Tanreqing injection demonstrates anti-dengue activity through the regulation of the NF-κB-ICAM-1/VCAM-1 axis.

BACKGROUND: Tanreqing injection (TRQ) has been employed in clinical practice as a treatment for dengue fever (DF). Nevertheless, the precise pharmacological mechanism underlying its efficacy remains elusive. METHOD: Network pharmacology, molecular docking, transcriptome sequencing, and experimental evaluation were employed to analyze and study the inhibitory potential of TRQ against dengue virus (DENV). RESULT: We found that TRQ inhibited the replication of DENV in human umbilical vein endothelial cells, Huh-7 cells, and Hep3B cells. In addition, TRQ prolonged the survival duration of AG129 mice infected with DF, decreased the viral load in serum and organs, and alleviated organ damage. Subsequently, ultra-high-performance liquid chromatography-tandem mass spectrometry analysis of TRQ was performed to identify 314 targets associated with 36 active compounds present in TRQ. Integration of multiple databases yielded 47 DF-related genes. Then, 15 hub targets of TRQ in DF were determined by calculating the network topology parameters (Degree). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these pathways were primarily enriched in the processes of cytokine activation and leukocyte cross-endothelial migration, with significant enrichment of cell adhesion molecules. Molecular docking revealed favorable binding affinity between TRQ's key active compounds and the predicted hub targets. Transcriptome sequencing results showed TRQ's ability to restore the expression of vascular cell adhesion molecule-1 (VCAM-1) post-DENV infection. Finally, TRQ was found to modulate the immune status by regulating the nuclear factor kappa-B (NF-κB)- intercellular cell adhesion molecule-1 (ICAM-1)/VCAM-1 axis, as well as reduce immune cell alterations, inflammatory factor secretion, vascular permeability, and bleeding tendencies induced by DENV infection. CONCLUSION: Our research suggests that TRQ exerts therapeutic effects on DF by regulating the NF-κB-ICAM-1/VCAM-1 axis.

Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

OBJECTIVE: To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. METHODS: Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 ß, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 ß were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. RESULTS: SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 ß, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). CONCLUSIONS: SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

Tanreqing injection inhibits dengue virus encephalitis by suppressing the activation of NLRP3 inflammasome.

BACKGROUND: Encephalitis caused by dengue virus (DENV) is considered a manifestation of severe dengue. Tanreqing injection (TRQ) is a well-known Chinese patented medicine, which has been used to treat brain-related disorders by inhibiting inflammation. Nevertheless, the effects of TRQ on DENV encephalitis have not been studied. The aim of this study was to evaluate the effects of TRQ on DENV encephalitis and to explore its potential mechanisms. METHODS: The cytotoxicity of TRQ was examined by MTT assay, and the anti-DENV activities of TRQ in BHK-21 baby hamster kidney fibroblast were evaluated through CCK-8 and plaque assays. The expression levels of NO, IL1B/IL-1ß, TNFα and IL6 were measured by qRT‒PCR and ELISA in the BV2 murine microglial cell line. The inhibitory effects of TRQ on NLRP3 inflammasome activation in BV2 cells were examined by Western blotting, qRT‒PCR and ELISA. The effects of TRQ on HT22 mouse hippocampal neuronal cells were examined by CCK-8 assay, morphology observation and flow cytometry. Moreover, a DENV-infected ICR suckling mouse model was developed to investigate the protective role of TRQ in vivo. RESULTS: TRQ decreased the release of NO, IL6, TNFα and IL1B from BV2 cells and inhibited the activation of NLRP3. The presence of the NLRP3 agonist nigericin reversed the anti-inflammatory activities of TRQ. Furthermore, TRQ inhibited the death of HT22 cells by decreasing IL1B in DENV-infected BV2 cells. In addition, TRQ significantly attenuated weight loss, reduced clinical scores and extended the survival in DENV-infected ICR suckling mice. Critically, TRQ ameliorated pathological changes in ICR suckling mice brain by inhibiting microglia and NLRP3 activation and decreasing the production of inflammatory factors and the number of dead neurons. CONCLUSION: TRQ exerts potent inhibitory effects on dengue encephalitis in vitro and in vivo by reducing DENV-2-induced microglial activation and subsequently decreasing the inflammatory response, thereby protecting neurons. These findings demonstrate the potential of TRQ in the treatment of dengue encephalitis.

TMT-based quantitative proteomics analysis of neuroprotective effects of Forsythoside A on the MPTP-induced Parkinson's disease mouse model.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characteristized by the presence of dyskinesia and the progressive loss of dopaminergic neurons. Although certain drugs can mitigate the symptoms of PD, they are unable to delay the disease progression, and their prolonged use may result in complications. Therefore, there exists an urgent necessity to identify potential agents that can effectively delay PD progression with fewer side effects. Recent research has unveiled that several traditional Chinese medicines (TCM) exhibit neuroprotective properties in various models pertinent to PD. Forsythoside A (FSA), the primary bioactive compound derived from TCM Lianqiao, has undergone extensive research in animal models of Alzheimer's disease and cerebral ischemia. However, the investigation into the impact of FSA on PD is limited in existing research. In this study, we aimed to evaluate the neuroprotective effects of FSA on MPTP-induced PD mouse model. FSA demonstrated significant improvements in the behavioral and neuropathological changes triggered by MPTP in mice. Furthermore, it exerted a suppressive effect on the activations of astrocyte and microglia. Meanwhile, Tandem mass tag (TMT)-based quantitative proteomics of striatal tissue and bioinformatics analysis were performed to elucidate the underlying mechanisms of FSA on PD mouse model. Proteomics demonstrated a total of 68 differentially expressed proteins (DEPs) were identified between HFSA and MPTP groups including 26 upregulated and 42 downregulated. Systematic bioinformatics analysis of the 68 DEPs illustrated that they were predominantly related to estrogen signaling pathway and calcium signaling pathway. The related DEPs (PLCß4, Grm2, HPAC and Cox4i1) expression levels were verified by Western blot. FSA effectively restored the altered expression of the four DEPs induced by MPTP. Summarily, FSA exerted remarkable neuroprotective effects in MPTP-induced mice. Further, our research may provide proteomics insights that contribute to the further exploration of FSA as a potential treatment for PD.

Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish.

OBJECTIVE: To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms. METHODS: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo. RESULTS: The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01). CONCLUSION: Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.

Liang-Ge-San: a classic traditional Chinese medicine formula, attenuates acute inflammation via targeting GSK3ß.

Sepsis is a serious life-threatening health disorder with high morbidity and mortality rates that burden the world, but there is still a lack of more effective and reliable drug treatment. Liang-Ge-San (LGS) has been shown to have anti-inflammatory effects and is a promising candidate for the treatment of sepsis. However, the anti-sepsis mechanism of LGS has still not been elucidated. In this study, a set of genes related to inflammatory chemotaxis pathways was downloaded from Encyclopedia of Genes and Genomes (KEGG) and integrated with sepsis patient information from the Gene Expression Omnibus (GEO) database to perform differential gene expression analysis. Glycogen synthase kinase-3ß (GSK-3ß) was found to be the feature gene after these important genes were examined using the three algorithms Random Forest, support vector machine recursive feature elimination (SVM-REF), and least absolute shrinkage and selection operator (LASSO), and then intersected with possible treatment targets of LGS found through the search. Upon evaluation, the receiver operating characteristic (ROC) curve of GSK-3ß indicated an important role in the pathogenesis of sepsis. Immune cell infiltration analysis suggested that GSK-3ß expression was associated with a variety of immune cells, including neutrophils and monocytes. Next, lipopolysaccharide (LPS)-induced zebrafish inflammation model and macrophage inflammation model was used to validate the mechanism of LGS. We found that LGS could protect zebrafish against a lethal challenge with LPS by down-regulating GSK-3ß mRNA expression in a dose-dependent manner, as indicated by a decreased neutrophils infiltration and reduction of inflammatory damage. The upregulated mRNA expression of GSK-3ß in LPS-induced stimulated RAW 264.7 cells also showed the same tendency of depression by LGS. Critically, LGS could induce M1 macrophage polarization to M2 through promoting GSK-3ß inactivation of phosphorylation. Taken together, we initially showed that anti-septic effects of LGS is related to the inhibition on GSK-3ß, both in vitro and in vivo.

Liang-Ge-San inhibits dengue virus serotype 2 infection by reducing caveolin1-induced cytoplasmic heat shock protein 70 translocation into the plasma membrane.

BACKGROUND: Dengue virus (DENV) is a major public health threat. However, there are no specific therapeutic drugs for DENV. Many Chinese heat-cleaning formulas, such as Liang-Ge-San (LGS), have been frequently used in the virus-induced diseases. The antiviral effect of LGS has not been reported yet. PURPOSE: In this study, the effect of LGS on the inhibition of dengue virus serotype 2 (DENV-2) was investigated and the relevant mechanism was explored. METHODS: High-performance liquid chromatography was applied to analyze the chemical characterization of LGS. The in vitro antiviral activities of LGS against DENV-2 were evaluated by time-of-drug-addition assay. The binding of heat shock protein 70 (Hsp70) and envelope (E) protein or caveolin1 (Cav1) were analyzed by immunofluorescence and immunoprecipitation assays. Then the role of Cav1 in the anti-DENV-2 effects of LGS was further examined. DENV-2 infected Institute of Cancer Research suckling mice (n = 10) and AG129 mice (n = 8) were used to examine the protective effects of LGS. RESULTS: It was found that geniposide, liquiritin, forsythenside A, forsythin, baicalin, baicalein, rhein, and emodin maybe the characteristic components of LGS. LGS inhibited the early stage of DENV-2 infection, decreased the expression levels of viral E and non-structural protein 1 (NS1) proteins. LGS also reduced E protein and Hsp70 binding and attenuated the translocation of Hsp70 from cytoplasm to the cell membrane. Moreover, LGS decreased the binding of Hsp70 to Cav1. Further study showed that the overexpression of Cav1 reversed LGS-mediated E protein and Hsp70 inhibition in the plasma membrane. In the in vivo study, LGS was highly effective in prolonging the survival time, reducing viral loads. CONCLUSION: This work demonstrates for the first time that LGS exerts anti-DENV-2 activity in vitro and in vivo. LGS decreases DENV-2-stimulated cytoplasmic Hsp70 translocation into the plasma membrane by Cav1 inhibition, thereby inhibiting the early stage of virus infection. These findings indicate that LGS may be a candidate for the treatment of DENV.

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. AIM OF THIS STUDY: The present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. METHODS: The structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. RESULTS: Two new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. CONCLUSION: Two new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

N-Butanol Extract of Glycyrrhizae Radix et Rhizoma Inhibits Dengue Virus through Targeting Envelope Protein.

BACKGROUND: At present, about half of the world's population is at risk of being infected with dengue virus (DENV). However, there are no specific drugs to prevent or treat DENV infection. Glycyrrhizae Radix et Rhizome, a well-known traditional Chinese medicine, performs multiple pharmacological activities, including exerting antiviral effects. The aim of this study was to investigate the anti-DENV effects of n-butanol extract from Glycyrrhizae Radix et Rhizome (GRE). METHODS: Compounds analysis of GRE was conducted via ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). The antiviral activities of GRE were determined by the CCK-8 assay, plaque assay, qRT-PCR, Western blotting, and the immunofluorescence assay. The DENV-infected suckling mice model was constructed to explore the antiviral effects of GRE in vivo. RESULTS: Four components in GRE were analyzed by UHPLC-MS/MS, including glycyrrhizic acid, glycyrrhetnic acid, liquiritigenin, and isoliquiritigenin. GRE inhibited the attachment process of the virus replication cycle and reduced the expression of the E protein in cell models. In the in vivo study, GRE significantly relieved clinical symptoms and prolong survival duration. GRE also significantly decreased viremia, reduced the viral load in multiple organs, and inhibited the release of pro-inflammatory cytokines in DENV-infected suckling mice. CONCLUSIONS: GRE exhibited significant inhibitory activities in the adsorption stage of the DENV-2 replication cycle by targeting the envelope protein. Thus, GRE might be a promising candidate for the treatment of DENV infection.

Liquiritin exhibits anti-acute lung injury activities through suppressing the JNK/Nur77/c-Jun pathway.

BACKGROUND: Licorice (Glycyrrhiza uralensis Fisch.), a well-known traditional medicine, is traditionally used for the treatment of respiratory disorders, such as cough, sore throat, asthma and bronchitis. We aim to investigate the effects of liquiritin (LQ), the main bioactive compound in licorice against acute lung injury (ALI) and explore the potential mechanism. METHODS: Lipopolysaccharide (LPS) was used to induce inflammation in RAW264.7 cells and zebrafish. Intratracheal instillation of 3 mg/kg of LPS was used for induction an ALI mice model. The concentrations of IL-6 and TNF-α were tested using the enzyme linked immunosorbent assay. Western blot analysis was used to detect the expression of JNK/Nur77/c-Jun related proteins. Protein levels in bronchoalveolar lavage fluid (BALF) was measured by BCA protein assay. The effect of JNK on Nur77 transcriptional activity was determined by luciferase reporter assay, while electrophoretic mobility shift assay was used to examine the c-Jun DNA binding activity. RESULTS: LQ has significant anti-inflammatory effects in zebrafish and RAW264.7 cells. LQ inhibited the expression levels of p-JNK (Thr183/Tyr185), p-Nur77 (Ser351) and p-c-Jun (Ser63), while elevated the Nur77 expression level. Inhibition of JNK by a specific inhibitor or small interfering RNA enhanced the regulatory effect of LQ on Nur77/c-Jun, while JNK agonist abrogated LQ-mediated effects. Moreover, Nur77-luciferase reporter activity was suppressed after JNK overexpression. The effects of LQ on the expression level of c-Jun and the binding activity of c-Jun with DNA were attenuated after Nur77 siRNA treatment. LQ significantly ameliorated LPS-induced ALI with the reduction of lung water content and BALF protein content, the downregulation of TNF-α and IL-6 levels in lung BALF and the suppression of JNK/Nur77/c-Jun signaling, which can be reversed by a specific JNK agonist. CONCLUSION: Our results indicated that LQ exerts significant protective effects against LPS-induced inflammation both in vivo and in vitro via suppressing the activation of JNK, and consequently inhibiting the Nur77/c-Jun signaling pathway. Our study suggests that LQ may be a potential therapeutic candidate for ALI and inflammatory disorders.

Identification of an effective fraction from Ampelopsis Radix with anti-dengue virus activities in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Dengue virus (DENV) infection is a global public health issue without effective therapeutic interventions. Chinese medicine with heat-clearing and detoxifying properties has been frequently used in the treatment of viral infection. Ampelopsis Radix (AR) is a traditional Chinese medicine for clearing heat and detoxification that has been widely used in the prevention and treatment of infectious diseases. However, no studies on the effects of AR against viral infection have been reported, thus far. AIM OF THE STUDY: To explore the anti-DENV activities of the fraction (AR-1) obtained from AR both in vitro and in vivo. MATERIALS AND METHODS: The chemical composition of AR-1 was identified by liquid chromatography-tandem MS (LC‒MS/MS). The antiviral activities of AR-1 were studied in baby hamster kidney fibroblast BHK-21 cells, ICR suckling mice and induction of interferon α/ß (IFN-α/ß) and IFN-γ R-/- (AG129) mice. RESULTS: Based on LC‒MS/MS analysis, 60 compounds (including flavonoids, phenols, anthraquinones, alkaloids and other types) were tentatively characterized from AR-1. AR-1 inhibited the cytopathic effect, the production of progeny virus and the synthesis of viral RNA and proteins by blocking DENV-2 binding to BHK-21 cells. Moreover, AR-1 significantly attenuated weight loss, decreased clinical scores and prolonged the survival of DENV-infected ICR suckling mice. Critically, the viral load in blood, brain and kidney tissues and the pathological changes in brain were remarkably alleviated after AR-1 treatment. Further study on AG129 mice showed that AR-1 obviously improved the clinical manifestations and survival rate, reduced viremia, attenuated gastric distension and relieved the pathological lesions caused by DENV. CONCLUSIONS: In summary, this is the first report that AR-1 exhibits anti-DENV effects both in vitro and in vivo, which suggests that AR-1 may be developed as a therapeutic candidate against DENV infection.

Quassinoids from Eurycoma longifolia with antiviral activities by inhibiting dengue virus replication.

BACKGROUND: Dengue caused by dengue virus (DENV) spreads rapidly around the world. However, there are no worldwide licensed vaccines or specific antivirals to combat DENV infection. Quassinoids are the most characteristic components of Eurycoma longifolia, which have been reported to display a variety of biological activities. However, whether quassinoids exert anti-DENV activities remains unknown. PURPOSE: To test the quassinoids of E. longifolia for their activity against DENV and to clarify the potential mechanisms. METHODS: The quassinoids from E. longifolia were isolated by chromatography techniques, and their chemical structures were elucidated by spectroscopic analysis. The anti-DENV activities of quassinoids on baby hamster kidney cells BHK-21 were determined by lactate dehydrogenase (LDH) assay. The synthesis of progeny virus was measured by plaque assay. The expression levels of envelope protein (E) and non-structural protein 1 (NS1) were evaluated by qRT-PCR, Western blot and immunofluorescence assays. Molecular docking was used to screen the potential targets of the most active quassinoid against DENV-2, and surface plasmon resonance analysis was employed to confirm the direct binding between the most active quassinoid and potential target. RESULTS: Twenty-four quassinoids, including three new quassinoids (1 - 3), were isolated from the ethanol extract of E. longifolia. Quassinoids 4, 5, 9, 11, 12, 15, 16, 17, 19 and 20 significantly reduced the LDH release at the stages of viral binding and entry or intracellular replication. Among them, 19 (6α-hydroxyeurycomalactone, 6α-HEL) exhibited the best anti-DENV-2 activities with an EC50 value of 0.39 ± 0.02 µM. Further experiments suggested that 6α-HEL remarkably inhibited progeny virus synthesis and mRNA and protein expression levels of E and NS1 of DENV-2. Time-of-drug-addition assay suggested that 6α-HEL inhibited intracellular replication of DENV-2 at an early stage. Moreover, 6α-HEL was shown to interact with NS5-RdRp domain at a binding affinity of -8.15 kcal/mol. SPR assay further verified 6α-HEL bound to RdRp protein with an equilibrium dissociation constant of 1.49 × 10-7 M. CONCLUSION: Ten quassinoids from E. longifolia showed anti-DENV activities at processes of virus binding and entry or intracellular replication. The most active quassinoid 6α-HEL exerts the anti-DENV-2 activities at intracellular replication stage by directly targeting the NS5-RdRp protein. These results suggest that 6α-HEL could be a promising candidate for the treatment of DENV-2 infection.

Vimentin Inhibits Dengue Virus Type 2 Invasion of the Blood-Brain Barrier.

Dengue virus (DENV) causes dengue fever, which is prevalent in the tropical and subtropical regions, and in recent years, has resulted in several major epidemics. Vimentin, a cytoskeletal component involved in DENV infection, is significantly reorganized during infection. However, the mechanism underlying the association between DENV infection and vimentin is still poorly understood. We generated vimentin-knockout (Vim-KO) human brain microvascular endothelial cells (HBMECs) and a Vim-KO SV129 suckling mouse model, combining the dynamic vimentin changes observed in vitro and differences in disease course in vivo, to clarify the role of vimentin in DENV-2 infection. We found that the phosphorylation and solubility of vimentin changed dynamically during DENV-2 infection of HBMECs, suggesting the regulation of vimentin by DENV-2 infection. The similar trends observed in the phosphorylation and solubility of vimentin showed that these characteristics are related. Compared with that in control cells, the DENV-2 viral load was significantly increased in Vim-KO HBMECs, and after DENV-2 infection, Vim-KO SV129 mice displayed more severe disease signs than wild-type SV129 mice, as well as higher viral loads in their serum and brain tissue, demonstrating that vimentin can inhibit DENV-2 infection. Moreover, Vim-KO SV129 mice had more disordered cerebral cortical nerve cells, confirming that Vim-KO mice were more susceptible to DENV-2 infection, which causes severe brain damage. The findings of our study help clarify the mechanism by which vimentin inhibits DENV-2 infection and provides guidance for antiviral treatment strategies for DENV infections.

Emodin Protects Against Lipopolysaccharide-Induced Acute Lung Injury via the JNK/Nur77/c-Jun Signaling Pathway.

Background: Acute lung injury (ALI) is a serious inflammatory disease with clinical manifestations of hypoxemia and respiratory failure. Presently, there is no effective treatment of ALI. Although emodin from Rheum palmatum L. exerts anti-ALI properties, the underlying mechanisms have not been fully explored. Purpose: This study aimed to investigate the therapeutic effect and mechanism of emodin on LPS-induced ALI in mice. Methods: RAW264.7 cells and zebrafish larvae were stimulated by LPS to establish inflammatory models. The anti-inflammatory effect of emodin was assessed by ELISA, flow cytometric analysis, and survival analysis. In vitro mechanisms were explored by using Western blotting, luciferase assay, electrophoretic mobility shift assay (EMSA), and small interfering RNA (siRNA) approach. The acute lung injury model in mice was established by the intratracheal administration of LPS, and the underlying mechanisms were assessed by detecting changes in histopathological and inflammatory markers and Western blotting in lung tissues. Results: Emodin inhibited the inflammatory factor production and oxidative stress in RAW264.7 cells, and prolonged the survival of zebrafish larvae after LPS stimulation. Emodin suppressed the expression levels of phosphorylated JNK at Thr183/tyr182 and phosphorylated Nur77 at Ser351 and c-Jun, and increased the expression level of Nur77 in LPS-stimulated RAW264.7 cells, while these regulatory effects of emodin on Nur77/c-Jun were counteracted by JNK activators. The overexpression of JNK dampened the emodin-mediated increase in Nur77 luciferase activity and Nur77 expression. Moreover, the inhibitory effect of emodin on c-Jun can be attenuated by Nur77 siRNA. Furthermore, emodin alleviated LPS-induced ALI in mice through the regulation of the JNK/Nur77/c-Jun pathway. Conclusions: Emodin protects against LPS-induced ALI through regulation on JNK/Nur77/c-Jun signaling. Our results indicate the potential of emodin in the treatment of ALI.

Anti-inflammatory, anti-angiogenetic and antiviral activities of dammarane-type triterpenoid saponins from the roots of Panax notoginseng.

Panax notoginseng has been used both as a traditional medicine and as a functional food for hundreds of years in Asia. However, the active constituents from P. notoginseng and their pharmacologic properties still need to be further explored. In this study, one new dammarane-type triterpenoid saponin (1), along with fourteen known analogs (2-15) were isolated and identified from the roots of P. notoginseng. The anti-inflammatory, anti-angiogenetic and anti-dengue virus effects of these isolated compounds were further evaluated. Compounds 1, 3, 5-7 and 10-12 exerted anti-inflammatory effects in two different zebrafish inflammatory models. Among them, 11, with the most significant activities, alleviated the inflammatory response by blocking the MyD88/NF-κB and STAT3 pathways. Moreover, compound 15 showed anti-angiogenetic activities in Tg(fli1:EGFP) and Tg(flk1:GFP) zebrafish, while 3 and 5 only inhibited angiogenesis in Tg(fli1:EGFP) zebrafish. Additionally, compounds 1, 3, 6, 8, 9 and 12 suppressed the replication of dengue virus either at the viral adsorption and entry stages or at the intracellular replication step. In conclusion, these findings enrich knowledge of the diversity of saponins in P. notoginseng and suggest that the dammarane-type triterpenoid saponins from P. notoginseng may be developed as potential functional foods to treat inflammation, angiogenesis or dengue-related diseases.

Screening of Bufadienolides from Toad Venom Identifies Gammabufotalin as a Potential Anti-inflammatory Agent.

Toad venom (Chansu) is used in the treatment of infectious and inflammatory diseases in China and East/Southeast Asian countries. However, the anti-inflammatory components of toad venom have not yet been systematically evaluated and clearly defined. To investigate the anti-inflammatory effects of toad venom and identify new anti-inflammatory ingredients, we used zebrafish, an alternative drug screening model, to evaluate the anti-inflammatory effects of 14 bufadienolides previously isolated from toad venom. Most of the bufadienolides were found to exert significant anti-inflammatory effects on lipopolysaccharide-, CuSO4-, or tail transection-induced zebrafish inflammatory models. Moreover, gammabufotalin ( 6: ) inhibits lipopolysaccharide-induced inflammation by suppressing the myeloid differentiation primary response 88/nuclear factor-kappa B and STAT3 signal pathways. This study confirms the potential of zebrafish in drug screening, clarifies the anti-inflammatory effects of bufadienolides from toad venom, and indicates that gammabufotalin may be developed as a novel therapeutic agent for inflammatory diseases in the future.

Optimal combination of anti-inflammatory components from Chinese medicinal formula Liang-Ge-San.

ETHNOPHARMACOLOGICAL RELEVANCE: Liang-Ge-San (LGS), a traditional Chinese medicine (TCM) formula, is usually used in acute inflammatory diseases in China. AIM OF THE STUDY: This study aims to detect the optimal combination of anti-inflammatory components from LGS. MATERIALS AND METHODS: Four mainly representative components (phillyrin, emodin, baicalin, and liquiritin) from LGS were chosen. The optimal combination was investigated by orthogonal design study. Zebrafish inflammation model was established by lipopolysaccharide (LPS)-yolk microinjection, and then the anti-inflammatory activities of different combinations were determined by survival analysis, changes on inflammatory cells infiltration, the MyD88/NF-κB and MAPK pathways and inflammatory cytokines production. RESULTS: The different combinations of bioactive ingredients from LGS significantly protected zebrafish from LPS-induced inflammation, as evidenced by decreased recruitment of macrophages and neutrophils, inhibition of the MyD88/NF-κB and MAPK pathways and down-regulation of TNF-α and IL-6. Among them, the combination group 8 most significantly protected against LPS. The combination of group 8 is: 0.1 µM of emodin, 2 µM of baicalin, 20 µM of phillyrin and 12.5 µM of liquiritin. CONCLUSION: The optimized combination group 8 exerts the most significant anti-inflammatory activity by inhibiting the recruitment of inflammatory cells, activation of the MyD88/NF-κB and MAPK pathways and the secretion of pro-inflammatory cytokines. This present study provides pharmacological evidences for the further development of new modern Chinese drug from LGS to treat acute inflammatory diseases, but indicated the use of zebrafish in the screening of components from formulas.

Sheng-Mai Yin exerts anti-inflammatory effects on RAW 264.7 cells and zebrafish.

ETHNOPHARMACOLOGICAL RELEVANCE: Sheng-Mai Yin (SMY), a famous traditional Chinese medicine formula, has been commonly used in China for centuries to treat various diseases, such as inflammation-related diseases. However, the anti-inflammatory activity of SMY and its potential mechanisms still have not yet been clearly understood. AIM OF THE STUDY: In this study, we aimed to determine the anti-inflammatory effect of SMY and explore its underlying mechanisms both on RAW 264.7 cells and zebrafish. MATERIALS AND METHODS: The levels of pro-inflammatory cytokines IL-6 and TNF-α secreted by RAW 264.7 cells were measured by ELISA. The protein expressions of IκBα, p-IκBα (Ser32), STAT3 and p-STAT3 (Tyr705) were determined by Western blotting. And the nuclear translocation of NF-κB p65 in LPS-induced RAW 264.7 macrophage cells was detected by confocal microscopy. Moreover, the in vivo anti-inflammatory effect of SMY and its potential mechanisms were further investigated by survival analysis, hematoxylin-eosin staining (H&E), observation of neutrophil migration and quantitative real-time PCR (qRT-PCR) analysis in zebrafish inflammatory models. RESULTS: SMY reduced the release of IL-6 and TNF-α, inhibited the phosphorylation of IκBα and STAT3 as well as the nuclear translocation of NF-κB p65 in LPS-induced RAW 264.7 cells. Furthermore, the increased survival, decreased infiltration of inflammatory cells and the attenuated migration of neutrophils together suggested the in vivo anti-inflammatory effects of SMY. More importantly, SMY reduced the gene expressions of pro-inflammatory cytokines and suppressed LPS-induced up-regulation of NF-κB, IκBα and STAT3 in zebrafish inflammatory models. CONCLUSION: SMY exerts significant anti-inflammatory effects with a potential mechanism of inhibiting the NF-κB and STAT3 signal pathways. Our findings suggest a scientific rationale of SMY to treat inflammatory diseases in clinic.

1ß-OH-arenobufagin induces mitochondrial apoptosis in hepatocellular carcinoma through the suppression of mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Chansu, dried secretions from Bufonidae, has long been used for cancer treatment as a traditional Chinese medicine. In searching for effective anti-hepatoma agents from Chansu, our preliminary drug screening found that a bufadienolide, namely 1ß-hydroxyl-arenobufagin (1ß-OH-ABF), displays anti-hepatoma activities. However, the anti-hepatoma effects and molecular mechanisms of 1ß-OH-ABF have not been defined. AIM OF THE STUDY: To evaluate the anti-hepatoma activity of 1ß-OH-ABF against liver cancer Hep3B and HepG2 cells in vitro and in vivo, as well as explore the underlying mechanisms. MATERIALS AND METHODS: The anti-proliferative effects of 1ß-OH-ABF on liver cancer Hep3B, HepG2, HuH7, SK-HEP-1 and normal hepatocyte LO2 cells were examined by MTT assay and colony formation assay. Hoechst 33258 staining and Annexin V-FITC/PI staining assay were used to analyze apoptosis induced by 1ß-OH-ABF. The collapse of the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining assay. Western blotting was used to examine the expression levels of targeted proteins. The role of mTOR in 1ß-OH-ABF-induced apoptosis was investigated using small interfering RNA (siRNA) transfection. Zebrafish xenograft model was established to evaluate the anti-hepatoma effects of 1ß-OH-ABF in vivo. RESULTS: We found that 1ß-OH-ABF inhibits the proliferation of Hep3B, HepG2, HuH7, SK-HEP-1 cells but has little cytotoxicity towards LO2 cells. 1ß-OH-ABF induces mitochondria dysfunction and triggers mitochondria apoptotic pathway, which is accompanied by the loss of ΔΨm, upregulation and translocation of Bax, as well as cleavages of caspase-9, caspase-3 and PARP. Mechanistically, 1ß-OH-ABF markedly decreases the expression level of p-AKT/AKT and p-mTOR (Ser2248 and Ser2481)/mTOR in a time-dependent manner. Inhibition of mTOR by siRNA strengthens 1ß-OH-ABF-mediated apoptosis. Critically, 1ß-OH-ABF shows a marked in vivo anti-hepatoma effect on human Hep3B cell xenografts in zebrafish model. CONCLUSION: 1ß-OH-ABF induces mitochondrial apoptosis through the suppression of mTOR signaling in vitro and in vivo, indicating that 1ß-OH-ABF may serve as a potential agent for the treatment of liver cancer.