基于蛋白质的化学修饰研究盐补骨脂醇提物所致肝损伤的作用机制

Mechanism of liver injury induced by alcohol extract of salt-processed Psoraleae Fructus based on chemical modification of protein

盐补骨脂是临床常用补益药,但其潜在毒性限制了应用,反应性代谢产物(reactive metabolites,RMs)对蛋白质的共价修饰是盐补骨脂肝毒性一个不可忽视的因素。该研究基于液相色谱-质谱联用(liquid chromatography-mass spectrometry,LCMS)技术探究盐补骨脂中主要毒性成分补骨脂素/异补骨脂素产生的RMs对蛋白质的共价修饰,探讨盐补骨脂所致肝损伤的作用机制。首先采用生化法检测小鼠丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、过氧化氢酶(catalase,CAT)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、还原型谷胱甘肽(reduced glutathione,GSH)、谷胱甘肽S转移酶(glutathione S-transferase,GST)的水平;通过苏木素-伊红(hematoxylin-eosin,HE)染色观察肝脏病理变化。随后,利用超高效液相色谱-四极杆飞行时间串联质谱(ultra performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry,UPLC-Q-TOF-MS)对盐补骨脂中的主要毒性成分补骨脂素/异补骨脂素以及产生的RMs进行鉴定,并对毒性成分/RMs与GSH及游离氨基酸的共价键加合物进行鉴定,进一步确定毒性成分对蛋白质上氨基酸的共价键修饰位点和修饰方式。将确定的RMs修饰方式添加到Proteome Discoverer的可变修饰中,结合液相色谱-四极杆轨道阱质谱联用技术,检测补骨脂素/异补骨脂素修饰的靶蛋白。最后,利用Label-free定量蛋白质组学技术,筛选差异蛋白,通过京都基因和基因组百科全书(KEGG)和基因本体论(GO)做进一步分析并进行qPCR实验验证。结果显示,与对照组比较,盐补骨脂组ALT、AST、MDA明显增高,SOD、CAT、GSH和GST均降低,并有剂量依赖性,小鼠肝细胞出现明显空泡化及炎性细胞浸润。此外,在给药组小鼠肝脏中鉴定到补骨脂素/异补骨脂素的5种RMs,2种与GSH的加合物及1种与半胱氨酸的加合物。并且鉴定到10个被补骨脂素/异补骨脂素RMs修饰的蛋白质。给药组小鼠肝脏内检测到133个差异蛋白,其中92个上调,41个下调,主要涉及蛋白质、核糖体、rRNA、谷胱甘肽,影响蛋白酶体通路;qPCR结果与差异蛋白趋势一致。综上可得,补骨脂素/异补骨素的RMs可以与GSH共价键结合,并且修饰肝脏蛋白质的半胱氨酸及赖氨酸残基,毒性成分对蛋白质的共价键修饰可进一步导致肝毒性。盐补骨脂产生的氧化应激损伤可能与蛋白酶体及其复合物异常、核糖体及其核蛋白复合体生物合成、rRNA结合、谷胱甘肽结合等有关。

Salt-processed Psoraleae Fructus is a commonly used tonic in clinical practice.However,its usage is restricted due to the inherent toxicity.The covalent modification of proteins by reactive metabolites(RMs)plays a role in the hepatotoxicity of saltprocessed Psoraleae Fructus.This study delves into the protein covalent modification by RMs generated from psoralen/isopsoralen,the primary toxic components of salt-processed Psoraleae Fructus,by liquid chromatography-mass spectrometry(LC-MS),aiming to elucidate the mechanism underlying the hepatic injury induced by salt-processed Psoraleae Fructus.Biochemical methods were utilized to measure the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),catalase(CAT),malondialdehyde(MDA),superoxide dismutase(SOD),reduced glutathione(GSH),and glutathione S-transferase(GST)in mice.The pathological changes in the liver were observed by hematoxylin-eosin(HE)staining.Subsequently,ultra performance liquid chromatographyquadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS)was employed to identify the primary toxic components of psoralen/isopsoralen and the RMs in salt-processed Psoraleae Fructus.Covalent bonding adducts of the toxic components/RMs with GSH and free amino acids were identified to investigate the effects of the toxic components on modification sites and patterns of amino acids.The modifications of RMs were incorporated into the variable modifications of Proteome Discoverer,and the target proteins of psoralen/isopsoralen were detected by liquid chromatography-quadrupole exactive-mass spectrometry.Lastly,Label-free quantitative proteomics was adopted to screen differential proteins,which were further subjected to KEGG and GO enrichment analyses and confirmed by qPCR.The results indicated that compared with the control group,salt-processed Psoraleae Fructus significantly elevated the ALT,AST,and MDA levels and lowered the SOD,CAT,GSH,and GST levels in a dose-dependent manner,while causing obvious vacuolization and inflammatory cell infiltration in mouse hepatocytes.Furthermore,the livers of mice in the salt-processed Psoraleae Fructus group showed the presence of five RMs of psoralen/isopsoralen,two adducts with GSH,and one adduct with cysteine.In addition,10 proteins modified by the RMs of psoralen/isopsoralen were identified.A total of 133 differential proteins were detected in the livers of mice in the salt-processed Psoraleae Fructus group,including 92 with up-regulated expression and 41 with down-regulated expression.These differential proteins mainly involved ribosomes,rRNAs,and glutathione,affecting the proteasome pathway.The qPCR results were consistent with the differential proteins.These findings suggest that the RMs of psoralen/isopsoralen can covalently bind to GSH and modify cysteine and lysine residues of liver proteins.This covalent modification of proteins by harmful substances can potentially result in liver damage.Therefore,it can be inferred that the oxidative stress damage induced by salt-processed Psoraleae Fructus may be associated with the abnormality of proteasome and its complex,biosynthesis of ribosomes and their nucleoprotein complex,rRNA binding,and glutathione binding.