香叶木苷对人肝癌HepG2细胞凋亡及Bcl-2/Bax基因表达的影响
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摘要
目的研究香叶木苷对人肝癌HepG2细胞增殖抑制及诱导凋亡的作用,并探讨其相关分子机制。方法将HepG2细胞分为对照组和香叶木苷组,香叶木苷组分别加入终质量浓度为1、5、25μg/mL的香叶木苷DMSO溶液,对照组加入等体积的DMSO。药物处理24 h后,台盼蓝染色及Live/Dead染色检测香叶木苷对HepG2细胞活性的影响;CCK-8及EdU染色检测香叶木苷对HepG2细胞增殖的影响;TUNEL染色检测香叶木苷对HepG2细胞凋亡的影响;实时荧光定量PCR(qRTPCR)及Western blotting法检测香叶木苷对Bcl-2、Bax mRNA和蛋白水平的影响。结果与对照组比较,香叶木苷降低HepG2细胞活力、抑制细胞增殖,5、25μg/mL组差异显著(P<0.05、0.01、0.001);促进HepG2细胞凋亡,各浓度组均差异显著(P<0.01、0.001),且均呈剂量相关性。经5μg/mL香叶木苷处理后,HepG2细胞中抗凋亡蛋白Bcl-2的mRNA水平显著降低(P<0.001),而促凋亡蛋白Bax的mRNA水平显著升高(P<0.001),Bcl-2/Bax蛋白水平显著降低(P<0.001)。结论香叶木苷抑制HepG2细胞活力和增殖、促进凋亡,作用机制与调控Bcl-2/Bax表达有关。
Objective To study the effects of diosmin on proliferation inhibition and apoptosis of human hepatoma HepG2 cells and to explore its molecular mechanism. Methods HepG2 cells were divided into control group and diosmin group. Diosmin group was added DMSO solution with the final concentration of 1, 5 and 25 μg/mL, and control group was added DMSO with the same volume. After treatment for 24 h,Trypan blue staining and Live/Dead staining were performed to test the effect of diosmin on the cell viability. CCK-8 assay and EdU staining were applied to test the influence of diosmin on the proliferation of HepG2. TUNEL staining was performed to detectthe effect of diosmin on apoptosis of HepG2. QRT-PCR and western blotting were applied to test the expressions of apoptotic-related proteins in each group. Results Compared with the control group, diosmin decreased the cell viability and inhibited the proliferation of HepG2 cells, and there were significant differences between 5 and 25 μg/mL groups(P <0.05, 0.01 and 0.001), and promoted the apoptosis of HepG2 cells in all concentration groups(P < 0.01 and 0.001), all were dose related. After 5 μg/mL diosmin treatment, the expression of anti-apoptotic protein Bcl-2 mRNA in HepG2 cells decreased significantly(P < 0.001), while the expression of pro-apoptotic protein Bax mRNA increased significantly(P < 0.001). The level of Bcl-2/Bax protein decreased significantly(P < 0.001). Conclusion Diosmin could decrease cell viability and proliferation, as well as promote apoptosis of HepG2 by regulating Bcl-2/Bax pathway. These findings may represent a new idea for drug therapy of liver cancer.
Objective To study the effects of diosmin on proliferation inhibition and apoptosis of human hepatoma HepG2 cells and to explore its molecular mechanism. Methods HepG2 cells were divided into control group and diosmin group. Diosmin group was added DMSO solution with the final concentration of 1, 5 and 25 μg/mL, and control group was added DMSO with the same volume. After treatment for 24 h,Trypan blue staining and Live/Dead staining were performed to test the effect of diosmin on the cell viability. CCK-8 assay and EdU staining were applied to test the influence of diosmin on the proliferation of HepG2. TUNEL staining was performed to detectthe effect of diosmin on apoptosis of HepG2. QRT-PCR and western blotting were applied to test the expressions of apoptotic-related proteins in each group. Results Compared with the control group, diosmin decreased the cell viability and inhibited the proliferation of HepG2 cells, and there were significant differences between 5 and 25 μg/mL groups(P <0.05, 0.01 and 0.001), and promoted the apoptosis of HepG2 cells in all concentration groups(P < 0.01 and 0.001), all were dose related. After 5 μg/mL diosmin treatment, the expression of anti-apoptotic protein Bcl-2 mRNA in HepG2 cells decreased significantly(P < 0.001), while the expression of pro-apoptotic protein Bax mRNA increased significantly(P < 0.001). The level of Bcl-2/Bax protein decreased significantly(P < 0.001). Conclusion Diosmin could decrease cell viability and proliferation, as well as promote apoptosis of HepG2 by regulating Bcl-2/Bax pathway. These findings may represent a new idea for drug therapy of liver cancer.
引文
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[8]Dung T D, Day C H, Binh T V, et al. PP2A mediates diosmin p53 activation to block HA22T cell proliferation and tumor growth in xenografted nude mice through PI3K-Akt-MDM2 signaling suppression[J]. Food ChemToxicol, 2012, 50(5):1802-1810.
[9]Srinivasan S, Pari L. Ameliorative effect of diosmin, a citrus flavonoid against streptozotocin-nicotinamide generated oxidative stress induced diabetic rats[J]. Chem Biol Interact, 2012, 195(1):43-51.
[10]Silambarasan T, Raja B.Diosmin, a bioflavonoid reverses alterations in blood pressure, nitric oxide, lipid peroxides and antioxidant status in DOCA-salt induced hypertensive rats[J]. Eur J Pharmacol, 2012, 679(1-3):81-89.
[11]Queenthy S S, John B. Diosmin exhibits antihyperlipidemic effects in isoproterenol induced myocardial infarcted rats[J]. Eur J Pharmacol, 2013, 718(1-3):213-218.
[12]梁凯,曹秉振.线粒体调控的细胞凋亡研究进展[J].生物医学工程与临床, 2014, 18(5):501-505.
[2]徐晶晶,徐超,刘斌.不同采收期薄荷中4个黄酮苷的含量测定[J].药物分析杂志, 2013, 33(12):2077-2081.
[3]姜艳艳,石任兵,刘斌,等.半边莲中黄酮类化学成分研究[J].北京中医药大学学报, 2009, 32(1):59-61.
[4]董坤,寇韩旭,宁馨,等.蓬子菜中香叶木苷抗急性血瘀作用及机制研究[J].药物评价研究, 2016, 39(2):207-210
[5]Buddhan R, Manoharan S. Diosminreduces cell viability of A431 skin cancer cells through apoptotic induction[J].Cancer Res Ther, 2017, 13(3):471-476.
[6]Lewinska A, Adamczyk-Grochala J, Kwasniewicz E, et al. Diosmin-induced senescence, apoptosis and autophagy in breast cancer cells of different p53 status and ERK activity[J]. Toxicol Lett, 2017, 265:117-130.
[7]Martínez Conesa C, Vicente Ortega V, Yá?ezgascón M J,et al. Treatment of metastatic melanoma B16F10 by the flavonoids tangeretin, rutin, and diosmin[J]. J Agric Food Chem, 2005, 53(17):6791-6797.
[8]Dung T D, Day C H, Binh T V, et al. PP2A mediates diosmin p53 activation to block HA22T cell proliferation and tumor growth in xenografted nude mice through PI3K-Akt-MDM2 signaling suppression[J]. Food ChemToxicol, 2012, 50(5):1802-1810.
[9]Srinivasan S, Pari L. Ameliorative effect of diosmin, a citrus flavonoid against streptozotocin-nicotinamide generated oxidative stress induced diabetic rats[J]. Chem Biol Interact, 2012, 195(1):43-51.
[10]Silambarasan T, Raja B.Diosmin, a bioflavonoid reverses alterations in blood pressure, nitric oxide, lipid peroxides and antioxidant status in DOCA-salt induced hypertensive rats[J]. Eur J Pharmacol, 2012, 679(1-3):81-89.
[11]Queenthy S S, John B. Diosmin exhibits antihyperlipidemic effects in isoproterenol induced myocardial infarcted rats[J]. Eur J Pharmacol, 2013, 718(1-3):213-218.
[12]梁凯,曹秉振.线粒体调控的细胞凋亡研究进展[J].生物医学工程与临床, 2014, 18(5):501-505.
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