Exogenous glutathione exerts a therapeutic effect in ischemic stroke rats by interacting with intrastriatal dopamine.

We previously showed that oral administration of exogenous glutathione (GSH) exerted a direct and/or indirect therapeutic effect on ischemic stroke rats, but the underlying mechanisms remain elusive. In the current study, we conducted a quantitative proteomic analysis to explore the pathways mediating the therapeutic effect of GSH in cerebral ischemia/reperfusion (I/R) model rats. Rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. The rats were treated with GSH (250 mg/kg, ig) or levodopa (L-dopa, 100 mg/kg, ig) plus carbidopa (10 mg/kg, ig). Neurologic deficits were assessed, and the rats were sacrificed at 24 h after cerebral I/R surgery to measure brain infarct sizes. We conducted a proteomic analysis of the lesion side striatum samples and found that tyrosine metabolism and dopaminergic synapse were involved in the occurrence of cerebral stroke and the therapeutic effect of GSH. Western blot assay revealed that tyrosine hydroxylase (TH) mediated the occurrence of I/R-induced ischemic stroke and the therapeutic effect of GSH. We analyzed the regulation of GSH on endogenous small molecule metabolites and showed that exogenous GSH had the most significant effect on intrastriatal dopamine (DA) in I/R model rats by promoting its synthesis and inhibiting its degradation. To further explore whether DA-related alterations were potential targets of GSH, we investigated the therapeutic effect of DA accumulation on ischemic brain injury. The combined administration of the precursor drugs of DA (L-dopa and carbidopa) significantly ameliorated neurological deficits, reduced infarct size, and oxidative stress, and decreased pro-inflammatory cytokines levels in the striatum of I/R injury rats. More interestingly, exogenous L-dopa/carbidopa could also greatly enhance the exposure of intracerebral GSH by upregulating GSH synthetases and enhancing homocysteine (HCY) levels in the striatum. Thus, administration of exogenous GSH exerts a therapeutic effect on ischemic stroke by increasing intrastriatal DA, and the accumulated DA can, in turn, enhance the exposure of GSH and its related substances, thus promoting the therapeutic effect of GSH.

Insights into the Authentic Active Ingredients and Action Sites of Oral Exogenous Glutathione in the Treatment of Ischemic Brain Injury Based on Pharmacokinetic-Pharmacodynamic Studies.

Glutathione (GSH) has been reported to be closely related to various diseases of the central nervous system, yet its authentic active ingredients and action sites remain unclear. In the present study, oral exogenous GSH significantly alleviated ischemic brain injury, but this result was inconsistent with its low bioavailability and blood-brain barrier (BBB) permeability. To ascertain the exposure of GSH-derived ingredients, including GSH, cysteine (CYS), glutamate (Glu), glycine (GLY), CYS-GLY, and γ-glutamylcysteine (γ-GC) were systematically studied both in vitro and in vivo. The outcomes demonstrated that oral GSH not only increases the GSH and CYS levels in rat striatum and cortex, but it also can decrease the rise of intracerebral Glu concentration caused by ischemia/reperfusion surgery. Then the influence of GSH on the BBB was investigated via measuring IgG leakage, intracerebral endotoxin, and tight-junction proteins. All indicators showed that GSH dosing can repair the destroyed BBB. Oral GSH greatly enhances the exposure of GSH, CYS, CYS-GLY, and γ-GC in rat duodenum, jejunum, ileum, and colon. Accumulating evidence reveals a close link between brain injury and gastrointestinal dysfunction. Our findings further suggest that oral GSH significantly improves intestinal inflammatory damage and barrier disruptions. In conclusion, oral GSH can have a direct therapeutic role in brain injury by stabilizing intracerebral levels of GSH, CYS, and Glu. It can also play an indirect therapeutic role by enhancing the intestinal exposure of GSH, CYS, CYS-GLY, and γ-GC and improving intestinal barrier disruptions. SIGNIFICANCE STATEMENT: The authentic active ingredients and action sites of exogenous glutathione (GSH) in the treatment of ischemic brain injury are unclear. We have shown that oral exogenous GSH not only stabilizes intracerebral levels of GSH, cysteine (CYS), and glutamate (Glu) to act directly on brain injury, but it can also exert an indirect therapeutic role by improving intestinal barrier disruptions. These findings have great significance for revealing the therapeutic effect of GSH on ischemic brain injury and for promoting its further development and clinical application.

Multiomics Profiling Reveals Protective Function of Schisandra Lignans against Acetaminophen-Induced Hepatotoxicity.

The action principles of traditional Chinese medicines (TCMs) feature multiactive components, multitarget sites, and weak combination with action targets. In the present study, we performed an integrated analysis of metabonomics, proteomics, and lipidomics to establish a scientific research system on the underlying mechanism of TCMs, and Schisandra lignan extract (SLE) was selected as a model TCM. In metabonomics, several metabolic pathways were found to mediate the liver injury induced by acetaminophen (APAP), and SLE could regulate the disorder of lipid metabolism. The proteomic study further proved that the hepatoprotective effect of SLE was closely related to the regulation of lipid metabolism. Indeed, the results of lipidomics demonstrated that SLE dosing has an obvious callback effect on APAP-induced lipidic profile shift. The contents of 25 diglycerides (DAGs) and 21 triglycerides (TAGs) were enhanced significantly by APAP-induced liver injury, which could further induce liver injury and inflammatory response by upregulating protein kinase C (PKCß, PKCγ, PKCδ, and PKCθ). The upregulated lipids and PKCs could be reversed to the normal level by SLE dosing. More importantly, phosphatidic acid phosphatase, fatty acid transport protein 5, and diacylglycerol acyltransferase 2 were proved to be positively associated with the regulation of DAGs and TAGs. SIGNIFICANCE STATEMENT: Integrated multiomics was first used to reveal the mechanism of APAP-induced acute liver failure (ALF) and the hepatoprotective role of SLE. The results showed that the ALF caused by APAP was closely related to lipid regulation and that SLE dosing could exert a hepatoprotective role by reducing intrahepatic diglyceride and triglyceride levels. Our research can not only promote the application of multicomponent technology in the study of the mechanism of traditional Chinese medicines but also provide an effective approach for the prevention and treatment of ALF.

Insights into Q-markers and molecular mechanism of Sanguisorba saponins in treating ulcerative colitis based on lipid metabolism regulation.

BACKGROUND: Sanguisorba saponin extract (SSE) is the main active part of Sanguisorba officinalis with various pharmacological activities such as anti-inflammatory, anti-bacterial and anti-oxidant. However, its therapeutic role and underlying mechanisms for ulcerative colitis (UC) still need to be elucidated. PURPOSE: This study aims to explore the therapeutic effect, effectiveness-material basis-quality markers (Q-markers) and prospective mechanism of function of SSE on UC. METHODS: Fresh 2.5% dextran sulfate sodium salt (DSS) solution was placed in drinking bottles for 7 days to induce a mouse model of UC. SSE and sulfasalazine (SASP) were supplemented to mice by gavage for consecutive 7 days to investigate the therapeutic role of SSE on UC. Mouse monocyte macrophages (RAW264.7) and human normal colonic epithelial (NCM460) cells were treated with LPS to induce inflammatory responses, followed by pharmacodynamic examination with different concentrations of SSE. Hematoxylin-eosin (HE) and Alcian blue staining were conducted to evaluate the pathological damage of mice colon. Lipidomic technology was conducted to explore the differential lipids closely related to the disease process of UC. Quantitative PCR analysis, immunohistochemistry and ELISA kit were used to measure the expression levels of the corresponding proteins and pro-inflammatory factors. RESULTS: SSE treatment could effectively reduce the elevated expressions of pro-inflammatory factors in RAW264.7 and NCM460 cells due to LPS stimulation. Intragastric administration of SSE was found to significantly alleviate the symptoms of DSS-induced colon injury and low-polar saponins in SSE. Low polarity saponins, especially ZYS-II, were proved to be the main active substances of SSE in treating UC. In addition, SSE could significantly ameliorate the aberrant lipid metabolism in UC mice. The role of phosphatidylcholine (PC)34:1 in the UC pathogenesis has been fully verified in our previous studies. Herein, SSE-dosing effectively reversed the metabolic disorder of PCs in UC mice, and increased the PC34:1 level to normal via up-regulating the expression of phosphocholine cytidylyltransferase (PCYT1α). CONCLUSION: Our data innovatively revealed that SSE could significantly alleviate the symptoms of UC by reversing the disorder of PC metabolism induced by DSS modeling. SSE was proved for the first time to be a promising and effective candidate for UC treatment.

The role of phosphatidylcholine 34:1 in the occurrence, development and treatment of ulcerative colitis.

Lipid homeostasis is considered to be related to intestinal metabolic balance, while its role in the pathogenesis and treatment of ulcerative colitis (UC) remains largely unexplored. The present study aimed to identify the target lipids related to the occurrence, development and treatment of UC by comparing the lipidomics of UC patients, mice and colonic organoids with the corresponding healthy controls. Here, multi-dimensional lipidomics based on LC-QTOF/MS, LC-MS/MS and iMScope systems were constructed and used to decipher the alteration of lipidomic profiles. The results indicated that UC patients and mice were often accompanied by dysregulation of lipid homeostasis, in which triglycerides and phosphatidylcholines were significantly reduced. Notably, phosphatidylcholine 34:1 (PC34:1) was characterized by high abundance and closely correlation with UC disease. Our results also revealed that down-regulation of PC synthase PCYT1α and Pemt caused by UC modeling was the main factor leading to the reduction of PC34:1, and exogenous PC34:1 could greatly enhance the fumarate level via inhibiting the transformation of glutamate to N-acetylglutamate, thus exerting an anti-UC effect. Collectively, our study not only supplies common technologies and strategies for exploring lipid metabolism in mammals, but also provides opportunities for the discovery of therapeutic agents and biomarkers of UC.

Global analysis of qualitative and quantitative metabolism of Notoginsenoside R1 in rat liver-brain-gut axis based on LC-IT-TOF/MS combing mMDF strategy.

BACKGROUND: The metabolism study of active components for traditional Chinese medicine (TCM) in target organs is conducive to clarify the authentic active ingredients. Notoginsenoside R1 (NG-R1), one of the bioactive components of Panax notoginsenoside (PNS), is commonly acknowledged as the characteristic marker of PNS. However, the metabolism of NG-R1 in target organs has not been clarified yet due to the lack of robust technique and approach. PURPOSE: The present study aimed to develop a reliable and efficient strategy and technology for revealing the qualitative and quantitative metabolism of active components of TCMs in target organs, and to clarify the biotransformation of NG-R1 in liver-brain-intestinal axis. METHODS: The metabolic transformation of NG-R1 in the brain gut axis was investigated in the in vitro incubation system of fresh rat brain, liver homogenate, and intestinal flora. To quickly lock the target metabolites, we set the mass defect filter (MDF) in different ranges to screen metabolites with different molecular weight (MW). This strategy was defined as multi-stage MDF (mMDF). In addition, we performed relative quantitative analysis on all metabolites according to the peak area acquired by LC-IT-TOF/MS to overcome the challenge that metabolites are difficult to be quantified due to the lack of standards. RESULTS: When MDF was set at 0.50 to 0.65 to screen metabolites with MW of 900 to 1200 Da, 6 novel metabolites were quickly found, and then identified as glucuronic acid binding, oxidation, dehydrogenation, methylation and hydrogenation products according to their LC and MS characteristics. When setting MDF at 0.42 - 0.52, 6 metabolites with MW of 600 to 900 Da were effectively screened and identified as Rg1, NG-R2, Rh1, Rg1+CH2+2H and Rg1+CH2. To screen the metabolites with MW of 300 to 600 Da, MDF was set at 0.25 - 0.42, and 4 novel metabolites were screened rapidly. The results of quantitative metabolism suggested that intestinal flora was the main metabolic site of NG-R1 in rat, and more than 60% of NG-R1 was converted to Rg1 by deglycosylation in the intestinal flora. CONCLUSION: The mMDF strategy can significantly improve the research efficiency of qualitative metabolism of saponins. Although NG-R1 could be transformed into a variety of metabolites in rat liver and brain homogenate, it still existed mainly in prototype form. In the rat flora, NG-R1 mainly existed in the form of deglycosylated metabolite Rg1.

Absolute quantitative analysis of endogenous neurotransmitters and amino acids by liquid chromatography-tandem mass spectrometry combined with multidimensional adsorption and collision energy defect.

Considering that neurotransmitters (NTs) and amino acids (AAs) exert pivotal roles in various neurological diseases, global detection of these endogenous metabolites is of great significance for the treatment of nervous system diseases. Herein, a workflow that could cope with various challenges was proposed to establish an extendable all-in-one injection liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for analyzing these small molecular metabolites with high coverage. To obtain a qualified blank biological matrix for the preparation of standard curves and quality control samples, different absorption solvents, including activated carbon (AC), calcite (Cal) and montmorillonite (Mnt) were systematically evaluated for efficient absorption of endogenous substances with minimum residue. We also firstly proposed a "Collision Energy Defect (CED)" strategy to solve the huge difference of mass signal strength caused by different properties and concentrations of 11 NTs and 17 AAs. The quantitative results were validated by LC-MS/MS. Sensitivity, accuracy, and recovery meeting generally accepted bioanalytic guidelines were observed in a concentration span of at least 100 to 500 times for each analyte. Then the temporal changes of intracerebral and peripheral NTs and AAs in ischemic stroke model and sham operated rats were successfully produced and compared using the described method. All these results suggested that the currently developed assay was powerful enough to simultaneously monitor a large panel of endogenous small molecule metabolites, which was expected to be widely used in the research of various diseases mediated by NTs and AAs.

Comparative analysis of constitutes and metabolites for traditional Chinese medicine using IDA and SWATH data acquisition modes on LC-Q-TOF MS.

Identification of components and metabolites of traditional Chinese medicines (TCMs) employing liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) techniques with information-dependent acquisition (IDA) approaches is increasingly frequent. A current drawback of IDA-MS is that the complexity of a sample might prevent important compounds from being triggered in IDA settings. Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) method where the instrument deterministically fragments all precursor ions within the predefined m/z range in a systematic and unbiased fashion. Herein, the superiority of SWATH on the detection of TCMs' components was firstly investigated by comparing the detection efficiency of SWATH-MS and IDA-MS data acquisition modes, and sanguisorbin extract was used as a mode TCM. After optimizing the setting parameters of SWATH, rolling collision energy (CE) and variable Q1 isolation windows were found to be more efficient for sanguisorbin identification than the fixed CE and fixed Q1 isolation window. More importantly, the qualitative efficiency of SWATH-MS on sanguisorbins was found significantly higher than that of IDA-MS data acquisition. In IDA mode, 18 kinds of sanguisorbins were detected in sanguisorbin extract. A total of 47 sanguisorbins were detected when SWATH-MS was used under rolling CE and flexible Q1 isolation window modes. Besides, 26 metabolites of sanguisorbins were identified in rat plasma, and their metabolic pathways could be deduced as decarbonylation, oxidization, reduction, methylation, and glucuronidation according to their fragmental ions acquired in SWATH-MS mode. Thus, SWATH-MS data acquisition could provide more comprehensive information for the component and metabolite identification for TCMs than IDA-MS.

The improved performance of MALDI-TOF MS on the analysis of herbal saponins by using DHB-GO composite matrix.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an excellent analytical technique for rapid analysis of a variety of molecules with straightforward sample pretreatment. The performance of MALDI-TOF MS is largely dependent on matrix type, and the development of novel MALDI matrices has aroused wide interest. Herein, we devoted to seek more robust MALDI matrix for herbal saponins than previous reported, and ginsenoside Rb1, Re, and notoginsenoside R1 were used as model saponins. At the beginning of the present study, 2,5-dihydroxybenzoic acid (DHB) was found to provide the highest intensity for saponins in four conventional MALDI matrices, yet the heterogeneous cocrystallization of DHB with analytes made signal acquisition somewhat "hit and miss." Then, graphene oxide (GO) was proposed as an auxiliary matrix to improve the uniformity of DHB crystallization due to its monolayer structure and good dispersion, which could result in much better shot-to-shot and spot-to-spot reproducibility of saponin analysis. The satisfactory precision further demonstrated that minute quantities of GO (0.1 µg/spot) could greatly reduce the risk of instrument contamination caused by GO detachment from the MALDI target plate under vacuum. More importantly, the sensitivity and linearity of the standard curve for saponins were improved markedly by DHB-GO composite matrix. Finally, the application of detecting the Rb1 in complex biological sample was exploited in rat plasma and proved it applicable for pharmacokinetic study quickly. This work not only opens a new field for applications of DHB-GO in herbal saponin analysis but also offers new ideas for the development of composite matrices to improve MALDI MS performance.