蝙蝠葛碱对肝癌Huh7细胞增殖和凋亡的作用及机制研究
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摘要
目的研究蝙蝠葛碱对肝癌Huh7细胞增殖和凋亡的作用,探索其抗肿瘤的作用机制和与Hedgehog信号通路的关系。方法不同质量浓度(2、4、8μg/mL)的蝙蝠葛碱处理Huh7细胞,采用四甲基偶氮唑蓝(MTT)法检测Huh7细胞增殖能力的变化;流式细胞术分析细胞凋亡;real-timePCR、Westernblotting法检测Hedgehog信号通路相关基因及蛋白表达水平。结果随着蝙蝠葛碱质量浓度及作用时间的增加,其对Huh7细胞增殖的抑制率升高。其中蝙蝠葛碱8μg/mL作用48h对细胞增殖抑制率最高(48.8%)。蝙蝠葛碱可明显诱导Huh7细胞发生凋亡。随着蝙蝠葛碱质量浓度的增大,细胞的凋亡率显著升高(P<0.05、0.01)。与对照组比较,蝙蝠葛碱各质量浓度组细胞Hedgehog信号通路中PTCH1、GLi1、SMO、SHHm RNA和蛋白表达水平显著降低,同时cleavedCaspase-3蛋白水平显著升高,而Bcl-2表达水平降低(P<0.05、0.01),并呈质量浓度依赖性。结论蝙蝠葛碱可明显抑制Huh7细胞的增殖,促进其凋亡,其可能通过抑制Hedgehog信号通路发挥作用。
Objective To study the effect of dauricine on the proliferation and apoptosis of hepatoma Huh7 cells, and explore its anti-tumor mechanism and its relationship with Hedgehog signaling pathway. Methods The effects of different concentrations of dauricine(2, 4, 8 μg/mL) on the proliferation of Huh7 cells were detected by MTT assay. Apoptosis of Huh7 cells was analyzed by flow cytometry. Real-time PCR and Western blotting were used to detect the levels of Hedgehog signaling pathway-related genes and proteins. Results With the increase of the concentration of dauricine and the duration of action, the inhibition rate of Huh7 cell proliferation was increased. Among them, 8 μg/mL dauricine had the highest inhibition rate(48.8%) at 48 h. Dauricine induced the apoptosis in Huh7 cells. With the increase of the concentration of dauricine, the apoptotic rate of cells was increased significantly(P <0.05, 0.01). The mRNA and protein expression levels of PTCH1, GLi1, SMO and SHH genes in Hedgehog signaling pathway were significantly decreased, while the level of cleaved Caspase-3 protein was significantly increased, accompany with the decreased expression of Bcl-2 in dauricine concentration-dependent pattern(P < 0.05, 0.01) in dauricine group compared with the control group.Conclusion Dauricine could significantly inhibit the proliferation and promote apoptosis of Huh7 cells, which may play a role by blocking Hedgehog signaling pathway.
Objective To study the effect of dauricine on the proliferation and apoptosis of hepatoma Huh7 cells, and explore its anti-tumor mechanism and its relationship with Hedgehog signaling pathway. Methods The effects of different concentrations of dauricine(2, 4, 8 μg/mL) on the proliferation of Huh7 cells were detected by MTT assay. Apoptosis of Huh7 cells was analyzed by flow cytometry. Real-time PCR and Western blotting were used to detect the levels of Hedgehog signaling pathway-related genes and proteins. Results With the increase of the concentration of dauricine and the duration of action, the inhibition rate of Huh7 cell proliferation was increased. Among them, 8 μg/mL dauricine had the highest inhibition rate(48.8%) at 48 h. Dauricine induced the apoptosis in Huh7 cells. With the increase of the concentration of dauricine, the apoptotic rate of cells was increased significantly(P <0.05, 0.01). The mRNA and protein expression levels of PTCH1, GLi1, SMO and SHH genes in Hedgehog signaling pathway were significantly decreased, while the level of cleaved Caspase-3 protein was significantly increased, accompany with the decreased expression of Bcl-2 in dauricine concentration-dependent pattern(P < 0.05, 0.01) in dauricine group compared with the control group.Conclusion Dauricine could significantly inhibit the proliferation and promote apoptosis of Huh7 cells, which may play a role by blocking Hedgehog signaling pathway.
引文
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[2]杜憬生,杜楚玲,邵长丽,等.HPLC测定不同产地北豆根中蝙蝠葛碱含量[J].中国中医药信息杂志,2017,24(6):68-70.
[3]周利龙,阎学斌,丁明,等.蝙蝠葛碱抗心脏机电反馈效应的初步研究[J].中国急救复苏与灾害医学杂志,2006,1(2):95-98.
[4]杨晓燕,周斌,蔡嘉宾,等.蝙蝠葛碱对大鼠局灶性脑缺血再灌注损伤的治疗作用[J].中国药理学通报,2005,21(9):1112-1115.
[5]李艳红,龚培力.蝙蝠葛碱对实验小鼠脑缺血的保护作用[J].中国药理学通报,2004,20(5):564-567.
[6]边文山,张英博,张朝颖,等.蝙蝠葛碱药理作用的研究现状[J].黑龙江科学,2014,5(7):10-11.
[7]吕桂帅,陈磊,王红阳.我国肝癌研究的现状与前景[J].生命科学,2015,27(3):237-248.
[8]朱笑生,刘文超.原发性肝癌全球流行情况和危险因素的新进展[J].现代肿瘤医学,2018,26(14):2297-2301.
[9]张晓媛,黄鹏,李燕京,等.晚期原发性肝癌治疗的研究进展[J].现代肿瘤医学,2017(10):1655-1659.
[10]孙婧,菅天孜,王珂,等.Hedgehog信号通路临床研究进展[J].医学综述,2017,23(4):625-628.
[11]刘满华.Hedgehog信号通路与肿瘤的关系[J].军医进修学院学报,2012,33(4):428-430.
[12]梅怡,殷佩浩,史永照.Hedgehog信号通路与肿瘤发生机制的研究进展[J].国际外科学杂志,2011,38(11):755-757.
[13]王凡,徐凌.Hedgehog信号通路在肝癌发生中的作用[J].同济大学学报:医学版,2013,34(2):120-124.
[14]孙全武,迟强,周逢海.Hedgehog信号通路在前列腺癌中的研究进展[J].国际肿瘤学杂志,2009,36(3):224-227.
[15]王磊,陈公琰.Hedgehog-gli信号通路在肺癌中的研究进展[J].实用肿瘤学杂志,2015,29(2):178-182.
[16]袁积汝,陈晓意,吴泽宇.Hedgehog信号通路与甲状腺癌[J].中华实验外科杂志,2018,35(3):589-592.
[17]陈剑辉,吴晖,马晋平,等.抑制胃癌hedgehog通路对转化生长因子β诱导的上皮间质化的影响[J].中华医学杂志,2013,93(26):2075-2078.
[18]胡晓丽,朱雪琼.Hedgehog信号通路在妇科恶性肿瘤中的研究[J].医学研究杂志,2016,45(10):19-23.
[19]Zhao J,Wang H M,Hsiao C H,et al.Simultaneous inhibition of hedgehog signaling and tumor proliferation remodels stroma and enhances pancreatic cancer therapy[J].Biomaterials,2018,doi:10.1016/j.biomaterials.2018.01.014.
[20]欧阳小波,王立.Hedgehog信号通路与肿瘤侵袭转移作用的研究进展[J].肿瘤,2011,31(2):173-177.
[21]张大伟,曹良启,卢海武,等.Hedgehog信号通路转录因子gli2在肝癌组织中的表达及意义[J].中华普通外科学文献:电子版,2013,7(1):12-16.
[22]路景涛,何伟,宋莎莎,等.Hedgehog-pat/smo-gli信号通路介导肿瘤的侵袭转移研究进展[J].中国药理学通报,2013,29(5):593-597.
[23]王歆光,王维民,杨尹默,等.Hedgehog信号传导通路相关基因在肝癌组织中的表达研究[J].中华普通外科杂志,2008,23(9):702-705.
[24]Che L,Yuan Y H,Jia J,et al.Activation of sonic hedgehog signaling pathway is an independent potential prognosis predictor in human hepatocellular carcinoma patients[J].Chin J Cancer Res,2012,24(4):323-331.
[25]Huang S,He J,Zhang X,et al.Activation of the hedgehog pathway in human hepatocellular carcinomas[J].Carcinogenesis,2006,27(7):1334-1340.
[26]李建华,秦凤绮.蝙蝠葛碱逆转k562/adm细胞多耐药性的研究[J].大连医科大学学报,2002,24(2):94-96.
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[29]Song M,Ou X P,Xiao C C,et al.Hedgehog signaling inhibitor cyclopamine induces apoptosis by decreasing gli2 and bcl2 expression in human salivary pleomorphic adenoma cells[J].Biomed Rep,2013,doi:10.3892/br.2013.61.
[30]Huang S L,Chao C C K.Silencing of taxol-sensitizer genes in cancer cells:Lack of sensitization effects[J].Cancers,2015,doi:10.3390/cancers7020824.
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