双氢青蒿素通过调控MAPK/PI3K/Akt信号通路抗结直肠癌作用研究  

Research of dihydroartemisinin on anti⁃colorectal cancer by regulating MAPK/PI3K/Akt signaling pathway

作　　者：徐嘉若 贾丰菁 陈佳靓 姚广涛[1] XU Jiaruo;JIA Fengjing;CHEN Jialiang;YAO Guangtao(Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Sichuan Pharmaceutical Inspection and Research Institue,Sichuan Medical Device Testing Center,Chengdu 611731,Sichuan,China;NMPA Key laboratory for Research on in Vitro-in Vivo Correlation Technology of Pharmaceutical Preparations,Chengdu 611731,Sichuan,China)

机构地区：[1]上海中医药大学,上海201203 [2]四川省药品检验研究院,四川省医疗器械检测中心,四川成都611731 [3]国家药品监督管理局药物制剂体内外相关性技术研究重点实验室,四川成都611731

出　　处：《上海中医药大学学报》2024年第2期83-92,共10页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：国家自然科学基金资助项目(82003641);国家科技重大专项(2019ZX09201004-002);上海市教委预算内项目(18LK019)。

摘　　要：目的:探讨双氢青蒿素(DHA)在体内对人结直肠癌(RKO)细胞的抗癌活性及体外机制。方法:将40只SPF级雄性裸鼠建立RKO荷瘤小鼠模型,随机分为模型组、顺铂组及DHA高剂量(200 mg/kg)、低剂量(100 mg/kg)组,每组10只;对比各组裸鼠体质量、肿瘤体积、肿瘤质量、抑瘤率、血清肿瘤坏死因子(TNF-α)水平等;苏木素-伊红(HE)染色观察各组裸鼠肿瘤组织病理学变化;噻唑蓝(MTT)比色法检测不同浓度(25、50、100μmol/L)DHA干预0、24、48、72 h内对RKO细胞增殖的影响;流式细胞仪、Hoechst 33258检测(0、50、95、150μmol/L)DHA干预48 h后对RKO细胞周期和细胞凋亡的影响;划痕实验观察95μmol/L DHA干预对细胞迁移能力的影响;实时荧光定量PCR(RT-qPCR)检测DHA对细胞内半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、Caspase-9、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax) mRNA表达水平的影响;蛋白质印迹法(Western blot)检测DHA对细胞内Caspase-3、Caspase-9、Bcl-2、Bax以及p38-丝裂原活化蛋白激酶(p38-MAPK)、p-p38-MAPK、磷脂酰肌醇3-激酶(PI3K)、p-PI3K、蛋白激酶B(Akt)、p-Akt、基质金属蛋白酶(MMP-9)等表达水平的影响。结果:200 mg/kg DHA显著降低肿瘤体积和质量,降低血清TNF-α水平,抑瘤率为41.45%;DHA可随浓度的增加而增强对RKO细胞增殖的抑制作用;(50、95、150μmol/L)DHA阻滞RKO细胞周期在G2/M期,且促进RKO细胞凋亡作用随DHA浓度增加而增强;95μmol/L DHA干预12、24 h后可显著降低RKO细胞迁移能力(P<0.01);95μmol/L DHA可显著上调RKO细胞内Caspase-3、Caspase-9mRNA表达水平(P<0.01)以及Bax/Bcl-2 mRNA表达比值(P<0.05);95μmol/L DHA可增加RKO细胞内剪切型Caspase-9(cleaved-Caspase-9)/Caspase-9、Bax/Bcl-2蛋白表达(P<0.01),同时降低MMP-9、p-P38 MAPK、p-PI3K、p-Akt及Akt蛋白表达水平(P<0.01)。结论:DHA有较好的抑制结直肠癌作用,其机制可能为抑制MAPK/PI3K/Akt信号通路,触发RKO细胞内源性凋亡,阻止其增殖�Objective:To investigate the anticancer activity of dihydroartemisinin(DHA)on RKO cells in vivo and its mechanism in vitro.Methods:40 RKO tumor-bearing mouse models were established in specific pathogen-free male nude mice and randomly divided into model group,cisplatin group,high dose DHA(200 mg/kg)group,and low dose DHA(100 mg/kg)group,with 10 mice in each group.The body weight,tumor volume,tumor mass,tumor inhibition rate and serum,tumor necrosis factor-α(TNF-α)level of each group were compared.Hematoxylin-eosin(H&E)staining was used to observe tumor histopathological changes in nude mice of each group.MTT assay was used to detect the effects of DHA at different concentrations(25,50,100μmol/L)on the proliferation of RKO cells within 0,24,48,and 72 h.Flow cytometry and Hoechst 33258 were used to detect the effects of DHA(0,50,95,150μmol/L)on the cell cycle and apoptosis of RKO cells after 48 h intervention.Wound healing assay was used to observe the effect of 95μmol/L DHA on cell migration.Real Time Quantitative PCR(RT-qPCR)was used to detect the effect of DHA on the mRNA expression levels of Caspase-3,Caspase-9,Bcl-2 and Bax.Western blot was used to detect the effect of DHA on the expression levels of Caspase-3,Caspase-9,Bcl-2,Bax,p38-MAPK,p-p38-MAPK,PI3K,p-PI3K,Akt,p-Akt and MMP-9.Results:Tumor volume and mass were significantly reduced and the serum level of TNF-αwas lowered by 200 mg/kg DHA,and the tumor inhibition rate was 41.45%.The inhibitory effect on RKO cell proliferation was enhanced with increasing concentration of DHA.RKO cell cycle was arrested in the G2/M phase by(50,95,150μmol/L)DHA,and the effect of promoting RKO cell apoptosis was enhanced with the increase of DHA concentration.The migration ability of RKO cells was significantly reduced after intervention with 95μmol/L DHA for 12 and 24 h(P<0.01).The mRNA expression levels of Caspase-3 and Caspase-9 in RKO cells were significantly up-regulated by 95μmol/L DHA(P<0.01),and Bax/Bcl-2 mRNA expression ratio was also significantly upregula

关 键 词：双氢青蒿素 结直肠癌 RKO细胞 细胞凋亡 MAPK/PI3K/Akt信号通路 

分 类 号：R285.5[医药卫生—中药学]