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

ABSTRACT

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 salt-processed 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 chromatography-quadrupole-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.