辣椒碱对肝癌SMMC-7721细胞增殖及HMGB1,IL-6表达的影响
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摘要
目的:观察辣椒碱对肝癌SMMC-7721细胞增殖的影响,并探讨其作用的分子机制。方法:设立辣椒碱(50,100,150,200,250,300μmol·L~(-1))组以及空白组,采用细胞计数试剂盒-8 (CCK-8)法检测辣椒碱(50,100,150,200,250,300μmol·L~(-1))作用SMMC-7721细胞24,48,72 h后的细胞活性;采用光学倒置显微镜观察辣椒碱(150,200,250μmol·L~(-1))作用SMMC-7721细胞形态的变化;实时荧光定量PCR(Real-time PCR)检测辣椒碱(150,200,250μmol·L~(-1))作用SMMC-7721细胞24 h后细胞中高迁移率族蛋白B1(HMGB1)和白细胞介素-6(IL-6) mRNA表达水平;采用酶联免疫吸附测定(ELISA)检测辣椒碱(150,200,250μmol·L~(-1))作用SMMC-7721细胞24 h后细胞培养上清中HMGB1和IL-6的水平。结果:与空白组比较,辣椒碱50,100μmol·L~(-1)组作用24,48,72 h差异均无统计学意义;其余辣椒碱(150,200,250,300μmol·L~(-1))作用不同时间点,增殖抑制率均明显升高(P <0. 05),且具有明显的浓度和时间依赖效应;与空白组比较,辣椒碱(150,200,250μmol·L~(-1))组SMMC-7721细胞均出现不同程度地形态学改变,表现为细胞变圆、皱缩,贴壁差,脱落增多,给药浓度越高,效果就越明显;与空白组比较,辣椒碱(150,200,250μmol·L~(-1))组SMMC-7721细胞中HMGB1,IL-6 mRNA表达明显下调(P <0. 05),上清中HMGB1和IL-6分泌明显降低(P <0. 05)。结论:辣椒碱对肝癌SMMC-7721细胞增殖具有抑制作用,潜在机制可能与其抑制细胞HMGB1和IL-6的表达与分泌有关。
Objective: To investigate the effect of capsaicin on proliferation in human hepatoma SMMC-7721 cells and its possible molecular mechanism. Method: Capsaicin( 50, 100, 150, 200, 250,300 μmol·L~(-1)) groups and blank group were set up. The cell viability was detected by cell counting kit-8( CCK-8) assay after SMMC-7721 cells were treated with capsaicin( 50,100,150,200,250,300 μmol·L~(-1))for 24,48,72 h. The morphological changes were observed under an inverted microscope after SMMC-7721 cells were treated with capsaicin( 150,200,250 μmol·L~(-1)) for 24 h. The mRNA expression levels of high mobility group box 1( HMGB1) and interleukin-6( IL-6) were measured by Real-time PCR after SMMC-7721 cells were treated with capsaicin( 150,200,250 μmol·L~(-1)) for 24 h. The levels of HMGB1 and IL-6 in cell culture supernatant were detected by enzyme-linked immunosorbent assay( ELISA) after SMMC-7721 cells were treated with capsaicin( 150,200,250 μmol·L~(-1)) for 24 h. Result: Compared with the blank group,there was no significant difference between 50 and 100 μmol·L~(-1) capsaicin groups treated for 24,48,72 h; after treated with the other concentrations of capsaicin( 150,200,250,300 μmol·L~(-1)) at different time points,the proliferation inhibition rate was statistically significant( P < 0. 05),with significant concentration and time-dependent effect;compared with the blank group,capsaicin( 150,200,250 μmol·L~(-1)) groups showed different degrees of morphological changes in SMMC-7721 cells,which became round and wrinkled,with a poor attachment and more exfoliation; compared with the blank group,the mRNA expressions of HMGB1 and IL-6 in SMMC-7721 cells of capsaicin( 150,200,250 μmol·L~(-1)) groups were significantly down-regulated( P < 0. 05),and the secretions of HMGB1 and IL-6 were significantly decreased in the supernatant( P < 0. 05). Conclusion: Capsaicin inhibits cell proliferation of SMMC-7721 cells,and the possible mechanism may be related to the down-regulation of HMGB1 and IL-6 at the mRNA and protein levels.
Objective: To investigate the effect of capsaicin on proliferation in human hepatoma SMMC-7721 cells and its possible molecular mechanism. Method: Capsaicin( 50, 100, 150, 200, 250,300 μmol·L~(-1)) groups and blank group were set up. The cell viability was detected by cell counting kit-8( CCK-8) assay after SMMC-7721 cells were treated with capsaicin( 50,100,150,200,250,300 μmol·L~(-1))for 24,48,72 h. The morphological changes were observed under an inverted microscope after SMMC-7721 cells were treated with capsaicin( 150,200,250 μmol·L~(-1)) for 24 h. The mRNA expression levels of high mobility group box 1( HMGB1) and interleukin-6( IL-6) were measured by Real-time PCR after SMMC-7721 cells were treated with capsaicin( 150,200,250 μmol·L~(-1)) for 24 h. The levels of HMGB1 and IL-6 in cell culture supernatant were detected by enzyme-linked immunosorbent assay( ELISA) after SMMC-7721 cells were treated with capsaicin( 150,200,250 μmol·L~(-1)) for 24 h. Result: Compared with the blank group,there was no significant difference between 50 and 100 μmol·L~(-1) capsaicin groups treated for 24,48,72 h; after treated with the other concentrations of capsaicin( 150,200,250,300 μmol·L~(-1)) at different time points,the proliferation inhibition rate was statistically significant( P < 0. 05),with significant concentration and time-dependent effect;compared with the blank group,capsaicin( 150,200,250 μmol·L~(-1)) groups showed different degrees of morphological changes in SMMC-7721 cells,which became round and wrinkled,with a poor attachment and more exfoliation; compared with the blank group,the mRNA expressions of HMGB1 and IL-6 in SMMC-7721 cells of capsaicin( 150,200,250 μmol·L~(-1)) groups were significantly down-regulated( P < 0. 05),and the secretions of HMGB1 and IL-6 were significantly decreased in the supernatant( P < 0. 05). Conclusion: Capsaicin inhibits cell proliferation of SMMC-7721 cells,and the possible mechanism may be related to the down-regulation of HMGB1 and IL-6 at the mRNA and protein levels.
引文
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[20] FAN H, JIANG C, ZHONG B, et al. Matrine ameliorates colorectal cancer in rats via inhibition of HMGB1 signaling and downregulation of IL-6, TNFalpha,and HMGB1[J]. J Immunol Res,2018,2018:5408324.
[21] Kumari N,Dwarakanath B S,Das A,et al. Role of interleukin-6 in cancer progression and therapeutic resistance[J]. Tumour Biol, 2016, 37(9):11553-11572.
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[24] Lanton T, Shriki A, Nechemia-Arbely Y, et al.Interleukin 6-dependent genomic instability heralds accelerated carcinogenesis following liver regeneration on a background of chronic hepatitis[J]. Hepatology,2017,65(5):1600-1611.
[25]沈丰,孙少华,吴红伟,等.薏苡仁提取物对C57小鼠肝癌模型IL-6抑制作用的实验研究[J].中国普外基础与临床杂志,2016,23(1):38-41.
[26]罗来育,陈斯泰,李常青,等.叶下珠复方Ⅱ号对二乙基亚硝胺诱导大鼠肝癌形成的抑制作用及机制[J].中国实验方剂学杂志,2016,22(8):126-132.
[2] El-Kott A F,Bin-Meferij M M. Suppressive effects of capsaicin against N-nitrosomethylurea-induced mammary tumorigenesis in rats[J]. Biomed Pharmacother,2018,98:673-679.
[3] Friedman J R,Nolan N A,Miles S L,et al. Anticancer activity of natural and synthetic capsaicin analogs[J]. J Pharmacol Exp Ther,2018,364(3):462-473.
[4]李伯和,袁磊.辣椒碱对乳腺癌MDA-MB-231细胞迁移和侵袭的抑制作用及其机制[J].生理学报,2017,69(2):183-188.
[5] Diaz-Laviada I, Rodriguez-Henche N. The potential antitumor effects of capsaicin[J]. Prog Drug Res,2014,68:181-208.
[6]谢智钦,李弘夏,唐才喜,等.辣椒素通过氧化应激诱导Hep G2肝癌细胞凋亡的研究[J].中国肿瘤,2018,27(6):470-475.
[7]肖潺潺,陈茂剑,梅凡彪,等.辣椒碱对肝癌SMMC-7721细胞迁移和侵袭的影响及机制[J].中国实验方剂学杂志,2018,24(18):124-129.
[8] Goodwin G H,Sanders C,Johns E W. A new group of chromatin-associated proteins with a high content of acidic and basic amino acids[J]. Eur J Biochem,1973,38(1):14-19.
[9] KANG R,XIE Y,ZHANG Q,et al. Intracellular HMGB1 as a novel tumor suppressor of pancreatic cancer[J]. Cell Res,2017,27(7):916-932.
[10] CHEN M,HUANG W,WANG C,et al. High-mobility group box 1 exacerbates CCl(4)-induced acute liver injury in mice[J]. Clin Immunol,2014,153(1):56-63.
[11] ZHAO X L,LIN Y,JIANG J,et al. High-mobility group box 1 released by autophagic cancer-associated fibroblasts maintains the stemness of luminal breast cancer cells[J]. J Pathol,2017,243(3):376-389.
[12]冯越,鲍秀琦.关于TNF-α和IL-6在肝癌患者血清中表达情况的研究[J].世界最新医学信息文摘,2018,18(2):140.
[13] Jones S A,Jenkins B J. Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer[J]. Nat Rev Immunol,2018,18:773-789.
[14] Surh Y J,Lee S S. Capsaicin,a double-edged sword:toxicity, metabolism, and chemopreventive potential[J]. Life Sci,1995,56(22):1845-1855.
[15] Shimizu S,Kouzaki H,Kato T,et al. HMGB1-TLR4signaling contributes to the secretion of interleukin 6 and interleukin 8 by nasal epithelial cells[J]. Am J Rhinol Allergy,2016,30(3):167-172.
[16] LIU F,ZHANG Y,PENG Z,et al. High expression of high mobility group box 1(hmgb1)predicts poor prognosis for hepatocellular carcinoma after curative hepatectomy[J]. J Transl Med,2012,10(1):135.
[17]贺新春,范学工,周蓉蓉. HMGB1对人肝癌细胞株Hep G2体外增殖的影响[J].中南大学学报:医学版,2010,35(5):451-457.
[18] CHENG P,DAI W,WANG F,et al. Ethyl pyruvate inhibits proliferation and induces apoptosis of hepatocellular carcinoma via regulation of the HMGB1-RAGE and Akt pathways[J]. Biochem Biophys Res Commun,2014,443(4):1162-1168.
[19] Yaser A M,HUANG Y,ZHOU R R,et al. The role of receptor for advanced glycation end products(RAGE)in the proliferation of hepatocellular carcinoma[J]. Int J Mol Sci,2012,13(12):5982-5997.
[20] FAN H, JIANG C, ZHONG B, et al. Matrine ameliorates colorectal cancer in rats via inhibition of HMGB1 signaling and downregulation of IL-6, TNFalpha,and HMGB1[J]. J Immunol Res,2018,2018:5408324.
[21] Kumari N,Dwarakanath B S,Das A,et al. Role of interleukin-6 in cancer progression and therapeutic resistance[J]. Tumour Biol, 2016, 37(9):11553-11572.
[22] Berasain C, Castillo J, Perugorria M J, et al.Inflammation and liver cancer:new molecular links[J]. Ann N Y Acad Sci,2009,1155:206-221.
[23] Ogata H,Kobayashi T,Chinen T,et al. Deletion of the SOCS3 gene in liver parenchymal cells promotes hepatitis-induced hepatocarcinogenesis[J].Gastroenterology,2006,131(1):179-193.
[24] Lanton T, Shriki A, Nechemia-Arbely Y, et al.Interleukin 6-dependent genomic instability heralds accelerated carcinogenesis following liver regeneration on a background of chronic hepatitis[J]. Hepatology,2017,65(5):1600-1611.
[25]沈丰,孙少华,吴红伟,等.薏苡仁提取物对C57小鼠肝癌模型IL-6抑制作用的实验研究[J].中国普外基础与临床杂志,2016,23(1):38-41.
[26]罗来育,陈斯泰,李常青,等.叶下珠复方Ⅱ号对二乙基亚硝胺诱导大鼠肝癌形成的抑制作用及机制[J].中国实验方剂学杂志,2016,22(8):126-132.