黄芩苷对体外氧化应激模型中肝型脂肪酸结合蛋白表达的影响及其意义

目的 研究黄芩苷对体外氧化应激模型中肝型脂肪酸结合蛋白(L-FABP)表达的影响及其意义. 方法 用终浓度为400 μ mol/L的过氧化氢(H2O2) 37℃避光孵育细胞20min,建立体外诱导氧化应激模型.应用甲基噻唑基四唑法(MTT)检测不同浓度黄芩苷作用细胞的存活率,确定24h、48 h黄芩苷的半数中毒浓度(TC50).流式细胞技术检测不同浓度黄芩苷(25、50、100μmol/L)作用后活性氧(ROS)的表达、细胞内超氧化物歧化酶(SOD)和谷胱甘肽(GSH)活性变化,实时PCR和Western blot检测肝细胞内L-FABP基因和蛋白表达.数据分析采用单因素方差分析.结果 根据MTT法得出25、50、100 μ mol/L的黄芩苷作用于细胞24h的存活率为83.60％±3.47％,72.36％±2.18％,70.16％±2.04％,F值为386.24,P＞0.05;作用于细胞48h的存活率为84.93％±3.11％,76.16％±2.45％,72.72％±2.31％,F值为475.92,P＞0.05.直线回归法得出黄芩苷持续作用24h和48h的TC50分别为153.2、170.6μmol/L.在此范围内,用25、50、100μmol/L浓度的黄芩苷分别作用Chang肝细胞24、48 h后,ROS含量24 h分别为37.0±3.30,22.90±3.84,29.60±2.52,F值为70.06,P＜0.05 ; 48h分别为35.77±2.35,21.80±3.10,23.87±1.98,F值为110.92,P＜0.05,而400μ mol/L H2O2组ROS含量24h和48h分别为45.50±3.47,48.80±2.70,以50μmol/L的黄芩苷作用48h效果最为显著.用50μmol/L黄芩苷处理48h后细胞内SOD活性为(51.53±1.91)μ g/mg,GSH为(49.85±1.45) U/mg;与对照组SOD为(26.36±1.23)μ g/mg,GSH为(25.11±1.74) U/mg,F值分别为93.81和92.51,P值均＜0.05).尽管50μmol/L黄芩苷处理48 h后细胞内L-FABP在mRNA水平并无明显变化,但50μmol/L黄芩苷处理48 h后细胞内L-FABP蛋白表达与对照组相比增加约80％.结论 黄芩苷能通过增强L-FABP蛋白表达,增加细胞内SOD和GSH的活性发挥抗氧化作用.

Effect of baicalin on liver fatty acid binding protein in oxidative stress model in vitro

Objective To investigate the effect of baicalin on liver fatty acid binding protein in oxidative stress model in vitro.Methods (1) Cellular oxidative stress in vitro was induced by incubating cells with 400μmol/L hydrogen peroxide (H2O2) for 20 minutes at 37℃ in the dark.After Chang liver cell line was treated with different dose of baicalin for 24,48 and 72 hours.MTT assay was employed to detect cell viability,and then the hydrogen peroxide (TC50) of the different dose of baicalin was calculated.(2) Based on MTT assay,cells were treated with three different doses of baicalin (25,50,100 μmol/L) for 24 and 48 hours before being exposed to 400 μmol/L H2O2 for 20 minutes at 37℃.Then,reactive oxygen species (ROS) assay and activity assays of superoxide dismutase (SOD) and reduced glutathione hormone (GSH) were evaluated.(3) Realtime PCR and Western blotting were applied to explore the influence of baicalin on the expression level of L-FABP.(4) One-way ANOVA was used for results statistical analysis.Reesult (1) MTT assay showed baicalin treatment at 25,50,100 μmol/L for 24 and 48 hours was feasible (83.60％ ± 3.47％,72.36％ ±2.18％,70.16％ ± 2.04％ for 24 hour; 84.93％ ± 3.11％o,76.16％ ± 2.45％,72.72％ ± 2.31％ for 48 hours,P ＞ 0.05,F=386.24,475.92 respectively).Meanwhile,we found by the linear regression model that the median toxic concentration of baicalin for 48 hours was 170.6 μmol/L,and the median toxic concentration of baicalin for 24 hours was 153.2 μmol/L.(2) ROS assay showed dichlorofluorescin in all baicalin-treated cells after stress was significantly reduced (37.0 ± 3.30,22.90 ± 3.84,29.60 ± 2.52 for 24 hours respectively,P ＜ 0.05,F =70.06; 35.77 ± 2.35,21.80 ± 3.10,23.87 ± 1.98 for 48 hours respectively,P ＜ 0.05,F =110.92) as compared with the H2O2-treated cells.Moreover,50 μmol/L baicalin treatment for 48 hours was the optimal condition against ROS generation (21.80 ± 3.10,P ＜ 0.01,F =110.92).Furthermore,the activities of intracellular SOD and GSH was increased significantly (51.53 ± 1.91 μg/mg for SOD,P＜ 0.05,F =93.81; 49.85 ± 1.45 U/mg for GSH,P ＜ 0.05,F =92.51).(3) Although realtime PCR analysis indicated 50 μmol/L baicalin treatment for 48 hours could have no changes of the level of L-FABP expression under the oxidative stress condition,western blotting analysis indicated 50 μmol/L baicalin treatment for 48 hours could increase up to about 80％ for the level of L-FABP expression.Conclusion Baicalin was suggested to be able to enhance both L-FABP expression and activity of intracellular SOD and GSH,and therefore protected hepatocytes from oxidative stress.