表没食子儿茶素没食子酸酯对肝细胞氧化损伤的初步研究
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摘要
目的观察表没食子儿茶素没食子酸酯(EGCG)对正常人HL7702肝细胞氧化损伤的作用。方法以正常人HL7702肝细胞为研究对象,用不同浓度EGCG (0μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL、80μg/mL、160μg/mL)处理细胞1 h、12 h、24 h,均以0μg/mL EGCG浓度组为对照组。倒置显微镜观察细胞形态,MTT法检测细胞存活率,DCFH-DA荧光探针法检测细胞内活性氧(ROS),生化分析检测细胞培养上清液中乳酸脱氢酶(LDH)的含量。结果 EGCG浓度在40μg/mL以下时,对肝细胞存活率下降无明显影响,差异均无统计学意义(P>0.05);且5μg/mL、20μg/mL EGCG处理24 h后的肝细胞存活率分别为(108.34±4.12)%、(109.59±3.14)%,与对照组的(100.00±3.40)%比较,细胞存活率明显提高,差异均有统计学意义(P<0.05);80μg/mL EGCG处理细胞1 h后,ROS为(5.36±0.68)%,与对照组的(3.88±0.10)%比较明显增多,差异有统计学意义(P<0.05);80μg/mL EGCG处理细胞24 h,与对照组的细胞存活率[(100.00±3.40)%]、LDH [(168.89±4.90) U/L]、ROS [(3.54±0.68)%]比较,细胞存活率[(40.05±7.83)%]明显降低,LDH [(195.48±11.23) U/L]、ROS [(10.99±1.54)%]显著升高,差异均有统计学意义(P<0.05);160μg/mL EGCG作用肝细胞1 h,与对照组的细胞存活率[(100.00±2.23)%]、LDH [(163.89±5.37) U/L]、ROS [(3.88±0.10)%]比较,肝细胞存活率[(79.87±4.72)%]明显降低,LDH [(200.72±13.45) U/L]、ROS [(8.14±0.29)%]明显升高,差异均有统计学意义(P<0.05);160μg/mL EGCG作用肝细胞12 h、24 h,细胞存活率分别为(16.00±0.99)%、(11.11±0.41)%,LDH分别为(199.21±13.11) U/L、(435.71±25.84) U/L,与对照组的细胞存活率[(100.00±0.34)%、(100.00±3.40)%]和LDH[(169.56±3.55) U/L、(168.89±4.90) U/L]比较差异均有统计学意义(P<0.05),同时显微镜下可见大量细胞死亡。结论低剂量EGCG (<40μg/mL)对正常人HL7702肝细胞无明显损伤作用,并具有一定的促增殖作用,高剂量下(>80μg/mL)可明显增加肝细胞氧化损伤,且随EGCG作用浓度、作用时间的增加损伤也增加。
Objective To observe the effect of epigallocatechin gallate(EGCG) on oxidative damage of human normal HL7702 liver cells. Methods Human normal HL7702 liver cells were treated with EGCG at different concentrations(0 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL, 160 μg/mL) for 1 h, 12 h and 24 h, respectively. The 0 μg/mL EGCG treated cells was used as the control for each treated cells. Cell morphology was observed by inverted microscope. MTT assay was used to detect cell viability. The reactive oxygen species(ROS) in the cells were measured by DCFH-DA fluorescence probe, and biochemical analysis was used to measure the contents of lactate dehydrogenase(LDH) in the supernatant of cells. Results When the treatment concentration was below 40 μg/mL, EGCG showed no obvious effect on the decrease of survival rate of hepatocytes(P>0.05). The survival rates of hepatocytes after treatment with 5 μg/mL and 20 μg/mL EGCG for 24 h were(108.34±4.12)% and(109.59±3.14)%, which were significantly improved as compared with(100.00 ± 3.40)% of the control(P<0.05). ROS after treatment with 80 μg/mL EGCG for 1 h was(5.36±0.68)%, which was significantly increased as compared with(3.88±0.10)% of the control(P<0.05). After treatment with 80 μg/mL EGCG more than 24 h, the cell survival rate was(40.05±7.83)%, significantly reduced as compared with(100.00±3.40)% of the control, and LDH, ROS were(195.48±11.23) U/L,(10.99±1.54)%, significantly increased as compared with(168.89±4.90) U/L,(3.54±0.68)% of control, all with statistically significant differences(P<0.05). After treatment with 160 μg/mL EGCG for 1 h, the survival rate of liver cells was(79.87±4.72)%, significantly reduced as compared with(100.00 ± 2.23)% of the control, and LDH, ROS were(200.72 ± 13.45) U/L,(8.14 ±0.29)%, which were significantly increased as compared with(163.89±5.37) U/L,(3.88±0.10)% of control, all with statistically significant differences(P<0.05). When cells were treated with 160 μg/mL EGCG for 12 h and 24 h, a large number of dead cells were observed under inverted microscope, and liver cell survival rate, LDH were(16.00±0.99)%and(11.11±0.41)%,(199.21±13.11) U/L and(435.71±25.84) U/L, versus(100.00±0.34)% and(100.00±3.40)%,(169.56±3.55) U/L and(168.89±4.90) U/L of the control cells(P<0.05). Conclusion Low dose EGCG(<40 μg/mL) has no obvious damage effect on normal hepatocytes and has a certain effect of promoting cell proliferation. High dose EGCG(>80 μg/mL) can significantly increase the oxidative damage of hepatocytes, and the damage also increases with the increase of time and drug concentration.
Objective To observe the effect of epigallocatechin gallate(EGCG) on oxidative damage of human normal HL7702 liver cells. Methods Human normal HL7702 liver cells were treated with EGCG at different concentrations(0 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL, 160 μg/mL) for 1 h, 12 h and 24 h, respectively. The 0 μg/mL EGCG treated cells was used as the control for each treated cells. Cell morphology was observed by inverted microscope. MTT assay was used to detect cell viability. The reactive oxygen species(ROS) in the cells were measured by DCFH-DA fluorescence probe, and biochemical analysis was used to measure the contents of lactate dehydrogenase(LDH) in the supernatant of cells. Results When the treatment concentration was below 40 μg/mL, EGCG showed no obvious effect on the decrease of survival rate of hepatocytes(P>0.05). The survival rates of hepatocytes after treatment with 5 μg/mL and 20 μg/mL EGCG for 24 h were(108.34±4.12)% and(109.59±3.14)%, which were significantly improved as compared with(100.00 ± 3.40)% of the control(P<0.05). ROS after treatment with 80 μg/mL EGCG for 1 h was(5.36±0.68)%, which was significantly increased as compared with(3.88±0.10)% of the control(P<0.05). After treatment with 80 μg/mL EGCG more than 24 h, the cell survival rate was(40.05±7.83)%, significantly reduced as compared with(100.00±3.40)% of the control, and LDH, ROS were(195.48±11.23) U/L,(10.99±1.54)%, significantly increased as compared with(168.89±4.90) U/L,(3.54±0.68)% of control, all with statistically significant differences(P<0.05). After treatment with 160 μg/mL EGCG for 1 h, the survival rate of liver cells was(79.87±4.72)%, significantly reduced as compared with(100.00 ± 2.23)% of the control, and LDH, ROS were(200.72 ± 13.45) U/L,(8.14 ±0.29)%, which were significantly increased as compared with(163.89±5.37) U/L,(3.88±0.10)% of control, all with statistically significant differences(P<0.05). When cells were treated with 160 μg/mL EGCG for 12 h and 24 h, a large number of dead cells were observed under inverted microscope, and liver cell survival rate, LDH were(16.00±0.99)%and(11.11±0.41)%,(199.21±13.11) U/L and(435.71±25.84) U/L, versus(100.00±0.34)% and(100.00±3.40)%,(169.56±3.55) U/L and(168.89±4.90) U/L of the control cells(P<0.05). Conclusion Low dose EGCG(<40 μg/mL) has no obvious damage effect on normal hepatocytes and has a certain effect of promoting cell proliferation. High dose EGCG(>80 μg/mL) can significantly increase the oxidative damage of hepatocytes, and the damage also increases with the increase of time and drug concentration.
引文
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[13]董峰,杨佳,李向阳,等.镉通过引发氧化应激和线粒体损伤诱导PK-15细胞凋亡[J/OL].中国生物化学与分子生物学报.https://doi.org/10.13865/j.cnki.cjbmb.2018.11.08
[14]QI S,FENG Z,LI Q,et al.Inhibition of ROS-mediated activation Src-MAPK/AKT signaling by orientin alleviates H2O2-induced apoptosis in PC12 cells[J].Drug Des Devel Ther,2018,12:3973-3984.
[15]郑冲,叶建方,刘泳廷.亚硫酸钠对人肝细胞L-02损伤机制的研究[J].微量元素与健康研究,2016,33(4):1-3.
[2]陈华,苗加伟,李晶,等.表没食子儿茶素没食子酸酯对脂多糖诱导胶质细胞炎症的保护作用[J].中国老年学杂志,2016,16:3913-3914.
[3]赵良渊,张秋华.表没食子儿茶素没食子酸酯对亚硫酸诱导的心肌细胞氧化损伤的抑制作用[J].中国药理学通报,2011,27(2):292-293.
[4]郑斌,李光宇,李佳睿,等.绿茶提取物EGCG对视神经节细胞RGC-5的促氧化和抗氧化作用[J].中国老年学杂志,2014,34(14):3924-3926.
[5]刘婷婷,孟馨.表没食子儿茶素没食子酸酯抗氧化作用机制的研究进展[J].现代药物与临床,2016,31(6):919-923.
[6]陈怡君,熊立瑰,黄建安,等.EGCG对细胞的促氧化作用研究进展[J].茶叶科学,2015,35(2):130-136.
[7]SANG S,LEE MJ,HOU Z,et al.Stability of tea polyphenol(-)-epigallocatechin-3-gallate and formation of dimers and epimers under common experimental conditions[J].J Agric Food Chem,2005,53(24):9478-9484.
[8]YOUNG PARK S,JEONG YJ,KIM SH,et al.Epigallocatechin gallate protects against nitric oxide-induced apoptosis via scavenging ROS and modulating the Bcl-2 family in human dental pulp cells[J].J Toxicol Sci,2013,38(3):371-378.
[9]ELBLING L,HERBACEK I,WEISS RM,et al.Hydrogen peroxide mediates EGCG-induced antioxidant protection in human keratinocytes[J].Free Radic Biol Med,2010,49(9):1444-1452.
[10]LAMBERT JD,ELIAS RJ.The antioxidant and pro-oxidant activities of green tea polyphenols:A role in cancer prevention[J].Arch Biochem Biophys,2010,501(1):65-72.
[11]GAO X,LI C,TANG Y,et al.Effect of hedyotis diffusa water extract on protecting human hepatocyte cells(LO2)from H2O2-induced cytotoxicity[J].Pharm Biol,2016,54(7):1148.
[12]SCHIEBER M,CHANDEL NS.ROS function in redox signaling and oxidative stress[J].Curr Biol,2014,24(10):R453-462
[13]董峰,杨佳,李向阳,等.镉通过引发氧化应激和线粒体损伤诱导PK-15细胞凋亡[J/OL].中国生物化学与分子生物学报.https://doi.org/10.13865/j.cnki.cjbmb.2018.11.08
[14]QI S,FENG Z,LI Q,et al.Inhibition of ROS-mediated activation Src-MAPK/AKT signaling by orientin alleviates H2O2-induced apoptosis in PC12 cells[J].Drug Des Devel Ther,2018,12:3973-3984.
[15]郑冲,叶建方,刘泳廷.亚硫酸钠对人肝细胞L-02损伤机制的研究[J].微量元素与健康研究,2016,33(4):1-3.