基于miR-124-3p_...调节炎症相关性结直肠癌研究_张磊.pdf

中草药 中草药 2023 年年 3 月月 第第 54 卷卷 第第 5 期期 Chinese Traditional and Herbal Drugs 2023 March Vol.54 No.5 1471 基于 miR-124-3p/IL-6 信号通路的槲皮素和表儿茶素协同调节炎症相关性结直肠癌研究 张 磊，罗 霄#，刘明靓，陈叶梓，袁成福，何毓敏，刘朝奇，周永芹*三峡大学 基础医学院，湖北 宜昌 423000 摘 要：目的 基于 miR-124-3p/白细胞介素-6（interleukin-6，IL-6）信号通路探究槲皮素与表儿茶素协同作用对炎症相关性结肠癌模型小鼠的干预作用及作用机制。方法 采用氧化偶氮甲烷（azoxymethane，AOM）/葡聚糖硫酸钠（dextran sodium sulfate，DSS）诱导小鼠建立炎症相关性结肠癌模型，同时单独或联合给予槲皮素和表儿茶素，待整个造模周期结束，测量各组小鼠结直肠长度，统计结直肠组织肿瘤数量；采用苏木素-伊红（HE）染色观察各组小鼠结肠组织病理变化；采用 qRT-PCR 检测结直肠组织中炎症相关基因Toll 样受体 4（Toll-like receptor 4，TLR4）、IL-1、IL-6、IL-17及肿瘤相关基因c-myc、血管内皮生长因子（vascular endothelial growth factor，VEGF）和 miR-124-3p 的 mRNA 表达；采用 Western blotting 检测结直肠组织中磷酸化信号转导与转录活化因子 3（phosphorylated signal transducer and activator of transcription 3，p-STAT3）和IL-6 蛋白表达。结果 与对照组比较，模型组小鼠结直肠肿瘤浸润数量增加（P0.001），结直肠组织病理形态发生改变；结直肠组织中 TLR4、IL-1、IL-6、IL-17、c-myc 和 VEGF mRNA 表达水平明显升高（P0.05），IL-6 和 p-STAT3 蛋白表达水平明显升高（P0.01）。与模型组比较，给药组结直肠肿瘤浸润数量减少（P0.01、0.001），结直肠组织病理形态明显得到改善，且联合给药组作用优于单独给药组；结直肠组织中 TLR4、IL-1、IL-6、IL-17、c-myc 和 VEGF mRNA 表达水平明显降低（P0.05、0.01），IL-6 和 p-STAT3 蛋白表达水平明显降低（P0.05、0.01）。通过生物信息学预测发现 miR-124-3p可靶向 IL-6 的 3-UTR 序列，与对照组比较，模型组 miR-124-3p 表达降低（P0.05）；与模型组比较，各给药组 miR-124-3p表达升高（P0.05）。结论 槲皮素联合表儿茶素通过 miR-124-3p/IL-6 的信号通路调节炎症反应，从而抑制结直肠癌。关键词：炎症相关性结肠癌；槲皮素；表儿茶素；IL-6/STAT3 信号通路；miR-124-3p 中图分类号：R285.5 文献标志码：A 文章编号：0253-2670(2023)05-1471-07 DOI:10.7501/j.issn.0253-2670.2023.05.013 Synergistic regulation of quercetin and epicatechin on inflammation-related colorectal cancer based on miR-124-3p/IL-6 signaling pathway ZHANG Lei,LUO Xiao,LIU Ming-jing,CHEN Ye-zi,YUAN Cheng-fu,HE Yu-min,LIU Chao-qi,ZHOU Yong-qin School of Basic Medicine,Three Gorges University,Yichang 423000,ChinaAbstract:Objective To explore the synergistic effect and mechanism of quercetin and epicatechin on inflammation-related colon cancer model mice based on miR-124-3p/interleukin-6(IL-6)signal pathway.Methods The model of inflammation-related colon cancer was induced by azomethane(AOM)/dextran sodium sulfate(DSS)in mice,quercetin and epicatechin were administered alone or in combination.After the end of whole modeling cycle,colon length of mice in each group was measured and number of colorectal tumors were counted;The histopathological changes of colon tissue were observed by hematoxylin-eosin(HE)staining;Inflammation-related genes Toll-like receptor 4(TLR4),IL-1,IL-6,IL-17,tumor-related genes c-myc,vascular endothelial growth factor(VEGF)and miR-124-3p mRNA expressions in colorectal tissue were detected by qRT-PCR;Western blotting was used to detect the expressions of phosphorylated signal transducer and activator of transcription 3(p-STAT3)and IL-6 protein in colorectal tissue.Results Compared with control group,number of colorectal tumors infiltration in model group was increased(P 0.001),pathological 收稿日期：2022-11-10 基金项目：国家自然科学基金资助项目（81673675）作者简介：张 磊，医学硕士，土家族，研究方向为中药在炎症相关疾病中作用机制。E-mail:*通信作者：周永芹，副教授，主要从事中药药理在感染及炎症相关疾病中的研究。E-mail:#共同第一作者：罗 霄，医学硕士，研究方向为中药干预消化系统疾病的相关作用机制。E-mail: 1472 中草药 中草药 2023 年年 3 月月 第第 54 卷卷 第第 5 期期 Chinese Traditional and Herbal Drugs 2023 March Vol.54 No.5 morphology of colorectal tissue was changed;TLR4,IL-1,IL-6,IL-17,c-myc and VEGF mRNA expressions in colorectal tissue were significantly increased(P 0.05),IL-6 and p-STAT3 protein expressions were significantly increased(P 0.01).Compared with model group,number of colorectal tumor infiltration in treatment group were decreased(P 0.01,0.001),pathological morphology of colorectal tissue was significantly improved,and the effect of combined administration group was better than that of single administration group;TLR4,IL-1,IL-6,IL-17,c-myc and VEGF mRNA expressions in colorectal tissue were significantly decreased(P 0.05,0.01),IL-6 and p-STAT3 protein expressions were significantly decreased(P 0.05,0.01).According to bioinformatics prediction,miR-124-3p could target the 3-UTR sequence of IL-6.Compared with control group,miR-124-3p expression in model group was decreased(P 0.05);Compared with model group,miR-124-3p expression in each treatment group was increased(P 0.05).Conclusion Quercetin combined with epicatechin can regulate the inflammatory response through miR-124-3p/IL-6 signal pathway,thus inhibiting colorectal cancer.Key words:inflammation-associated colon cancer;quercetin;epicatechin;IL-6/STAT3 signal pathway;miR-124-3p 结直肠癌是消化内科疾病中发病率最高的恶性肿瘤性疾病，近年来其发病年龄越来越趋于年轻化，且发病率逐年稳步上升，且发现晚1。慢性炎症已被公认为多种癌症的重要危险因素，癌变发生的各个阶段都受到炎症反应的影响，肠道炎症是已知的结直肠癌危险因素，炎症过程在结直肠癌的发展中起着重要的作用，且患有结肠炎的患者患上结肠癌的风险更高2-4。肠道组织中的细胞因子网络是组织稳态和炎症的关键介质，白细胞介素（interleukin，IL）在结直肠癌的发生机制中发挥中介作用5。炎症相关性结直肠癌（colitis associated cancer，CAC）是与炎症性肠病（inflammatory bowel disease，IBD）相关的结直肠癌亚型，由长期反复的肠道炎症所引起6-7。氧化偶氮甲烷（azoxymethane，AOM）/葡聚糖硫酸钠（dextran sodium sulfate，DSS）模型是一种可重复性高且相对便宜的 CAC 动物模型。AOM 是一种致癌物，它可以通过细胞色素 P450（cytochrome P450，CYP）以及同工酶 CYP2E1 代谢，转化为甲基苯丙醇，这是一种高活性的烷基化物种，可诱导DNA产生O6-甲基鸟嘌呤化合物导致GA转变8，在排入胆汁后，可被结肠上皮细胞所吸收并诱发DNA 损伤。DSS 是一种类似肝素的多糖，可对结肠上皮细胞造成损伤，并诱发结肠炎，从而模仿 IBD的病理特征9。AOM/DSS 模型的主要特点是造模时间短和对 CAC 的精确建模，肿瘤的发展可以在短至 10 周内发生。此外，AOM/DSS 诱导的肿