何首乌肝毒性客观性、临床标志物及损伤机制的初步研究
|
摘要
何首乌为蓼科植物何首乌Polygonum multiflorum Thunb的块根,是著名的补益类中药,广泛用于补肝肾、乌发的中药制剂、保健食品、洗护发用品,以及大众日常餐饮、食疗保健等。一些商家炒作“人形何首乌”延年益寿,近来备受民众追捧。然而,自2006年英国MHRA(药品与健康产品管理局)通报了何首乌制剂的肝损害案例并指出其具有安全性问题以来,国内外陆续出现了大量关于何首乌导致肝损害的报道,甚至有致死或肝移植案例。加拿大、英国、澳大利亚等国药品监管部门相继出台了对何首乌及含何首乌的制剂进行监管甚至限用的政策。
但是,何首乌肝毒性的客观真实性如何?是药物自身具有毒性,还是不合理用药造成的?能否在临床上早期辨识,进而早期干预,从而减少或降低毒副反应的发生?由于目前尚无对何首乌肝脏不良反应的系统性研究,更没有针对其早期诊断方法和指标的研究,制约了对何首乌及其制剂安全性进行科学监管及合理用药。考虑到何首乌广泛的使用人群以及其可能造成的肝脏严重不良反应,对何首乌肝毒性评价及其早期诊断进行深入研究已迫在眉睫。
为此,本课题充分发挥医院病例资源的优势,以临床病例资料和患者标本为主要研究对象,结合代谢组学、细胞毒理学及网络毒理学等方法,阐明何首乌肝毒性的客观真实性,筛选和研究何首乌肝毒性的可能成分、临床代谢标志物及其肝毒性的损伤机制,为评价何首乌肝毒性提供直接的临床数据,为何首乌肝毒性的早期诊断提供灵敏的辅助诊断指标,也为进一步指导何首乌临床合理用药减少其肝毒性的发生提供参考依据。主要研究内容和结论如下：
1.何首乌致肝毒性的现象客观存在,不规范使用是何首乌产生肝毒性的重要原因之一,市场流通的药材质量对何首乌肝毒性的发生也有一定的影响。
充分利用医院临床病例资料,结合何首乌肝损伤的文献报道,开展何首乌肝损伤的临床流行病学调查,深入调查分析何首乌产生肝毒性的客观原因,为阐释何首乌肝毒性的客观真实性及毒性特征提供基本的临床信息。主要结果显示：(1)何首乌致肝毒性的现象客观存在。(2)何首乌肝毒性存在特异质现象。(3)何首乌肝损害危险因素包括：何首乌本身存在可能会导致肝毒性成分,市场流通药材质量存在一定问题；另外民众过分夸大何首乌的功效,购买使用何首乌缺少监管；另外,处方中生首乌、制首乌书写不规范等也会影响何首乌临床合理应用。
为了加强何首乌合理用药的指导,减少肝毒性的发生,建议有关部门加强实施对何首乌正确使用的科普宣传和监管。
2.建立了基于临床标本和代谢组学技术的何首乌肝毒性诊断标志物研究方法和筛选模式,分析了何首乌致肝毒性的体内代谢通路变化,为何首乌的临床早期诊断和预后提供科学参考。
以何首乌肝损伤患者的血清为研究对象,采用LC-MS/MS Q-TOF技术表征临床何首乌肝毒性患者血清与正常人血清的代谢图谱差异,结合代谢组学分析手段,筛选了能准确评估何首乌致肝毒性引发代谢紊乱的特征标志物40个,代谢通路分析推测何首乌肝毒性可引发肝脏损伤和肝胆淤积,其机制可能与脂质过氧化有关,并发现1个与患者治疗转归过程相关的标志物四氢皮质酮,可望用于何首乌肝损伤患者的预后,且在典型患者和何首乌致肝损伤的大鼠中被验证,标志物的发现对科学评估何首乌肝毒性的客观真实性和早期辅助诊断提供临床有价值的参考。但由于病例数量的限制,该标志物的普适性和准确性还需要开展大样本量的前瞻性研究来进一步证实。
3.初步阐明了何首乌致肝损伤大鼠血清和胆汁中重要胆汁酸的变化规律,探讨了何首乌改变胆汁酸代谢及其与胆汁淤积型肝损伤的相关性。
何首乌在临床上引起的肝损伤部分表现为胆汁淤积的症状,为了探讨何首乌致胆汁淤积的内在规律,我们在大鼠上复制何首乌致肝损伤的模型,通过检测反应胆汁淤积的灵敏指标总胆汁酸(TBA)、总胆红素(TBIL)、碱性磷酸酶(ALP)等,判断肝损伤的基本类型与胆汁淤积的内在关系,结果显示生首乌组大鼠的TBA在血清和胆汁中的浓度都显著下降,在肝脏中显著升高；血清、胆汁中ALP和TBIL的变化情况在一定程度上表现了胆汁淤积的生化特征。生首乌致肝损伤大鼠血清和胆汁中TBA的含量降低,进一步采用质谱检测胆汁和血清中几种主要的胆汁酸含量,结果任廊清和胆汁中大部分胆汁酸的含量呈降低趋势,特别在胆汁中除了TCA,所检测的其他8种胆汁酸均呈下降趋势,胆汁酸的下降会影响胆汁对食物的消化,特别是对脂类物质,临床何首乌肝中毒的患者经常出现的食各欠不振、厌油等症状是否与几种主要胆汁酸含量的降低有关,还需要进一步验证。
4.考察了何首乌及主要单体成分对体外培养的人正常肝细胞系L02细胞毒性作用,并明确了何首乌及主要单体成分对肝细胞凋亡的影响,在细胞水平上揭示其引起肝细胞毒性的主要机制。
给药24h后,何首乌、大黄素、大黄酸、没食子酸及三个单体联合给药组均出现Caspase-3和Bax表达量的提高,较对照组相比大黄素对Caspase-3和Bax表达量具有显著的促进作用,没食子酸作用最小：大黄酸组Caspase-9蛋白表达提高,其他实验组则出现表达下降的结果,其中大黄素对Caspase-9蛋白表达的下调作用最大,没食子酸和何首乌对Caspase-9的作用次之；何首乌组Bcl-2表达量下降,其他各实验组Bcl-2表达量均提高,大黄酸对Bcl-2的促进表达作用最强,大黄素较弱。何首乌及其主要成分与细胞凋亡信号通路Caspase通路有关,能调节Bcl-2和Bax两个凋亡相关蛋白的表达。本实验结果为解释何首乌引起肝毒性的类型及相关机制提供了分子水平的证据。除大黄素、大黄酸和没食子酸以外,何首乌中其他成分对人肝细胞的影响需进-步研究。
5.采用网络毒理学方法,基于“疾病-基因-靶点-药物”相互作用网络,筛选预测何首乌可能的肝毒性成分4个及其可能作用的酶靶点11个。
为了更深入全面认识何首乌产生肝毒性的内在规律及作用物质,我们借鉴网络药理学的研究思路和方法,在前期研究的基础上,通过构建“疾病-基因-靶点-药物”相互作用网络的基础上,分析基因网络库、蛋白网络库、疾病网络库、药物网络库等现有数据库的信启、资料,结合从前期实验中获得的谱图数据,利用专业网络分析软件及算法整体预测何首乌产生肝毒性的成分和毒性机制。结果预测出与何首乌肝毒性密切相关的成分4个,其中涉及11个主要的酶,何首乌的肝毒性机制可能是通过影响这些酶的变化产生的。预测出的何首乌毒性成分和可能的机制还需要进一步实验验证,验证工作本课题组正在进行。
6.根据本课题对何首乌肝毒性客观性的认识,提出何首乌合理用药建议。
何首乌导致患者肝损害的现象客观存在,肝胆系统损伤是其最重要和最主要的不良反应,少数患者发生严重肝损害,患者的总体预后较好。基于本课题研究发现,对何首乌临床合理用药提出以下建议：(1)生首乌不宜用于保健食品,制首乌慎用于保健食品；(2)何首乌使用中严格控制剂量和适应症;(3)取缔非法售药途径,避免劣质药材和饮片流入市场；(4)完善何首乌质量控制标准,增加生物毒价评控指标；(5)尽快发布何首乌肝毒性警示信息,提高群众安全意识。
综上所述,本研究针对何酋乌的肝脏严重不良反应问题,采用临床病例标本、代谢组学、细胞毒理学及网络毒理学研究方法,用临床证据和实验数据证实了何首乌肝毒性的客观真实性,查实了盲目滥用和不规范使用是造成何首乌肝毒性产生较多的主要原因,筛选并鉴定了一些可用于何首乌肝毒性早期辅助临床诊断的生物标志物,提出了临床合理用药方案,以期减少何首乌肝损害的发生,避免患者不必要的身心和经济损失。同时,本研究基于临床标本和转化毒理学的中药毒副作用研究模式,对其他中药毒性客观评价和研究也有参考意义。
Polygoni Multiflori Radix (PM) is the tuberous root of Polygonum multiflorum Thunb., which is famous as a tonic TCM. PM is widely used in liver-and-kidney-nourishing and hair-blacking TCM preparations, health-care foods, hairdressing and care products, and the general public diet. Recently, many people are chasing the "humanoid Polygonum multiflorum" which is hyped of miraculous efficacies, such as prolonging life. However, since the British MHRA (Medicines and Healthcare Products Regulatory Agency) reported the liver injury cases of PM and pointed out its safety problems in2006, several follow-up reports on PM or its ingredients causing liver injury appeared all over the world. Canada, Britain, Australia and other countries'drug regulatory departments promulgated the policies to warn and limit the use of PM and its preparations.
However, wether the PM-induced liver injury is objective in clinic?Is PM itself toxic? Or the liver adverse effect is caused by irrational use of PM? Is there possibility to reduce the incidence of liver adverse effect of PM by means of early diagnosis and identification of its intoxication? The scientific regulatory and rational use for PM and its preparations are restricted by the absence of systematic study on liver adverse reaction caused by PM and its early diagnosis methods and indices. Considering the broad populations using PM and its serious adverse reaction to liver, there is a great need to assess the hepatotoxicity of PM and establish early diagnosis methods.
In this paper, we collected the clinical intoxication cases of PM and used the patients'specimens as the main research materials to clarify the objectivity of PM-induced hepatotoxicity, utilized metabolomics, cell toxicology and network toxicology methods, etc. The potential toxic components in PM and the clinical metabolic biomarker were screened. The injury mechanism of PM was investigated as well. The results provided direct clinical evidences in evaluating PM-induced hepatotoxicity and candidate indices in early diagnosis for PM intoxication in clinic.
The study also offered references in reducing hepatotoxicity occurence of PM. The main results and conclusions are as follows:
1. There is clinical objectivity of PM-induced hepatotoxicity which is usually caused by misuse or abuse for the main reasons and poor quality of PM for the additional reasons as well.
Based on the epidemiological surveys on clinical cases and literature reports of PM-induced liver injury, the objective causes for PM hepatotoxicity were summarized:(1) the PM-induced hepatotoxicity do existed in clinic;(2) The hepatotoxicity of PM might be idiosyncratic;(3) The hepatotoxicity risk factors included the hepatotoxic components of PM and the poor quality of drugs in market circulation as well. Moreover, the efficacy of PM is great exaggerated which misleads to abuse uses of the herb. The misuse or abuse is the most important reason of PM-related adverse events. The nonnormative writing of crude or processed PM in prescription contributed to the misuse of PM.
2. The diagnosis methods and biomarkers for PM hepatotoxicity were established based on metabonomics approach and clinical specimens. It was also analyzed for the metabolic pathway related to the PM hepatotoxicity.
The metabonomic research is performed by LC-MS/MS Q-TOF using patient serum samples of PM intoxication. The data was processed with multivariate analysis. Totally40biomarkers corresponding to the metabolic disorder caused by PM hepatotoxicity were discovered. Metabolic pathway analysis indicated that this hepatotoxicity might lead to liver injury and cholestasis. Tetrahydronaphthalene, one of the biomarkers, was found to be correlated with the recovery stage of patients or mice. The discovery of these biomarkers provided valuable reference for the early diagnosis and identification of PM-induced hepatotoxicity. The universality and accuracy of these biomarkers need further verification with enlargd sample size.
3. The major change pattern of bile acids in PM-induced liver injury rats serum and bile was clarified preliminary and the correlation of bile acid change and PM-induced cholestasis were also discussed.
Cholestasis was often occurred in PM-induced hepatotoxicity in patients. In order to explore the mechanism of cholestasis induced by PM, we tested the liver intoxication of PM in rats, determining the total bile acids (TBA), total bilirubin (TBIL),alkaline phosphatase (ALP), and internal relationship between the basic types of liver injury and cholestasis. The results showed that TBA concentration significantly decreased in serum and bile in crude PM-injured rats, while obviously increased in liver, compared to the normal rats. The changes of ALP and TBIL in serum and bile illustrated the biochemical characters of cholestasis. TBA concentration in the serum and bile decreased in liver injury rats induced by raw PM, then further detection of several major bile acids in serum and bile with mass spectrometric was done, and the results were that:the bile acid content in serum and bile decreased mostly, especially in bile except for TCA, the other eight kinds of bile acid showed a declining trend. The decrement of bile acid will affect bile in food digestion, particularly for lipid substances. Clinical hepatotoxicity patients induced by PM always showed such symptoms as recurrent inappetence, tired of oil, etc.
4. The toxic action of PM and its main components was investigated in cultured human normal liver cell line L02, regarding the effect to liver apopotosis.
The total extract of PM as well emodin, rhein, gallic acid and their mixture were tested in cultured human normal liver cell line L02. The expression quantity of Caspase-3and Bax increased in all the drug-treatment groups twenty four hours after administration. Emodin had a more significant promotion effect on Caspase-3and Bax compared to control group, whilegallic acid had the weakest effect. The expression quantity of Caspase-9increased in rhein group, while decreased in the other groups. Emodin had the strongest effect on decreasing the expression quantity of Caspase-9. The expression quantity of bal-2decreased in PM group, while increased in the other groups. Rhein had the strongest effect, while emodin was the weakest. The toxicity of PM and its main components were related in the caspase pathway of apoptosis. They could regulate the expression of apoptosis-related proteins such as Bcl-2and Bax. The results of this experiment provided the molecular level proof for explaining the types of hepatotoxicity caused by PM. Except for emodin, rhein and gallic acid, further research on the toxicity of the other components in PM should be carried out.
5. It was predicted of5possible hepatotoxic components in PM and11possible enzyme targets, using network pharmacology methods and "disease-gene-target-drug" interactive networks.
To comprehensively understand the hepatotoxicity of PM, we used the network pharmacology approach, constructed "disease-genes-targets-drugs" interactive networks on the basis of preliminary studies, and then analyzed existing database information such as gene network bank, protein network bank, disease network bank and drug network bank, and combined with spectral data obtained from the previous experiments, then predicted entirely the components and mechanisms of PM hepatotoxicity by professional network analysis software and algorithms. The results predicted5components closely related to PM hepatotoxicity, and11major enzymes, from which we could know hepatotoxicity mechanism may generate from the impact of enzymes changes. The PM hepatotoxicity components and mechanisms predicted needs further experimental verification, and such work will be carried out in our research group.
6. The rational use guideline for PM was proposed according to the results of PM hepatotoxicity.
According to the abovementiioned results, it could be concluded that the PM-induced hepatotoxicity exists objectively in clinic. The hepatobiliary system damage is the most important and leading adverse reactions. A small number of patients may show severe liver injury, but the patient's overall prognosis is very well. Based on this research, we present guidance for the rational use of PM as follows:(1) Raw PM should not be used in health food, the processed one should be used with caution in health food;(2) The dose and indications should be strictly controlled in use of PM;(3) The illegal sale of PM should be prohibited to avoid poor quality herbs flowing into market;(4) quality control standard of PM should be perfected with increment of biological noxious value assessment and control indexes;(5) The warning information of hepatotoxicity caused by PM should be announced as soon as possible, to improve the people's awareness of the risk.
In summary, aiming at serious adverse reaction problems caused by PM, we confirmed the objectivity of PM-induced hepatotoxicity by clinical evidence and experimental data. The main reason of PM-induced hepatotoxicity refers to misuse or abuse without physicians' guidance. Also, some candidate clinical biomarkers for early diagnosis of PM-induced hepatotoxicity were screened and identified in this study. We also proposed the guidance for the rational use of PM to reduce the occurence of liver injury, and avoid unnecessary hurts and economic losses of patients. At the same time, the translational research model of this study would provide references for objective evaluation and research on the toxicity of other TCMs.
但是,何首乌肝毒性的客观真实性如何?是药物自身具有毒性,还是不合理用药造成的?能否在临床上早期辨识,进而早期干预,从而减少或降低毒副反应的发生?由于目前尚无对何首乌肝脏不良反应的系统性研究,更没有针对其早期诊断方法和指标的研究,制约了对何首乌及其制剂安全性进行科学监管及合理用药。考虑到何首乌广泛的使用人群以及其可能造成的肝脏严重不良反应,对何首乌肝毒性评价及其早期诊断进行深入研究已迫在眉睫。
为此,本课题充分发挥医院病例资源的优势,以临床病例资料和患者标本为主要研究对象,结合代谢组学、细胞毒理学及网络毒理学等方法,阐明何首乌肝毒性的客观真实性,筛选和研究何首乌肝毒性的可能成分、临床代谢标志物及其肝毒性的损伤机制,为评价何首乌肝毒性提供直接的临床数据,为何首乌肝毒性的早期诊断提供灵敏的辅助诊断指标,也为进一步指导何首乌临床合理用药减少其肝毒性的发生提供参考依据。主要研究内容和结论如下：
1.何首乌致肝毒性的现象客观存在,不规范使用是何首乌产生肝毒性的重要原因之一,市场流通的药材质量对何首乌肝毒性的发生也有一定的影响。
充分利用医院临床病例资料,结合何首乌肝损伤的文献报道,开展何首乌肝损伤的临床流行病学调查,深入调查分析何首乌产生肝毒性的客观原因,为阐释何首乌肝毒性的客观真实性及毒性特征提供基本的临床信息。主要结果显示：(1)何首乌致肝毒性的现象客观存在。(2)何首乌肝毒性存在特异质现象。(3)何首乌肝损害危险因素包括：何首乌本身存在可能会导致肝毒性成分,市场流通药材质量存在一定问题；另外民众过分夸大何首乌的功效,购买使用何首乌缺少监管；另外,处方中生首乌、制首乌书写不规范等也会影响何首乌临床合理应用。
为了加强何首乌合理用药的指导,减少肝毒性的发生,建议有关部门加强实施对何首乌正确使用的科普宣传和监管。
2.建立了基于临床标本和代谢组学技术的何首乌肝毒性诊断标志物研究方法和筛选模式,分析了何首乌致肝毒性的体内代谢通路变化,为何首乌的临床早期诊断和预后提供科学参考。
以何首乌肝损伤患者的血清为研究对象,采用LC-MS/MS Q-TOF技术表征临床何首乌肝毒性患者血清与正常人血清的代谢图谱差异,结合代谢组学分析手段,筛选了能准确评估何首乌致肝毒性引发代谢紊乱的特征标志物40个,代谢通路分析推测何首乌肝毒性可引发肝脏损伤和肝胆淤积,其机制可能与脂质过氧化有关,并发现1个与患者治疗转归过程相关的标志物四氢皮质酮,可望用于何首乌肝损伤患者的预后,且在典型患者和何首乌致肝损伤的大鼠中被验证,标志物的发现对科学评估何首乌肝毒性的客观真实性和早期辅助诊断提供临床有价值的参考。但由于病例数量的限制,该标志物的普适性和准确性还需要开展大样本量的前瞻性研究来进一步证实。
3.初步阐明了何首乌致肝损伤大鼠血清和胆汁中重要胆汁酸的变化规律,探讨了何首乌改变胆汁酸代谢及其与胆汁淤积型肝损伤的相关性。
何首乌在临床上引起的肝损伤部分表现为胆汁淤积的症状,为了探讨何首乌致胆汁淤积的内在规律,我们在大鼠上复制何首乌致肝损伤的模型,通过检测反应胆汁淤积的灵敏指标总胆汁酸(TBA)、总胆红素(TBIL)、碱性磷酸酶(ALP)等,判断肝损伤的基本类型与胆汁淤积的内在关系,结果显示生首乌组大鼠的TBA在血清和胆汁中的浓度都显著下降,在肝脏中显著升高；血清、胆汁中ALP和TBIL的变化情况在一定程度上表现了胆汁淤积的生化特征。生首乌致肝损伤大鼠血清和胆汁中TBA的含量降低,进一步采用质谱检测胆汁和血清中几种主要的胆汁酸含量,结果任廊清和胆汁中大部分胆汁酸的含量呈降低趋势,特别在胆汁中除了TCA,所检测的其他8种胆汁酸均呈下降趋势,胆汁酸的下降会影响胆汁对食物的消化,特别是对脂类物质,临床何首乌肝中毒的患者经常出现的食各欠不振、厌油等症状是否与几种主要胆汁酸含量的降低有关,还需要进一步验证。
4.考察了何首乌及主要单体成分对体外培养的人正常肝细胞系L02细胞毒性作用,并明确了何首乌及主要单体成分对肝细胞凋亡的影响,在细胞水平上揭示其引起肝细胞毒性的主要机制。
给药24h后,何首乌、大黄素、大黄酸、没食子酸及三个单体联合给药组均出现Caspase-3和Bax表达量的提高,较对照组相比大黄素对Caspase-3和Bax表达量具有显著的促进作用,没食子酸作用最小：大黄酸组Caspase-9蛋白表达提高,其他实验组则出现表达下降的结果,其中大黄素对Caspase-9蛋白表达的下调作用最大,没食子酸和何首乌对Caspase-9的作用次之；何首乌组Bcl-2表达量下降,其他各实验组Bcl-2表达量均提高,大黄酸对Bcl-2的促进表达作用最强,大黄素较弱。何首乌及其主要成分与细胞凋亡信号通路Caspase通路有关,能调节Bcl-2和Bax两个凋亡相关蛋白的表达。本实验结果为解释何首乌引起肝毒性的类型及相关机制提供了分子水平的证据。除大黄素、大黄酸和没食子酸以外,何首乌中其他成分对人肝细胞的影响需进-步研究。
5.采用网络毒理学方法,基于“疾病-基因-靶点-药物”相互作用网络,筛选预测何首乌可能的肝毒性成分4个及其可能作用的酶靶点11个。
为了更深入全面认识何首乌产生肝毒性的内在规律及作用物质,我们借鉴网络药理学的研究思路和方法,在前期研究的基础上,通过构建“疾病-基因-靶点-药物”相互作用网络的基础上,分析基因网络库、蛋白网络库、疾病网络库、药物网络库等现有数据库的信启、资料,结合从前期实验中获得的谱图数据,利用专业网络分析软件及算法整体预测何首乌产生肝毒性的成分和毒性机制。结果预测出与何首乌肝毒性密切相关的成分4个,其中涉及11个主要的酶,何首乌的肝毒性机制可能是通过影响这些酶的变化产生的。预测出的何首乌毒性成分和可能的机制还需要进一步实验验证,验证工作本课题组正在进行。
6.根据本课题对何首乌肝毒性客观性的认识,提出何首乌合理用药建议。
何首乌导致患者肝损害的现象客观存在,肝胆系统损伤是其最重要和最主要的不良反应,少数患者发生严重肝损害,患者的总体预后较好。基于本课题研究发现,对何首乌临床合理用药提出以下建议：(1)生首乌不宜用于保健食品,制首乌慎用于保健食品；(2)何首乌使用中严格控制剂量和适应症;(3)取缔非法售药途径,避免劣质药材和饮片流入市场；(4)完善何首乌质量控制标准,增加生物毒价评控指标；(5)尽快发布何首乌肝毒性警示信息,提高群众安全意识。
综上所述,本研究针对何酋乌的肝脏严重不良反应问题,采用临床病例标本、代谢组学、细胞毒理学及网络毒理学研究方法,用临床证据和实验数据证实了何首乌肝毒性的客观真实性,查实了盲目滥用和不规范使用是造成何首乌肝毒性产生较多的主要原因,筛选并鉴定了一些可用于何首乌肝毒性早期辅助临床诊断的生物标志物,提出了临床合理用药方案,以期减少何首乌肝损害的发生,避免患者不必要的身心和经济损失。同时,本研究基于临床标本和转化毒理学的中药毒副作用研究模式,对其他中药毒性客观评价和研究也有参考意义。
Polygoni Multiflori Radix (PM) is the tuberous root of Polygonum multiflorum Thunb., which is famous as a tonic TCM. PM is widely used in liver-and-kidney-nourishing and hair-blacking TCM preparations, health-care foods, hairdressing and care products, and the general public diet. Recently, many people are chasing the "humanoid Polygonum multiflorum" which is hyped of miraculous efficacies, such as prolonging life. However, since the British MHRA (Medicines and Healthcare Products Regulatory Agency) reported the liver injury cases of PM and pointed out its safety problems in2006, several follow-up reports on PM or its ingredients causing liver injury appeared all over the world. Canada, Britain, Australia and other countries'drug regulatory departments promulgated the policies to warn and limit the use of PM and its preparations.
However, wether the PM-induced liver injury is objective in clinic?Is PM itself toxic? Or the liver adverse effect is caused by irrational use of PM? Is there possibility to reduce the incidence of liver adverse effect of PM by means of early diagnosis and identification of its intoxication? The scientific regulatory and rational use for PM and its preparations are restricted by the absence of systematic study on liver adverse reaction caused by PM and its early diagnosis methods and indices. Considering the broad populations using PM and its serious adverse reaction to liver, there is a great need to assess the hepatotoxicity of PM and establish early diagnosis methods.
In this paper, we collected the clinical intoxication cases of PM and used the patients'specimens as the main research materials to clarify the objectivity of PM-induced hepatotoxicity, utilized metabolomics, cell toxicology and network toxicology methods, etc. The potential toxic components in PM and the clinical metabolic biomarker were screened. The injury mechanism of PM was investigated as well. The results provided direct clinical evidences in evaluating PM-induced hepatotoxicity and candidate indices in early diagnosis for PM intoxication in clinic.
The study also offered references in reducing hepatotoxicity occurence of PM. The main results and conclusions are as follows:
1. There is clinical objectivity of PM-induced hepatotoxicity which is usually caused by misuse or abuse for the main reasons and poor quality of PM for the additional reasons as well.
Based on the epidemiological surveys on clinical cases and literature reports of PM-induced liver injury, the objective causes for PM hepatotoxicity were summarized:(1) the PM-induced hepatotoxicity do existed in clinic;(2) The hepatotoxicity of PM might be idiosyncratic;(3) The hepatotoxicity risk factors included the hepatotoxic components of PM and the poor quality of drugs in market circulation as well. Moreover, the efficacy of PM is great exaggerated which misleads to abuse uses of the herb. The misuse or abuse is the most important reason of PM-related adverse events. The nonnormative writing of crude or processed PM in prescription contributed to the misuse of PM.
2. The diagnosis methods and biomarkers for PM hepatotoxicity were established based on metabonomics approach and clinical specimens. It was also analyzed for the metabolic pathway related to the PM hepatotoxicity.
The metabonomic research is performed by LC-MS/MS Q-TOF using patient serum samples of PM intoxication. The data was processed with multivariate analysis. Totally40biomarkers corresponding to the metabolic disorder caused by PM hepatotoxicity were discovered. Metabolic pathway analysis indicated that this hepatotoxicity might lead to liver injury and cholestasis. Tetrahydronaphthalene, one of the biomarkers, was found to be correlated with the recovery stage of patients or mice. The discovery of these biomarkers provided valuable reference for the early diagnosis and identification of PM-induced hepatotoxicity. The universality and accuracy of these biomarkers need further verification with enlargd sample size.
3. The major change pattern of bile acids in PM-induced liver injury rats serum and bile was clarified preliminary and the correlation of bile acid change and PM-induced cholestasis were also discussed.
Cholestasis was often occurred in PM-induced hepatotoxicity in patients. In order to explore the mechanism of cholestasis induced by PM, we tested the liver intoxication of PM in rats, determining the total bile acids (TBA), total bilirubin (TBIL),alkaline phosphatase (ALP), and internal relationship between the basic types of liver injury and cholestasis. The results showed that TBA concentration significantly decreased in serum and bile in crude PM-injured rats, while obviously increased in liver, compared to the normal rats. The changes of ALP and TBIL in serum and bile illustrated the biochemical characters of cholestasis. TBA concentration in the serum and bile decreased in liver injury rats induced by raw PM, then further detection of several major bile acids in serum and bile with mass spectrometric was done, and the results were that:the bile acid content in serum and bile decreased mostly, especially in bile except for TCA, the other eight kinds of bile acid showed a declining trend. The decrement of bile acid will affect bile in food digestion, particularly for lipid substances. Clinical hepatotoxicity patients induced by PM always showed such symptoms as recurrent inappetence, tired of oil, etc.
4. The toxic action of PM and its main components was investigated in cultured human normal liver cell line L02, regarding the effect to liver apopotosis.
The total extract of PM as well emodin, rhein, gallic acid and their mixture were tested in cultured human normal liver cell line L02. The expression quantity of Caspase-3and Bax increased in all the drug-treatment groups twenty four hours after administration. Emodin had a more significant promotion effect on Caspase-3and Bax compared to control group, whilegallic acid had the weakest effect. The expression quantity of Caspase-9increased in rhein group, while decreased in the other groups. Emodin had the strongest effect on decreasing the expression quantity of Caspase-9. The expression quantity of bal-2decreased in PM group, while increased in the other groups. Rhein had the strongest effect, while emodin was the weakest. The toxicity of PM and its main components were related in the caspase pathway of apoptosis. They could regulate the expression of apoptosis-related proteins such as Bcl-2and Bax. The results of this experiment provided the molecular level proof for explaining the types of hepatotoxicity caused by PM. Except for emodin, rhein and gallic acid, further research on the toxicity of the other components in PM should be carried out.
5. It was predicted of5possible hepatotoxic components in PM and11possible enzyme targets, using network pharmacology methods and "disease-gene-target-drug" interactive networks.
To comprehensively understand the hepatotoxicity of PM, we used the network pharmacology approach, constructed "disease-genes-targets-drugs" interactive networks on the basis of preliminary studies, and then analyzed existing database information such as gene network bank, protein network bank, disease network bank and drug network bank, and combined with spectral data obtained from the previous experiments, then predicted entirely the components and mechanisms of PM hepatotoxicity by professional network analysis software and algorithms. The results predicted5components closely related to PM hepatotoxicity, and11major enzymes, from which we could know hepatotoxicity mechanism may generate from the impact of enzymes changes. The PM hepatotoxicity components and mechanisms predicted needs further experimental verification, and such work will be carried out in our research group.
6. The rational use guideline for PM was proposed according to the results of PM hepatotoxicity.
According to the abovementiioned results, it could be concluded that the PM-induced hepatotoxicity exists objectively in clinic. The hepatobiliary system damage is the most important and leading adverse reactions. A small number of patients may show severe liver injury, but the patient's overall prognosis is very well. Based on this research, we present guidance for the rational use of PM as follows:(1) Raw PM should not be used in health food, the processed one should be used with caution in health food;(2) The dose and indications should be strictly controlled in use of PM;(3) The illegal sale of PM should be prohibited to avoid poor quality herbs flowing into market;(4) quality control standard of PM should be perfected with increment of biological noxious value assessment and control indexes;(5) The warning information of hepatotoxicity caused by PM should be announced as soon as possible, to improve the people's awareness of the risk.
In summary, aiming at serious adverse reaction problems caused by PM, we confirmed the objectivity of PM-induced hepatotoxicity by clinical evidence and experimental data. The main reason of PM-induced hepatotoxicity refers to misuse or abuse without physicians' guidance. Also, some candidate clinical biomarkers for early diagnosis of PM-induced hepatotoxicity were screened and identified in this study. We also proposed the guidance for the rational use of PM to reduce the occurence of liver injury, and avoid unnecessary hurts and economic losses of patients. At the same time, the translational research model of this study would provide references for objective evaluation and research on the toxicity of other TCMs.
引文
[1]Kleiner, D. E. The pathology of drug-induced liver injury. Semin. Liver Dis.29, 364-372 (2009).
[2]Lewis, J. H. Liver biopsy differentiates DILI from autoimmune hepatitis. Nat. Rev. Gastroenterol. Hepatolgy.8,540-542 (2011).
[3]Danan, G.& Benichou, C. Causality assessment of adverse drug reactions to drugs-Ⅰ. A novel method based on the conclusions of international consensus meetings:application to drug-induced liver injuries. J. Clin. Epidemiol.46, 1323-1330(1993).
[4]Suzuki, A. et al. The use of liver biopsy evaluation in discrimination of idiopathic autoimmune hepatitis versus drug-induced liver injury. Hepatology doi:10.1002/ hep.24481.
[5]Wang, J.B., Kong, W.J., Wang, H.J., et al.. Toxic effects caused by rhubarb (Rheum palmatum L.) are reversed on immature and aged rats. Journal of Ethnopharmacology.134,216-220 (2011)
[6]Wang, J.B., Zhao, Y.L., Xiao XH. Assessment of the renal protection and hepatotoxicity of rhubarb extract in rats. Journal of Ethnopharmaclogy.121:18-25 (2009)
[7]Rockey, D. C. et al. Causality assessment in drug-induced liver injury using a structured expert opinion process:comparison to the Roussel-Uclef causality assessment method. Hepatology 51,2117-2126 (2010).
[8]Liss, G., Rattan, S.& Lewis, J. H. Predicting and preventing acute drug-induced liver injury:what's new in 2010? Expert Opin. Drug Metab. Toxicol.6, 1047-1061 (2010).
[9]Fontana, R. J. et al. Standardization of nomenclature and causality assessment in drug-induced liver injury:summary of a clinical research workshop. Hepatology 52, 730-742 (2010).
[10]Gabrielsson J, Jonsson H, Airiau C,et al.. OPLS methodology for analysis of preprocessing effectson spectroscopic data. Chem om In tell Lab Syst,84(1-2): 153-158(2006).
[11]Wang Y, Tang H, Nicholson JK, et al.. A metabonomic strategy for the detection of themetabolic effects of Chamo-mile ingestion. J Agric Food Chem,53(2): 191-196(2005).
[12]Wang Y, Holmes E, Tang H, et al.. Experimental metabonomic model of dietary variation and stress interactions. J Proteome Res,5(7):1535-1542 (2006).
[13]Marchesi JR, Holmes E, Khan F, et al.. Rapid and noninvasive metabonomic characterization of inflammatory bowel disease.J Proteome Res,2007,6(2): 546-551 (2007).
[14]圈家药典委员会.中华人民共和国药典2010年版一部[M].北京：中国医药科技出版社,2010：164-165.
[15]刘芷青,赵桂云.何首乌过量中毒1例[J].医生进修杂志,1988,3：31.
[16]Hinshaw LB. Sepsis/septic shock:Participation of the microcirculation:An abbreviated review[J]. Crit Care Med,1996,249(6):1072-1078.
[17]国家食品药品监督管理局药品评价中心.英国MHRA警告何首乌的肝损害不良反应[J].中国药物警戒,2006,3(5)：313-314.
[18]Panis B, Wong D R, H ooymans P M, et al. Recurrent toxic hepatitis in a Cau casian girl related to the use of Shou Wu Pian, a Chinese herb alpreparat ion [J]. J P ediatr Gastroenterol Nutr,2005,41:256-258.
[19]Kyoung Ah Jung,Hyun Ju Min,Seung Suk Yoo,et al.Drug-induced liver injury:twenty five cases of acute hepatitis following ingestion of Polygonum multiflorum Thunb[J].Gut and Liver,2011,5(4):493-499
[20]王秀娟,许利平,王敏.常用中药及复方制剂的肝毒性[J].首都医科大学学报,2007,28(2)：221.
[21]叶亲华服用何首乌致急性中毒性肝病1例[J].中国中西医结合杂志,1996,16(12)：732.
[22]盛家琦.中药首乌所致家族性急性肝损伤[J],中华肝脏病杂志,1988,6：59
[23]S. P. Ip, A. S. M. Tse, M. K. T. Poon, K. M. Ko, C. Y. Ma.Antioxidant Activities of Polygonum multiflorum Thunb., In Vivo and In Vitro[J].Phytotherapy Research,1997, (11):42-44.
[24]刘煜德,黄小洵,吴丽莎,孙丹邈,卢冬彦.何首乌肝毒性实验研究[J].亚太传统医药,2007,(4)：71.
[25]Toshihiro N, Takehiro Y, Tomoko O, Tomohiro O,Takashi O.Tomoki A,Toshio Kazuo M,Keiko S.Hot-water-extracts of Polygonum Multiflorum Do Not Induce Any Toxicity but Elicit Beneficial Effects on the Liver in Mice [J]. Jo urnal of Health Science,2009,55 (5):720-725.
[26]胡锡琴,杨晓青,刑玉瑞.制何首乌致肝损害的实验研究[J],陕西中医,2006,27(5)625.
[27]胡锡琴,禚君,李娅琳.何首乌中二苯乙烯苷对大鼠肝酶及蛋白的影响[J],辽宁中医杂志,2011,38(5)：988.
[28]S. P. Ip, A. S. M. Tse, M. K. T. Poon, C. Y. Ma.Antioxidant Activities of Polygonum multiflorum Thunb., In Vivo and In Vitro[J].Phytotherapy Research, 1997, (11):42-44.
[29]孙向红,孙玉维,李红.何首乌主要成分大黄素、大黄酸和二苯乙烯苷对肝细胞、肝癌细胞的影响[J].现代中西医结合杂志,2010,19(11)：1315-1319.
[30]张瑞晨,刘斌,孙震晓.何首乌提取物对人正常肝细胞L02周期阻滞及凋亡的影响[J],中西医结合学报,2010,8(6)：554.
[31]展玉涛,李定国,魏红山,等.大黄素对大鼠肝纤维化形成的影响[J].中国中西医结合杂志.2000；20(4)：276-278.
[32]张欣,党双锁,程延安,等.大黄素和黄芪多糖对实验性肝损伤大鼠肝脏超微结构的影响[J].西安交通大学学报(医学版).2009；30(4)：502-505.
[33]Bironaite D, Ollinger K.The hepatotoxicity of rhein involves impairment of mitochondrial functions [J] Chem BiolInteract,1997,103(1):35-50.
[34]闰向勇,孟庆常,燕忠生,何首乌致肝损害1例[J].疑难病杂志,2009.8(7)：399.
[35]赵文艳,白蚀丸致药物性肝炎2例[J].齐齐哈尔医学院学报,2004.25(7)：766.
[36]孙钧,补益中药致多次严重肝损害1例[J].中国中西医结合消化杂志,2002.10(5)：292.
[37]燕玉清,持续小剂量口服何首乌致肝损害一例[J].解放军医药杂志,2011.23(5)：41.
[38]陈晨,赵春九,余鑫之,口服何首乌致肝损害1例[J].安徽医药,2004.9(3)：204.
[39]袁伟东,中药何首乌致急性肝损伤一例[J].中国药物与临床,2002.2(6)：416.
[40]刘文洲,滕有明,雷蕾,中药何首乌丸致急性肝损伤1例[J].汕头大学医学院学报,2010.23(3)：172.
[41]范冬梅,周志莲,首乌片引起肝损害1例报告㈨白求恩医科大学学报,1998.24(3)：292.
[42]付鹏,于燕,何首乌致肝功能损害1例[J].中国民间疗法,2007.15(3)：41.
[43]牛建海.口服首乌片出现严重肝损害1例[J].新药与临床,1996.15(6)：382.
[44]陈盛鹏.生何首乌致急性肝损害1例[J].肝脏,2012,17(1)：66-68.
[45]盛家琦,中药首乌所致家族性急性肝损伤[J].中华肝脏病杂志,1998.3(1)：59.
[46]曹明雪,刘淼,吴荣荣等,制何首乌致肝损害1例[J].中国药物警戒,2011.8(8)： 510.
[47]张晓刚,吕志平,汇仁肾宝口服液致急性肝炎[J].药物不良反应杂志,2005.3：213.
[48]杨德全,何首乌致重症肝损伤[J].药物不良反应杂志,2005.7(6)：449.
[49]赵向东,首乌丸致肝功能损害1例[J].中国中药杂志,2007.32(5)444.
[50]霍敏英,凌世华,首乌片、养血生发胶囊同服引起肝炎[J].药物不良反应杂志,2004.5：335-336.
[51]杨军,李治国,何首乌致药物性肝炎两例[J].山西医药杂志,2008.37(11)：973.
[52]胡永成,李惠珍.何首乌致药物性肝炎1例[J].医药导报,2012,31(4)：98
[53]郑联喜,曾朝珠.何首乌致药物性肝炎1例报道[J].中国民族民间医药杂志,2012,21(21)：87.
[54]何汉明,郑新杰.何首乌致急性肝损害2例[J].陕西中医,1996,17(5)：230.
[55]李宗华,盖德美,庄学仁,何首乌致肝脏损害[J].山东中医杂志,1998.17(7)：311.
[56]邵艳玲,李倩.何首乌致肝损伤2例[J].齐鲁护理杂志,2012,18(22)：147
[57]冷梅,内服常规剂量首乌致急性重症肝损伤1例[J].河南职工医学院学报,2002.14(3)：277.
[58]朱邦胜,口服何首乌致药物性肝炎1例[J].淮海医药,2008.26(4)：320.
[59]张文芳,活力苏口服液引起肝损害1例报道[J].实用中医药杂志,1997.13(2)： 41.
[60]孙少梅,何首乌致肝损伤2例[J].中国医药指南,2011.9(26)：136-137.
[61]申国庆,江丽,龚春燕,何首乌致肝损害案例追溯分析与临床监控[J].中国医院用药评价与分析,2010.11：1040-1042.
[62]王芳芳,金朝辉,管枚,何首乌致肝损害3例[J].华西医学,2008.23(3)：631.
[63]邵泽勇,何首乌致肝损害2例[J].浙江中西医结合杂志,2003.13(11)：716.
[64]何蓉,何首乌致产后肝损害1例[J].西部医学,2009.21(3)：774.
[65]李智,何首乌致肝损害2例[J].中国误诊学杂志,2006.6(10)：2033.
[66]韩俊玲,何首乌引起肝炎[J].药物不良反应杂志,2006.8(5)：390.
[67]戴淑琴,李永梅,何首乌片致肝损害1例[J].宁夏医学杂志,2002.24(8)：501.
[68]叶亲华,服用何首乌致急性中毒性肝病1例[J].中国中西医结合杂志,1996.16(12)：732.
[69]杨军兰,董玉娟,服首乌片致肝损害2例[J].第四军医大学学报,2007.28(2)： 162.
[70]李方香,服首乌片致肝损害1例报告[J].新中医,1997.29(12)：42.
[71]张栩,丁向春,杨岩,口服何首乌致肝脏损害6例报告[J].宁夏医学院学报,2000.22(2)：115-116.
[72]Park GJH, Mann SP, Ngu MC. Acute hepatitis induced by Shou-Wu-Pian, a herbal product derived from Polygonum multiforum [J]. J Gastroenterol Hepatol, 2001,16(1):115-117.
[73]Panis B, Wong DR, Hooymans PM, et al. Recurrent toxic hepatitis in a Caucasian girl related to the use of Shou-Wu-Pian, a Chinese herbal preparation [J]. J Pediatr Gastroenterol Nutr,2005,41:256-258.
[74]杨兴祥,江南,林建梅.何首乌致肝损害13例临床分析[J].四川医学,2008,29(12)： 1619-1620
[75]徐静,汪茂荣,何长伦,隋云华,乔飞.口服何首乌致肝损害40例临床分析[J].东南国防医药,2009,11(3)：209-210
[76]张栩,丁向春,杨岩,张平,武菲菲.口服何首乌致肝损害6例报告[J].宁夏医学院学报,2000,22(2)：115-116
[77]周祥慧,邱雄.中药何首乌所致药物性肝炎11例临床分析[J].川北医学院学报,2005,20(12)：107-108
[78]刘国强.何首乌致肝损害9例临床分析[J].当代医学,2009,，15(30)：149
[79]李剑.中药何首乌致药物性肝炎11例临床分析[J]_内蒙古中医药,2010，,21：89-90
[80]孙震晓,张力.何首乌及其制剂相关肝损害国内文献回顾与分析[J].药物不良反应杂志,2010,12(1)：26-30
[81]赵军宁.中药及植物药的肝靶毒性与安全性评价.药品技术审评论坛,2003,(3)：203-208
[82]补血类中药-何首乌http://220.228.1.69/X1Chinese/D32Health/H419WellSuppply 12.htm
[83]李军,徐国钧,徐珞珊,等.中药首乌类的研究：原植物调查和商品鉴定.中草药,1995,26(1)：33-35
[84]陈吉炎,涂汉军,涂自良,李光富.中国武当中草药志[M],湖北科学技术出版社
[85]贾伟.医学代谢组学[M].上海：上海科学技术出版社,2011：2-9.
[86]王伽伯,马永刚,肖小河,等.对应分析在大黄炮制减毒“量-毒”规律研究中的应用.中国中药杂志,34(19)：2498-2502(2009)
[87]Fang Fang, Jia-Bo Wang, Xiaohe Xiao, et al. A comparative study on the tissue distributions of rhubarb anthraquinones in normal and CC14-injured rats orally administered rhubarb extract. Journal of Ethnopharmacology,137:1492-1497(2011)
[88]Weijun Kong, Xinhua Xia, Xiaohe Xiao, et al. Solid-phase extraction and ultra high performance liquid chromatography tandem mass spectrometry analysis of the gastrointestinal absorption of emodin in different digestive segments of rats. Journal of Separation Science,34:260-267 (2011)
[89]Wang, J.B., Li, H.F., Jin, Ch., et al.. Development and validation of a UPLC method for quality control of rhubarb-based medicine:Fast simultaneous determination of five anthraquinone derivatives. Journal of Pharmaceutical and Biomedical Analysis.47,765-770 (,2008)
[90]WANG Jia-bo, Hai-ping Zhao, XIAO Xiao-he, et al. Hepatotoxicity or Hepatoprotection? Pattern Recognition for the Paradoxical Effect of the Chinese Herb Rheum palmatum L. in Treating Rat Liver Injury. PLoS ONE 2011,6(9): e24498
[91]方芳,王伽伯,赵海平,等,生熟大黄总提取物灌胃给药后游离蒽醌在SD大鼠组织中的分布差异研究,药学学搦,46(3)：350-354(2011).
[92]Wang, J., Zhao, H., Kong, W., et al.. Microcalorimetric assay on the antimicrobial property of five hydroxyanthraquinone derivatives in rhubarb(Rheum palmatum L.) to Bifidobacterium adolescentis. Phytomedicine. 17,684-689 (2010)
[93]Wang, J.B., Ma, Y.G., Zhang, P., et al.. Effect of processing on the chemical contents and hepatic and renal toxicity of rhubarb studied by canonical correlation analysis. Yao Xue Xue Bao.44,885-890, (2009).
[94]杨红莲.氯丙嗪致大鼠胆汁淤积效应及其作用机制研究[J].军事医学科学院博士学位论文.
[95]丁敏,叶炼,王猛,邵勇.高效液相色谱-串联质谱法同时测定血清中游离和结合型胆汁酸[J].分析化学(FENXIHUAXUE)研究简报,35(10),1506-1508(2007).
[96]叶炼.高效液相色谱一串联质谱法测定血清中游离和结合型胆汁酸的方法学研究和临床应用.重庆医科大学2006届硕士学位论文.
[97]李梢.网络靶标：中药方剂网络药理学研究的一个切入点[J].中国中药杂志,2011,36,(15),2017-2020.
[98]吴磊宏,高秀梅,王林丽,等.附子多成分作用靶点预测及网络药理学研究[J].中国中药杂志,2011,36(21),2907-2910.
[2]Lewis, J. H. Liver biopsy differentiates DILI from autoimmune hepatitis. Nat. Rev. Gastroenterol. Hepatolgy.8,540-542 (2011).
[3]Danan, G.& Benichou, C. Causality assessment of adverse drug reactions to drugs-Ⅰ. A novel method based on the conclusions of international consensus meetings:application to drug-induced liver injuries. J. Clin. Epidemiol.46, 1323-1330(1993).
[4]Suzuki, A. et al. The use of liver biopsy evaluation in discrimination of idiopathic autoimmune hepatitis versus drug-induced liver injury. Hepatology doi:10.1002/ hep.24481.
[5]Wang, J.B., Kong, W.J., Wang, H.J., et al.. Toxic effects caused by rhubarb (Rheum palmatum L.) are reversed on immature and aged rats. Journal of Ethnopharmacology.134,216-220 (2011)
[6]Wang, J.B., Zhao, Y.L., Xiao XH. Assessment of the renal protection and hepatotoxicity of rhubarb extract in rats. Journal of Ethnopharmaclogy.121:18-25 (2009)
[7]Rockey, D. C. et al. Causality assessment in drug-induced liver injury using a structured expert opinion process:comparison to the Roussel-Uclef causality assessment method. Hepatology 51,2117-2126 (2010).
[8]Liss, G., Rattan, S.& Lewis, J. H. Predicting and preventing acute drug-induced liver injury:what's new in 2010? Expert Opin. Drug Metab. Toxicol.6, 1047-1061 (2010).
[9]Fontana, R. J. et al. Standardization of nomenclature and causality assessment in drug-induced liver injury:summary of a clinical research workshop. Hepatology 52, 730-742 (2010).
[10]Gabrielsson J, Jonsson H, Airiau C,et al.. OPLS methodology for analysis of preprocessing effectson spectroscopic data. Chem om In tell Lab Syst,84(1-2): 153-158(2006).
[11]Wang Y, Tang H, Nicholson JK, et al.. A metabonomic strategy for the detection of themetabolic effects of Chamo-mile ingestion. J Agric Food Chem,53(2): 191-196(2005).
[12]Wang Y, Holmes E, Tang H, et al.. Experimental metabonomic model of dietary variation and stress interactions. J Proteome Res,5(7):1535-1542 (2006).
[13]Marchesi JR, Holmes E, Khan F, et al.. Rapid and noninvasive metabonomic characterization of inflammatory bowel disease.J Proteome Res,2007,6(2): 546-551 (2007).
[14]圈家药典委员会.中华人民共和国药典2010年版一部[M].北京：中国医药科技出版社,2010：164-165.
[15]刘芷青,赵桂云.何首乌过量中毒1例[J].医生进修杂志,1988,3：31.
[16]Hinshaw LB. Sepsis/septic shock:Participation of the microcirculation:An abbreviated review[J]. Crit Care Med,1996,249(6):1072-1078.
[17]国家食品药品监督管理局药品评价中心.英国MHRA警告何首乌的肝损害不良反应[J].中国药物警戒,2006,3(5)：313-314.
[18]Panis B, Wong D R, H ooymans P M, et al. Recurrent toxic hepatitis in a Cau casian girl related to the use of Shou Wu Pian, a Chinese herb alpreparat ion [J]. J P ediatr Gastroenterol Nutr,2005,41:256-258.
[19]Kyoung Ah Jung,Hyun Ju Min,Seung Suk Yoo,et al.Drug-induced liver injury:twenty five cases of acute hepatitis following ingestion of Polygonum multiflorum Thunb[J].Gut and Liver,2011,5(4):493-499
[20]王秀娟,许利平,王敏.常用中药及复方制剂的肝毒性[J].首都医科大学学报,2007,28(2)：221.
[21]叶亲华服用何首乌致急性中毒性肝病1例[J].中国中西医结合杂志,1996,16(12)：732.
[22]盛家琦.中药首乌所致家族性急性肝损伤[J],中华肝脏病杂志,1988,6：59
[23]S. P. Ip, A. S. M. Tse, M. K. T. Poon, K. M. Ko, C. Y. Ma.Antioxidant Activities of Polygonum multiflorum Thunb., In Vivo and In Vitro[J].Phytotherapy Research,1997, (11):42-44.
[24]刘煜德,黄小洵,吴丽莎,孙丹邈,卢冬彦.何首乌肝毒性实验研究[J].亚太传统医药,2007,(4)：71.
[25]Toshihiro N, Takehiro Y, Tomoko O, Tomohiro O,Takashi O.Tomoki A,Toshio Kazuo M,Keiko S.Hot-water-extracts of Polygonum Multiflorum Do Not Induce Any Toxicity but Elicit Beneficial Effects on the Liver in Mice [J]. Jo urnal of Health Science,2009,55 (5):720-725.
[26]胡锡琴,杨晓青,刑玉瑞.制何首乌致肝损害的实验研究[J],陕西中医,2006,27(5)625.
[27]胡锡琴,禚君,李娅琳.何首乌中二苯乙烯苷对大鼠肝酶及蛋白的影响[J],辽宁中医杂志,2011,38(5)：988.
[28]S. P. Ip, A. S. M. Tse, M. K. T. Poon, C. Y. Ma.Antioxidant Activities of Polygonum multiflorum Thunb., In Vivo and In Vitro[J].Phytotherapy Research, 1997, (11):42-44.
[29]孙向红,孙玉维,李红.何首乌主要成分大黄素、大黄酸和二苯乙烯苷对肝细胞、肝癌细胞的影响[J].现代中西医结合杂志,2010,19(11)：1315-1319.
[30]张瑞晨,刘斌,孙震晓.何首乌提取物对人正常肝细胞L02周期阻滞及凋亡的影响[J],中西医结合学报,2010,8(6)：554.
[31]展玉涛,李定国,魏红山,等.大黄素对大鼠肝纤维化形成的影响[J].中国中西医结合杂志.2000；20(4)：276-278.
[32]张欣,党双锁,程延安,等.大黄素和黄芪多糖对实验性肝损伤大鼠肝脏超微结构的影响[J].西安交通大学学报(医学版).2009；30(4)：502-505.
[33]Bironaite D, Ollinger K.The hepatotoxicity of rhein involves impairment of mitochondrial functions [J] Chem BiolInteract,1997,103(1):35-50.
[34]闰向勇,孟庆常,燕忠生,何首乌致肝损害1例[J].疑难病杂志,2009.8(7)：399.
[35]赵文艳,白蚀丸致药物性肝炎2例[J].齐齐哈尔医学院学报,2004.25(7)：766.
[36]孙钧,补益中药致多次严重肝损害1例[J].中国中西医结合消化杂志,2002.10(5)：292.
[37]燕玉清,持续小剂量口服何首乌致肝损害一例[J].解放军医药杂志,2011.23(5)：41.
[38]陈晨,赵春九,余鑫之,口服何首乌致肝损害1例[J].安徽医药,2004.9(3)：204.
[39]袁伟东,中药何首乌致急性肝损伤一例[J].中国药物与临床,2002.2(6)：416.
[40]刘文洲,滕有明,雷蕾,中药何首乌丸致急性肝损伤1例[J].汕头大学医学院学报,2010.23(3)：172.
[41]范冬梅,周志莲,首乌片引起肝损害1例报告㈨白求恩医科大学学报,1998.24(3)：292.
[42]付鹏,于燕,何首乌致肝功能损害1例[J].中国民间疗法,2007.15(3)：41.
[43]牛建海.口服首乌片出现严重肝损害1例[J].新药与临床,1996.15(6)：382.
[44]陈盛鹏.生何首乌致急性肝损害1例[J].肝脏,2012,17(1)：66-68.
[45]盛家琦,中药首乌所致家族性急性肝损伤[J].中华肝脏病杂志,1998.3(1)：59.
[46]曹明雪,刘淼,吴荣荣等,制何首乌致肝损害1例[J].中国药物警戒,2011.8(8)： 510.
[47]张晓刚,吕志平,汇仁肾宝口服液致急性肝炎[J].药物不良反应杂志,2005.3：213.
[48]杨德全,何首乌致重症肝损伤[J].药物不良反应杂志,2005.7(6)：449.
[49]赵向东,首乌丸致肝功能损害1例[J].中国中药杂志,2007.32(5)444.
[50]霍敏英,凌世华,首乌片、养血生发胶囊同服引起肝炎[J].药物不良反应杂志,2004.5：335-336.
[51]杨军,李治国,何首乌致药物性肝炎两例[J].山西医药杂志,2008.37(11)：973.
[52]胡永成,李惠珍.何首乌致药物性肝炎1例[J].医药导报,2012,31(4)：98
[53]郑联喜,曾朝珠.何首乌致药物性肝炎1例报道[J].中国民族民间医药杂志,2012,21(21)：87.
[54]何汉明,郑新杰.何首乌致急性肝损害2例[J].陕西中医,1996,17(5)：230.
[55]李宗华,盖德美,庄学仁,何首乌致肝脏损害[J].山东中医杂志,1998.17(7)：311.
[56]邵艳玲,李倩.何首乌致肝损伤2例[J].齐鲁护理杂志,2012,18(22)：147
[57]冷梅,内服常规剂量首乌致急性重症肝损伤1例[J].河南职工医学院学报,2002.14(3)：277.
[58]朱邦胜,口服何首乌致药物性肝炎1例[J].淮海医药,2008.26(4)：320.
[59]张文芳,活力苏口服液引起肝损害1例报道[J].实用中医药杂志,1997.13(2)： 41.
[60]孙少梅,何首乌致肝损伤2例[J].中国医药指南,2011.9(26)：136-137.
[61]申国庆,江丽,龚春燕,何首乌致肝损害案例追溯分析与临床监控[J].中国医院用药评价与分析,2010.11：1040-1042.
[62]王芳芳,金朝辉,管枚,何首乌致肝损害3例[J].华西医学,2008.23(3)：631.
[63]邵泽勇,何首乌致肝损害2例[J].浙江中西医结合杂志,2003.13(11)：716.
[64]何蓉,何首乌致产后肝损害1例[J].西部医学,2009.21(3)：774.
[65]李智,何首乌致肝损害2例[J].中国误诊学杂志,2006.6(10)：2033.
[66]韩俊玲,何首乌引起肝炎[J].药物不良反应杂志,2006.8(5)：390.
[67]戴淑琴,李永梅,何首乌片致肝损害1例[J].宁夏医学杂志,2002.24(8)：501.
[68]叶亲华,服用何首乌致急性中毒性肝病1例[J].中国中西医结合杂志,1996.16(12)：732.
[69]杨军兰,董玉娟,服首乌片致肝损害2例[J].第四军医大学学报,2007.28(2)： 162.
[70]李方香,服首乌片致肝损害1例报告[J].新中医,1997.29(12)：42.
[71]张栩,丁向春,杨岩,口服何首乌致肝脏损害6例报告[J].宁夏医学院学报,2000.22(2)：115-116.
[72]Park GJH, Mann SP, Ngu MC. Acute hepatitis induced by Shou-Wu-Pian, a herbal product derived from Polygonum multiforum [J]. J Gastroenterol Hepatol, 2001,16(1):115-117.
[73]Panis B, Wong DR, Hooymans PM, et al. Recurrent toxic hepatitis in a Caucasian girl related to the use of Shou-Wu-Pian, a Chinese herbal preparation [J]. J Pediatr Gastroenterol Nutr,2005,41:256-258.
[74]杨兴祥,江南,林建梅.何首乌致肝损害13例临床分析[J].四川医学,2008,29(12)： 1619-1620
[75]徐静,汪茂荣,何长伦,隋云华,乔飞.口服何首乌致肝损害40例临床分析[J].东南国防医药,2009,11(3)：209-210
[76]张栩,丁向春,杨岩,张平,武菲菲.口服何首乌致肝损害6例报告[J].宁夏医学院学报,2000,22(2)：115-116
[77]周祥慧,邱雄.中药何首乌所致药物性肝炎11例临床分析[J].川北医学院学报,2005,20(12)：107-108
[78]刘国强.何首乌致肝损害9例临床分析[J].当代医学,2009,，15(30)：149
[79]李剑.中药何首乌致药物性肝炎11例临床分析[J]_内蒙古中医药,2010，,21：89-90
[80]孙震晓,张力.何首乌及其制剂相关肝损害国内文献回顾与分析[J].药物不良反应杂志,2010,12(1)：26-30
[81]赵军宁.中药及植物药的肝靶毒性与安全性评价.药品技术审评论坛,2003,(3)：203-208
[82]补血类中药-何首乌http://220.228.1.69/X1Chinese/D32Health/H419WellSuppply 12.htm
[83]李军,徐国钧,徐珞珊,等.中药首乌类的研究：原植物调查和商品鉴定.中草药,1995,26(1)：33-35
[84]陈吉炎,涂汉军,涂自良,李光富.中国武当中草药志[M],湖北科学技术出版社
[85]贾伟.医学代谢组学[M].上海：上海科学技术出版社,2011：2-9.
[86]王伽伯,马永刚,肖小河,等.对应分析在大黄炮制减毒“量-毒”规律研究中的应用.中国中药杂志,34(19)：2498-2502(2009)
[87]Fang Fang, Jia-Bo Wang, Xiaohe Xiao, et al. A comparative study on the tissue distributions of rhubarb anthraquinones in normal and CC14-injured rats orally administered rhubarb extract. Journal of Ethnopharmacology,137:1492-1497(2011)
[88]Weijun Kong, Xinhua Xia, Xiaohe Xiao, et al. Solid-phase extraction and ultra high performance liquid chromatography tandem mass spectrometry analysis of the gastrointestinal absorption of emodin in different digestive segments of rats. Journal of Separation Science,34:260-267 (2011)
[89]Wang, J.B., Li, H.F., Jin, Ch., et al.. Development and validation of a UPLC method for quality control of rhubarb-based medicine:Fast simultaneous determination of five anthraquinone derivatives. Journal of Pharmaceutical and Biomedical Analysis.47,765-770 (,2008)
[90]WANG Jia-bo, Hai-ping Zhao, XIAO Xiao-he, et al. Hepatotoxicity or Hepatoprotection? Pattern Recognition for the Paradoxical Effect of the Chinese Herb Rheum palmatum L. in Treating Rat Liver Injury. PLoS ONE 2011,6(9): e24498
[91]方芳,王伽伯,赵海平,等,生熟大黄总提取物灌胃给药后游离蒽醌在SD大鼠组织中的分布差异研究,药学学搦,46(3)：350-354(2011).
[92]Wang, J., Zhao, H., Kong, W., et al.. Microcalorimetric assay on the antimicrobial property of five hydroxyanthraquinone derivatives in rhubarb(Rheum palmatum L.) to Bifidobacterium adolescentis. Phytomedicine. 17,684-689 (2010)
[93]Wang, J.B., Ma, Y.G., Zhang, P., et al.. Effect of processing on the chemical contents and hepatic and renal toxicity of rhubarb studied by canonical correlation analysis. Yao Xue Xue Bao.44,885-890, (2009).
[94]杨红莲.氯丙嗪致大鼠胆汁淤积效应及其作用机制研究[J].军事医学科学院博士学位论文.
[95]丁敏,叶炼,王猛,邵勇.高效液相色谱-串联质谱法同时测定血清中游离和结合型胆汁酸[J].分析化学(FENXIHUAXUE)研究简报,35(10),1506-1508(2007).
[96]叶炼.高效液相色谱一串联质谱法测定血清中游离和结合型胆汁酸的方法学研究和临床应用.重庆医科大学2006届硕士学位论文.
[97]李梢.网络靶标：中药方剂网络药理学研究的一个切入点[J].中国中药杂志,2011,36,(15),2017-2020.
[98]吴磊宏,高秀梅,王林丽,等.附子多成分作用靶点预测及网络药理学研究[J].中国中药杂志,2011,36(21),2907-2910.