靛玉红通过下调Cyclin A2阻滞细胞周期抑制小鼠Lewis肺癌的体内外及网络药理学研究  

Network pharmacology,in vivo and in vitro research on indirubin inhibiting Lewis lung carcinoma by down‑regulating Cyclin A2 and arresting cell cycle

作　　者：苏琳 李伟 邹纯朴[1,2] 朱杨壮壮 刘洺希 陈晓[1] 胥孜杭 SU Lin;LI Wei;ZOU Chunpu;ZHU Yangzhuangzhuang;LIU Mingxi;CHEN Xiao;XU Zihang(School of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Shanghai Key Laboratory of Health Assessment and Identification,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Department of Medical Cosmetic Center,Shanghai Skin Disease Hospital,Shanghai 200050,China)

机构地区：[1]上海中医药大学中医学院,上海201203 [2]上海中医药大学上海市健康辨识与评估重点实验室,上海201203 [3]上海市皮肤病医院医学美容科,上海200050

出　　处：《上海中医药杂志》2024年第8期80-87,100,共9页Shanghai Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82274229);上海市科委青年科技启明星项目(23QA1409100);上海中医药大学杏林青年学者人才计划项目(B1-GY21-409-04-37)。

摘　　要：目的研究中药成分靛玉红在体内外对小鼠Lewis肺癌(LLC)的抑制效果及对细胞周期的调节作用。方法通过细胞毒性实验探究靛玉红在体外对小鼠LLC细胞的抑制作用。构建肺癌原位小鼠模型,随机分为模型组(磷酸盐缓冲液灌胃,每天1次)和靛玉红低、中、高剂量组(25 mg/kg、50 mg/kg、100 mg/kg灌胃,每天1次)、顺铂组(2 mg/kg腹腔注射,每3天1次),连续3周,记录生存期和体质量。通过活体成像法记录小鼠肺癌进展,取肺肿瘤组织,称重并计算抑瘤率。通过网络药理学预测靛玉红治疗肺癌的核心靶点和通路。Western blot法分别验证靛玉红在体外对LLC细胞及在体内对小鼠肺癌组织中细胞周期蛋白A2(Cyclin A2)表达的影响;免疫组织化学染色(IHC)法和实时荧光定量逆转录聚合酶链式反应(RT-qPCR)法分别检测靛玉红对小鼠肺癌组织中Cyclin A2蛋白及其编码基因CCNA2表达的影响。流式细胞术和IHC法分别检测靛玉红对小鼠肺癌肿瘤细胞的细胞周期和肺癌肿瘤增殖抗原Ki-67表达的影响。结果靛玉红在体外对小鼠LLC细胞的抑制作用呈剂量和时间依赖性,24 h、48 h、72 h的半抑制浓度(IC_(50))分别为188.7μmol/L、109.0μmol/L、58.3μmol/L。与模型组比较,靛玉红中剂量组小鼠肺部荧光表达降低(P<0.05),生存率提高(中位生存期延长7 d),肿瘤抑制率较高(37.76%),且对小鼠体质量没有明显影响(P>0.05)。网络药理学分析表明,靛玉红抑制肺癌可能通过调控细胞周期及Cyclin A2实现。进一步研究证明,靛玉红通过抑制Cyclin A2,导致细胞周期S期阻滞,抑制肿瘤细胞增殖。结论靛玉红体外抑制小鼠LLC细胞,体内通过下调肺癌肿瘤细胞Cyclin A2导致细胞周期阻滞,抑制肿瘤进展,延长肺癌小鼠生存期。Objective Study the inhibitory effect of the traditional Chinese medicine component indirubin on Lewis lung carcinoma(LLC)in vivo and in vitro and its regulatory effect on the cell cycle.Methods The inhibitory effect of indirubin on mouse LLC cells in vitro was explored by cytotoxicity experiments.An orthotopic mouse model of lung cancer was constructed and randomly divided into a model group(phosphate buffer by gavage,once a day),indirubin low-,medium-,and high-dose groups(25 mg/kg,50 mg/kg,and 100 mg/kg by gavage),cisplatin group(2 mg/kg intraperitoneal injection,once every 3 days),for 3 consecutive weeks,the survival period and body mass were recorded.The progression of lung cancer was recorded by in vivo imaging,the lung tumor tissue was weighed,and the tumor inhibition rate was calculated.The core targets and pathways of indirubin for the treatment of lung cancer were predicted through network pharmacology.Western blot was used to verify the effect of indirubin on LLC cells in vitro and on the expression of Cyclin A2 in mouse lung cancer tissue in vivo;immunohistochemical staining(IHC)and real‑time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the effect of indirubin on the expression of Cyclin A2 protein and its encoding gene CCNA2 in mouse lung cancer tissues.Flow cytometry and IHC were used to detect the effects of indirubin on the cell cycle of mouse lung cancer tumor cells and the expression of lung cancer tumor proliferation antigen Ki-67,respectively.Results The inhibitory effect of indirubin on LLC cells in vitro was dose-and time-dependent,the IC_(50) at 24,48,and 72 hours were 188.7μmol/L,109.0μmol/L,and 58.3μmol/L,respectively.Compared with the model group,the medium-dose indirubin group had lower lung fluorescence expression(P<0.05),increased survival rate(MST increased 7 days),higher tumor inhibition rate(37.76%),and no significant effect on the body mass of the mice(P>0.05).Network pharmacology and biological analysis showed that indir

关 键 词：肺癌 靛玉红 抗肿瘤 细胞周期蛋白A2 细胞周期阻滞 网络药理学 中药研究 

分 类 号：R285.5[医药卫生—中药学]