Identification of a Small Molecule Compound Active against Antibiotic-Tolerant Staphylococcus aureus by Boosting ATP Synthesis.

Antibiotic tolerance poses a threat to current antimicrobial armamentarium. Bacteria at a tolerant state survive in the presence of antibiotic treatment and account for persistence, relapse and recalcitrance of infections. Antibiotic treatment failure may occur due to antibiotic tolerance. Persistent infections are difficult to treat and are often associated with poor prognosis, imposing an enormous burden on the healthcare system. Effective strategies targeting antibiotic-tolerant bacteria are therefore highly warranted. In this study, small molecule compound SA-558 was identified to be effective against Staphylococcus aureus that are tolerant to being killed by conventional antibiotics. SA-558 mediated electroneutral transport across the membrane and led to increased ATP and ROS generation, resulting in a reduction of the population of antibiotic-tolerant bacteria. In a murine chronic infection model, of which vancomycin treatment failed, we demonstrated that SA-558 alone and in combination with vancomycin caused significant reduction of MRSA abundance. Our results indicate that SA-558 monotherapy or combinatorial therapy with vancomycin is an option for managing persistent S. aureus bacteremia infection and corroborate that bacterial metabolism is an important target for counteracting antibiotic tolerance.

Linoleic acid metabolism activation in macrophages promotes the clearing of intracellular Staphylococcus aureus.

Multidrug-resistant bacterial pathogens pose an increasing threat to human health. Certain bacteria, such as Staphylococcus aureus, are able to survive within professional phagocytes to escape the bactericidal effects of antibiotics and evade killing by immune cells, potentially leading to chronic or persistent infections. By investigating the macrophage response to S. aureus infection, we may devise a strategy to prime the innate immune system to eliminate the infected bacteria. Here we applied untargeted tandem mass spectrometry to characterize the lipidome alteration in S. aureus infected J774A.1 macrophage cells at multiple time points. Linoleic acid (LA) metabolism and sphingolipid metabolism pathways were found to be two major perturbed pathways upon S. aureus infection. The subsequent validation has shown that sphingolipid metabolism suppression impaired macrophage phagocytosis and enhanced intracellular bacteria survival. Meanwhile LA metabolism activation significantly reduced intracellular S. aureus survival without affecting the phagocytic capacity of the macrophage. Furthermore, exogenous LA treatment also exhibited significant bacterial load reduction in multiple organs in a mouse bacteremia model. Two mechanisms are proposed to be involved in this progress: exogenous LA supplement increases downstream metabolites that partially contribute to LA's capacity of intracellular bacteria-killing and LA induces intracellular reactive oxygen species (ROS) generation through an electron transport chain pathway in multiple immune cell lines, which further increases the capacity of killing intracellular bacteria. Collectively, our findings not only have characterized specific lipid pathways associated with the function of macrophages but also demonstrated that exogenous LA addition may activate lipid modulator-mediated innate immunity as a potential therapy for bacterial infections.

Broad-spectrum antiviral activity of Spatholobus suberectus Dunn against SARS-CoV-2, SARS-CoV-1, H5N1, and other enveloped viruses.

The current COVID-19 pandemic caused by SARS-Cov-2 is responsible for more than 6 million deaths globally. The development of broad-spectrum and cost-effective antivirals is urgently needed. Medicinal plants are renowned as a complementary approach in which antiviral natural products have been established as safe and effective drugs. Here, we report that the percolation extract of Spatholobus suberectus Dunn (SSP) is a broad-spectrum viral entry inhibitor against SARS-CoV-1/2 and other enveloped viruses. The viral inhibitory activities of the SSP were evaluated by using pseudotyped SARS-CoV-1 and 2, HIV-1ADA and HXB2 , and H5N1. SSP effectively inhibited viral entry and with EC50 values ranging from 3.6 to 5.1 µg/ml. Pre-treatment of pseudovirus or target cells with SSP showed consistent inhibitory activities with the respective EC50 value of 2.3 or 2.1 µg/ml. SSP blocked both SARS-CoV-2 spike glycoprotein and the host ACE2 receptor. In vivo studies indicated that there was no abnormal toxicity and behavior in long-term SSP treatment. Based on these findings, we concluded that SSP has the potential to be developed as a drug candidate for preventing and treating COVID-19 and other emerging enveloped viruses.

Phosphatidic acid phosphatase 1 impairs SARS-CoV-2 replication by affecting the glycerophospholipid metabolism pathway.

Viruses exploit the host lipid metabolism machinery to achieve efficient replication. We herein characterize the lipids profile reprogramming in vitro and in vivo using liquid chromatography-mass spectrometry-based untargeted lipidomics. The lipidome of SARS-CoV-2-infected Caco-2 cells was markedly different from that of mock-infected samples, with most of the changes involving downregulation of ceramides. In COVID-19 patients' plasma samples, a total of 54 lipids belonging to 12 lipid classes that were significantly perturbed compared to non-infected control subjects' plasma samples were identified. Among these 12 lipid classes, ether-linked phosphatidylcholines, ether-linked phosphatidylethanolamines, phosphatidylcholines, and ceramides were the four most perturbed. Pathway analysis revealed that the glycerophospholipid, sphingolipid, and ether lipid metabolisms pathway were the most significantly perturbed host pathways. Phosphatidic acid phosphatases (PAP) were involved in all three pathways and PAP-1 deficiency significantly suppressed SARS-CoV-2 replication. siRNA knockdown of LPIN2 and LPIN3 resulted in significant reduction of SARS-CoV-2 load. In summary, these findings characterized the host lipidomic changes upon SARS-CoV-2 infection and identified PAP-1 as a potential target for intervention for COVID-19.

Targeting ACLY efficiently inhibits SARS-CoV-2 replication.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the biggest public health challenge the world has witnessed in the past decades. SARS-CoV-2 undergoes constant mutations and new variants of concerns (VOCs) with altered transmissibility, virulence, and/or susceptibility to vaccines and therapeutics continue to emerge. Detailed analysis of host factors involved in virus replication may help to identify novel treatment targets. In this study, we dissected the metabolome derived from COVID-19 patients to identify key host factors that are required for efficient SARS-CoV-2 replication. Through a series of metabolomic analyses, in vitro, and in vivo investigations, we identified ATP citrate lyase (ACLY) as a novel host factor required for efficient replication of SARS-CoV-2 wild-type and variants, including Omicron. ACLY should be further explored as a novel intervention target for COVID-19.

A trifunctional peptide broadly inhibits SARS-CoV-2 Delta and Omicron variants in hamsters.

The emergence of highly transmissible SARS-CoV-2 variants has led to the waves of the resurgence of COVID-19 cases. Effective antivirals against variants are required. Here we demonstrate that a human-derived peptide 4H30 has broad antiviral activity against the ancestral virus and four Variants of Concern (VOCs) in vitro. Mechanistically, 4H30 can inhibit three distinct steps of the SARS-CoV-2 life cycle. Specifically, 4H30 blocks viral entry by clustering SARS-CoV-2 virions; prevents membrane fusion by inhibiting endosomal acidification; and inhibits the release of virions by cross-linking SARS-CoV-2 with cellular glycosaminoglycans. In vivo studies show that 4H30 significantly reduces the lung viral titers in hamsters, with a more potent reduction for the Omicron variant than the Delta variant. This is likely because the entry of the Omicron variant mainly relies on the endocytic pathway which is targeted by 4H30. Moreover, 4H30 reduces syncytia formation in infected hamster lungs. These findings provide a proof of concept that a single antiviral can inhibit viral entry, fusion, and release.

Bacillus Calmette-Guérin-induced trained immunity protects against SARS-CoV-2 challenge in K18-hACE2 mice.

SARS-CoV-2 has been confirmed in over 450 million confirmed cases since 2019. Although several vaccines have been certified by the WHO and people are being vaccinated on a global scale, it has been reported that multiple SARS-CoV-2 variants can escape neutralization by antibodies, resulting in vaccine breakthrough infections. Bacillus Calmette-Guérin (BCG) is known to induce heterologous protection based on trained immune responses. Here, we investigated whether BCG-induced trained immunity protected against SARS-CoV-2 in the K18-hACE2 mouse model. Our data demonstrate that i.v. BCG (BCG-i.v.) vaccination induces robust trained innate immune responses and provides protection against WT SARS-CoV-2, as well as the B.1.617.1 and B.1.617.2 variants. Further studies suggest that myeloid cell differentiation and activation of the glycolysis pathway are associated with BCG-induced training immunity in K18-hACE2 mice. Overall, our study provides the experimental evidence that establishes a causal relationship between BCG-i.v. vaccination and protection against SARS-CoV-2 challenge.

SARS-CoV-2 exploits host DGAT and ADRP for efficient replication.

Coronavirus Disease 2019 (COVID-19) is predominantly a respiratory tract infection that significantly rewires the host metabolism. Here, we monitored a cohort of COVID-19 patients' plasma lipidome over the disease course and identified triacylglycerol (TG) as the dominant lipid class present in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced metabolic dysregulation. In particular, we pinpointed the lipid droplet (LD)-formation enzyme diacylglycerol acyltransferase (DGAT) and the LD stabilizer adipocyte differentiation-related protein (ADRP) to be essential host factors for SARS-CoV-2 replication. Mechanistically, viral nucleo capsid protein drives DGAT1/2 gene expression to facilitate LD formation and associates with ADRP on the LD surface to complete the viral replication cycle. DGAT gene depletion reduces SARS-CoV-2 protein synthesis without compromising viral genome replication/transcription. Importantly, a cheap and orally available DGAT inhibitor, xanthohumol, was found to suppress SARS-CoV-2 replication and the associated pulmonary inflammation in a hamster model. Our findings not only uncovered the mechanistic role of SARS-CoV-2 nucleocapsid protein to exploit LDs-oriented network for heightened metabolic demand, but also the potential to target the LDs-synthetase DGAT and LDs-stabilizer ADRP for COVID-19 treatment.

Restoring perturbed oxylipins with Danqi Tongmai Tablet attenuates acute myocardial infarction.

Salvianolic acids have a special synergic effect on panax notoginsenosides in acute myocardial infarction (AMI) and have been developed into a new drug as Danqi Tongmai Tablet (DQTT). To explore candidate targets and mechanisms of DQTT on AMI, a network pharmacology-based analysis was performed on absorbed prototype compounds of DQTT in rat plasma. Target prediction from network analysis indicated that the arachidonic acid pathway might contribute to the therapeutic effects of DQTT on AMI, and the regulatory effects on cyclooxygenase (COX) and lipoxygenase (LOX) were validated using an oxygen-glucose deprivation/reoxygenation model established on H9c2 cardiomyocytes. To further explore the action mechanisms of DQTT, 38 oxylipins were quantitatively analyzed among high, medium, and low doses of DQTT using a rat AMI model with an ultra high performance liquid chromatograph coupled with a triple quadrupole mass spectrometry (UHPLC-QqQ/MS) detection system. As attenuation was observed in AMI with DQTT treatment, the perturbed arachidonic acid metabolome was partly restored in a dose-dependent fashion with a significant elevation of anti-inflammatory metabolites, while pro-inflammatory lipids were decreased. Cytokine array analysis also supported the anti-inflammatory effects of DQTT, as significant down-regulation of pro-inflammatory cytokines was observed. The analysis of ischemic heart tissues demonstrated that COX and LOX, the inflammation-induced catalytic enzymes of arachidonic acid metabolism, were inhibited on both gene expression and protein level. These results confirmed that DQTT could restore the arachidonic acid metabolome to maintain an anti-inflammatory profile against the ischemic tissue injury and support that DQTT can be a promising medicinal therapy against AMI.

Differential role of sphingomyelin in influenza virus, rhinovirus and SARS-CoV-2 infection of Calu-3 cells.

Host cell lipids play a pivotal role in the pathogenesis of respiratory virus infection. However, a direct comparison of the lipidomic profile of influenza virus and rhinovirus infections is lacking. In this study, we first compared the lipid profile of influenza virus and rhinovirus infection in a bronchial epithelial cell line. Most lipid features were downregulated for both influenza virus and rhinovirus, especially for the sphingomyelin features. Pathway analysis showed that sphingolipid metabolism was the most perturbed pathway. Functional study showed that bacterial sphingomyelinase suppressed influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, but promoted rhinovirus replication. These findings suggest that sphingomyelin pathway can be a potential target for antiviral therapy, but should be carefully evaluated as it has opposite effects on different respiratory viruses. Furthermore, the differential effect of sphingomyelinase on rhinovirus and influenza virus may explain the interference between rhinovirus and influenza virus infection.

Metabolic Profiling Reveals Significant Perturbations of Intracellular Glucose Homeostasis in Enterovirus-Infected Cells.

Enterovirus A71 (EV-A71) is a common cause of hand, foot, and mouth disease. Severe EV-A71 infections may be associated with life-threatening neurological complications. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Metabolites are known to play critical roles in multiple stages of the replication cycles of viruses. The metabolic reprogramming induced by viral infections is essential for optimal virus replication and may be potential antiviral targets. In this study, we applied targeted metabolomics profiling to investigate the metabolic changes of induced pluripotent human stem cell (iPSC)-derived neural progenitor cells (NPCs) upon EV-A71 infection. A targeted quantitation of polar metabolites identified 14 candidates with altered expression profiles. A pathway enrichment analysis pinpointed glucose metabolic pathways as being highly perturbed upon EV-A71 infection. Gene silencing of one of the key enzymes of glycolysis, 6-phosphofructo-2-kinase (PFKFB3), significantly suppressed EV-A71 replication in vitro. Collectively, we demonstrated the feasibility to manipulate EV-A71-triggered host metabolic reprogramming as a potential anti-EV-A71 strategy.

Exploring the protective effects of Danqi Tongmai tablet on acute myocardial ischemia rats by comprehensive metabolomics profiling.

BACKGROUND: Danqi Tongmai tablet (DQTM), a combination of salvianolic acids (SA) and panax notoginsenosides (PNS), is now in phase II clinical trial developed for the treatment of cardiovascular diseases. However, the mechanisms of its protective effects through regulating endogenous metabolites remain unclear. PURPOSE: The purpose of this study was to explore the protective effects of DQTM on acute myocardial ischemia rats by comprehensive metabolomics profiling. STUDY DESIGN: The rats were divided into three groups: sham-operating, acute myocardial ischemia (AMI) and DQTM groups. The plasma and heart were collected and profiled by LC-MS based metabolomics and lipidomics. Based on the identified differential metabolites, the pathway analysis results were obtained and further validated using the network pharmacology approach. METHODS: The AMI model was induced by ligating the left anterior descending coronary artery. The metabolomics and lipidomics profiling were based on two established LC-QTOF/MS analysis methods. The raw data were processed using XCMS Online, then the differential metabolites with nonparametric t-test p value less than 0.05 were selected and identified using HMDB and METLIN. The pathway analysis was conducted using MetaboAnalyst and validated with the predicted network results obtained by BATMAN-TCM. RESULTS: The metabolomics and lipidomics profiles of plasma and heart in response to AMI and DQTM were significantly different. The AMI operation had a serious influence on metabolites in heart ischemia region, while DQTM had a greater impact on lipids in heart non-ischemia region. A total of 151 differential metabolites were identified, including mainly amino acids and fatty acids. Multiple metabolic pathways were disturbed after AMI and could be restored by DQTM, of which arachidonic acid metabolism was further validated with the predicted results of network pharmacology. CONCLUSION: The protective effects of DQTM on acute myocardial ischemia rats could be achieved through the regulation of multiple metabolic pathways.

Lipidomic Profiling Reveals Significant Perturbations of Intracellular Lipid Homeostasis in Enterovirus-Infected Cells.

Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the most common causes of hand, foot, and mouth disease. Severe EV-A71 and CV-A16 infections may be associated with life-threatening complications. However, the pathogenic mechanisms underlying these severe clinical and pathological features remain incompletely understood. Lipids are known to play critical roles in multiple stages of the virus replication cycle. The specific lipid profile induced upon virus infection is required for optimal virus replication. The perturbations in the host cell lipidomic profiles upon enterovirus infection have not been fully characterized. To this end, we performed ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS)-based lipidomics to characterize the change in host lipidome upon EV-A71 and CV-A16 infections. Our results revealed that 47 lipids within 11 lipid classes were significantly perturbed after EV-A71 and CV-A16 infection. Four polyunsaturated fatty acids (PUFAs), namely, arachidonic acid (AA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), were consistently upregulated upon EV-A71 and CV-A16 infection. Importantly, exogenously supplying three of these four PUFAs, including AA, DHA, and EPA, in cell cultures significantly reduced EV-A71 and CV-A16 replication. Taken together, our results suggested that enteroviruses might specifically modulate the host lipid pathways for optimal virus replication. Excessive exogenous addition of lipids that disrupted this delicate homeostatic state could prevent efficient viral replication. Precise manipulation of the host lipid profile might be a potential host-targeting antiviral strategy for enterovirus infection.

SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target.

Viruses are obligate intracellular microbes that exploit the host metabolic machineries to meet their biosynthetic demands, making these host pathways potential therapeutic targets. Here, by exploring a lipid library, we show that AM580, a retinoid derivative and RAR-α agonist, is highly potent in interrupting the life cycle of diverse viruses including Middle East respiratory syndrome coronavirus and influenza A virus. Using click chemistry, the overexpressed sterol regulatory element binding protein (SREBP) is shown to interact with AM580, which accounts for its broad-spectrum antiviral activity. Mechanistic studies pinpoint multiple SREBP proteolytic processes and SREBP-regulated lipid biosynthesis pathways, including the downstream viral protein palmitoylation and double-membrane vesicles formation, that are indispensable for virus replication. Collectively, our study identifies a basic lipogenic transactivation event with broad relevance to human viral infections and represents SREBP as a potential target for the development of broad-spectrum antiviral strategies.

Study on the herb-herb interaction of Danqi Tongmai Tablet based on the pharmacokinetics of twelve notoginsenoides in acute myocardial ischemia and sham rats.

Danqi Tongmai tablet (DQTT), an innovative TCM formula under clinical trials, is composed of salvianolic acids (SA) and panax notoginsenosides (PNE) for the treatment of coronary heart disease and angina pectoris. However, the in vivo herb-herb interaction of DQTT remains unclear. In the present research, a rapid, reliable and sensitive method for quantitative analysis of multi-notoginsenoside in rat plasma based on ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-TQ/MS) was established and then applied to explore the herb-herb interaction mechanism of DQTT based on the pharmacokinetics in acute myocardial ischemia (AMI) and sham rats after oral administration of DQTT and PNE. Compared with sham rats after oral administration of PNE, the values of AUC0-t for Rf and Rb2 were significantly higher in DQTT group. Compared with AMI rats after oral PNE, AUC0-t for NR1, Rg1, Re, Rb1, Rd, Rg2, Rb2, NR2, Rh1, F1 and F2 were significantly increased after oral administration of DQTT. These results hinted that SA could improve the bioavailability of notoginsenosides in AMI rats, which provides scientific information for better understanding the herb-herb interaction mechanism and offers a reference for clinical administration of DQTT. Additionally, the presently developed methodology was simple, robust, accurate, precise, and would be useful for the pharmacokinetic studies for all kinds of notoginsenosides and other herbal saponins.

Characterization of the Lipidomic Profile of Human Coronavirus-Infected Cells: Implications for Lipid Metabolism Remodeling upon Coronavirus Replication.

Lipids play numerous indispensable cellular functions and are involved in multiple steps in the replication cycle of viruses. Infections by human-pathogenic coronaviruses result in diverse clinical outcomes, ranging from self-limiting flu-like symptoms to severe pneumonia with extrapulmonary manifestations. Understanding how cellular lipids may modulate the pathogenicity of human-pathogenic coronaviruses remains poor. To this end, we utilized the human coronavirus 229E (HCoV-229E) as a model coronavirus to comprehensively characterize the host cell lipid response upon coronavirus infection with an ultra-high performance liquid chromatography-mass spectrometry (UPLC⁻MS)-based lipidomics approach. Our results revealed that glycerophospholipids and fatty acids (FAs) were significantly elevated in the HCoV-229E-infected cells and the linoleic acid (LA) to arachidonic acid (AA) metabolism axis was markedly perturbed upon HCoV-229E infection. Interestingly, exogenous supplement of LA or AA in HCoV-229E-infected cells significantly suppressed HCoV-229E virus replication. Importantly, the inhibitory effect of LA and AA on virus replication was also conserved for the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV). Taken together, our study demonstrated that host lipid metabolic remodeling was significantly associated with human-pathogenic coronavirus propagation. Our data further suggested that lipid metabolism regulation would be a common and druggable target for coronavirus infections.

Suppression of Staphylococcus aureus virulence by a small-molecule compound.

Emerging antibiotic resistance among bacterial pathogens has necessitated the development of alternative approaches to combat drug-resistance-associated infection. The abolition of Staphylococcus aureus virulence by targeting multiple-virulence gene products represents a promising strategy for exploration. A multiplex promoter reporter platform using gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus-virulence-associated genes was used to identify compounds that modulate the expression of virulence factors. One small-molecule compound, M21, was identified from a chemical library to reverse virulent S. aureus into its nonvirulent state. M21 is a noncompetitive inhibitor of ClpP and alters α-toxin expression in a ClpP-dependent manner. A mouse model of infection indicated that M21 could attenuate S. aureus virulence. This nonantibiotic compound has been shown to suppress the expression of multiple unrelated virulence factors in S. aureus, suggesting that targeting a master regulator of virulence is an effective way to control virulence. Our results illustrate the power of chemical genetics in the modulation of virulence gene expression in pathogenic bacteria.

A Strategy Combining Higher Energy C-Trap Dissociation with Neutral Loss- and Product Ion-Based MSn Acquisition for Global Profiling and Structure Annotation of Fatty Acids Conjugates.

Fatty acids conjugates (FACs) are ubiquitous but found in trace amounts in the natural world. They are composed of multiple unknown substructures and side chains. Thus, FACs are difficult to be analyzed by traditional mass spectrometric methods. In this study, an integrated strategy was developed to global profiling and targeted structure annotation of FACs in complex matrix by LTQ Orbitrap. Dicarboxylic acid conjugated bufotoxins (DACBs) in Venenum bufonis (VB) were used as model compounds. The new strategy (abbreviated as HPNA) combined higher-energy C-trap dissociation (HCD) with product ion- (PI), neutral loss- (NL) based MSn (n ≥ 3) acquisition in both positive-ion mode and negative-ion mode. Several advantages are presented. First, various side chains were found under HCD in negative-ion mode, which included both known and unknown side chains. Second, DACBs with multiple side chains were simultaneously detected in one run. Compared with traditional quadrupole-based mass method, it greatly increased analysis throughput. Third, the fragment ions of side chain and steroids substructure could be obtained by PI- and NL-based MSn acquisition, respectively, which greatly increased the accuracy of the structure annotation of DACBs. In all, 78 DACBs have been discovered, of which 68 were new compounds; 25 types of substructure formulas and seven dicarboxylic acid side chains were found, especially five new side chains, including two saturated dicarboxylic acids [(azelaic acid (C9) and sebacic acid (C10)] and three unsaturated dicarboxylic acids (u-C8, u-C9, and u-C10). All these results greatly enriched the structures of DACBs in VB. Graphical Abstract ᅟ.

Profiling and identification of metabolites of isorhynchophylline in rats by ultra high performance liquid chromatography and linear ion trap Orbitrap mass spectrometry.

The searching of potentially bioactive metabolites in the biological body is an interesting and meaningful work for the drug study. However, the structural clarification of possible metabolites is one of the most challenging tasks in drug metabolism studies because of the variety of metabolic reactions and complexity of metabolites in vivo. Here, an ultra high performance liquid chromatography/linear ion trap-Orbitrap mass spectrometry (U-HPLC/LTQ-Orbitrap-MS) with combination of data post-processing techniques, including extracted ion chromatogram (EIC) and multiple mass defect filters (MMDF), was established for profiling and identification of metabolites of isorhynchophylline (IR) in vivo and in vitro, and the possible metabolic pathways were subsequently proposed after the oral dose of 20mg/kg; A total of 47 metabolites of IR were tentatively identified, including 47, 21, 18, and 25 metabolites in rat urine, plasma, liver and rat liver microsomes (RLM) samples, respectively. To our knowledge, most of them were reported for the first time. Seven metabolic pathways, including dehydrogenation, oxidation, hydrolysis, reduction, demethylation, hydroxylation and glucuronide conjugation were involved in the metabolism. Among them, dehydrogenation, hydrolysis, hydroxylation and oxidation were considered as the main metabolic pathway of metabolism according to metabolic profile of in vivo and in vitro. The relative percentage of each metabolite and main metabolite types were also determined to better understand the metabolic behavior of IR in rats. The newly discovered IR metabolites significantly expanded our understanding and were going to be greatly helpful for the further pharmacokinetic study of IR in vivo.

Venenum Bufonis induces rat neuroinflammation by activiating NF-κB pathway and attenuation of BDNF.

ETHNOPHARMACOLOGICAL RELEVANCE: Venenum Bufonis (VB), also called toad venom, has been widely used in clinic as a cardiotonic, anohyne and antineoplastic agents both in China and other Asian countries. However, its neurotoxicity and cardiotoxicity limit its wide clinical application. Compared with extensive attention attracted with cardiotoxicity, the toxic effect of VB on Central Nervous System (CNS) is much less studied. AIM OF THE RESEARCH: This study was performed to examine the neurotoxicity caused by VB on Sprague Dawley (SD) rats, then to clarify the mechanism in vivo by investigating its action on the neuroinflammation which possibly attributed to the activation of nuclear factor κB (NF-κB) pathway and the attenuation of brain-derived neurotrophic factor (BDNF). MATERIALS AND METHODS: Rats administrated with 0.5% carboxymethyl cellulose sodium salt (CMC-Na) aqueous solution and VB (100mg/kg, 200mg/kg and 400mg/kg) were sacrificed at 2h, 4h, 6h, 8h, 24h and 48h. The brain level of neurotransmitters and their corresponding receptors, pro-inflammatory cytokines, BDNF/TrkB and NF-κB pathway-related proteins were examined, respectively. RESULTS: VB administration induced severe neurologic damage and neuroinflammation, as indicated by the disordered 5-hydroxytryptamine (5-HT), dopamine (DA) and their corresponding receptors, together with the over production of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). VB also notably promoted the expression of p-NF-κBp65, p-IκBα, p-IKKα and p-IKKß and down-regulated the expression of BDNF and TrkB. CONCLUSION: This study demonstrates that VB triggers neurotoxicity which probably is induced by neuroinflammation via activating of NF-κB pathway and attenuating the expression of BDNF.