基于IL-6/STAT3信号通路探讨miR-34a-5p过表达对溃疡性结肠炎的影响及小檗碱的干预作用  

Effect of miR-34a-5p overexpression on ulcerative colitis and the intervention of berberine based on IL-6/STAT3 signaling pathway

作　　者：陈琴 张志云[1] 石西南 万春平 朱云婴[1] 娄龙 徐瑞 CHEN Qin;ZHANG Zhiyun;SHI Xi'nan;WAN Chunping;ZHU Yunying;LOU Long;XU Rui(Department of Proctology,Kunming Hospital of Traditional Chinese Medicine,Kunming 650011,China;不详)

机构地区：[1]昆明市中医医院肛肠科,昆明650011 [2]云南中医药大学基础医学院 [3]云南中医药大学第一附属医院中心实验室

出　　处：《山东医药》2024年第1期1-5,共5页Shandong Medical Journal

基　　金：云南省中医药应用基础研究联合专项-面上项目(202101AZ070001-268);昆明市卫生健康委员会卫生科研课题(2022-04-01-009);昆明市卫生科技人才培养项目暨“十百千”工程[2022-SW(后备)-60]。

摘　　要：目的基于白细胞介素6(IL-6)/信号转导和转录激活因子3(STAT3)信号通路探讨微小RNA-34a-5p(miR-34a-5p)过表达对溃疡性结肠炎(UC)的影响及小檗碱(BBR)的干预作用。方法体外传代培养人结肠癌HT-29细胞。取传3代、对数生长期、生长状态良好的HT-29细胞,随机分为空白对照组、NC mimics组、miR-34a-5p mimics组,NC mimics组和miR-34a-5p mimics组分别转染NC mimics、miR-34a-5p mimics,空白对照组不予转染。转染6 h更换新鲜培养基继续培养48 h,收集细胞,采用RT-qPCR法验证转染效率。收集三组转染后细胞,采用CCK-8法检测细胞活力;分离三组培养上清液,采用ELISA法检测IL-6、IL-1β、TNF-α含量;收集三组转染后细胞,分别采用RT-qPCR法、Western blotting法检测IL-6、STAT3 mRNA和蛋白表达。取HT-29细胞,加入LPS刺激48 h,建立UC细胞模型。取UC细胞,分别加入0、2.5、5、10、20、40、80、160、320μg/mL BBR干预24 h,采用CCK-8法检测细胞活力。随机将UC细胞分为模型组和BBR组,BBR组予BBR干预24 h,模型组不予BBR干预。分离两组培养上清液,采用ELISA法检测IL-6、IL-1β、TNF-α含量;取两组干预24 h细胞,采用RT-qPCR法检测miR-34a-5p以及IL-6、STAT3mRNA表达。结果miR-34a-5p mimics组miR-34a-5p相对表达量高于NC mimics组和空白对照组(P均<0.05),NC mimics组与空白对照组比较差异无统计学意义(P>0.05)。miR-34a-5p mimics组细胞活力低于NC mimics组和空白对照组(P均<0.05),NC mimics组与空白对照组比较差异无统计学意义(P>0.05)。miR-34a-5p mimics组培养上清液IL-6、IL-1β、TNF-α含量均低于NC mimics组和空白对照组(P均<0.05),NC mimics组与空白对照组比较差异均无统计学意义(P均>0.05)。miR-34a-5p mimics组IL-6、STAT3 mRNA以及IL-6蛋白相对表达量均低于NC mimics组和空白对照组(P均<0.05),NC mimics组与空白对照组比较差异均无统计学意义(P均>0.05)。40、80、160、320μg/mL BBR干预24 h UC细胞活力均高于0�Objective To explore the influence of microRNA-34a-5p(miR-34a-5p)overexpression on ulcerative colitis(UC)and the intervention mechanism of berberine(BBR)based on interleukin-6(IL-6)/signal transduction and activator of transcription 3(STAT3)signaling pathway.Methods HT-29 cells were cultured in vitro.HT-29 cells in the third passage,which were in logarithmic growth phase and had good growth status,were taken and randomly divided into the blank control group,NC mimics group and miR-34a-5p mimics group,respectively.Cells in the NC mimics group and miR-34a-5p mimics group were transfected with NC mimics and miR-34a-5p mimics,respectively,while cells in the blank control group were not transfected.After transfection for 6 h,we replaced the medium with the fresh medium and continued to culture for 48 h.The transfection efficiency was verified by RT-qPCR.The transfected cells of the three groups were collected and cell viability was detected by CCK-8.The culture supernatants of the three groups were separated and the content of IL-6,IL-1βand TNF-αwas detected by ELISA.The transfected cells of the three groups were collected,and the mRNA and protein expression levels of IL-6 and STAT3 were detected by RT-qPCR and Western blotting.The UC cell model was established by taking HT-29 cells and adding LPS for simulation of 48 h.UC cells were taken and treated with 0,2.5,5,10,20,40,80,160,320μg/mL BBR for 24 h,and cell viability was detected by CCK-8.UC cells were randomly divided into the model group and BBR group.The BBR group was treated with BBR for 24 h,while the model group was not treated with BBR.The culture supernatant of the two groups was separated and the content of IL-6,IL-1βand TNF-αwas detected by ELISA.The cells of the two groups were intervened for 24 h and the mRNA expression levels of miR-34a-5p,IL-6 and STAT3 were detected by RT-qPCR.Results The relative expression of miR-34a-5p was higher in the mimics group than in the NC mimics group and the blank control group(both P<0.05),but there was no signific

关 键 词：溃疡性结肠炎 小檗碱 微小RNA-34a-5p 白细胞介素6/信号转导和转录激活因子3信号通路 

分 类 号：R574.6[医药卫生—消化系统]