黄芪甲苷调控肿瘤外泌体生成与分泌抑制结直肠癌转移的作用机制  

Mechanism of astragalosideⅣregulating exosome production and secretion and inhibiting metastasis of colorectal cancer

作　　者：浦匀舟 李昊泽 李玲[1] 周晶 季青[1] PU Yunzhou;LI Haoze;LI Ling;ZHOU Jing;JI Qing(Department of Medical Oncology&Cancer Institute of Integrative Medicine,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Department of Liver Disease,Ningbo No.2 Hospital,Ningbo,Zhejiang 315010,China)

机构地区：[1]上海中医药大学附属曙光医院肿瘤科/肿瘤研究室,上海201203 [2]宁波市第二医院中西医结合肝病科,浙江宁波315010

出　　处：《上海中医药杂志》2023年第6期41-49,共9页Shanghai Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82074225,82274297,82030118);上海市卫健委上海市进一步加快中医药事业发展三年行动计划项目(ZY〔2021-2023〕-0208);上海市炎癌转化病证生物学前沿研究基地项目(2021KJ03-12);浙江省自然科学基金项目(LQ23H290003);中国博士后基金面上项目(2022M722159);上海市中医临床重点实验室项目(20DZ2272200)。

摘　　要：目的从调控外泌体分泌角度,探讨黄芪甲苷(ASⅣ)抑制结直肠癌转移的作用及机制。方法①细胞实验:使用低、中、高剂量ASⅣ干预MC38细胞,收集细胞上清提取外泌体;透射电镜(TEM)观察外泌体形态;纳米颗粒跟踪分析仪检测外泌体密度;免疫荧光法和实时荧光定量逆转录聚合酶链式反应(RT-qPCR)法检测ASⅣ对外泌体生成、分泌相关基因中性鞘磷脂酶-2(nSMase2)、Ras相关蛋白27a(Rab27a)表达的影响;划痕和Transwell实验检测ASⅣ对肿瘤细胞迁移能力的影响。②动物实验:构建小鼠结直肠癌肝转移模型,随机分为模型组、ASⅣ组和鞘磷脂酶抑制剂(GW4869)组,28d后处死小鼠,观察小鼠肝转移情况。苏木素-伊红(HE)染色法观察肝组织病理学变化,免疫荧光法检测肝转移灶内nSMase2、Rab27a的表达。结果①细胞实验结果显示:与对照组比较,ASⅣ干预不改变外泌体形态,但能够通过浓度和时间依赖方式抑制MC38细胞外泌体分泌(P<0.05);与对照组比较,ASⅣ能够减少MC38细胞中nSMase2、Rab27a的mRNA和蛋白表达水平(P<0.05),且呈浓度依赖性;与对照组比较,ASⅣ能够抑制MC38细胞侵袭迁移能力(P<0.05),且呈浓度依赖性。②动物实验结果显示:与模型组比较,ASⅣ能够显著降低小鼠结直肠癌肝转移模型肝组织质量(P<0.05),减少肝转移瘤数量(P<0.05),降低nSMase2、Rab27a蛋白表达(P<0.05);且ASⅣ抑制结直肠癌肝转移的作用与外泌体生成抑制剂GW4869相当(P>0.05)。结论ASⅣ能够调控肿瘤外泌体生成与分泌,进而抑制结直肠癌肝转移。Objective To investigate the effect and mechanism of AstragalosideⅣ(ASⅣ)in inhibiting colorectal cancer metastasis through regulating exosome secretion.Methods①Cell experiments:MC38 cells were treated with ASⅣat low,medium and high doses,and cell supernatants were collected to extract exosomes.The morphology of exosomes was observed by transmission electron microscopy(TEM).Nanoparticle tracking analyzer was used to detect exosome density.Immunofluorescence assay and RT-qPCR were used to detect the effect of ASⅣon the expression of nSMase2 and Rab27a,which were genes related to exosome production and secretion.The effect of ASⅣon tumor cell migration was detected by cell scrath assay and Transwell assay.②Animal experiments:A mouse model of colorectal cancer liver metastasis was established.The mice were randomly divided into model group,ASⅣgroup and GW4869 group,and were sacrificed after 28 d to observe the liver metastasis.HE staining was used to observe the histopathological changes in the liver tissue,and immunofluorescence was used to detect the expression of nSMase2 and Rab27a in the liver metastases.Results①Cell experiments showed that compared with the condition in control group,ASⅣintervention did not change the morphology of exosomes but could inhibit exosome secretion from MC38 cells in a concentration-and time-dependent manner(P<0.05);ASⅣcould reduce the mRNA and protein expression levels of nSMase2 and Rab27a in MC38 cells in a concentration-dependent manner(P<0.05);and ASⅣcould inhibit the invasion and migration of MC38 cells in a concentration-dependent manner(P<0.05).②Animal experiments showed that compared with the condition in model group,ASⅣsignificantly reduced the liver tissue mass(P<0.05),decreased the number of liver metastases(P<0.05),and inhibited the protein expression levels of nSMase2 and Rab27a(P<0.05)in the mouse model of colorectal cancer liver metastasis;Moreover,the inhibitory effect of ASⅣon liver metastasis of colorectal cancer was comparable to that

关 键 词：结直肠癌 黄芪甲苷 外泌体 转移 小鼠模型 作用机制 中药研究 

分 类 号：R285[医药卫生—中药学]