基于氧化应激和TLR-4/NF-κB通路研究算盘子根总黄酮保肝作用及其机制
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摘要
目的:研究算盘子根总黄酮对四氯化碳(CCl4)致急性肝损伤小鼠的保护作用及其作用机制。方法:60只小鼠随机分为正常组、模型组、水飞蓟素组(150 mg/kg)、算盘子根总黄酮(300 mg/kg、150 mg/kg、75mg/kg)剂量组,每日以10 ml/kg灌胃给药1次,连续给药10d。末次给药2 h后,除正常组外,其余各组小鼠均腹腔注射0. 12%CCl4花生油溶液(10 ml/kg)建立急性肝损伤模型,16h后,眼球取血,收集肝组织。生化法测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、总超氧化物歧化酶(T-SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)及一氧化氮(NO)活性或含量;酶联免疫吸附法(ELISA)检测肝组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)水平; HE染色肝切片,观察肝组织病理变化;蛋白免疫印迹法(Western blot)分析肝组织中Toll样受体4(TLR-4)和核转录因子-κB(NF-κB)蛋白表达情况。结果:算盘子根总黄酮各剂量均可显著降低肝损伤小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、一氧化氮(NO)及丙二醛(MDA)活性或含量,增强T-SOD和GSH-Px活性;下调肝组织IL-1β、IL-6、TNF-α水平并抑制肝组织TLR-4及NF-κB蛋白表达;同时病理切片显示算盘子根总黄酮各剂量均对小鼠肝损伤有改善作用。结论:算盘子根总黄酮对CCl4致急性肝损伤小鼠有良好保护作用,其作用机制可能与抑制氧化应激、抑制炎症反应以及调控TLR-4/NF-κB通路有关。
Objective: To study the protective effects and related mechanismof total flavonoids from the root of Glochidionpuberum(Linn.) Hutch.on mice with acute liver injury induced by carbon tetrachloride(CCl4). Methods: Sixty mice were randomly divided into normal group,model group,silymarin(150 mg/kg) group,total flavonoids from the root of Glochidionpuberum(Linn.) Hutch.(TFRG)(300 mg/kg,150 mg/kg,75 mg/kg) groups. Each treatment group was administered once a day for 10 days. After the last administration for 2 hours,except for the normal group,all other mice were injected intraperitoneally with 0. 12% CCl4 peanut oil solution(10 ml/kg) to establish acute liver injury model in mice. After 16 hours,the eyeballs were taken to separate serum and the liver tissues were collected. The levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),total-superoxide dismutase(T-SOD),malondialdehyde(MDA),glutathione peroxidase(GSH-Px) andnitric oxide(NO) in serum were measured by biochemical method. The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β) and interleukin-6(IL-6) in liver were detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of liver tissue were observed after HE staining. The expressions of Toll-like receptor-4(TLR-4) and nuclear factor-κB(NF-κB) protein in liver tissue were analyzed by Western blot. Results: Compared with the model group,the levels of ALT,AST,NO and MDA in the serum of the TFRG groups(300 mg/kg,150 mg/kg,75 mg/kg) obviously were decreased,while the the activities of T-SOD and GSH-Px were increased,and the expressions of TNF-α,IL-1β,IL-6,TLR-4 and NF-κB were decreased in the TFRG groups. At the same time,the pathological section showed that the pathological changes of liver tissue in the TFRG groups improved significantly. Conclusion: TFRG could prevent CCl4-induced acute liver injury in mice,and its mechanism may be related to inhibition of oxidative stress,inflammatory reaction and TLR-4/NF-κB pathway.
Objective: To study the protective effects and related mechanismof total flavonoids from the root of Glochidionpuberum(Linn.) Hutch.on mice with acute liver injury induced by carbon tetrachloride(CCl4). Methods: Sixty mice were randomly divided into normal group,model group,silymarin(150 mg/kg) group,total flavonoids from the root of Glochidionpuberum(Linn.) Hutch.(TFRG)(300 mg/kg,150 mg/kg,75 mg/kg) groups. Each treatment group was administered once a day for 10 days. After the last administration for 2 hours,except for the normal group,all other mice were injected intraperitoneally with 0. 12% CCl4 peanut oil solution(10 ml/kg) to establish acute liver injury model in mice. After 16 hours,the eyeballs were taken to separate serum and the liver tissues were collected. The levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),total-superoxide dismutase(T-SOD),malondialdehyde(MDA),glutathione peroxidase(GSH-Px) andnitric oxide(NO) in serum were measured by biochemical method. The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β) and interleukin-6(IL-6) in liver were detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of liver tissue were observed after HE staining. The expressions of Toll-like receptor-4(TLR-4) and nuclear factor-κB(NF-κB) protein in liver tissue were analyzed by Western blot. Results: Compared with the model group,the levels of ALT,AST,NO and MDA in the serum of the TFRG groups(300 mg/kg,150 mg/kg,75 mg/kg) obviously were decreased,while the the activities of T-SOD and GSH-Px were increased,and the expressions of TNF-α,IL-1β,IL-6,TLR-4 and NF-κB were decreased in the TFRG groups. At the same time,the pathological section showed that the pathological changes of liver tissue in the TFRG groups improved significantly. Conclusion: TFRG could prevent CCl4-induced acute liver injury in mice,and its mechanism may be related to inhibition of oxidative stress,inflammatory reaction and TLR-4/NF-κB pathway.
引文
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15 Yue S,Hu B,Wang Z,et al. Salvia miltiorrhiza compounds protect the liver from acute injury by regulation of p38 and NFκB signaling in Kupffer cells. Pharm Biol,2014; 52(10)∶1278~1285
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17 Wang X,Rui Sun,Wei H,et al. High-mobility group box 1(HMGB1)-toll-like receptor(TLR)4-interleukin(IL)-23-IL-17A axis in drug-induced damage-associated lethal hepatitis∶Interaction ofγδT cells with macrophages. Hepatology,2013; 57(1)∶373~384
18 Chaochao Q,Lou G,Yang Y,et al. Macrophage Inflammatory Protein-2 in High Mobility Group Box 1 Secretion of Macrophage Cells Exposed to Lipopolysaccharide. Cell Physiol Biochem,2017; 42(3)∶913~928
2 赵雪巍,刘培玉,刘丹,等.黄酮类化合物的构效关系研究进展.中草药,2015; 46(21)∶3264~3271
3 丁水平,丁水生,李涵志.算盘子对溃疡性结肠炎大鼠细胞因子的影响.医药导报,2002; 21(2)∶76~77
4 黄爱军.算盘子提取物抗炎镇痛作用的实验研究.湖北民族学院学报(医学版),2010; 27(4)∶17~19
5 肖怀,张桢,何文姬.药用植物算盘子化学成分的初步研究.大理学院学报,2009; 8(10)∶1~2
6 赵文惠,曾诚,秦冬梅.维药昆仑雪菊多糖对四氯化碳所致小鼠急性肝损伤的预防作用及机制研究.中国药房,2017; 28(31)∶4407~4411
7 乔靖怡,李汉伟,付双楠,等.山楂总黄酮对四氯化碳致大鼠急性肝损伤的保护作用.中药药理与临床,2016; 32(5)∶52~55
8 Mantawy E M,Tadros M G,Awad A S,et al. Insights antifibrotic mechanism of methyl palmitate∶impact on nuclear factor kappa B and proinflammatory cytokines. Toxicology Applied Pharmacology,2012; 258(1)∶134~144
9王跃峰,张可锋,周雨晴,等.拳卷地钱总黄酮对四氯化碳致急性肝损伤大鼠的保护作用及其作用机制.时珍国医国药,2017; 28(2)∶277~279
10黄思茂,曹后康,高雅,等.金花茶多糖对四氯化碳致小鼠急性肝损伤的保护作用及其机制的研究.中药药理与临床,2016; 32(6)∶117~120
11 Hussain F,Malik A,Ayyaz U,et al. Efficient hepatoprotective activity of cranberry extract against CCl4-induced hepatotoxicity in Wistar albino rat model∶Down-regulation of liver enzymes and strong antioxidant activity. Asian Pac J Trop Med,2017; 10(11)∶1054~1058
12 Molehin O R,Adeyanju A A,Adefegha S A,et al. Sildenafil,a phosphodiesterase-5 inhibitor,offers protection against carbon tetrachloride-induced hepatotoxicity in rat. J Basic Clin Physiol Pharmacol,2018; 29(1)∶29~35
13 黄静,李胜立,赵宏伟,等.霍山石斛多糖对四氯化碳致急性肝损伤小鼠的保护作用.中国中药杂志,2013; 38(4)∶528~532
14 王春妍,范玉强,迟宝荣,等.核因子-κB及其下游因子TNF-α、Bcl-2在急性肝损伤中的作用及机制.世界华人消化杂志,2008; 16(25)∶2804~2808
15 Yue S,Hu B,Wang Z,et al. Salvia miltiorrhiza compounds protect the liver from acute injury by regulation of p38 and NFκB signaling in Kupffer cells. Pharm Biol,2014; 52(10)∶1278~1285
16 Zhang W B,Zhang H Y,Zhang Q,et al. Glutamine ameliorates lipopolysaccharide-induced cardiac dysfunction by regulating the toll-like receptor4 /mitogen-activated protein kinase/nuclear factor-kB signaling pathway. Exp Ther Med,2017; 14(6)∶5825~5832
17 Wang X,Rui Sun,Wei H,et al. High-mobility group box 1(HMGB1)-toll-like receptor(TLR)4-interleukin(IL)-23-IL-17A axis in drug-induced damage-associated lethal hepatitis∶Interaction ofγδT cells with macrophages. Hepatology,2013; 57(1)∶373~384
18 Chaochao Q,Lou G,Yang Y,et al. Macrophage Inflammatory Protein-2 in High Mobility Group Box 1 Secretion of Macrophage Cells Exposed to Lipopolysaccharide. Cell Physiol Biochem,2017; 42(3)∶913~928