ABSTRACT
Wilson disease (WD) is an inherited disorder characterized by abnormal copper metabolism with complex pathological features. Currently, this mechanism of copper overload-induced hepatic injury remains unclear. In this study, male toxic milk (TX) mice were selected as experimental subjects. Copper levels and biochemical indices were measured by atomic absorption spectroscopy (AAS) and kits. Liver tissue ultrastructure was observed by hematoxylin-eosin (H&E), sirius red staining and transmission electron microscopy. Plasma and liver metabolic profiles of TX mice were characterized by untargeted metabolomics. In addition, the expression of enzymes related to arachidonic acid metabolism in liver tissue was detected by Western blotting. The results showed the excessive copper content, concomitant oxidative stress, and hepatic tissue structural damage in TX mice. Seventy-eight metabolites were significantly different in WD, mainly involved in the metabolism of arachidonic acid, glycerophospholipids, sphingolipids, niacin and nicotinamide, and phenylalanine. Furthermore, the arachidonic acid metabolic pathway is an important pathway involved in WD metabolism. The level of arachidonic acid in the liver of TX mice was significantly lower (p < 0.01) compared to the control group. The expression of cytoplasmic phospholipase A2 (cPLA2) and arachidonic acid 12-lipoxygenase (ALOX12), related to the arachidonic acid metabolic pathway, was significantly different in the liver of TX mice (p < 0.01). Modulation of the arachidonic acid metabolic pathway could be a potential therapeutic strategy to alleviate WD symptoms.
Subject(s)
Copper , Disease Models, Animal , Hepatolenticular Degeneration , Liver , Metabolomics , Animals , Hepatolenticular Degeneration/metabolism , Mice , Liver/metabolism , Male , Metabolomics/methods , Copper/metabolism , Arachidonic Acid/metabolism , Oxidative Stress , Milk/metabolismABSTRACT
Gandouling tablets are used in a clinical agent for the treatment of hepatocellular degeneration; however, their chemical constituents have not been elucidated. Here, we screened and identified the chemical constituents of Gandouling tablets using ultra-high-performance liquid chromatography (UHPLC)-quadrupole time of flight/mass spectrometry. A method for the quality evaluation of Gandouling tablets was developed by combining the UHPLC fingerprints and the simultaneous quantitative analysis of multiple active ingredients. For fingerprint analysis, 20 shared peaks were identified to assess the similarities among the 10 batches of Gandouling tablets and the similarity was >0.9. The levels of nine representative active ingredients were simultaneously determined to ensure consistency in quality. A total of 99 chemical components were identified, including 18 alkaloids, 20 anthraquinones, 13 flavonoids, 11 phenolic acids, 9 polyphenols, 7 phenanthrenes, 5 sesquiterpenes, 3 curcuminoids, 2 lignans, 2 isoflavones, 2 dianthranones, and 7 other components. The retention times, molecular formulae, and secondary fragmentation information of these compounds were analyzed, and the cleavage pathways and characteristic fragments of some of the representative compounds were elucidated. This systematic analysis used to identify the chemical components of Gandouling tablets lays the foundation for its further quality control and research on their pharmacodynamic substances.
Subject(s)
Alkaloids , Drugs, Chinese Herbal , Chromatography, High Pressure Liquid/methods , Tandem Mass Spectrometry/methods , Drugs, Chinese Herbal/analysis , Alkaloids/chemistry , TabletsABSTRACT
BACKGROUND: Hepatolenticular degeneration (HLD) is an autosomal recessive disorder concerning copper metabolism. Copper overload is also accompanied by iron overload in HLD patients, which can lead to ferroptosis. Curcumin, the active component in turmeric, has the potential to inhibit ferroptosis. PURPOSE: The current study proposed a systematic investigation of the protective effects of curcumin against HLD and the underlying mechanisms. METHODS: The protective effect of curcumin on toxic milk (TX) mice was studied. Liver tissue was observed via hematoxylin-eosin (H&E) staining and the ultrastructure of the liver tissue was observed through transmission electron microscopy. Copper levels in the tissues, serum, and metabolites were measured by atomic absorption spectrometry (AAS). In addition, serum and liver indicators were evaluated. In cellular experiments, the effect of curcumin on the viability of rat normal liver cells (BRL-3A) was determined via the 3-[4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell and mitochondrial morphology were observed in curcumin-mediated HLD model cells. The intracellular copper ion fluorescence intensity was observed via fluorescence microscopy, and intracellular copper iron content was detected using AAS. Further, oxidative stress indicators were evaluated. Cellular reactive oxygen species (ROS) and cellular mitochondrial membrane potential were examined via flow cytometry. Furthermore, the expression levels of nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutathione peroxidase 4 (GPX4) were determined via western blotting (WB). RESULTS: The histopathology of the liver confirmed the hepatoprotective effects of curcumin. Curcumin improved copper metabolism in TX mice. Both serum liver enzyme markers and antioxidant enzyme levels indicated the protective effect of curcumin against HLD-related liver injury. The MTT assay results showed that curcumin was protective against excess copper-induced injury. Curcumin improved the morphology of HLD model cells and their mitochondrial morphology. The Cu2+ fluorescent probe and the AAS results indicated that curcumin reduced Cu2+ content in HLD hepatocytes. In addition, curcumin improved oxidative stress levels and prevented the decline of mitochondrial membrane potential in HLD model cells. The ferroptosis inducer Erastin reversed these effects of curcumin. WB revealed that curcumin promoted Nrf2, HO-1, and GPX4 protein expression in HLD model cells, and the Nrf2 inhibitor ML385 reversed the effects of curcumin. CONCLUSION: Curcumin demonstrates a protective role by expelling copper and inhibiting ferroptosis, activating the Nrf2/HO-1/GPX4 signaling pathway in HLD.
Subject(s)
Curcumin , Ferroptosis , Hepatolenticular Degeneration , Mice , Rats , Animals , Curcumin/pharmacology , Copper/pharmacology , NF-E2-Related Factor 2/metabolismABSTRACT
Gandou decoction (GDD) is a classic prescription for the treatment of hepatolenticular degeneration (HLD) in China; however, the liver-protecting mechanism of this prescription needs further evaluation. In the present study, we explored the protective mechanisms of GDD in a copper-laden HLD model using integrated pharmacology and cellular metabolomics in vitro. The results revealed that GDD could significantly promote copper excretion in copper-laden HLD model cells and improve the ultrastructural changes in hepatocytes. In addition, GDD could decrease the extent of lipid peroxidation, levels of reactive oxygen species, and the release rate of lactate dehydrogenase while increasing the activity of superoxide dismutase and the ratio of glutathione to oxidized glutathione in the copper-laden HLD model cells. On conducting statistical analysis of significant metabolic changes, 47 biomarkers and 30 related metabolic pathways were screened as pharmacological reactions induced by GDD in HLD model cells. d-glutamate and d-glutamine metabolic pathways showed the highest importance and significance among the 30 metabolic pathways, and the differential expression levels of the glutamine synthetase (GS) and the renal type and liver type GLS (GLS1 and GLS2) proteins were verified by Western blotting. Collectively, our data established the underlying mechanism of GDD therapy, such as the promotion of copper excretion and improvement in oxidative stress by regulating the expressions of GS, GLS1, and GLS2 protein to protect hepatocytes from injury.
ABSTRACT
Hepatolenticular degeneration (HLD) is an inherited disorder associated with human copper metabolism. Gandou decoction (GDD), a traditional Chinese medicinal formula, has been used as a therapeutic agent for the treatment of HLD in China for decades. Recent pharmacological evaluation in our laboratory has demonstrated that GDD exerts positive and beneficial effects on HLD model rats. However, its underlying therapeutic mechanisms are not yet well understood. To explore the potential therapeutic effects of GDD against HLD, liver and urine metabolomics approach combined with histopathological examination were performed to reveal the underlying mechanisms. Changes in metabolic profiles were estimated by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) coupled with multivariate statistical analyses. The results indicated that GDD could significantly improve liver pathological variations. Moreover, 19 and 11 significantly altered metabolites were found in the liver and urine between the normal and model groups, respectively. After GDD treatment, the levels of all these disordered metabolites showed different degrees of improvement compared with the model group, including lysoPC(18:2), lysoPE(20:2/0:0), PC(18:1/14:1), alpha-linolenic acid, sphinganine, taurochenodesoxycholic acid, tetracosahexaenoic acid, 13-OxoODE, and 13-L-hydroperoxyl inoleic acid. Metabolic pathway enrichment suggested that lipid and oxidative stress metabolism were the two main pathways that participated in copper-laden rat models with GDD administration. This work indicates that GDD could achieve a therapeutic effect on HLD by ameliorating the associated metabolic disturbances.
Subject(s)
Drugs, Chinese Herbal , Hepatolenticular Degeneration/metabolism , Liver/drug effects , Metabolome/drug effects , Protective Agents , Animals , Biomarkers/analysis , Biomarkers/metabolism , Biomarkers/urine , Chromatography, High Pressure Liquid , Disease Models, Animal , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Liver/metabolism , Liver/pathology , Male , Mass Spectrometry , Metabolomics , Protective Agents/chemistry , Protective Agents/pharmacology , Rats , Rats, Wistar , Reproducibility of ResultsABSTRACT
Objective: This study was designed to investigate the therapeutic efficacy and underlying mechanisms of Gandou Decoction (GDD) in copper-laden hepatolenticular degeneration (HLD) model rats. Methods: In this study, high-performance liquid chromatography (HPLC) fingerprint analysis and eight representative active components were simultaneously measured for quality control of GDD. The therapeutic effect of GDD in HLD was studied by constructing a rat model of copper-laden HLD. The copper levels in the liver, serum, urine, and feces were quantified by atomic absorption spectrophotometry (AAS). Subsequently, UV-Vis spectrophotometry was used to study the coordination ability of copper ion (Cu2+) with six representative active components in GDD to explore its potential copper expulsion mechanism. Serological indexes including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AKP) were evaluated. Hepatic indicators including superoxide dismutase (SOD), glutathione (GSH), and the total antioxidant capacity (T-AOC) were determined. Moreover, the liver tissue was stained with hematoxylin-eosin to observe the histological changes. Results: Thirty characteristic fingerprint peaks were used to assess the similarities among 10 samples and showed the similarity was >0.98, indicating a good correlation among the common peaks. Simultaneous quantification of eight markers in GDD was then performed to determine the consistency of quality. GDD could decrease the serum and hepatic copper levels by increasing the urinary and fecal copper content in copper-laden rats. Meanwhile, the results of UV-Vis absorption studies show that six representative active ingredients in GDD can coordinate with Cu2+, indicating that complexing copper removal may be a potential mechanism for GDD to play a role in copper removal. Serum hepatic enzyme markers AST, ALT, and AKP activities and antioxidant enzyme SOD, T-AOC activities, and GSH level in hepatic tissue showed the protection of GDD against liver injury induced by excessive copper. Additionally, the hepatoprotective effect of GDD was also evidenced by the results of the liver histological evaluation. Conclusions: This study suggested that GDD could reduce the serum and hepatic copper levels through promoting urinary and fecal copper excretion in copper-laden rats. At the same time, GDD could alleviate hepatic injury by inhibition of oxidative stress.
ABSTRACT
We aim to investigate the function and mechanism of GanDouLing combinated with Penicillamine on cerebrovascular injury in Wilson's disease (WD). ELISA was performed to analyze the expression of vascular injury factors. Pathological changes of cerebral vessels were observed by HE stain. Immunohistochemistry assays were performed to analyze the expression of ICAM-1, VCAM-1, and GRP78. Western blotting was measured to analyze the expression of caspase-3, caspase-12, PERK, eIF2α, and CHOP. Apoptosis was detected with TUNEL assay. The expression of vascular injury factors and ICAM-1, VCAM-1 was significantly increased by WD and markedly decreased in GanDouLing-Penicillamine group. The expression of caspase-3, caspase-12, PERK, eIF2α, and CHOP were obviously expressed in Wilson group, GanDouLing-Penicillamine suppressed apoptosis and endoplasmic reticulum (ER) stress. Our findings suggested that GanDouLing-Penicillamine improved cerebrovascular injury through PERK/eIF2α/CHOP ER stress pathway in the mouse model of WD.