雷公藤多苷致大鼠肝损伤“时—效关系”的免疫毒理机制研究

【作者】 杨雪；

【导师】 高永翔；

【作者基本信息】 成都中医药大学 ， 中西医结合基础， 2007， 硕士

【摘要】 目的:探讨雷公藤多苷临床等效剂量致大鼠肝损伤“时—效关系”的免疫毒理机制。观测不同给药时段大鼠肝损伤后恢复期的损伤可逆程度。方法:给予健康成年大鼠雷公藤多苷片临床常用等效剂量的水溶液灌胃,分五个时间点给药,检测大鼠一般情况及免疫相关指标,包括体重、肝脏、胸腺、脾脏脏器指数,血清ALT、AST酶;免疫细胞活性（NK细胞活性、T细胞增值转化功能、B细胞增值转化功能）;免疫组织化学方法,检测肝脏Kupffer细胞CD₆₈表达情况,对大鼠肝Kupffer细胞活化程度进行定性定量分析;酶联免疫方法（ELISA）检测肝脏组织（匀浆）中TNF-α的含量;免疫组织化学方法标记大鼠肝组织中凋亡蛋白Fas的表达,并通过图像分析系统定量;流式细胞仪检测大鼠肝细胞凋亡率（指数）和凋亡周期;激光共聚焦显微镜检测大鼠肝细胞内钙离子浓度。以期对临床等效剂量雷公藤多苷导致肝损伤的免疫毒性的可能机制进行探讨和研究,结合给药时间分析肝损伤的时—效相关性。并探讨不同时段临床等效剂量雷公藤多苷导致肝损伤后恢复期的损伤可逆程度。结果:1、与空白组比较,临床等效剂量雷公藤多苷给药五周后大鼠体重下降（p＜0.05）、肝脏指数下降（p＜0.05）、脾脏指数下降（p＜0.05）;给药三周、四周和五周后大鼠胸腺指数均下降（p＜0.05）;给药五周后大鼠的血清ALT酶升高（p＜0.05）;给药四周后大鼠血清AST酶升高（p＜0.05）,给药五周后大鼠血清AST酶升高（p＜0.01）;肝组织病理形态学观察结果提示:给药三周内未见肝组织的明显病理改变。给药三周后大鼠肝组织开始出现肝小叶失去正常结构,肝细胞索排列紊乱,肝细胞混浊肿胀,空泡变性,血栓,脂肪变性以及溶解坏死,肝组织中有淋巴细胞和中性粒细胞浸润,出现凋亡小体,细胞点片状坏死,给药五周后更为明显。2、与空白组相比较,给药四周和五周后大鼠的NK细胞活性下降（p＜0.05）;给药四周后大鼠的T淋巴细胞增殖转化能力下降（p＜0.05）,给药五周后明显下降（p＜0.01）:给药四周后大鼠的B淋巴细胞增殖转化能力下降（p＜0.05）,给药五周后明显下降（p＜0.01）。3、与空白组比较,给药三周、四周后大鼠肝脏组织中Kupffer细胞的活化标志CD₆₈表达增加（p＜0.05）;五周后CD₆₈表达明显增加（p＜0.01）;给药四周后大鼠肝组织匀浆中TNF-α的合成和分泌增加（p＜0.05）;给药五周后明显增加（p＜0.01）;给药四周后大鼠肝脏组织中Fas的表达上调（p＜0.05）;给药五周后明显上调（p＜0.01）;给药四周后大鼠肝组织中Ca²⁺浓度增加（p＜0.05）;给药五周后明显增加（p＜0.01）;给药四周后大鼠肝细胞的凋亡率（指数）增加,（P＜0.05）;给药五周后明显增加（p＜0.01）。4、恢复期大鼠通过检测肝功能和肝组织病理切片,与空白组相比,给药四周、五周组恢复期肝功基本正常（p＞0.05）;给药三周、四周、五周组恢复期肝组织病理切片细胞形态基本恢复正常。结论:1、雷公藤多苷对大鼠体重、脏器指数、肝功能和肝脏组织病理形态具有抑制/损伤作用,且抑制/损伤程度与用药时间相关。2、雷公藤多苷对大鼠的免疫细胞（NK细胞;T淋巴细胞、B淋巴细胞）的活性有抑制作用,且抑制作用与用药时间相关。3、雷公藤多苷导致大鼠肝损伤机制的可能途径:a,活化肝脏Kupffer细胞并诱导TNF-α的合成与分泌增加;b,诱导凋亡信号Fas的表达上调;c,诱导肝细胞内Ca²⁺浓度增加。d,此外,雷公藤多苷可能作为抗原/半抗原在体内与相应抗体结合形成IC,沉积于肝内血管壁,诱导Ⅲ型超敏反应,导致肝组织损伤。4、雷公藤多苷能诱导大鼠肝细胞凋亡率（指数）增加,且与用药时间相关;各用药组大鼠肝细胞凋亡周期主要集中于G₁期（DNA合成前期）。5、雷公藤多苷导致大鼠肝损伤在一定时间范围内具有可逆性,且其恢复效果与用药时间相关。更多还原

【Abstract】 Objective:To reserch the relationship between time and effect of immunotoxicology in the liver of SD rat with the clinical dosage of Tripterygium Glycosides,and to study the recovery of the injuried liver at intervals.Methods:The model rats were intragastric administration with clinical dosage of Tripterygium Glycosides,the rat were divided into five groupes based on different times.To detect the general state of health and the basic immunological index inculding:body weight,index number of the liver、thymus and spleen;The glutamic-pyruvic transaminase（GPT/ALT） and the glutamic-oxal（o）acetic transaminase,（GOT/AST）;immune activity cells（natural killer cell,T-cell and B-cell activity ）function were investigated too.Immunohistochemistry technique was employed to study the CD₆₈ antigen at the macrophage and Fas expression in liver, through ELISA to investigate the TNF-αin the homogenate of liver and use the flow cytometer to detection the apoptosis index of the liver cell cycle and Fas expression heptocyte,to analyze the concentration of Ca²⁺ in the liver with confocal microscope.In order to approach the clinical dosage of Tripterygium Glycosides induction the liver injured mechanism,the results incorporate with the time-effect relationship,At the same time,we also do the observation of injury groups,and investigate the convalescent period of injury groups.Reslut:1.The weight of experimental groups decreased significantly（p＜0.05）,the index number of the liver increased in the 5th group（p＜0.05）,spleen decreased in the 5th group（p＜0.05）,and the index number of the thymus decreased（p＜0.05） too;The glutamic-pyruvic transaminase（GPT/ALT） increased（p＜0.05） at the 5th week;The glutamic-oxal（o）acetic transaminase,（GOT/AST） increased（p＜0.05） at the 4th week, and increased signifcantly（p＜0.01） at the 5th week.HE showed the hepatocyte began to loss the original structure,even with swollen,necrosis,vacuolar degeneration, thrombus,fatty degeneration,apoptotic body and inflritation of netrophlic cell and lymphcyte from the 3nd week,obviously at the 5th week.2.The activity of NK decreased（p＜0.05） at the 4th and 5th week in the experimental group;the proliferation of experimental T and B cell decreased（p＜0.05） at 4th week; and decreased signifcantly（p＜0.01） at the 5th week.3.The expression of the CD₆₈ in the liver Kupffer cell increased（p＜0.05） at the 3nd and 4th week,signifcantly（p＜0.01） in the 5th week.The expression of the TNF-αin the liver increased（p＜0.05） at the 4th week,signifcantly（p＜0.01） in the 5th week;The expression of Fas in liver increased（p＜0.05） at the 4th week, signifcantly（p＜0.01） at the 5th week;The expression of Ca²⁺ and apoptosis index of liver cells increased（p＜0.05） at the 4th week,signifcantly（p＜0.01） at the 5th week.4.The liver function of the 4th and 5th group are almost normal（p＞0.05）,and morphology of liver are closed to normal too.Conclusion:1.The weight,index of organ,the liver function and the pathomorphology of rats liver can be impressed by Tripterygium Glycosides based on morphological and functional test,which is related with time.2.The activity of NK cell,T-cell and B-cell immune function were depressant effect by Tripterygium Glycosides too,and also concerned with the administration time.3.Tripterygium Glycosides lead to liver injury with the high secretion of TNF-α, from macrophage,high expression of Fas,and the accumulation of Ca²⁺ in the hepatocyte.4.Tripterygium Glycosides lead to apoptosis index of hepatocyte increased,and also concerned with the administration time.The periodicity ofhepatocyte apoptosis which mostly stayed at the stage of G₁（DNA presynthetic phase）.5.The liver injury caused by Tripterygium Glycosides were recoverable at the certain time,and it depends on the intragastric administration time.更多还原