祁州漏芦通过下调JNK和NFκB抑制H2O2致肝细胞凋亡(1)
[摘要]研究祁州漏芦对H2O2所致HepG2细胞凋亡的抑制作用机制。建立H2O2诱导的人HepG2细胞损伤模型，采用MTT法检测细胞存活率；采用化学比色法检测LDH，ALT，AST活性；采用黄嘌呤氧化酶法检测细胞SOD活性，二硫代二硝基苯甲酸法检测GSH含量，采用硫代巴比妥酸法检测MDA生成量，比色法测定Caspase3，8，9的相对活性；蛋白印迹法测定Cleaved Caspase3(Casp3)，细胞色素c(Cyto c)和NFκB，ERK，JNK，p38 MAPK及其磷酸化蛋白的表达。结果显示，祁州漏芦在质量浓度25～400 mg·L-1对HepG2细胞活力无显著影响。H2O2降低细胞存活率，造成细胞损伤，并上调Casp3，胞浆Cyto c，pJNK以及核NFκB蛋白水平。与模型组比较，祁州漏芦组细胞存活率升高；培养液中LDH，ALT和AST活性降低；细胞内MDA含量降低，SOD活性和GSH含量升高，Caspase3，8，9相对活性降低，细胞Casp3和胞浆Cyto c蛋白表达降低，细胞pJNK及核NFκB蛋白水平降低。提示，祁州漏芦对H2O2所致HepG2细胞凋亡具有抑制作用，其作用可能与其抑制JNK激活和NFκB核转位作用有关。
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[关键词]祁州漏芦；H2O2；氧化应激；凋亡；HepG2
[Abstract]To study the inhibitory effect of Rhaponticum uniflorum on apoptosis induced by H2O2 in HepG2 cells Human HepG2 cells injury models were established by H2O2， then cell survival rate was assayed by MTT method； levels of LDH， ALT， and AST were detected by chemical colorimetric method；SOD activity was detected by xanthine oxidase method； GSH content was detected by dithiobisnitrobenzoic acid(DTNB)； MDA level was detected by thiobarbituric acid (TBA) method；and the relative activities of Caspase3， 8 and 9 were measured by Colorimetry The expression levels of Cleaved Caspase3(Casp3)， cytochrome(Cyto c)， NFκB， ERK， JNK， p38 MAPK， as well as the phospharylated proteins were determined with Western blotting method The results showed that R unifloru had no significant effect on cell viabilities of HepG2 cells at the concentrations of 25400 mg·L-1 However， H2O2decreased the cell viabilities， increased the cellular oxidative stress， and upregulated the protein expressions of Casp3， cytoplasmic Cyto c， pJNK and nuclear NFκB As compared with the model group，R unifloru could increase the cell viability， reduce LDH， ALT and AST leakage， reduce the MDA formation， increase the SOD and GSH levels，reduce the relative activities of Caspase3， 8 and 9， downregulated the protein expressions of Casp3 and cytoplasmic Cyto c， and downregulate the pJNK and nuclear NFκB levelsThe results indicated that R unifloru had the inhibitory effect on apoptosis induced by H2O2in HepG2 cells， and the mechanism maybe associated with inhibiting JNK activation and NFκB nuclear translocation
, http://www.100md.com
[Key words]Rhaponticum unifloru； H2O2； oxidative stress； apoptosis； HepG2
近年來肝病发生率的逐年上升已经引起社会的关注，而活性氧引发的肝细胞氧化应激是多种肝病发生的重要机制之一，因此，探讨如何有效缓解氧化应激损伤对肝病的防治具有重要意义[1]。祁州漏芦是菊科植物Rhaponticum uniflorum (L) DC的干燥根，是一味常用中药。广泛分布于我国内蒙古、东北、山东等地区，药源丰富。它性味咸、苦、寒，能清热解毒，消肿排毒，通乳汁；主治于乳汁不通、淋巴结结核、风湿性关节炎、痔疮等[23]。近年来研究发现，祁州漏芦具有提高机体免疫、抗动脉硬化、抗脂质过氧化、抗衰老、抗炎等作用[46]。课题组研究还发现，祁州漏芦具有良好的保肝作用，能预防多种因素引起的肝损伤，如，可抑制四氯化碳、氨基半乳糖所致化学性肝损伤以及对乙酰氨基酚所致药物性肝损伤等[79]。本实验通过建立H2O2致HepG2细胞损伤模型，预防性给药后观察祁州漏芦水提物对氧化损伤所致凋亡以及凋亡相关蛋白表达的变化，以便探讨祁州漏芦肝细胞损伤的保护作用机制，为祁州漏芦的保肝作用研究提供参考。, http://www.100md.com(何鑫 刘春彦 尹基峰 金爱花 尹学哲 全吉淑)
, http://www.100md.com
[关键词]祁州漏芦；H2O2；氧化应激；凋亡；HepG2
[Abstract]To study the inhibitory effect of Rhaponticum uniflorum on apoptosis induced by H2O2 in HepG2 cells Human HepG2 cells injury models were established by H2O2， then cell survival rate was assayed by MTT method； levels of LDH， ALT， and AST were detected by chemical colorimetric method；SOD activity was detected by xanthine oxidase method； GSH content was detected by dithiobisnitrobenzoic acid(DTNB)； MDA level was detected by thiobarbituric acid (TBA) method；and the relative activities of Caspase3， 8 and 9 were measured by Colorimetry The expression levels of Cleaved Caspase3(Casp3)， cytochrome(Cyto c)， NFκB， ERK， JNK， p38 MAPK， as well as the phospharylated proteins were determined with Western blotting method The results showed that R unifloru had no significant effect on cell viabilities of HepG2 cells at the concentrations of 25400 mg·L-1 However， H2O2decreased the cell viabilities， increased the cellular oxidative stress， and upregulated the protein expressions of Casp3， cytoplasmic Cyto c， pJNK and nuclear NFκB As compared with the model group，R unifloru could increase the cell viability， reduce LDH， ALT and AST leakage， reduce the MDA formation， increase the SOD and GSH levels，reduce the relative activities of Caspase3， 8 and 9， downregulated the protein expressions of Casp3 and cytoplasmic Cyto c， and downregulate the pJNK and nuclear NFκB levelsThe results indicated that R unifloru had the inhibitory effect on apoptosis induced by H2O2in HepG2 cells， and the mechanism maybe associated with inhibiting JNK activation and NFκB nuclear translocation
, http://www.100md.com
[Key words]Rhaponticum unifloru； H2O2； oxidative stress； apoptosis； HepG2
近年來肝病发生率的逐年上升已经引起社会的关注，而活性氧引发的肝细胞氧化应激是多种肝病发生的重要机制之一，因此，探讨如何有效缓解氧化应激损伤对肝病的防治具有重要意义[1]。祁州漏芦是菊科植物Rhaponticum uniflorum (L) DC的干燥根，是一味常用中药。广泛分布于我国内蒙古、东北、山东等地区，药源丰富。它性味咸、苦、寒，能清热解毒，消肿排毒，通乳汁；主治于乳汁不通、淋巴结结核、风湿性关节炎、痔疮等[23]。近年来研究发现，祁州漏芦具有提高机体免疫、抗动脉硬化、抗脂质过氧化、抗衰老、抗炎等作用[46]。课题组研究还发现，祁州漏芦具有良好的保肝作用，能预防多种因素引起的肝损伤，如，可抑制四氯化碳、氨基半乳糖所致化学性肝损伤以及对乙酰氨基酚所致药物性肝损伤等[79]。本实验通过建立H2O2致HepG2细胞损伤模型，预防性给药后观察祁州漏芦水提物对氧化损伤所致凋亡以及凋亡相关蛋白表达的变化，以便探讨祁州漏芦肝细胞损伤的保护作用机制，为祁州漏芦的保肝作用研究提供参考。, http://www.100md.com(何鑫 刘春彦 尹基峰 金爱花 尹学哲 全吉淑)