中药特异质肝损伤易感因素的代谢组学研究:以何首乌制剂为例
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摘要
药物特异质肝损伤的易感机制是当前临床毒理学研究的难点.本研究以何首乌制剂——润燥止痒胶囊为例,结合临床病例分析,采用代谢组学探讨其肝损伤可能的易感机制.病例分析表明润燥止痒胶囊相关肝损伤具有较典型的特异质属性,且与免疫异常活化有关.在正常小鼠上,灌胃润燥止痒胶囊对肝功能生化指标和肝组织病理无显著影响;在易感模型小鼠,免疫应激造模因素并未引起肝损伤表型,仅伴随肝组织少量炎性免疫细胞浸润,但从代谢组学可见组氨酸和丙氨酸等代谢通路显著上调(P<0.01)和色氨酸等代谢通路显著下调(P<0.01),提示代谢重编程可能是其肝损伤易感因素的重要内在机制;而在免疫应激易感因素基础上,灌胃润燥止痒胶囊引起了显著的肝损伤表型,并伴有肝组织大量炎性免疫细胞浸润和炎症反应,同时代谢组学上亦表现为花生四烯酸、亚油酸、甘油磷脂、胆汁酸等与炎症相关代谢通路的显著改变.综合提示代谢重编程可能是药物特异质肝损伤易感性的重要机制之一.基于易感因素相关的代谢紊乱通路,筛选发现了亚油酸、骨化二醇、18-羟基皮质酮等10个生物标志物可较好地识别易感模型动物(ROC分析中AUC均大于0.9),对临床识别易感人群具有潜在的应用价值.
The underlying mechanism(s) that govern idiosyncratic drug-induced liver injury(IDILI) have remained poorly defined. Several studies have shown that IDILI was caused by body diathesis, environment, metabolism, immunization, etc. However, the underlying mechanism(s) of IDILI was still ambiguous, which led to an unsolved issue in clinical toxicology. Understanding of such mechanisms is critical not only for the prevention of such DILI adverse effect as well as the clinical diagnosis, intervention or management of the affected individuals. In this study, we carried out a metabolomic study on mice treated with Runzao Zhiyang Capsule(RZZY), since Polygonum multiflorum, a key ingredient in this preparation, is known to cause DILI. Clinical case analysis showed that a small number of patients developed drug-induced liver injury(DILI) in a dose-independent manner. Meanwhile, the affected individuals often exhibited skin conditions associated with features of abnormal inflammatory responses. In normal mice, treatment with RZZY alone had no significant effects on biochemical indices of liver function, including aspartate aminotransferase(AST) and alanine aminotransferase(ALT) activities. At the same time, it had no significant effects on liver histopathology either. Moreover, we used non-toxic dose of lipopolysaccharide(LPS) pretreatment to produce mild inflammatory stress mice model, which showed no significant liver injury phenotype, except for a small number of inflammatory immune cells infiltration in portal areas of liver. However, by metabolomics profiling, we found significant alterations of plasma metabolites in the LPS treated mice, including up-regulated histidine and alanine metabolic pathways(P<0.01) and significantly down-regulated tryptophan metabolic pathways(P<0.01), etc. These results suggested that metabolic reprogramming might be an important underlying mechanism for susceptibility of liver injury. On the other side, the mice co-treated with RZZY and LPS induced significant liver injury phenotype, accompanying with a large number of inflammatory immune cells infiltration in portal areas of liver. Meanwhile, there were significant changes in several pathways related to inflammation by serum metabolome analysis, including the arachidonic, linoleic acid, glycerophospholipid, bile acid metabolic pathways, etc. The results above comprehensively suggested that metabolic reprogramming may play an important role in susceptibility mechanism of IDILI. Furthermore, based on those disordered metabolic pathways related to the susceptible factors, a receiver operating characteristic(ROC) curves analysis was used for identifying biomarkers effected by susceptible factors. Eventually, 10 biomarkers were found to be able to identify susceptible model mice from normal ones(ROC AUC all greater than 0.9), involving linoleic acid, calcifediol, beta-alanine, l-histidine, cortexolone, cortisone, dihydrocortisol, melatonine and 18-hydroxycorticosterone, which may provide potential application value for recognition of susceptible individuals in clinical.
The underlying mechanism(s) that govern idiosyncratic drug-induced liver injury(IDILI) have remained poorly defined. Several studies have shown that IDILI was caused by body diathesis, environment, metabolism, immunization, etc. However, the underlying mechanism(s) of IDILI was still ambiguous, which led to an unsolved issue in clinical toxicology. Understanding of such mechanisms is critical not only for the prevention of such DILI adverse effect as well as the clinical diagnosis, intervention or management of the affected individuals. In this study, we carried out a metabolomic study on mice treated with Runzao Zhiyang Capsule(RZZY), since Polygonum multiflorum, a key ingredient in this preparation, is known to cause DILI. Clinical case analysis showed that a small number of patients developed drug-induced liver injury(DILI) in a dose-independent manner. Meanwhile, the affected individuals often exhibited skin conditions associated with features of abnormal inflammatory responses. In normal mice, treatment with RZZY alone had no significant effects on biochemical indices of liver function, including aspartate aminotransferase(AST) and alanine aminotransferase(ALT) activities. At the same time, it had no significant effects on liver histopathology either. Moreover, we used non-toxic dose of lipopolysaccharide(LPS) pretreatment to produce mild inflammatory stress mice model, which showed no significant liver injury phenotype, except for a small number of inflammatory immune cells infiltration in portal areas of liver. However, by metabolomics profiling, we found significant alterations of plasma metabolites in the LPS treated mice, including up-regulated histidine and alanine metabolic pathways(P<0.01) and significantly down-regulated tryptophan metabolic pathways(P<0.01), etc. These results suggested that metabolic reprogramming might be an important underlying mechanism for susceptibility of liver injury. On the other side, the mice co-treated with RZZY and LPS induced significant liver injury phenotype, accompanying with a large number of inflammatory immune cells infiltration in portal areas of liver. Meanwhile, there were significant changes in several pathways related to inflammation by serum metabolome analysis, including the arachidonic, linoleic acid, glycerophospholipid, bile acid metabolic pathways, etc. The results above comprehensively suggested that metabolic reprogramming may play an important role in susceptibility mechanism of IDILI. Furthermore, based on those disordered metabolic pathways related to the susceptible factors, a receiver operating characteristic(ROC) curves analysis was used for identifying biomarkers effected by susceptible factors. Eventually, 10 biomarkers were found to be able to identify susceptible model mice from normal ones(ROC AUC all greater than 0.9), involving linoleic acid, calcifediol, beta-alanine, l-histidine, cortexolone, cortisone, dihydrocortisol, melatonine and 18-hydroxycorticosterone, which may provide potential application value for recognition of susceptible individuals in clinical.
引文
1 Zhu Y,Liu S H,Wang J B,et al.Clinical analysis of drug-induced liver injury caused by Polygonum multiflorum and its preparations(in Chinese).Chin J Integr Tradl Western Med,2015,35:1442-1447[朱云,刘树红,王伽伯,等.何首乌及其制剂导致药物性肝损伤的临床分析.中国中西医结合杂志,2015,35:1442-1447]
2 Pang J Y,Bai Z F,Niu M,et al.The toxic and protective effects of Polygonum multiflorum on normal and liver injured rats based on the symptom-based prescription theory.Acta Pharm Sin,2015,50:973-979
3 SiTu X X,Zhang H.Study on curative effect of desloratadine combined with runzaozhiyang capsule on senile skin pruritus(in Chinese).China Med Pharm,2018,8:95-97[司徒欣欣,张红.地氯雷他定联合润燥止痒胶囊对老年皮肤瘙痒症的疗效研究.中国医药科学,2018,8:95-97]
4 Wang D C,Zhou R.A case of liver injury caused by Runzaozhiyang capsule was reported repeatedly and the literature was reviewed(in Chinese).Chin J Clin Ratl Drug Use,2018,4:19[王冬初,周荣.润燥止痒胶囊反复导致肝损伤1例并文献复习.临床合理用药杂志,2018,4:19]
5 Tian L L,Zhou T R,Wu J,et al.A case of liver injury induced by oral Runzaozhiyang capsule(in Chinese).Shanghai Med Pharm J,2017,38:35-37[田璐璐,周陶然,吴涓,等.润燥止痒胶囊致药物性肝损1例.上海医药,2017,38:35-37]
6 Lin F,Li K.Acute liver failure induced by Runzaozhiyang capsules(in Chinese).Adverse Drug React J,2014,5:319-320[林芳,李克.润燥止痒胶囊致急性肝衰竭.药物不良反应杂志,2014,5:319-320]
7 Zhu Y,Niu M,Chen J,et al.Hepatobiliary and pancreatic:Comparison between Chinese herbal medicine and Western medicine-induced liver injury of 1985 patients.J Gastroen Hepatol,2016,31:1476-1482
8 Li C Y,Li X F,Tu C,et al.The idiosyncratic hepatotoxicity of Polygonum multiflorum based on endotoxin model(in Chinese).Acta Pharm Sin,2015,50:28-33[李春雨,李晓菲,涂灿,等.基于内毒素模型的何首乌特异质肝损伤评价.药学学报,2015,50:28-33]
9 Li C,Niu M,Bai Z,et al.Screening for main components associated with the idiosyncratic hepatotoxicity of a tonic herb,Polygonum multiflorum.Front Med,2017,11:1-13
10 Bai Z F,Yuan G,Zuo X B,et al.Progress in research on the pathogenesis of immune regulation and idiosyncratic drug-induced liver injury(in Chinese).Acta Pharm Sin,2017,52:1019-1027[柏兆方,高源,左晓彬,等.免疫调控与特异质型药物性肝损伤发生机制研究进展.药学学报,2017,52:1019-1027]
11 Tang J F,Wang X Y,Wen Q,et al.Idiosyncratic hepatotoxicity evaluation of Zhuangguguanjie wan mediated by immune stress(in Chinese).Acta Pharm Sin,2017,52:1033-1040[唐进法,王晓艳,温强,等.免疫应激介导的壮骨关节丸致特异质肝损伤评价.药学学报,2017,52:1033-1040]
12 Wang J B,Chui H R,Bai Z F,et al.Precision medicine-oriented safety assessment strategy for traditional Chinese medicines:Disease-syndrome-based toxicology(in Chinese).Acta Pharm Sin,2016,51:1681-1688[王伽伯,崔鹤蓉,柏兆方,等.精准医学下的中药安全性评价策略和方法:病证毒理学.药学学报,2016,51:1681-1688]
13 Li C Y,He Q,Gao D,et al.Idiosyncratic drug-induced liver injury linked to Polygonum multiflorum:A case study by pharmacognosy.Chin J Integr Med,2017,23:625-630
14 Próchnicki T,Latz E.Inflammasomes on the crossroads of innate immune recognition and metabolic control.Cell Metab,2017,26:71-93
15 Kishton R J,Sukumar M,Restifo N P.Metabolic regulation of T cell longevity and function in tumor immunotherapy.Cell Metab,2017,26:94-109
16 Buck M D,Sowell R T,Kaech S M,et al.Metabolic instruction of immunity.Cell,2017,169:570-586
17 Ghesquière B,Wong B W,Kuchnio A,et al.Metabolism of stromal and immune cells in health and disease.Nature,2014,511:167-176
18 Murray P J,Rathmell J,Pearce E.SnapShot:Immunometabolism.Cell Metab,2015,22:190
19 Wang K,Wang F,Yu H X.Metabolic reprogramming of immune cells and its effects on immune function(in Chinese).Current Immunol,2017,37:146-151[王可,王芳,余红秀.免疫细胞的代谢重编程及其对免疫功能的影响.现代免疫学,2017,37:146-151]
20 Jha A K,Huang S C,Sergushichev A,et al.Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization.Immunity,2015,42:419-430
21 Pearce E L,Pearce E J.Metabolic pathways in immune cell activation and quiescence.Immunity,2013,38:633-643
22 Jin M Y,Liu B J,Sun J Y,et al.Amino acid metabolism and immune response(in Chinese).Chin J Anim Sci,2007,43:37-39[井明艳,刘波静,孙建义,等.氨基酸代谢与免疫反应.中国畜牧杂志,2007,43:37-39]
23 Vandevyver S,Dejager L,Vandenbroucke R E,et al.An acute phase protein ready to go therapeutic for sepsis.EMBO Mol Med,2014,6:2-3
24 Reeds P J,Fjeld C R,Jahoor F.Do the differences between the amino acid compositions of acute-phase and muscle proteins have a bearing on nitrogen loss in traumatic states?J Nutr,1994,124:906-908
25 Yamaki K,Thorlacius H,Xie X L,et al.Characteristics of histamine-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery.Brit J Pharmacol,2010,123:390-399
26 Wang H,Sheng Y X,Wei W,et al.Effects of melatonin on free radical and cytokine of immunological liver injury mice(in Chinese).Chin Pharmacol Bull,2002,18:331-333[王华,沈玉先,魏伟,等.褪黑素对免疫性肝损伤小鼠自由基和细胞因子的影响.中国药理学通报,2002,18:331-333]
27 Wada J.Reprogramming of metabolism in immune-mediated cells.Diabetol Inter,2017,8:1-4
28 Uetrecht J.Immunoallergic drug-induced liver injury in humans.Semin Liver Dis,2009,29:383-392
29 Grant G E,Rokach J,Powell W S.5-Oxo-ETE and the OXE receptor.Prostag Oth Lipid M,2009,89:98-104
30 Khazen W,M’Bika J P,Collinet M,et al.Differentiation-dependent expression of interferon gamma and toll-like receptor 9 in3T3-F442A adipocytes.Biochimie,2007,89:669-675
31 Ramsden C E,Ringel A,Feldstein A E,et al.Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans.Prostag Leukotr Ess,2012,87:135
32 Zein C O,Lopez R,Fu X,et al.Pentoxifylline decreases oxidized lipid products in nonalcoholic steatohepatitis:New evidence on the potential therapeutic mechanism.Hepatology,2012,56:1291
33 Feldstein A E,Lopez R,Tamimi T A,et al.Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.J Lipid Res,2010,51:3046-3054
2 Pang J Y,Bai Z F,Niu M,et al.The toxic and protective effects of Polygonum multiflorum on normal and liver injured rats based on the symptom-based prescription theory.Acta Pharm Sin,2015,50:973-979
3 SiTu X X,Zhang H.Study on curative effect of desloratadine combined with runzaozhiyang capsule on senile skin pruritus(in Chinese).China Med Pharm,2018,8:95-97[司徒欣欣,张红.地氯雷他定联合润燥止痒胶囊对老年皮肤瘙痒症的疗效研究.中国医药科学,2018,8:95-97]
4 Wang D C,Zhou R.A case of liver injury caused by Runzaozhiyang capsule was reported repeatedly and the literature was reviewed(in Chinese).Chin J Clin Ratl Drug Use,2018,4:19[王冬初,周荣.润燥止痒胶囊反复导致肝损伤1例并文献复习.临床合理用药杂志,2018,4:19]
5 Tian L L,Zhou T R,Wu J,et al.A case of liver injury induced by oral Runzaozhiyang capsule(in Chinese).Shanghai Med Pharm J,2017,38:35-37[田璐璐,周陶然,吴涓,等.润燥止痒胶囊致药物性肝损1例.上海医药,2017,38:35-37]
6 Lin F,Li K.Acute liver failure induced by Runzaozhiyang capsules(in Chinese).Adverse Drug React J,2014,5:319-320[林芳,李克.润燥止痒胶囊致急性肝衰竭.药物不良反应杂志,2014,5:319-320]
7 Zhu Y,Niu M,Chen J,et al.Hepatobiliary and pancreatic:Comparison between Chinese herbal medicine and Western medicine-induced liver injury of 1985 patients.J Gastroen Hepatol,2016,31:1476-1482
8 Li C Y,Li X F,Tu C,et al.The idiosyncratic hepatotoxicity of Polygonum multiflorum based on endotoxin model(in Chinese).Acta Pharm Sin,2015,50:28-33[李春雨,李晓菲,涂灿,等.基于内毒素模型的何首乌特异质肝损伤评价.药学学报,2015,50:28-33]
9 Li C,Niu M,Bai Z,et al.Screening for main components associated with the idiosyncratic hepatotoxicity of a tonic herb,Polygonum multiflorum.Front Med,2017,11:1-13
10 Bai Z F,Yuan G,Zuo X B,et al.Progress in research on the pathogenesis of immune regulation and idiosyncratic drug-induced liver injury(in Chinese).Acta Pharm Sin,2017,52:1019-1027[柏兆方,高源,左晓彬,等.免疫调控与特异质型药物性肝损伤发生机制研究进展.药学学报,2017,52:1019-1027]
11 Tang J F,Wang X Y,Wen Q,et al.Idiosyncratic hepatotoxicity evaluation of Zhuangguguanjie wan mediated by immune stress(in Chinese).Acta Pharm Sin,2017,52:1033-1040[唐进法,王晓艳,温强,等.免疫应激介导的壮骨关节丸致特异质肝损伤评价.药学学报,2017,52:1033-1040]
12 Wang J B,Chui H R,Bai Z F,et al.Precision medicine-oriented safety assessment strategy for traditional Chinese medicines:Disease-syndrome-based toxicology(in Chinese).Acta Pharm Sin,2016,51:1681-1688[王伽伯,崔鹤蓉,柏兆方,等.精准医学下的中药安全性评价策略和方法:病证毒理学.药学学报,2016,51:1681-1688]
13 Li C Y,He Q,Gao D,et al.Idiosyncratic drug-induced liver injury linked to Polygonum multiflorum:A case study by pharmacognosy.Chin J Integr Med,2017,23:625-630
14 Próchnicki T,Latz E.Inflammasomes on the crossroads of innate immune recognition and metabolic control.Cell Metab,2017,26:71-93
15 Kishton R J,Sukumar M,Restifo N P.Metabolic regulation of T cell longevity and function in tumor immunotherapy.Cell Metab,2017,26:94-109
16 Buck M D,Sowell R T,Kaech S M,et al.Metabolic instruction of immunity.Cell,2017,169:570-586
17 Ghesquière B,Wong B W,Kuchnio A,et al.Metabolism of stromal and immune cells in health and disease.Nature,2014,511:167-176
18 Murray P J,Rathmell J,Pearce E.SnapShot:Immunometabolism.Cell Metab,2015,22:190
19 Wang K,Wang F,Yu H X.Metabolic reprogramming of immune cells and its effects on immune function(in Chinese).Current Immunol,2017,37:146-151[王可,王芳,余红秀.免疫细胞的代谢重编程及其对免疫功能的影响.现代免疫学,2017,37:146-151]
20 Jha A K,Huang S C,Sergushichev A,et al.Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization.Immunity,2015,42:419-430
21 Pearce E L,Pearce E J.Metabolic pathways in immune cell activation and quiescence.Immunity,2013,38:633-643
22 Jin M Y,Liu B J,Sun J Y,et al.Amino acid metabolism and immune response(in Chinese).Chin J Anim Sci,2007,43:37-39[井明艳,刘波静,孙建义,等.氨基酸代谢与免疫反应.中国畜牧杂志,2007,43:37-39]
23 Vandevyver S,Dejager L,Vandenbroucke R E,et al.An acute phase protein ready to go therapeutic for sepsis.EMBO Mol Med,2014,6:2-3
24 Reeds P J,Fjeld C R,Jahoor F.Do the differences between the amino acid compositions of acute-phase and muscle proteins have a bearing on nitrogen loss in traumatic states?J Nutr,1994,124:906-908
25 Yamaki K,Thorlacius H,Xie X L,et al.Characteristics of histamine-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery.Brit J Pharmacol,2010,123:390-399
26 Wang H,Sheng Y X,Wei W,et al.Effects of melatonin on free radical and cytokine of immunological liver injury mice(in Chinese).Chin Pharmacol Bull,2002,18:331-333[王华,沈玉先,魏伟,等.褪黑素对免疫性肝损伤小鼠自由基和细胞因子的影响.中国药理学通报,2002,18:331-333]
27 Wada J.Reprogramming of metabolism in immune-mediated cells.Diabetol Inter,2017,8:1-4
28 Uetrecht J.Immunoallergic drug-induced liver injury in humans.Semin Liver Dis,2009,29:383-392
29 Grant G E,Rokach J,Powell W S.5-Oxo-ETE and the OXE receptor.Prostag Oth Lipid M,2009,89:98-104
30 Khazen W,M’Bika J P,Collinet M,et al.Differentiation-dependent expression of interferon gamma and toll-like receptor 9 in3T3-F442A adipocytes.Biochimie,2007,89:669-675
31 Ramsden C E,Ringel A,Feldstein A E,et al.Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans.Prostag Leukotr Ess,2012,87:135
32 Zein C O,Lopez R,Fu X,et al.Pentoxifylline decreases oxidized lipid products in nonalcoholic steatohepatitis:New evidence on the potential therapeutic mechanism.Hepatology,2012,56:1291
33 Feldstein A E,Lopez R,Tamimi T A,et al.Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.J Lipid Res,2010,51:3046-3054
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