Screening and identification of reactive metabolic compounds of Cortex Periplocae based on glutathione capture-mass spectrometry.

As a traditional Chinese medicine (TCM), Cortex Periplocae (CP) has a wide range of pharmacological effects, as well as toxic side effects. The main toxic components of it are cardiac glycosides, which tend to cause cardiotoxicity. Currently, it has also been reported in studies to cause hepatotoxicity, but it is not clear whether the hepatotoxicity is related to the toxicity caused by the reactive metabolites. This study aims to investigate the target components of CP that generate reactive metabolic toxicity. The fluorescent probe method was used to detect glutathione (GSH)-trapped reactive metabolites in a co-incubation system of CP extract with rat liver microsomes. Identification of GSH conjugates was performed by LC-MS/MS and that of the possible precursor components that produce reactive metabolites was conducted by UPLC-Q-TOF/MS. Cell viability assays were performed on HepG2 and L02 cells to determine the cytotoxicity of the target components. The findings of our study demonstrate that the extract derived from CP has the ability to generate metabolites that exhaust the intracellular GSH levels, resulting in the formation of GSH conjugates and subsequent cytotoxic effects. Through the utilization of the UPLC-Q-TOF/MS technique, we were able to accurately determine the molecular weight of the precursor compound in CP to be 355.1023. The primary evidence to determining the GSH conjugetes relies on the appearance of characteristic product ions resulting from central neutral loss (CNL) scanning of 129 Da and product scanning of m/z 660 in the positive MS/MS spectrum. Through analysis, it was ultimately ascertained that the presence of chlorogenic acid (CGA) and its isomers, namely neochlorogenic acid (NCGA) and cryptochlorogenic acid (CCGA), could lead to the production of GSH conjugates, resulting in cytotoxicity at elevated levels. Taking these findings into consideration, the underlying cause for the potential hepatotoxicity of CP was initially determined.

Convolutional Neural Network-Based Pattern Recognition of Partial Discharge in High-Speed Electric-Multiple-Unit Cable Termination.

Partial discharge detection is considered a crucial technique for evaluating insulation performance and identifying defect types in cable terminals of high-speed electric multiple units (EMUs). In this study, terminal samples exhibiting four typical defects were prepared from high-speed EMUs. A cable discharge testing system, utilizing high-frequency current sensing, was developed to collect discharge signals, and datasets corresponding to these defects were established. This study proposes the use of the convolutional neural network (CNN) for the classification of discharge signals associated with specific defects, comparing this method with two existing neural network (NN)-based classification models that employ the back-propagation NN and the radial basis function NN, respectively. The comparative results demonstrate that the CNN-based model excels in accurately identifying signals from various defect types in the cable terminals of high-speed EMUs, surpassing the two existing NN-based classification models.

The Molecular Basis of the Anti-Inflammatory Property of Astragaloside IV for the Treatment of Diabetes and Its Complications.

Astragali Radix is a significant traditional Chinese medication, and has a long history of clinical application in the treatment of diabetes mellitus (DM) and its complications. AS-IV is an active saponin isolated from it. Modern pharmacological study shows that AS-IV has anti-inflammatory, anti-oxidant and immunomodulatory activities. The popular inflammatory etiology of diabetes suggests that DM is a natural immune and low-grade inflammatory disease. Pharmacological intervention of the inflammatory response may provide promising and alternative approaches for the prevention and treatment of DM and its complications. Therefore, this article focuses on the potential of AS-IV in the treatment of DM from the perspective of an anti-inflammatory molecular basis. AS-IV plays a role by regulating a variety of anti-inflammatory pathways in multiple organs, tissues and target cells throughout the body. The blockade of the NF-κB inflammatory signaling pathway may be the central link of AS-IV's anti-inflammatory effect, resulting in a reduction in the tissue structure and function damage stimulated by inflammatory factors. In addition, AS-IV can delay the onset of DM and its complications by inhibiting inflammation-related oxidative stress, fibrosis and apoptosis signals. In conclusion, AS-IV has therapeutic prospects from the perspective of reducing the inflammation of DM and its complications. An in-depth study on the anti-inflammatory mechanism of AS-IV is of great significance for the effective use of Chinese herbal medicine and the promotion of its status and influence on the world.

Comprehensive investigation on the metabolism of emodin both in vivo and in vitro.

Emodin is a natural anthraquinone, which displays numerous pharmacological activities, including anti-tumor, anti-inflammation and immunosuppression. However, there was no comprehensive study on its absorption, metabolism, distribution, and excretion. In order to further evaluation on the possibility of drug development of emodin, both in vivo and in vitro experiments were fulfilled in this study. The results showed that the absolute bioavailability of emodin is approximately 3.2%. Furthermore, about 56% of emodin was unabsorbed and mainly excreted into feces as prototype. The absorb constituent could be rapidly metabolized as hydroxylated and glucuronidated metabolites. Both prototype and metabolites of emodin absorbed into the body circulation were predominantly distributed in kidney. Hydroxyed metabolites were predominantly excreted via urine and feces and glucuronidated metabolites were predominantly excreted via urine and bile. CYP1A2, CYP2E1, UGT1A1, UGT1A9, and UGT2B7 played a key role in the metabolism of emodin in liver microsomes of rats. To the best of our knowledge, this is the first comprehensive study on the absorption, metabolism, distribution, and excretion of emodin, and our results could help to understand both pharmaceutical and toxicological effects of emodin greatly.

Characterization of volatile organic compounds in Polygonum multiflorum and two of its processed products based on multivariate statistical analysis for processing technology monitoring.

Herein we describe a comprehensive analysis of the volatile organic compounds (VOCs) of raw Polygonum multiflorum Thunb. (PM) and two of its processed products, as well as an effective and simple method based on volatile markers to determine to which extent the PM had been processed. Sixty-five VOCs were identified by headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), along with headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). Principal component analysis (PCA) of the HS-SPME-GC-MS spectra and fingerprint analysis of the HS-GC-IMS spectra allowed the identification of raw PM from its processed products based the VOCs identified. Furthermore, the content and distribution of VOCs in the samples were easily analyzed visually based on clustering-kernel density estimation (Cluster-KDE). Finally, exploratory factor analysis (EFA) allowed the screening of significant markers to identify the processing method and consequently distinguish the three studied groups of PM.

Studies on Chemical Characterization of Ginkgo Amillaria Oral Solution and Its Drug-Drug Interaction With Piceatannol 3'-O-ß-D-Glucopyranoside for Injection.

Ginkgo Amillaria oral solution (GAO) is commonly used for the treatment of cardiovascular and cerebrovascular diseases in China. Piceatannol-3'-O-ß-D-glucopyranoside for injection (PGI) is mainly used for the prevention and treatment of ischemic cerebrovascular diseases. With the spread of cerebrovascular disease, the possibility of combining the two drugs has increased; however, there is no research on the drug-drug interaction (DDI) between these two medicines. In this paper, an ultrahigh-performance liquid chromatography/quadrupole-orbitrap mass spectrometry (UHPLC/Q-Orbitrap MS) method was established to characterize the chemical constituents of GAO first; 62 compounds were identified or tentatively identified based on their retention time (RT), MS, and MS/MS data. Nine main compounds were determined by ultrahigh-performance liquid chromatography/triple quadrupole mass spectrometry (UPLC-QQQ-MS). Furthermore, incubation with liver microsomes in vitro was fulfilled; the results showed that GAO had a significant inhibitory effect on UGT1A9 and UGT2B7 (p < 0.05), and PGI was mainly metabolized by UGT1A9. The identification results of in vivo metabolites of PGI showed that PGI mainly undergoes a phase II binding reaction mediated by UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) in vivo. Therefore, pharmacokinetic studies were performed to investigate the DDI between GAO and PGI. The results showed that the AUC (p < 0.05) and T1/2 (p < 0.05) of PGI in vivo were significantly increased when administered together with GAO, whereas the CL was significantly decreased (p < 0.05). The exploration of in vitro and in vivo experiments showed that there was a DDI between GAO and PGI.

Biodistribution and pharmacokinetic profile of berberine and its metabolites in hepatocytes.

BACKGROUND: Berberine has been shown in clinical studies to have many health benefits, including anti-inflammatory and antioxidant properties, along with gut-flora balancing properties. However, its clinical efficacy is hindered by its low oral bioavailability and rapid metabolism. PURPOSE: This study aims to identify the berberine metabolites' forms and characterize their biodistribution patterns in and out of HepG2 cells. METHODS: The qualitative analysis of metabolites of berberine in HepG2 cells was performed using the LC/MSn-IT-TOF method. Subsequent cellular pharmacokinetics characterization of intracellular and extracellular berberine and its metabolites was performed by LC-MS/MS analysis. RESULTS: Berberine's metabolites of phase I metabolism were demethyleneberberine, jatrorrhizine, columbamine, berberrubine, etc., while its phase II metabolites were sulfate and glucuronide conjugates of phase I metabolites. Among the phase I metabolites of berberine, jatrorrhizine+columbamine accounted for over two-thirds of the total, followed by demethyleneberberine, which accounted for about a quarter. The intracellular demethyleneberberine is 25.14 times more enriched than extracellular demethyleneberberine. On the other hand, jatrorrhizine+columbamine and berberrubine were primarily distributed extracellularly, and their extracellular concentrations were 7.13 times and 15.61 times of their intracellular concentrations, respectively. Berberine metabolites produced in phase II metabolism are predominantly sulfate conjugates. CONCLUSION: Our results show that demethyleneberberine is highly concentrated intracellularly in HepG2, possibly because it is an essential metabolite of berberine that likely contributes to berberine's efficacy. In light of our findings, berberine's poor plasma concentration-effectiveness characteristics have been partially explained.

The interaction of alkaloids in Coptis chinensis Franch -Tetradium ruticarpum (A. Juss.) T.G. Hartley with hOCT1 and hOCT2.

ETHNOPHARMACOLOGICAL RELEVANCE: Zuojin Pill, a traditional poly-herbal drug, comprises Coptis chinensis Franch - Tetradium ruticarpum (A. Juss.) T.G. Hartley (6:1). The significant quantity of alkaloids found in the participating herbs is a key aspect of the Zuojin Pill. According to traditional Chinese medicine (TCM), these numerous alkaloidal compounds within Zuojin Pill have various essential therapeutic effects. AIM OF THE STUDY: The alkaloids in Tetradium are mainly indole alkaloids, while the alkaloids in Coptis are mostly isoquinoline alkaloids with low bioavailability. Alkaloids and their metabolites are nitrogen-containing compounds or weakly alkaline substances that can be partially ionized under physiological pH conditions. Fortunately, organic cation transporters (OCTs) play a crucial role in the cellular uptake of weakly alkaline compounds. Therefore, we speculated that the alkaloidal compounds might interact with liver cation transporters hOCT1 and kidney cation transporters hOCT2 to alter cell drug disposal. In order to clarify our hypothesis, a series of alkaloids-OCTs interaction experiments were conducted. MATERIALS AND METHODS: HEK293 cells stably expressing hOCT1 and hOCT2 were modeled and evaluated. Afterward, high-content screening (HCS) was conducted to analyze whether the main alkaloids and their metabolites of Coptis - Tetradium were inhibitors of hOCT1 and hOCT2 transporters. Meanwhile, LC-MS/MS was used to investigate whether the alkaloidal compounds were substrates of hOCT1 and hOCT2 transporters. Finally, drug interactions at the cellular level were assessed by LC-MS/MS after co-administration of berberine and rutacorine. RESULTS: Berberine, jateorhizine, coptisine, epiberberine, columbamine, demethyleneberberine, and berberrubine could significantly inhibit hOCT1 and hOCT2 activity. Isoquinoline alkaloids, including berberine, jateorhizine, coptisine, epiberberine, columbamine, and palmatine, were substrates of hOCT1 and hOCT2, but not the indole alkaloids evodiamine and rutaecarpine. Furthermore, evodiamine at a concentration of 20 µmol/L had a trivial effect on berberine accumulation in HEK293-hOCT2 cells. CONCLUSIONS: These results support the idea that alkaloidal compounds within Coptis and Tetradium have hOCT1 and hOCT2 inhibitory activity or be their substrates, and the increased oral bioavailability of berberine in vivo was closely related to the potential interactions of small molecules in Coptis- Tetradium. Overall, our study provides a framework for investigating the potential interactions of small molecules in Coptis- Tetradium.

An integrated strategy for the systematic chemical characterization of Salvianolate lyophilized injection using four scan modes based on the ultra-high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry.

Salvianolate lyophilized injection (SLI), a freeze-dried powder injection derived from aqueous extract of S. miltiorrhiza, is therapeutically used to treat the syndrome of blood stasis and collateral blockage during the recovery period after stroke. To date, it has remained a significant challenge to comprehensively characterize the compounds of SLI, particularly the minor components with potential bioactivities, in one sample injection analysis. Using an integrative four scan modes approach coupled with ultra-high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (UHPLC-QTRAP-MS/MS), we propose a novel, sensitive, and simple strategy for systematic and rapid profiling of the chemical components of SLI. First, an in-house database of constituents from the water-soluble extract of Danshen was created. Second, the fragmentation behaviors of the representative components in SLI were obtained using the untargeted scan mode enhanced MS (EMS)-information dependent acquisition (IDA)-enhanced product ion (EPI). The specific fragments acquired were then utilized to conduct precursor ion (Prec) and neutral loss (NL)-IDA-EPI scans. Following that, a sensitive predictive multiple reaction monitoring (pMRM)-IDA-EPI scan method with 454 transitions was developed based on the prominent fragment ions and plausible predictions. A total of 171 compounds were tentatively identified from SLI. Among them, 27 minor components have not been previously reported. This strategy allows most isomeric compounds at trace levels to be readily distinguished and annotated. Finally, 15 batches of 13 representative components in SLI selected by the qualitative results were accurately quantified. Salvianolic acid A (Sal A), Sal B, Sal D, lithospermic acid (LA), and rosmarinic acid (RA) were proved to be the predominant constituents. Sal B had the highest amount (195.08-350.46 µg·mg-1), followed by LA, Sal A, Sal D, and RA. Moreover, these 15 batches of samples showed good uniformity, and no abnormal batches existed. These results suggest that this novel strategy can accelerate the identification of undiscovered chemical components and serve as an alternative method for in-depth profiling of compounds in other traditional Chinese medicines (TCMs).

Rapid discovery of potentially vasodilative compounds from Uncaria by UHPLC/Q-Orbitrap-MS based metabolomics and correlation analysis.

The genus Uncaria belongs to the family of Rubiaceae, which contains approximately 34 species. It has been widely used as a traditional Chinese medicine (TCM) in China to treat hypertension, fevers, headaches, gastrointestinal illness, epilepsy, wounds, and ulcers. Uncaria rhynchophylla. (Miq.) Miq. ex Hvail.(URM) and Uncaria hirsuta Havil.(UHH) are mainly used as remedies for hypertension, which both belong to the resource of "Gou-teng" in the Chinese Pharmacopoeia. However, the authentic antihypertensive components of Uncaria still have not been fully elucidated until now. In this work, we firstly explored and compared the vasorelaxation effect of URM and UHH on the isolated rat mesenteric artery ring. Then, the variations of metabolite profiles between URM and UHH samples were investigated by UHPLC/Q-Orbitrap-MS, and 16 different metabolites have been found through multivariate statistical analysis. Further, the potential vasodilative compounds which include corynoxeine, isocorynoxeine, isorhynchophylline, rhynchophylline, hirsuteine and hirsutine were screened through the correlation analysis between metabolites and anti-hypertension activities. And the relaxation effects of the six compounds on the mesenteric artery have verified. The results indicated that metabolomics combined with correlation analysis could be effective strategies to rapid explore the active compounds from TCM.

An effective strategy for distinguishing the processing degree of Polygonum multiflorum based on the analysis of substance and taste by LC-MS, ICP-OES and electronic tongue.

The efficacy of raw and processed products of Polygonum multiflorum (PM) varies greatly. "Nine cycles of steaming and sunning" (NCSS) is recognized as an effective technology in enhancing efficacy and reducing toxicity for PM. In this paper, PM was prepared differently into three groups (including group R, M, and "9"), which represent raw PM, PM processed using the method of Chinese Pharmacopoeia (ChP) and PM processed using traditional NCSS, respectively. The purpose is to establish an effective method to distinguish raw PM from different processed products and highlight the rationality of processing technology. The main organic compounds that could distinguish these three groups of samples were identified by in-depth mining of mass spectral information and various chemometric methods. Level of related metal cations have been quantified and used as another important distinguishing markers. The electronic tongue was utilized to determine the taste traits of aqueous extract from PM. Furthermore, the material basis that caused the difference in taste was discovered according to correlation analysis. In detail, saltiness has the most important contribution associated with the concentrations of K+ and Na+, however, bitterness and astringency were mainly associated with the contents of epicatechin gallate, catechin, procyanidin B1, procyanidin B2 and epicatechin. This study proposed a novel and effective strategy for identification of processing technology of PM. It lays the foundation for clarifying the modern scientific recommendations of processing technology to PM. On the other hand, it also provides a reference for related researches on other traditional Chinese medicine (TCM).

Polygonum multiflorum: Recent updates on newly isolated compounds, potential hepatotoxic compounds and their mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum multiflorum Thunb.(PM), (known as Heshouwu () in China) is one of the most important and well mentioned Chinese medicinal herbs in the literature for its use in blackening hair, nourishing liver and kidney, anti-aging, anti-hyperlipidemia, antioxidant, anti-inflammatory, anticancer, hepatoprotection, cardio-protection and improving age-related cognitive dysfunction. The purpose of this review is to give a comprehensive and recent update on PM: new compounds or isolated for the first time, potential hepatotoxic compounds and their mechanisms. Moreover, future perspectives and challenges in the future study of this plant are conversed which will make a new base for further study on PM. MATERIALS AND METHODS: A comprehensive review of relevant published literature on PM using the scientific databases SCOPUS, PubMed, and Science Direct was done. RESULTS: PM is broadly produced in many provinces of China and well known in other Eastern Asian Countries for its ethno-medical uses. Previous phytochemical investigation of PM had led to the isolation of more than 175 compounds including recently isolated 70 new compounds. Most of the new compounds isolated after 2015 are majorly dianthrone glycosides and stilbene glycosides. Processing has also a significant effect on chemical composition, pharmacological activities, and toxicity of PM. PM-induced liver injury is increasing after the first report in Hong Kong in 1996. Hepatotoxicity of PM was constantly reported in Japan, Korea, China, Australia, Britain, Italy, and other countries although its toxicity is related to idiosyncratic hepatotoxicity. More interestingly, although there is indispensable interest to predict idiosyncratic hepatotoxicity of PM and understand its mechanisms, the responsible hepatotoxic compounds and mechanisms of liver damage induced by PM are still not clear. There is a big controversy on the identification of the most responsible constituent. Anthraquinone and stilbene compounds in PM, mainly emodine and TSG are mentioned in the literature to be the main responsible hepatotoxic compounds. However, comparing the two compounds, which one is the more critical toxic agent for PM-induced hepatotoxicity is not well answered. Affecting different physiological and metabolic pathways such as oxidative phosphorylation and TCA cycle pathway, metabolic pathways, bile acid excretion pathway and genetic polymorphisms are among the mechanisms of hepatotoxicity of PM. CONCLUSION: Deeper and effective high throughput experimental studies are still research hotspots to know the most responsible constituent and the mechanism of PM-induced hepatotoxicity.

The composition differences between small black beans and big black beans from different habitats and its effects on the processing of Polygonum multiflorum.

INTRODUCTION: The roots of Polygonum multiflorum (PM) serve as a classical traditional Chinese medicine (TCM), which has multiple biological activities. However, many cases of hepatotoxicity in PM have been reported in recent years. Processing PM with black beans decoction is one of the typical processing methods to reduce the hepatotoxicity of PM since ancient times. OBJECTIVES: To find potential effective constituents, as well as the optimal variety and origin of black beans for the processing of PM. METHODS: Based on ultrahigh-performance liquid chromatography Q-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) analysis, we measured the contents of the two potential toxic compounds (emodin-8-O-glucoside and torachrysone-O-hexose) in raw PM (R-PM), PM processed with big black beans (B-PM) and PM processed with small black beans (S-PM). The flow cytometry method analysed the effects of different processed products of PM on apoptosis of L02 cells in different drug concentration. Proton nuclear magnetic resonance (1 H-NMR) and UHPLC-Q-Orbitrap-MS together with multivariate statistical analysis were used to systematically analyse the different components between small black beans (Small-BB) and big black beans (Big-BB) from 30 different habitats. RESULTS: The toxicity was ranked from small to large: S-PM < B-PM < R-PM. Processing PM with black beans could significantly decrease the apoptosis rate of L02 cells, especially when the drug concentration is 80 µg/mL. Besides, we find five differential compounds (α-arabinose, α-galactose, proline, isomer of daidzein and isomer of genistein) may be potential active ingredients. In terms of the black beans collected from 30 producing areas, we find that Small-BB from Weifang in Shandong province was optimum to processing PM, followed by Shangqiu in Henan province, Jilin and Liaoning province. CONCLUSION: The ingredients that affect the processing of PM may be attributed to α-arabinose, α-galactose, proline, isomer of daidzein and isomer of genistein in black beans. When the drug concentration is higher, the effect of reducing the hepatotoxicity of PM is better. Besides, Small-BB was more effective than Big-BB for reducing the toxicity of PM, especially Small-BB from Weifang in Shandong, Shangqiu in Henan province and northeast China.

Thirteen bisbenzylisoquinoline alkaloids in five Chinese medicinal plants: Botany, traditional uses, phytochemistry, pharmacokinetic and toxicity studies.

RELEVANCE: Bisbenzylisoquinoline (BBIQ) alkaloids are generally present in plants of Berberidaceae, Monimiaceae and Ranunculaceae families in tropical and subtropical regions. Some species of these families are used in traditional Chinese medicine, with the effects of clearing away heat and detoxification, promoting dampness and defecation, and eliminating sores and swelling. This article offers essential data focusing on 13 representative BBIQ compounds, which are mainly extracted from five plants. The respective botany, traditional uses, phytochemistry, pharmacokinetics, and toxicity are summarized comprehensively. In addition, the ADME prediction of the 13 BBIQ alkaloids is compared and analyzed with the data obtained. MATERIALS AND METHODS: We have conducted a systematic review of the botanical characteristics, traditional uses, phytochemistry, pharmacokinetics and toxicity of BBIQ alkaloids based on literatures collected from PubMed, Web of Science and Elsevier during 1999-2020. ACD/Percepta software was utilized to predict the pharmacokinetic parameters of BBIQ alkaloids and their affinity with enzymes and transporters. RESULTS: Botany, traditional uses, phytochemistry, pharmacokinetic and toxicity of 13 alkaloids, namely, tetrandrine, dauricine, curine, trilobine, isotrilobine, cepharanthine, daurisoline, thalicarpine, thalidasine, isotetrandrine, liensinine, neferine and isoliensinine, have been summarized in this paper. It can't be denied that these alkaloids are important material basis of pharmacological effects of family Menispermaceae and others, and for traditional and local uses which has been basically reproduced in the current studies. The 13 BBIQ alkaloids in this paper showed strong affinity and inhibitory effect on P-glycoprotein (P-gp), with poor oral absorption and potent binding ability with plasma protein. BBIQ alkaloids represented by tetrandrine play a key role in regulating P-gp or reversing multidrug resistance (MDR) in a variety of tumors. The irrationality of their usage could pose a risk of poisoning in vivo, including renal and liver toxicity, which are related to the formation of quinone methide during metabolism. CONCLUSION: Although there is no further clinical evaluation of BBIQ alkaloids as MDR reversal agents, their effects on P-gp should not be ignored. Considering their diverse distribution, pharmacokinetic characteristics and toxicity reported during clinical therapy, the quality standards in different plant species and the drug dosage remain unresolved problems.

Structure-Based Reactivity Profiles of Reactive Metabolites with Glutathione.

Therapeutic agents can be transformed into reactive metabolites under the action of various metabolic enzymes in vivo and then covalently combine with biological macromolecules (such as protein or DNA), resulting in increasing toxicity. The screening of reactive metabolites in drug discovery and development stages and monitoring of biotransformation in post-market drugs has become an important research field. Generally, reactive metabolites are electrophilic and can be captured by small nucleophiles. Glutathione (GSH) is a small peptide composed of three amino acids (i.e., glutamic acid, cysteine, and glycine). It has a thiol group which can react with electrophilic groups of reactive metabolic intermediates (such as benzoquinone, N-acetyl-p-benzoquinoneimine, and Michael acceptor) to form a stable binding conjugate. This paper aims to provide a review on structure-based reactivity profiles of reactive metabolites with GSH. Furthermore, this review also reveals the relationship between drugs' molecular structures and reactive metabolic toxicity from the perspective of metabolism, giving a reference for drug design and development.

Protecting Intestinal Microenvironment Alleviates Acute Graft-Versus-Host Disease.

Acute gut graft-versus-host disease (aGVHD) is a leading threat to the survival of allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Abnormal gut microbiota is correlated with poor prognosis in allo-HSCT recipients. A disrupted intestinal microenvironment exacerbates dysbiosis in GVHD patients. We hypothesized that maintaining the integrity of the intestinal barrier may protect gut microbiota and attenuate aGVHD. This hypothesis was tested in a murine aGVHD model and an in vitro intestinal epithelial culture. Millipore cytokine array was utilized to determine the expression of proinflammatory cytokines in the serum. The 16S rRNA sequencing was used to determine the abundance and diversity of gut microbiota. Combining Xuebijing injection (XBJ) with a reduced dose of cyclosporine A (CsA) is superior to CsA alone in improving the survival of aGVHD mice and delayed aGVHD progression. This regimen also reduced interleukin 6 (IL-6) and IL-12 levels in the peripheral blood. 16S rRNA analysis revealed the combination treatment protected gut microbiota in aGVHD mice by reversing the dysbiosis at the phylum, genus, and species level. It inhibited enterococcal expansion, a hallmark of GVHD progression. It inhibited enterococcal expansion, a hallmark of GVHD progression. Furthermore, Escherichia coli expansion was inhibited by this regimen. Pathology analysis revealed that the combination treatment improved the integrity of the intestinal tissue of aGVHD mice. It also reduced the intestinal permeability in aGVHD mice. Besides, XBJ ameliorated doxorubicin-induced intestinal epithelial death in CCK-8 assay. Overall, combining XBJ with CsA protected the intestinal microenvironment to prevent aGVHD. Our findings suggested that protecting the intestinal microenvironment could be a novel strategy to manage aGVHD. Combining XBJ with CsA may reduce the side effects of current aGVHD prevention regimens and improve the quality of life of allo-HSCT recipients.

Xuebijing Injection Maintains GRP78 Expression to Prevent Candida albicans-Induced Epithelial Death in the Kidney.

Sepsis and septic shock threaten the survival of millions of patients in the intensive care unit. Secondary fungal infections significantly increased the risk of mortality in sepsis patients. Chinese medicine Xuebijing injection (XBJ) has been routinely used as an add-on treatment to sepsis and septic shock in China. Our network pharmacology analysis predicted that XBJ also influences fungal infection, consisting with results of pioneer clinical studies. We conducted in vivo and in vitro experiments to verify this prediction. To our surprise, XBJ rescued mice from lethal Candida sepsis in a disseminated Candida albicans infection model and abolished the colonization of C. albicans in kidneys. Although XBJ did not inhibit the growth and the virulence of C. albicans in vitro, it enhanced the viability of 293T cells upon C. albicans insults. Further RNA-seq analysis revealed that XBJ activated the endoplasmic reticulum (ER) stress pathway upon C. albicans infection. Western blot confirmed that XBJ maintained the expression of GRP78 in the presence of C. albicans. Interestingly, key active ingredients in XBJ (C0127) mirrored the effects of XBJ. C0127 not only rescued mice from lethal Candida sepsis and prevented the colonization of C. albicans in kidneys, but also sustained the survival of kidney epithelial cells partially by maintaining the expression of GRP78. These results suggested that XBJ may prevent fungal infection in sepsis patients. Pre-activation of ER stress pathway is a novel strategy to control C. albicans infection. Network pharmacology may accelerate drug development in the field of infectious diseases.

Network Pharmacology-Guided Development of a Novel Integrative Regimen to Prevent Acute Graft-vs.-Host Disease.

Lapses in the graft-vs.-host disease (GVHD) prophylaxis and side effects of current standard care following allogeneic hematopoietic stem cell transplantation (allo-HSCT) call for novel regimens. Traditional approaches targeting T cells showed limited success in preventing acute GVHD (aGVHD). System medicine showed promising results treating complex diseases such as sepsis and multi-organ dysfunction syndrome (MODS). Adapting established network pharmacology analysis methods, we aimed to develop novel integrative regimens to prevent aGVHD. Our network pharmacology analysis predicted that Xuebijing injection (XBJ) targets a series of key node proteins in aGVHD network. It also unveiled that Salviae miltiorrhizae (Danshen), an herb in Xuebijing formula, which prevented aGVHD in rats, shares five out of six key GVHD node proteins targeted by XBJ. Interestingly, network pharmacology analysis indicated Xuebijing may share multiple aGVHD targets with Cyclosporin A (CsA), a first-line drug for preventing aGVHD in the clinic. Based on current information, we hypothesized that combination of XBJ and CsA may yield superior results in aGVHD prevention than either drug alone. We performed in vitro and in vivo assays to validate the predictions by the network pharmacology analysis. In vitro assays revealed XBJ prevented platelet aggregation and NF-κB nuclear translocation in macrophages. XBJ also promoted angiogenesis in tube-formation assay. Importantly, the combination of CsA and XBJ was effective in rescuing mice subjected to lethal GVHD. XBJ contributed to the rescue through preventing NF-κB nuclear translocation, attenuating inflammation and maintaining viability of macrophages. Overall, network pharmacology is a powerful tool to develop novel integrative regimens. Combination of XBJ and CsA may shed light on preventing aGVHD.

Anti-sepsis protection of Xuebijing injection is mediated by differential regulation of pro- and anti-inflammatory Th17 and T regulatory cells in a murine model of polymicrobial sepsis.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuebijing injection (XBJ), a Chinese herbal medicine containing extracts from 5 herbs, is frequently used as an add-on with standard therapies to treat sepsis or septic shock with fewer side effects in China. Nonetheless, its mechanism of action on septic shock remains to be unveiled. We explored the differential effects of XBJ on subtypes of CD4+ T cell differentiation and septic shock protection in a murine model to understand the contribution of XBJ to regulation of the inflammation-immune axis function. MATERIALS AND METHODS: In vitro T cell differentiation assays were performed to determine the effect of XBJ on CD4+ regulatory T cell and T helper cell differentiation. Besides, 2ml/kg, 6ml/kg- and 18ml/kg of XBJ were administered to different groups of septic mice once/day for 5 days after cecal ligation and puncture (CLP) surgeries. 36h after CLP, serum levels of pro-inflammatory cytokine TNF-α and IL-6 were determined with Elisa. Frequencies of CD4+ T cells were analyzed after staining with Tregs and T helper cell lineage specific antibodies by flow cytometer. RESULTS: XBJ at 18ml/kg stimulated Treg differentiation and moderately inhibited Th17 differentiation in vitro. Accordingly, 18ml/kg XBJ facilitated the expansion of IL-10+ Tregs and normalized pro-inflammatory Th17 population in septic mice. This regimen also significantly reduced serum levels of inflammatory cytokines TNF-α and IL-6 in septic mice. Additionally, 18ml/kg XBJ injection effectively prevented neutrophil infiltration into the lung and kidney and improved survival in this septic shock model. CONCLUSIONS: In summary, XBJ improves survival in septic shock partially through preventing cytokine storm, inhibiting inflammation and regulating the balance of Tregs and Th17 cells. Thus, higher dose of XBJ is a potential regimen to benefit septic shock patients.