机构地区:[1]烟台市中医医院肿瘤科,山东烟台264000 [2]烟台市中医医院肿瘤四科,山东烟台264000 [3]烟台市中医医院肿瘤一科,山东烟台264000
出 处:《广州中医药大学学报》2024年第3期719-728,共10页Journal of Guangzhou University of Traditional Chinese Medicine
基 金:山东省重点研发项目(编号:2019GSF108245);烟台市科技创新发展计划项目(编号:2021MSGY050);山东省中医药科技项目(编号:2020M132)。
摘 要:【目的】观察益肠散结颗粒对结肠癌小鼠肠道菌群及免疫功能的调节作用及机制。【方法】将60只小鼠随机分为6组,即正常组,模型组,益肠散结颗粒低、中、高剂量组,过表达黑色素瘤缺失基因2(AIM2)质粒(pcDNA-AIM2)干预组,每组10只。除正常组,其他各组采用氧化偶氮甲烷(AOM)/葡聚糖硫酸钠(DSS)诱导法制备结肠癌模型。给药后,绘制各组小鼠生存曲线,计算肿瘤体积;酶联免疫吸附分析(ELISA)检测血清中免疫球蛋白(Ig)G、IgM、白细胞介素(IL)-1β和IL-18水平;流式细胞仪检测外周血T细胞CD3^(+)、CD4^(+)、CD8^(+)水平;测定脾指数;苏木素-伊红(HE)染色法观察结肠组织病理学变化;16S-rDNA肠道菌群测序检测肠道菌群α多样性及肠道菌群群落结构;蛋白免疫印迹(Wetsern Blot)法检测结肠组织中AIM2、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶1(caspase-1)蛋白表达。【结果】与正常组比较,模型组小鼠生存率,血清中IgG、IgM水平,外周血中CD3^(+)、CD4^(+)水平和CD4^(+)/CD8^(+)比值,结肠组织AIM2、ASC、caspase-1蛋白表达水平显著降低,肿瘤体积,血清中IL-1β、IL-18水平,外周血CD8^(+)水平,脾指数显著增加(均P<0.05),HE染色结果可见结肠癌组织特征表现;与模型组比较,益肠散结颗粒低、中、高剂量组和pcDNA-AIM2组小鼠生存率,血清中IgG、IgM水平,外周血中T细胞CD3^(+)、CD4^(+)水平和CD4^(+)/CD8^(+)比值,结肠组织AIM2、ASC、caspase-1蛋白表达水平显著升高,肿瘤体积,血清中IL-1β、IL-18水平,外周血CD8^(+)水平,脾指数显著降低(均P<0.05),HE染色结果可见结肠癌组织病理改变减轻。与正常组比较,模型组小鼠Observed指数、Chao1指数、Shannon指数,拟杆菌门、变形菌门、杆菌科、毛螺菌属、瘤胃梭菌属菌落相对丰度显著降低,厚壁菌门、放线菌门和Patescibateria、乳杆菌属、臭杆菌属、别样杆菌属、未培养的瘤胃球菌科和拟杆菌属菌落相对丰Objective To observe the regulating effect and mechanism of Yichang Sanjie Granules on intestinal flora and immune function in mice with colon cancer.Methods Sixty mice were randomly divided into six groups,i.e.,the normal group,the model group,the low-,medium-and high-dose groups of Yichang Sanjie Granules,and the overexpression of melanoma absent gene 2(AIM2)plasmid(pcDNA-AIM2)intervention group,with 10 mice in each group.Colorectal cancer model was prepared by oxidized azomethine(AOM)/dextran sulfate sodium(DSS)induction method in all groups except normal group.After drug administration,the survival curves of mice in each group were plotted and the tumor volume was calculated;serum levels of immunoglobulin(Ig)G,IgM,interleukin(IL)-1βand IL-18 were detected by enzyme-linked immunosorbent assay(ELISA);peripheral blood levels of CD3^(+),CD4^(+),CD8^(+)T cells were detected by flow cytometry;the splenic index was determined;Hematoxylin-eosin(HE)staining was used to observe the pathological changes in colon tissues;16S-rDNA intestinal flora sequencing was used to detect theα-diversity of intestinal flora and the structure of intestinal flora communities;and protein immunoblotting(Wetsern Blot)was used to detect the protein expressions of AIM2,apoptosis-associated speckled-like protein containing a CARD(ASC),and cystatinase-1(caspase-1)in colon tissues.Results Compared with the normal group,the survival rate,serum levels of IgG and IgM,peripheral blood levels of CD3^(+)and CD4^(+)and CD4^(+)/CD8^(+)ratio,protein expression levels of colon tissue AIM2,ASC and caspase-1 in the model group were significantly decreased,and the tumor volume,serum levels of IL-1βand IL-18,peripheral blood level of CD8^(+),and splenic index were significantly increased(all P<0.05),and the HE staining results showed the characteristic manifestations of colon cancer;compared with the model group,the survival rate,serum levels of IgG and IgM,peripheral blood levels of CD3^(+)and CD4^(+)and CD4^(+)/CD8^(+)ratio,protein expression levels of
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