柚皮苷对L02肝细胞氧化损伤的保护作用及机制

Protection and mechanism of naringin on oxidative damage of L02 liver cells

目的探讨柚皮苷(naringin)对L02肝细胞氧化损伤的保护作用及机制。方法设立空白组、naringin组、过氧化氢(H2O2)损伤组(损伤组)和保护组,其中空白组为L02肝细胞,naringin组为L02肝细胞加入终浓度25μmol/L的naringin,损伤组为L02肝细胞加入终浓度200μmol/L的H2O2,保护组为L02肝细胞加入终浓度200μmol/L的H2O2和25μmol/L的naringin。通过检测细胞吸光度值(A488)测定细胞活性,流式细胞术检测细胞凋亡率,用四氯四乙基苯并咪唑基羰花青碘化物(JC-1)检测线粒体膜电位(Ψm),通过检测细胞吸光度值(A532)测定丙二醛(MDA),流式细胞术检测荧光强度测定活性氧自由基(ROS),改良黄嘌呤氧化酶法检测超氧化物歧化酶(SOD)比活力。各组数据比较采用单因素方差分析和LSD-t检验。结果损伤组平均细胞活性(A488)、细胞凋亡率、Ψm分别为0.702±0.010、(25.0±0.9)%、0.95±0.03,保护组相应为0.839±0.032、(14.8±1.7)%、1.28±0.07,空白组相应为0.992±0.013、(6.2±0.5)%、1.71±0.08。损伤组细胞活性、细胞凋亡率、Ψm与空白组比较,差异有统计学意义(LSD-t=-27.44,34.98,-19.88;P<0.05)。保护组细胞活性、细胞凋亡率、Ψm与损伤组比较,差异有统计学意义(LSD-t=12.94,-18.91,8.66;P<0.05)。损伤组MDA(A532)、ROS(荧光强度)、SOD分别为0.610±0.051、1 772±69、(67±2)U/g,保护组相应为0.346±0.008、588±24、(111±6)U/g,naringin组相应为0.121±0.003、337±17、(159±5)U/g,空白组相应为0.123±0.007、375±16、(142±5)U/g。naringin组ROS、SOD与空白组比较,差异有统计学意义(LSD-t=-2.24,5.55;P<0.05)。损伤组MDA、ROS、SOD与空白组比较,差异有统计学意义(LSD-t=31.66,77.85,-24.12;P<0.05)。保护组MDA、ROS、SOD与损伤组比较,差异有统计学意义(LSD-t=-17.12,-65.97,14.03;P<0.05)。结论 naringin通过提高L02肝细胞的抗氧化能力发挥对细胞氧化损伤的保护作用。

Objective To investigate the protective effect and mechanism of naringin on oxidative damage of L02 liver cells. Methods Blank group （L02 liver cells only）, naringin group （L02 liver cells with naringin 25 pmol/L）, H202 damage group （damage group, L02 liver cells with H202 200μmol/L）and protection group （L02 liver cells with H202 200μmol/L and naringin 25 μmol/L） were assigned. Cell viability was evaluated by detecting cell absorbance value （A488）. Cell apoptosis rate was detected by flow cytometry. Mitochondrial membrane potential （ △ψm） was detected by using 5, 5＇, 6, 6＇-tetrachloro-1, 1＇, 3, 3＇- tetrethyl benzimidalyl carbocyanine iodide （JC-1）. Malondialdehyde （MDA） was evaluated by detecting cell absorbance value （A532）. Reactive oxygen species （ROS） was evaluated by detecting fluorescence intensity by flow cytometry. Superoxide dismutase （SOD） specific activity was detected by using improved xanthine oxidase detection method. Data of these groups were compared by one-way analysis of variance and LSD-t test. Results The average cell viability （A488）, cell apoptosis rate, △ψm were 0.702±0.010, （25.0±0.9）%, 0.95±0.03 respectively in damage group, 0.839±0.032, （14.8±1.7）%, 1.28±0.07 respectively in protection group, and 0.992±0.013, （6.2±0.5）%,1.71±0.08 respectively in blank group. There were significant differences in cell viability, cell apoptosis rate,△ψm between damage group and blank group （LSD-t=- 27.44, 34.98, -19.88; P〈0.05）. There were significant differences in cell viability, cell apoptosis rate, △ψm between damage group and protection group （LSD-t=12.94, -18.91, 8.66; P〈0.05）. The MDA （A532）, ROS （fluorescence intensity） and SOD specific activity were 0.6100±051, 1 772±69, （67±2）U/g respectively in damage group, 0.346±0.008, 588±24, （11±6）U/g respectively in protective group, 0.121±0.003, 337±17, （159±5）U/g respectively in naringin group, and 0.123±0.007, 375±16, （142±5）U/g respectively in blank group. There were significant differences in ROS and SOD specific activity between naringin group and blank group （LSD-t= -2.24, 5.55; P〈0.05）. There were significant differences in MDA, ROS and SOD specific activity between damage group and blank group （LSD-t=31.66, 77.85, -24.12; P〈0.05）. There were significant differences in MDA, ROS and SOD specific activity between protective group and damage group （LSD-t= -17.12,-65.97, 14.03; P〈0.05）. Conclusion Naringin can protect L02 liver cell against oxidative damage by improving its ant^oxidant capacity.