nodosin 促进大鼠部分肝移植肝细胞再生的实验研究

摘要

目的：探讨溪黄草有效成分 nodosin 对大鼠减体积肝移植术后肝细胞再生的影响。方法以Wistar 大鼠作为供体，SD 大鼠作为受体，采用改良的二袖套法建立大鼠部分肝移植模型，将24只受体大鼠随机分为 nodosin 组和对照组。nodosin 组于肝移植术后经尾静脉注射 nodosin 100μg／ml。在术后3 d 和7 d 分别处死大鼠取外周血浆及肝脏组织标本。分光光度法检测外周血浆中丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和白蛋白（ALB）含量；光学显微镜下观察肝组织形态学改变；采用免疫组织化学法检测肝组织增殖细胞核抗原（PCNA）抗体的表达水平；采用蛋白印迹法（Western blot）检测肝组织中磷酸化蛋白激酶（p-AKT）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、细胞周期素 D1（cyclin D1）、血红素加氧酶（HO）-1蛋白的表达水平；采用磷脂酰丝氨酸外翻分析（Annexin V 法）和 dUTP 缺口末端标记（TUNEL）法检测肝细胞的凋亡情况。结果术后3 d 和7 d，与对照组比较，nodosin 组大鼠血清中 ALT、AST 含量较低，ALB 含量较高（均为 P ＜0.05），同时 nodosin 能够减轻大鼠肝移植术后肝组织病理损伤。nodosin 组的 PCNA 阳性表达细胞数明显多于对照组（P ＜0.05）。nodosin 组增殖相关蛋白 p-AKT、p-mTOR、cyclin D1和 HO-1蛋白表达水平均明显高于对照组（均为 P ＜0.05）。nodosin 组发生凋亡的肝细胞数量和比例明显低于对照组（P ＜0.05）。结论大鼠肝移植术后应用 nodosin 具有减少肝细胞凋亡数量，促进肝细胞增殖的作用。%Objective To evaluate the effect of nodosin,as an effective element extracted from rabdosiae serrae, on hepatocyte regeneration after partial liver transplantation.Methods Wistar rats were used as donors and SD rats as recipients.Rat models with partial liver transplantation were established by modified two-cuff technique.Twenty-four recipient rats were randomly assigned into the nodosin and control groups.In the nodosin group,nodosin at a dosage of 1 00 μg/ml was administered via tail venous route after liver transplantation.Peripheral plasma and liver specimen were obtained at postoperative 3 and 7 d.The levels of alanine transaminase (ALT),aspartate aminotransferase (ALT)and albumin (ALB)in the peripheral plasma were measured by spectrophotometry.Hepatic histomorphological changes were observed under light microscope.The positive cell count of proliferating cell nuclear antigen (PCNA)antibody in the liver tissue was detected by immunohistochemistry. The expression levels of phosphorylated protein kinase (p-AKT ), phosphorylated mammalian target of rapamycin (p-mTOR),cyclin D1 and heme oxygenase (HO)-1 proteins were measured by western blot.The apoptosis of liver cells was detected by Annexin V method and TdT mediated-dUTP nick end labeling (TUNEL).Results Compared with the control group,the serum levels of ALT and AST were significantly lower at 3 d and 7 d after operation,whereas the ALB content was significantly higher in the nodosin group (all in P <0.05).And nodosin could alleviate the pathological injury of rat liver tissue after transplantation.The positive cell count of PCNA in the nodosin group was significantly higher than that in the control group (P <0.05).In the nodosin group,the expression levels of p-AKT,p-mTOR,cyclin D1 and HO-1 proteins were significantly higher than those in the control group (all in P <0.05).The quantity and percentage of apoptotic hepatocytes in the nodosin group were significantly lower than those in the control group (both in P <0.05).Conclusions Application of nodosin can decrease the quantity of apoptotic hepatocytes and accelerate hepatocyte proliferation after liver transplantation in rat models.