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1、陕西中医学院硕士学位论文何首乌对肝脏损害的物质基础及其机制研究姓名：张敏申请学位级别：硕士专业：中药学指导教师：卫培峰2011-04摘要目的：本课题通过何首乌不同成分及制首乌对肝细胞凋亡及P450含量的影响，以及何首乌不同炮制品对肝脏CYP2E1基因mRNA表达及CYP2E1酶活性的影响，旨在探究何首乌致肝损害的物质基础及其作用机制。也为进一步探索何首乌炮制方法与导致肝损害之间的关系提供科学依据。方法：用制首乌不同成分提取物大黄素、大黄酸、大黄酚，以及制首乌分别按不同剂量给予大鼠灌胃，每天一次，观察大鼠一般情况，连续给药3个月后，摘眼球采血检测ALT和AST指标，颈总动脉处死检测肝细胞凋亡指数
2、及细胞色素P450含量。筛选出何首乌致肝损害的化学成分，制何首乌和此种物质按一定剂量给予小鼠灌胃，连续给药4周，摘眼球采血检测ALT和AST指标，颈总动脉处死检测肝细胞凋亡率。用何首乌的不同炮制品即清蒸首乌、黑豆汁制首乌A、B、C分别给予大鼠灌胃，观察大鼠一般情况，连续给药3个月后，摘眼球采血检测ALT和AST指标，颈总动脉处死检测肝脏CYP2E1基因mRNA表达水平、CYP2E1酶活性及肝脏的病理学变化。结果：给药期间，与对照组比较，各给药组食、水摄入量没有差异。在何首乌不同成分致肝损害的实验研究中，各给药组大鼠血清中 ALT 均有不同程度的升高，与空白组比较，大黄酚组大鼠血清 ALT 升高
3、具有显著的统计学意义（P0.05），大黄素组大鼠血清 AST 升高具有显著的统计学意义（P0.05）；制首乌及大黄酚组肝细胞凋亡指数明显增加有非常显著的统计学意义（P0.05）；各给药组肝脏细胞色素 P450 含量无明显变化（P0.05）。在制首乌及大黄酚致小鼠肝细胞凋亡实验中，发现各给药组小鼠血清 ALT、AST 均有不同程度的升高，但与空白组比较无统计学意义（P0.05）；制首乌组、大黄酚组肝细胞凋亡率均升高，与空白组比较，肝细胞凋亡率均有显著的统计学意义（P0.05）。在何首乌不同炮制品致肝损害的实验研究中，清蒸组大鼠血清中 AST 升高具有非常显著的统计学意义（P0.01），黑豆汁制首
4、乌 A、B、C 组大鼠血清 AST 升高具有显著的统计学意义（P0.05）；与空白组比较，各给药组CYP2E1 基因 mRNA 表达水平明显降低(P0.05)；与空白组比较，黑豆汁制首乌 B 组、黑豆汁制首乌 C 组对大鼠肝微粒体 CYP2E1 酶活性有显著性诱导作用（P0.05)；病理观察显示：与空白组比较，各给药组出现了明显的病理改变。结论：何首乌中导致肝细胞凋亡的主要成分很有可能是大黄酚等某种成分，并且会对肝脏造成一定程度的损害。何首乌不同炮制品均能够显著抑制 CYP2E1 mRNA 的表达；黑豆汁制首乌 B、C 组能够诱导大鼠肝微粒体 CYP2E1 酶活性，清蒸及黑豆汁制首乌 A 组对
5、其影响不显著；各给药组肝脏的病理结果主要表现为肝细胞脂肪变性，色素颗粒沉积，炎细胞浸润，由此得知何首乌不同炮制方法与导致肝损害之间存在一定的关系。关键词：何首乌；肝脏损害；物质基础；作用机制Study of Material Foundation and Mechanism ofmultiflorum Thunb on liver injuryAbstractPolygonumObjectiveThrougheffectofdifferentcomponentsofPolygonummultiflorum and Radix Polygoni Multiflori prepared on He
6、patocyte Apoptosisand Liver Cytochrome P450 and effect of different Radix Polygoni multifloriPreparata on the enzyme activity and mRNA level of CYP2E1 insubject were designed to explore the material base and mechanismliver ,thisof Polygonummultiflorum Thunb .Providing a scientific basiswith further
7、explore torelationship between processing methods of Polygonum multiflorum Thunb andthe causing of hepatic damage.Methods The rats were infused with different component extract of Polygonummultiflorum Thunbsuch as emodin, rhein, chrysophanoland prepared RadixPolygoli Multiflori ,different dosages on
8、ce a day.Watch the general situationof rats;It was successively administered for 3 months, collect blood from eyein order to detection of ALT and AST parameters,sacrificed rats from carotidartery to testing Hepatocyte Apoptosis indexand liver Cytochrome P450;Afterscreening the chemical composition o
9、f Polygonummultiflorum Thunb whichcaused liver damage,the mice were infused with prepared Radix PolygoliMultiflori and this chemicals , certain dosages once a day, it was successivelyadministered for 4 weeks, collect blood from eye in order to detection of ALTand AST parameters,sacrificed mice from
10、carotid artery to testing HepatocyteApoptosis rate. The rats were infused with different Radix Polygoni Multifloriprepared such as the steamedand Polygonummultiflorum Thunb (A、B、C)，different dosages once a day.Watch the general situation of rats;It wassuccessively administered for 3 months, collect
11、blood from eye in order todetection of ALT and AST parameters，sacrificed rats from carotid artery totesting on the enzyme activity and mRNA level of CYP2E1 inliver and thehepatocytes changing of rats in shape.Results During the experiment,water and food took in by test team andthe control ones had n
12、o differences.In the experiment of different componentsof Polygonummultiflorum causing Hepatic damage,each of the rats serum ALTof the administration droupselevated in different degree,compared withcontrol group,the serum ALT of chrysophanol group elevated has a significantstatistical significance（P
13、0.05），the serum AST of emodin groupelevatedhas a significant statistical significance（P0.05）; The hepatocyte apoptosisindex of Radix Polygoni Multiflori prepared and chrysophanol groupsignificantelevated,andithasaverysignificantstatisticalsignificance(P0.05),each of the liverCytochromeP450oftheadministrationdroupshasnosignificantdifference(P0.05). In the experiment ofRadix Polygoni Multiflori preparedand chrysophanolcausinghepatocyte apoptosis of the mouse, it is foundthat each of the mice seru
