L-肉碱对幼鼠丙戊酸肝毒性的保护作用及其机理探讨

Protective function of L-carnitine against valproate-associated hepatotoxicity in infant rats and its mechanism

目的:研究L-肉碱对丙戊酸(VPA)肝毒性的保护作用及相关机制。方法:采用VPA及苯巴比妥钠(PB)灌胃制作幼鼠VPA肝损伤的动物模型,同时以L-肉碱作为干预措施,观察其对实验动物肝脏,尤其肝线粒体功能的保护作用。化学比色法测定血氨、L-肉碱、肝脏凝血因子合成功能、线粒体呼吸酶系等;高效液相色谱、流式细胞仪分别检测VPA与PB血药浓度及肝线粒体跨膜电位(MMP);采用Oil-Red-O染色观察肝细胞脂肪变性。结果:(1)VPA/VPA+PB组肝线粒体呼吸链关键酶琥珀酸脱氢酶(SDH)及细胞色素氧化酶(CCO)活性与对照组均存在非常显著性差异(P<0.01),而补充L-肉碱对SDH和CCO活性均有明显保护作用;(2)VPA+PB组MMP下降21.47%,与对照组差异显著(P<0.05)。L-肉碱能有效维护MMP正常;(3)在VPA+PB组,凝血因子PT、TT、APTT、Fbg,以及血氨和L-肉碱含量均有显著改变。补充L-肉碱改善肝脏凝血因子合成、使血氨正常;(4)Oil-Red-O染色显示,VP/VPA+PB组均出现门管区为主的肝细胞脂肪变性,脂肪细胞数分别高出对照组9.2倍和15.7倍,差异非常显著(P<0.01)。L-肉碱干预后脂肪变性肝细胞数均有非常显著减少(P<0.01),表明L-肉碱能有效的阻止肝细胞脂肪变性。结论:L-肉碱对VPA肝损伤有较好防治作用,这一作用主要是通过阻止肝脂肪沉积和保护线粒体功能而实现。另外,当VPA与肝酶诱导剂联合运用时,虽然VPA血药浓度值可能不高,但由于肝酶诱导剂使具有非常强肝毒性的VPA中间代谢产物明显增加,所以仍应密切监测其肝毒副作用。

Objective：To explore the protective function of L-carnitine against valproate-associated hepatotoxicity in infant rats and its mechanism.Methods：Rat models with VPA liver lesion were established by oral administration of VPA and PB. L-carnitine was adopted as intervention measure：to observe its protection of experiment rat livers,especially the function of liver mitochondrion.Levels of plasma ammonia,L-carnitine and coagulation factors in sera as well as the changes of respiratory enzymes in hepatic mitochondria were measured by chemical colorimetry. Mitochondrial membrane potential （MMP）,and VPA and PB blood drug levels in liver were determined by flow cytometer and HPLC,respectively.and morphological changes of hepatecytes were observed under microscope with Oil-Red-O staining.Results （1）In the groups affected by VPA or VPA added with PB,the activities of SDH and CCO significantly decreased compared with control group （P〈 0.01 ）;While activities of SDH and CCO were effectively protected by L-carnitine; （2）MMP was reduced by 21.47% in infant rats treated with VPA added with PB,Which was significantly different from control group （P 〈0.05）.This reduction could be effectively reversed by L-carnitine;（3）In all rats treated with VPA added with PB,there were significant abnormalities in function of blood coagulation and serum fibrinogen,especially reflected on the content change of clotting factors PT、TT、APTT and Fbg,and the levels of plasma ammonia and L-carnitine also changed significantly. Furthermore,compared with controls,no alteration of coagulation function and ammonia levels in blood had been found since L-carnifine supplementation; （4）Oil-Red-O staining showed that significant lipid vacuoles primarily occurred in periportal areas of liver lobule in rats treated with VPA or VPA added with PB.The number of bepatocyte containing lipid droplets in infant rats was 9.2 times higher than that in control,and 15.7 times higher than that in VPA added with PB group. The difference was significant （P〈 0.01）.These indicated that lipid droplets in liver cells induced by VPA could effectively prevented with coadministration of L-carnitine.Conclusion：Protective effects of L-carnitine against VPA-asseciated hepatotoxicity were well demonstrated in the study,which might be achieved by preventing the formation of lipid droplets in hepatocyte and protecting the function of mitochondria. In addition,VPA serum levels may not be high because of the coadministration with liver enzyme inducers,but we still should pay more attention to its hepatotoxicity especially in infant because of its incompleted substrata.