中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2011年
12期
927-931
,共5页
活性氧%氧化性应激%黄芩苷%肝型脂肪酸结合蛋白
活性氧%氧化性應激%黃芩苷%肝型脂肪痠結閤蛋白
활성양%양화성응격%황금감%간형지방산결합단백
Reactive oxygen species%Oxidative stress%Baicalin%Liver fatty acid binding protein
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.
目的 研究黃芩苷對體外氧化應激模型中肝型脂肪痠結閤蛋白(L-FABP)錶達的影響及其意義. 方法 用終濃度為400 μ mol/L的過氧化氫(H2O2) 37℃避光孵育細胞20min,建立體外誘導氧化應激模型.應用甲基噻唑基四唑法(MTT)檢測不同濃度黃芩苷作用細胞的存活率,確定24h、48 h黃芩苷的半數中毒濃度(TC50).流式細胞技術檢測不同濃度黃芩苷(25、50、100μmol/L)作用後活性氧(ROS)的錶達、細胞內超氧化物歧化酶(SOD)和穀胱甘肽(GSH)活性變化,實時PCR和Western blot檢測肝細胞內L-FABP基因和蛋白錶達.數據分析採用單因素方差分析.結果 根據MTT法得齣25、50、100 μ mol/L的黃芩苷作用于細胞24h的存活率為83.60％±3.47％,72.36％±2.18％,70.16％±2.04％,F值為386.24,P＞0.05;作用于細胞48h的存活率為84.93％±3.11％,76.16％±2.45％,72.72％±2.31％,F值為475.92,P＞0.05.直線迴歸法得齣黃芩苷持續作用24h和48h的TC50分彆為153.2、170.6μmol/L.在此範圍內,用25、50、100μmol/L濃度的黃芩苷分彆作用Chang肝細胞24、48 h後,ROS含量24 h分彆為37.0±3.30,22.90±3.84,29.60±2.52,F值為70.06,P＜0.05 ; 48h分彆為35.77±2.35,21.80±3.10,23.87±1.98,F值為110.92,P＜0.05,而400μ mol/L H2O2組ROS含量24h和48h分彆為45.50±3.47,48.80±2.70,以50μmol/L的黃芩苷作用48h效果最為顯著.用50μmol/L黃芩苷處理48h後細胞內SOD活性為(51.53±1.91)μ g/mg,GSH為(49.85±1.45) U/mg;與對照組SOD為(26.36±1.23)μ g/mg,GSH為(25.11±1.74) U/mg,F值分彆為93.81和92.51,P值均＜0.05).儘管50μmol/L黃芩苷處理48 h後細胞內L-FABP在mRNA水平併無明顯變化,但50μmol/L黃芩苷處理48 h後細胞內L-FABP蛋白錶達與對照組相比增加約80％.結論 黃芩苷能通過增彊L-FABP蛋白錶達,增加細胞內SOD和GSH的活性髮揮抗氧化作用.
목적 연구황금감대체외양화응격모형중간형지방산결합단백(L-FABP)표체적영향급기의의. 방법 용종농도위400 μ mol/L적과양화경(H2O2) 37℃피광부육세포20min,건입체외유도양화응격모형.응용갑기새서기사서법(MTT)검측불동농도황금감작용세포적존활솔,학정24h、48 h황금감적반수중독농도(TC50).류식세포기술검측불동농도황금감(25、50、100μmol/L)작용후활성양(ROS)적표체、세포내초양화물기화매(SOD)화곡광감태(GSH)활성변화,실시PCR화Western blot검측간세포내L-FABP기인화단백표체.수거분석채용단인소방차분석.결과 근거MTT법득출25、50、100 μ mol/L적황금감작용우세포24h적존활솔위83.60％±3.47％,72.36％±2.18％,70.16％±2.04％,F치위386.24,P＞0.05;작용우세포48h적존활솔위84.93％±3.11％,76.16％±2.45％,72.72％±2.31％,F치위475.92,P＞0.05.직선회귀법득출황금감지속작용24h화48h적TC50분별위153.2、170.6μmol/L.재차범위내,용25、50、100μmol/L농도적황금감분별작용Chang간세포24、48 h후,ROS함량24 h분별위37.0±3.30,22.90±3.84,29.60±2.52,F치위70.06,P＜0.05 ; 48h분별위35.77±2.35,21.80±3.10,23.87±1.98,F치위110.92,P＜0.05,이400μ mol/L H2O2조ROS함량24h화48h분별위45.50±3.47,48.80±2.70,이50μmol/L적황금감작용48h효과최위현저.용50μmol/L황금감처리48h후세포내SOD활성위(51.53±1.91)μ g/mg,GSH위(49.85±1.45) U/mg;여대조조SOD위(26.36±1.23)μ g/mg,GSH위(25.11±1.74) U/mg,F치분별위93.81화92.51,P치균＜0.05).진관50μmol/L황금감처리48 h후세포내L-FABP재mRNA수평병무명현변화,단50μmol/L황금감처리48 h후세포내L-FABP단백표체여대조조상비증가약80％.결론 황금감능통과증강L-FABP단백표체,증가세포내SOD화GSH적활성발휘항양화작용.
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.
