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

作　　者：陈文雅 李璐 郭尔楚 容少玲 陈奔[2] CHEN Wenya;LI Lu;GUO Erchu;RONG Shaoling;CHEN Ben(School of Pharmacy,Guangxi University of Traditional Chinese Medicine,Guangxi Nanning 530001,China;Guangxi Sci-ence Experimental Center of Traditional Chinese Medicine,Guangxi University of Traditional Chinese Medicine,Guangxi Nanning 530001,China;Teaching Training Center,Guangxi University of Traditional Chinese Medicine,Guangxi Nanning 530001,China)

机构地区：[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

出　　处：《中国医院药学杂志》2024年第16期1851-1859,共9页Chinese Journal of Hospital Pharmacy

基　　金：广西中医药大学自然科学研究项目(编号:XP018091);广西中医药大学自然科学研究项目(编号:2017QN016);广西高校中青年教师科研基础能力提升项目(编号:2021KY0319);中药学广西一流学科(编号:桂教科研[2022]1号)。

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:0.496 g·kg^(–1))、中(YGZY-M:0.991 g·kg^(–1))、高(YGZY-H:1.982 g·kg^(–1))剂量组,连续给药14 d。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-κB1、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。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(YGZYL:0.496 g·kg–1),medium(YGZY-M:0.991 g·kg–1)and high(YGZY-H:1.982 g·kg–1)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-γand TNF-αand 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 downregulated 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 imp

关 键 词：余甘子叶总黄酮 抗肿瘤 免疫功能调节 肿瘤微环境 

分 类 号：R967[医药卫生—药理学]