1、ObjectiveTo study the protective effect and its mechanism of Yulangsan polysaccharide(YLS) on primary cultured rat hepatocytes injury induced by H2O2. MethodsThe primary rat hepatocytes were isolated by perfusion with IV collaganase and injured by H2O2. The contents of malondialdehyde(MDA) and gluta
2、thion(GSH) in hepatocytes and the levels of AST and ALT in cultural supernatant were determined by general methods. Cell viability was assayed by MTT method. ResultsThe elevation of MDA content in hepatocytes and AST and ALT levels in supernatant of cultural hepatocytes , and the decrease in cell vi
3、ability and GSH content induced by H2O2 were restored remarkably by the treatment with YLS.ConclusionThe results suggest that YLS possesses direct protective action on primary hepatocyte injury induced by H2O2. This might be associated with the anti-oxidative activity of YLS.Key words:Yulangsan Poly
4、saccharide(YLS); Hepatocyte; H2O2 ; Anti-oxidative activity 玉郎伞是一种尚未开发利用的民间草药,为蝶型花科植物疏叶岩豆 Millettia pulchra Kurz var. laxior(Dunn)Z Wei 的块根。具有散淤、消肿止痛之功能,民间用于高血压、肝炎、消化不良等的治疗,本课题组经多年研究证实玉郎伞提取物具有抗氧化和免疫调节作用,并对急、慢性实验性肝损伤均有明显的保护作用1,2。玉郎伞多糖是玉郎伞提取物中主要的有效成分,为进一步研究YLS对肝损伤的作用,本文采用IV型胶原酶灌流法分离肝实质细胞进行体外培养,用过氧化氢(H
5、2O2)诱导肝细胞损伤模型进行体外研究,观察YLS对体外肝细胞损伤的保护作用及其机制。1 器材动物 Wistar大鼠,雌雄不拘,体重(23030)g;由广西医科大学实验动物中心提供。实验动物生产许可证:SCXK 桂2003 0003,实验动物使用许可证:SYXK 桂2003 0005。药物及试剂 YLS,由本室自行提取;IV型胶原酶(Sigma公司);RPMI 1640(Gibco公司);胎牛血清(杭州四季青公司);H2O2(重庆川江化学试剂厂);维生素E(Sigma公司);台盼蓝(SigmaT 6146,北京拜尔迪生物公司分装);噻唑蓝;D Hanks缓冲液;Hanks缓冲液;二甲基亚砜;天
6、门冬氨酸转换酶(AST)、丙氨酸氨基转换酶(ALT)试剂盒;丙二醛(MDA)试剂盒;谷胱甘肽(GSH)试剂盒。仪器 HL 2型恒流泵;CO2恒温培养箱;TDL80 2B台式离心机;XD-101倒置显微镜; SZX型超净工作台;722 s型可见分光光度计;450型酶标仪。2 方法大鼠原代肝细胞的分离及原代培养 3,4大鼠用20%乌拉坦腹腔麻醉后,固定四肢,腹部消毒后,无菌操作下,打开腹腔,将肠管推向左侧,暴露门静脉和下腔静脉,小心分离穿线各一根,门静脉穿刺静脉留置针,退出针芯,结扎牢固。将留置管连接输液管,开启恒流泵,灌流37预热D Hanks液,流速15 ml/min,迅速剪开下腔静脉放血。同
7、时打开胸腔暴露并结扎上腔静脉。灌流约10 min后,肝脏颜色呈黄白色。下腔静脉插入硅胶管,结扎牢固。改用含% IV型胶原酶的Hanks液15 ml/min,约15 min后,肝脏软化,压之凹陷不易恢复,迅速取下完整肝脏,连同含胶原酶灌流液置于平皿中,剔除肝包膜,轻柔摆动肝组织,让细胞慢慢游离出来,200目筛过滤得细胞悬液。600 r/min离心,弃上清,沉淀加1640培养液吹打混匀,800 r/min离心,共洗涤两次。用血球计数板测定细胞活率及细胞密度。用1640培养液调整细胞密度为5105/ml,肝细胞活力大于90%。将上述肝细胞悬液加入96孔和24孔培养板中,置37,5%CO2培养箱中培养
8、12 h后,肝细胞全部贴壁生长,供试验用。YLS对H2O2诱导肝细胞损伤的作用5,6维生素E作阳性对照,用少量DMSO溶解,加入到培养液后其终浓度为50 mol/ml。取上述贴壁生长的肝细胞,设H2O2模型组、YLSmg/ml 4个剂量组和空白对照组、维生素E组,每组至少设6个复孔。分别加入H2O2mmol/L,不同浓度的YLS,维生素E和溶媒,继续培养24 h后,收集培养上清检测AST,ALT活性;弃肝细胞培养上清,加入 ml Triton-100水溶液 ml,混匀,2 500 r/min离心10 min,取上清测定肝细胞MDA和GSH含量,同时用MTT比色法,测定肝细胞的活率。MTT比色法
9、待测细胞悬液于96孔培养板培养24 h后,各孔加入20 l MTT试剂,轻轻混匀,继续37培养4 h,吸出全部液体,加200 l DMSO,微量振荡器上振荡15 min,待溶解完全,于酶标仪570 nm波长比色。赖氏法测定AST和ALT按试剂盒说明测定。MDA和GSH含量测定按试剂盒说明测定。数据处理 所有数据均以s表示,采用t检验进行统计处理。3 结果结果见表1。YLS对H2O2损伤肝细胞ALT和AST活性的影响 H2O2损伤肝细胞后,促进肝细胞内酶的释放,模型组的ALT,AST水平较正常组明显升高,YLS在mg/ml剂量范围内,能显着抑制H2O2损伤肝细胞后ALT,AST水平的上升,并呈明
10、显的剂量依赖性;VitE也能明显抑制H2O2损伤肝细胞后ALT,AST水平的上升。YLS对H2O2损伤肝细胞MDA和GSH的影响 H2O2损伤肝细胞后,肝细胞中的MDA明显升高,GSH则显着降低,除 mg/ml外,其余YLS各剂量组均能显着提高H2O2损伤肝细胞中的GSH含量和降低H2O2损伤肝细胞中的MDA含量,并呈明显的剂量依赖性;VitE对H2O2损伤肝细胞也具有提高GSH含量和降低MDA含量的作用。YLS对H2O2损伤肝细胞活性的影响 MTT法测定结果表明,H2O2损伤后模型组细胞活性明显下降,YLS在mg/ml剂量范围内,能显着恢复和升高H2O2损伤肝细胞后的细胞活性,并呈明显的剂量
11、依赖性;VitE也能恢复和升高H2O2损伤肝细胞后的细胞活性。表1 YLS对H2O2诱导肝细胞损伤的作用与H2O2组比较, *,*,与对照组比较,;n=64 讨论在肝细胞损伤过程中,氧自由基起了重要作用。OFR及其诱发的脂质过氧化物(LPO)可引起激烈的链式反应,产生几十种毒性分子,再次诱发成 1 焦 杨,段小群,黄仁彬,等. 玉郎伞提取物对超氧阴离子自由基和羟自由基的抑制和清除作用J.广西医科大学报,2004,21(1):22. 2 黄仁彬,段小群,焦 杨,等.玉郎伞提取物对小鼠急性化学性肝损伤保护作用及其机制的研究J.广西医科大学学报,2003,20(6):874.3 Solis-Herr
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