an inoculation of H22 cell,50 Kunming mice were randomized into five groups of Control,5-fluorouracil(5-FU)and low(YGZY-L:0.496 g·kg-1),medium(YGZY-M:0.991 g·kg-1)and high(YGZY-H:1.982 g·kg1)doses of PEL total flavonoids(n=10 each).Tumor weight and tumor inhibition rate(TIF)were recorded after 14-day dosing.The levels of interleukin-2/6/10(IL-2/6/10),interferon-gamma(IFN-γ)and tumor necrosis factor-alpha(TNF-α)in tumor tissues were detected by enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin(HE)stain was utilized for observing the morphology of tumor tissues and immunohistochemistry for detecting the ratio of CD4+T/CD8+T cell in tumor tissues.Single-cell suspension of tumor tissues was prepared for isolating lymphocytes and the proportion of infiltrating lymphocytes in tumor tissues was observed by flow cytom-etry.Western blot was utilized for observe the expressions of NF-κB1,FLT3,PD-1,BTLA and CTLA-4.RESULTS TIF was 62.2％in 5-FU group,13.3％in YGZY-L group,40.7％in YGZY-M group and 53.5％in YGZY-H group.HE stain indi-cated that PEL total flavonoids induced cellular necrosis and nuclear condensation.Immunohistochemistry revealed that PEL total flavonoids and 5-FU could activate the number of CD4+T/CD8+T cells in tumor microenvironment(P＜0.01).PEL total flavo-noids significantly up-regulated the levels of IL-2,IL-6,IFN-y and TNF-α and down-regulated IL-10 in tumor microenviron-ment(P＜0.01).The proportions of CD4+CD25+Foxp3+Treg,CD4+PD-1+TIL and CD8+PD-1+TIL dropped while the pro-portion of CD8+CD28+CTL rose(P＜0.05 or P＜0.01).As compared with Control group,PEL total flavonoids down-regulated markedly the expressions of NF-κB1/FLT3(P＜0.01)and PD-1/CTLA-4/BTLA(P＜0.05 or P＜0.01)in tumor microenvironment.CONCLUSION PEL total flavonoids may treat liver cancer tumors through improving tumor immune micro-environment and modulating immune function of tumor-bearing mice.
关键词
余甘子叶总黄酮/抗肿瘤/免疫功能调节/肿瘤微环境Key words
Phyllanthus emblica L.total flavonoids/anti-tumor/immunomodulatory effects/tumor microenvironment引用本文复制引用
基金项目
广西中医药大学自然科学研究项目(XP018091)
广西中医药大学自然科学研究项目(2017QN016)
广西高校中青年教师科研基础能力提升项目(2021KY0319)
中药学广西一流学科(桂教科研[2022]1号)
出版年
2024
NETL
NSTL
万方数据