Strong Binding Activity Research Articles

Antioxidant, antimicrobial, α-amylase and α-glucosidase inhibitory activities of methanolic leaf extract of Breynia vitis-idaea using in vitro and in silico techniques

Breynia vitis-idaea (Phyllanthaceae) is used in the Chinese traditional medicinal system, to treat chronic lung disease and wound healing. This study was aimed to identify the chemical constituents of B. vitis-idaea having biological properties by in-vitro and in-silico methods. Breynia vitis-idaea leaf extract was screened for the presence of phenol, flavonoid contents, antibacterial, antioxidant, and antidiabetic activities. Molecular docking of secondary metabolites with target proteins and ADMET analysis was done. Preliminary phytochemical screening of B. vitis-idaea contains a variety of phytoconstituents such as alkaloids, flavonoids, carbohydrates, tannin, saponin, terpenoids, phenol, protein, steroids and glycosides. The antibacterial activity of methanol leaf extract of B. vitis-idaea evaluated against Escherichia coli (17 mm), Klebsiella pneumoniae (16 mm), Bacillus subtilis (22 mm), and Staphylococcus aureus (18 mm) showed high activity. Antioxidant activities were assessed by ABTS and FRAP assays and their IC50 values were 41.06 µg/mL and 46.40 µL/mL, respectively. α-amylase and α-glucosidase activity showed IC50 values of 20.42 µg/mL and 14.60 µg/mL, respectively. Molecular docking study showed strong binding activity of γ-sitosterol to α-amylase and α-glucosidase, followed by guanosine and arbutin. ADMET analysis shows that γ-sitosterol, guanosine, and arbutin are good drug candidates. This work demonstrates the anti-diabetic potential of B. vitis-idaea.

Abstract B123: Mechanism of action of ivonescimab (AK112/SMT112): a first-in-class tetravalent Fc-silent bispecific antibody with dual blockade of PD-1 and VEGF that promotes cooperative biological effects

Abstract Background: Combination treatments using anti-PD-1/PD-L1 antibodies with other VEGF antagonists have shown enhanced clinical antitumor activities1. The expression of PD-1 and VEGF are found to be frequently upregulated and co-expressed in solid tumors. Importantly, VEGF promotes tumor angiogenesis and suppresses antitumor immune response2,3. Consequently, we characterized the mechanism-of-action of a novel first-in-class anti-PD-1/VEGF bispecific antibody, ivonescimab, designed to simultaneously inhibit PD-1-mediated immunosuppression and block tumor angiogenesis in the tumor microenvironment. Methods: Binding activity of ivonescimab to PD-1/VEGF were assessed by ELISA. Blockade of PD-1/VEGF signaling pathways was determined in luciferase reporter cell assays. Ivonescimab-VEGF complex formation was detected by SEC-HPLC. Cooperative binding of ivonescimab-VEGF complex to PD-1 or ivonescimab-PD-1 complex to VEGF was measured by Octet BLI. The enhanced PD-1 blockade bioactivity of ivonescimab with VEGF was evaluated in hPBMC and engineered cell-line co-culture/luciferase-reporter cell assays. Antitumor activity of ivonescimab was investigated in hPBMC-humanized SCID/Beige mice implanted with HCC827 (mEGFR lung adenocarcinoma) or U87MG (glioblastoma). Immuno-safety was assessed by FcγR binding, ADCC, ADCP assays, and reported clinical irAEs. Results: Ivonescimab displayed strong binding activity to human PD-1 and VEGF alone or simultaneously, effectively blocking interactions with ligands and the downstream signaling effects. In presence of VEGF, ivonescimab forms soluble complexes with VEGF dimers, leading to over 10-fold enhanced binding affinity (KD) of ivonescimab to PD-1. The avidity increase was consistent with reduced cell surface PD-1 expression on human T-cell lines, increased potency on blockade of PD-1/PD-L1 signaling and subsequent enhanced T cell activation in-vitro. Likewise, PD-1 binding enhanced ivonescimab binding to VEGF which was associated with enhanced VEGF-signaling blockade. Also, ivonescimab treatment demonstrated statistically significant dose-dependent antitumor response in hPBMC-humanized murine HCC827 and U87MG tumor models. Additionally, ivonescimab enhanced antitumor response in combination with anti-CD47 (AK117) or anti-CD73 (AK119) in mouse models. Finally, ivonescimab contains Fc-silencing mutations abrogating FcγRI/IIIa binding and showed significantly reduced ADCC, ADCP activities and cytokine release in-vitro. Clinically, this is consistent with the safety profile in Phase 1/2 studies of ivonescimab in advanced solid tumors4,5. Conclusions: Ivonescimab is a novel tetravalent anti-PD-1/VEGF bispecific antibody displaying unique cooperative binding to each of its intended targets consistent with increased in-vitro functional bioactivities compared with bevacizumab or PD-1 inhibitors alone. Importantly, the Fc-null IgG1 design resulted in reduced FcgR interactions and minimal ADCC, ADCP activities consistent with its clinical immunosafety profile. Ivonescimab is currently in Ph3 NSCLC trials in the US and EU. Citation Format: Tingting Zhong, Zhaoliang Huang, Xinghua Pang, Chunshan Jin, Xinrong He, Jose G Montoya, Betty Y Chang, Michelle Xia, Baiyong Li, Jing Min. Mechanism of action of ivonescimab (AK112/SMT112): a first-in-class tetravalent Fc-silent bispecific antibody with dual blockade of PD-1 and VEGF that promotes cooperative biological effects [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B123.

GRP94 in cerebrospinal fluid may contribute to a potential biomarker of depression: Based on proteomics

The present study was designed to investigate potential biomarkers of depression and targets of antidepressants from the perspective of hippocampal endoplasmic reticulum stress (ERS) based on cerebrospinal fluid (CSF) proteomics. Firstly, a six-week depression model was established and treated with fluoxetine (FLX). We found antidepressant-FLX could ameliorate depression-like behaviors and cognition in depressed rats caused by chronic unpredictable mild stress (CUMS). FLX significantly increased neuronal numbers in dentate gyrus (DG) and CA3 regions of hippocampus. CSF proteome data revealed thirty-seven differentially expressed proteins (DEPs) co-regulated by CUMS and FLX, including GRP94 and EIF2α. Results of Gene Oncology (GO) annotation and KEGG pathway enrichment for DEPs mainly included PERK-mediated unfolded protein response, endoplasmic reticulum, and translational initiation. The expression levels of GRP94, p-PERK, p-EIF2α, CHOP and Caspase-12 were increased in hippocampus of CUMS rats, and FLX worked the opposite way. FLX had strong affinity and binding activity with GRP94 protein, and four key proteins on the PERK pathway (PERK, EIF2α, p-EIF2α, CHOP). We proposed that FLX may exert antidepressant effects and neuroprotective action by alleviating excessive activation of the hippocampal PERK pathway and reducing neuronal deficits in depressed rats. PERK, EIF2α, p-EIF2α, and CHOP may be potential targets for antidepressant-FLX. GRP94 in CSF may be a potential biomarker of depression and the therapeutic effects of antidepressants.

Compound musk injection in the treatment of ischemic stroke: A network analysis of the mechanism of action.

Ischemic stroke (IS) is affected by a wide range of factors and has certain treatment limitations. Studies have reported that compound musk injection (CMI) is effective in the treatment of IS, however, its mechanism of action is still unclear. The main active ingredients in CMI were retrieved from HERB, TCMSP and BATMAN databases, and the relevant targets were predicted by Swiss Target Prediction platform. MalaCards, OMIM, DrugBank, DisGeNET, Genecards and TTD databases were used to obtain the genes related to IS. The intersection of drugs and disease targets was used to construct protein-protein interaction networks, and gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed. AutoDock Vina software was used for molecular docking, and cell experiments were conducted to verify the results. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression level of relative mRNA in cells. Network analysis and molecular docking results showed that the key targets of CMI in the treatment of IS were SRC, TP53, PIK3R1, MAPK3, PIK3CA, MAPK1, etc. KEGG pathway enrichment analysis mainly involved PI3K/Akt signaling pathway, Rap1 signaling pathway and MAPK signaling pathway. The molecular docking results all showed that the key ingredients were strong binding activity with the key targets. The quantitative RT-PCR results indicated that CMI may increase the expression of PIK3CA, MAPK3 mRNA and decrease the expression of SRC mRNA. CMI can treat IS by regulating pathways and targets related to inflammatory response and apoptosis in a multi-component manner.

Generation of a Novel SORT1×HER2 Bispecific Antibody-Drug Conjugate Targeting HER2-Low-Expression Tumor.

Human epidermal growth factor receptor 2 (HER2) is considered an ideal antibody-drug conjugate (ADC) target because the gene is overexpressed in many tumors compared to normal tissues. Multiple anti-HER2 ADCs conjugated with different toxic payloads bring benefits to patients with high HER2 expression. However, HER2-targeted ADC technology needs further optimization to improve its effect for the treatment of patients with low HER2 expression. We hypothesized that bispecific antibody-drug conjugate (bsADC) targeting HER2 and Sortilin-1 (SORT1) would overcome this limitation. SORT1 is a suitable target for pairing with HER2 to generate a bispecific antibody (BsAb) since the gene is co-expressed with HER2 in tumors and possesses rapid internalization. We developed a BsAb (bsSORT1×HER2) that exhibited strong binding and internalization activity on HER2-low-expression tumor cells and facilitated higher HER2 degradation. The bsSORT1×HER2 was further conjugated with DXd to generate a bsADC (bsSORT1×HER2-DXd) that showed strong cytotoxicity on HER2-low-expression tumor cells and antitumor efficacy in an MDA-MB-231 xenograft mice model. These results demonstrated that employment of a SORT1×HER2-targeted bsADC may be promising to improve the antitumor efficacy of HER2-targeted ADC for the treatment of tumors with low HER2 expression.

Identification of the Metabolites of Both Formononetin in Rat Hepatic S9 and Ononin in Rat Urine Samples and Preliminary Network Pharmacology Evaluation of Their Main Metabolites.

Astragalus membranaceus is a traditional Chinese medicine derived from the roots of Astragalus membranaceus (Fisch.) Bge., which has the same medicinal and edible uses in China. It is also widely used in daily food, and its pharmacological effects mainly include antioxidant effects, vascular softening effects, etc. Currently, it is increasingly widely used in the prevention of hypertension, cerebral ischemia, and stroke in China. Formononetin and its glucopyranoside (ononin) are both important components of Astragalus membranaceuss and may play important roles in the treatment of cardiovascular diseases (CVDs). This study conducted metabolic studies using formononectin and its glucopyranoside (ononin), including a combination of the in vitro metabolism of Formonetin using rat liver S9 and the in vivo metabolism of ononin administered orally to rats. Five metabolites (Sm2, 7, 9, 10, and 12) were obtained from the solution incubated with formononetin and rat hepatic S9 fraction using chromatographic methods. The structures of the five metabolites were elucidated as (Sm2)6,7,4'-trihydroxy-isoflavonoid; (Sm7)7,4'-dihydroxy-isoflavonoid; (Sm9)7,8,4'-trihydroxy-isoflavonoid; (Sm10)7,8,-dihydroxy-4'-methoxy-isoflavonoid; and (Sm12)6,7-dihydroxy-4'-methoxy- isoflavonoid on the basis of UV, NMR, and MS data. Totally, 14 metabolites were identified via HPLC-DAD-ESI-IT-TOF-MSn analysis, from which the formononetin was incubated with rat hepatic S9 fraction, and the main metabolic pathways were hydroxylation, demethylation, and glycosylation. Then, 21 metabolites were identified via HPLC-DAD-ESI-IT-TOF-MSn analysis from the urine samples from SD rats to which ononin was orally administered, and the main metabolic pathways were glucuronidation, hydroxylation, demethylation, and sulfonation. The main difference between the in vitro metabolism of formononetin and the in vivo metabolism of ononin is that ononin undergoes deglycemic transformation into Formonetin in the rat intestine, while Formonetin is absorbed into the bloodstream for metabolism, and the metabolic products also produce combined metabolites during in vivo metabolism. The six metabolites obtained from the aforementioned separation indicate the primary forms of formononetin metabolism, and due to their higher contents of similar isoflavone metabolites, they are considered the main active compounds that are responsible for pharmacological effects. To investigate the metabolites of the active ingredients of formononetin in the rat liver S9 system, network pharmacology was used to evaluate the cardiovascular disease (CVD) activities of the six primary metabolites that were structurally identified. Additionally, the macromolecular docking results of six main components and two core targets (HSP90AA1 and SRC) related to CVD showed that formononetin and its main metabolites, Sm10 and Sm12, may have roles in CVD treatment due to their strong binding activities with the HSP90AA1 receptor, while the Sm7 metabolite may have a role in CVD treatment due to its strong binding activity with the SRC receptor.

Identification of biomarkers of acne based on transcriptome analysis and combined with network pharmacology to explore the therapeutic mechanism of Jinhuang ointment.

The incidence of acne is on the rise due to unhealthy diet and living habits. Jinhuang ointment (JHO) is a classic prescription composed of 10 kinds of commonly used Chinese herbal medicine, which has been widely used in clinical prevention and treatment of skin inflammatory diseases since ancient times. However, the pharmacological mechanism and target of JHO are not clear. The acne microarray dataset was downloaded from gene expression omnibus database to identify differentially expressed genes (DEG). Immune infiltration was analyzed by CiberSort algorithm. HUB gene was identified by protein-protein interaction network. The gene expression omnibus dataset validates the biomarkers of acne with high diagnostic value. The potential active components and targets of JHO were obtained through Traditional Chinese Medicine Systems Pharmacology database, and the therapeutic targets were obtained by crossing with disease targets. R-packet is used for enrichment analysis. Molecular docking using Auto Dock Tools. A total of 202 DEGs were identified from 12 skin samples in the GSE6475. Immune infiltration analysis showed that there were a large number of macrophages and mast cells in acne skin. Gene set enrichment analysis analysis showed that DEGS was mainly involved in bacterial reaction, inflammatory reaction and so on. Six central genes and gene cluster modules were identified by Cytoscape software. A total of 185 JHO active components and 220 targets were obtained, of which 10 targets were potential targets for JHO in the treatment of acne. Kyoto encyclopedia of genes and genomes enrichment analysis showed that JHO treatment of acne was mainly related to Toll-like receptors, IL-17 and other signal pathways. The results of molecular docking showed that 5 active compounds in JHO had strong binding activity to the core protein receptor. IL-1 β, CXCL8, toll-like receptor 2, CXCL2, LCN2, and secretory phosphoprotein 1 may be potential biomarkers for early diagnosis of acne. JHO active components may regulate skin cell metabolism and inflammatory response and improve cellular immune microenvironment by acting on core targets (CXCL8, ESR1, IL-1 β, MMP1, MMP3, secretory phosphoprotein 1), thus achieving the purpose of treating acne. This is the result of the joint action of multiple targets and multiple pathways. It provides an idea for the development of a new combination of drugs for the treatment of acne.

Network pharmacology-based analysis of Jin-Si-Wei on the treatment of Alzheimer's disease

Ethnopharmacological relevanceJin-Si-Wei (JSW), a traditional Chinese medicine (TCM) formula, have cognitive enhancing effect and delay the memory decline in an animal model of AD， which has been reported. However, the therapeutic mechanism of JSW in the treatment of AD remains unclear. Aim of the studyThis study aimed to verify the pharmacodynamics of JSW in the treatment of AD, and to explore its potential mechanism based on network pharmacology, molecular docking and experimental validation both in vitro and in vivo. Materials and methodsIn this study, the underlying mechanism of JSW against AD was investigated by the integration of network pharmacology. Then, the core pathways and biological process of JSW were verified by experiment, including behavioral test and pathological and biochemical assays with 6-month-old APPswe/PS1ΔE9 transgenic (APP/PS1) mice in vivo and verified with Aβ1-42-stimulated SH-SY5Y cells in vitro. At last, molecular docking was used to show the binding activity of each active ingredient to the core genes of JSW treatment in AD. ResultsA Drug-Ingredient-Target network was established, which included 363 ingredients and 116 targets related to the JSW treatment of AD. The main metabolic pathway of JSW treatment for AD is neuroactive ligand-receptor interaction pathway, and biological processes are mainly involved in Aβ metabolic process. In vivo experiments, compared with APP/PS1 mice, the cognitive and memory ability of mice was significantly improved after JSW administration. In brain tissue of APP/PS1 mice, JSW could increase the contents of low-density lipoprotein receptor-related protein 1 (LRP-1), enkephalinase (NEP) and Acetyl choline (ACh), and decrease the contents of Aβ1-42, amyloid precursor protein (APP) and receptor for advanced glycation end products (RAGE), decrease the vitality of cholinesterase (AChE) and choline acetyltransferase (ChAT). Besides, JSW could increase α-secretase expression and decrease β/γ-secretase expression, and improve the number and morphology of synapses in CA1 region of the hippocampus of APP/PS1 mice. In vitro experiments, Drug-Containing Serum (JSW-serum) has a neuroprotective effect by reducing the apoptosis on Aβ1-42-stimulated SH-SY5Y cells. Molecular docking results showed that 2-Isopropyl-8-methylphenanthrene-3,4-dione had strong binding activity with PTGS2, which maybe a potential ingredient for the treatment of AD. ConclusionsJSW improves AD in APP/PS1 mice, and this therapeutic effect may be achieved in part by altering the neuroactive ligand-receptor interaction pathway.

Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

Exploring the pharmacological mechanisms of Biyan Qingdu Granula in the treatment after nasopharyngeal carcinoma radiotherapy based on UPLC/Q-TOF MS, network pharmacology and molecular docking

BackgroundBiyan Qingdu Granula (BYQD) is a traditional Chinese medicine (TCM) formula commonly used for post-radiotherapy treatment of nasopharyngeal carcinoma (NPC). Despite its extensive use, the underlying pharmacological mechanisms have yet to be fully elucidated. MethodsUPLC/Q-TOF MS was used to comprehensively analyze the chemical composition of BYQD. Additionally, an everted gut sac model, coupled with UPLC/Q-TOF MS, was used to screen and identify the active ingredients. Subsequently, we conducted a network pharmacological analysis to delve into the potential mechanisms of these active ingredients. Molecular docking experiments were also performed to assess the interactions between active ingredients and potential core targets. ResultsThe findings revealed the identification of 62 identical ingredients upon comparing the sample solution and intestinal absorbed solution of BYQD. We constructed a protein-protein interaction (PPI) network, which led to the identification of five core targets, namely, TP53, STAT3, MAPK1, SRC and AKT1. Through the construction of a drug-active ingredient-intersection target network, we identified Quercetin, Luteolin, Eupatilin, Magnoflorine, Acacetin and other compound as potential active ingredients. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested that pathways in cancer, PI3K-Akt signaling pathway, lipid and atherosclerosis, proteoglycans in cancer, and the MAPK signaling pathway might play the key roles in the treatment of NPC after radiotherapy using BYQD. Molecular docking results corroborated strong binding activity between the putative core targets and the corresponding key active ingredients. ConclusionThis study provides a preliminary revelation of the active ingredients and potential pharmacological mechanisms of BYQD in the post-radiotherapy treatment of NPC. These findings establish a vital theoretical basis and serve as a scientific reference for the future investigating the pharmacological mechanisms and clinical application of BYQD.

Integration of metabolomics and network pharmacology to reveal the protective mechanism underlying Qibai Pingfei capsule on chronic obstructive pulmonary disease.

In this study, we have employed metabolomics technology in combination with network pharmacology to ascertain the key metabolites and hub genes. The objective was to explore the pathway of Qibai Pingfei Capsule (QBPF) in treating COPD through metabolomics. We identified 96 differential metabolites in the lung tissues of rats belonging to control and model groups, out of which 47 were observed to be critical (VIP >2, p < 0.05). Furthermore, 16 important differential metabolites were reversed after QBPF treatment. Using network pharmacology, we identified 176 core targets of 81 drug-active ingredients. Our comprehensive analysis of network pharmacology and metabolomics enabled us to identify a core target, prostaglandin-endoperoxide synthase 2 (PTGS2), and a core metabolic pathway for glutathione metabolism. Finally, the result of molecular docking showed that PTGS2 had strong binding activity to 18 compounds including Fumarine and Kaempferol, etc.. PTGS2 is a marker of ferroptosis, so we wanted to explore whether QBPF could inhibit ferroptosis in COPD. The results showed that ferroptosis was involved in the pathogenesis of COPD, and QBPF could inhibit the occurrence of ferroptosis. In conclusion, the mechanism of QBPF for treating COPD may be related to PTGS2 expression, glutathione metabolism and ferroptosis.

Possible molecular exploration of herbal pair Haizao-Kunbu in the treatment of Graves' disease by network pharmacology, molecular docking, and molecular dynamic analysis.

To promote the development and therapeutic application of new medications, it is crucial to conduct a thorough investigation into the mechanism by which the traditional Chinese herb pair of Haizao-Kunbu (HK) treats Graves' disease (GD). Chemical ingredients of HK, putative target genes, and GD-associated genes were retrieved from online public databases. Using Cytoscape 3.9.1, a compound-gene target network was established to explore the association between prosperous ingredients and targets. STRING, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analyses visualized core targets and disease pathways. Additionally, we conducted a refined analysis of the binding interactions between active ingredients and their respective targets. To visualize these findings, we employed precise molecular docking techniques. Furthermore, we carried out molecular dynamics simulations to gain insights into the formation of more tightly bound complexes. We found that there were nine key active ingredients in HK, which mainly acted on 21 targets. These targets primarily regulated several biological processes such as cell population proliferation, protein phosphorylation, and regulation of kinase activity, and acted on PI3K-AKT and MAPK pathways to treat GD. Analysis of the molecular interaction simulation under computer technology revealed that the key targets exhibited strong binding activity to active ingredients, and Fucosterol-AKT1 and Isofucosterol-AKT1 complexes were highly stable in humans. This study demonstrates that HK exerts therapeutic effects on GD in a multi-component, multi-target, and multi-pathway manner by regulating cell proliferation, differentiation, inflammation, and immunomodulatory-related targets. This study provides a theoretical foundation for further investigation into GD.

Unveiling the potential mechanisms of Amomi fructus against gastric ulcers via integrating network pharmacology and in vivo experiments

Ethnopharmacological relevanceAs a well-known traditional Chinese medicine, Amomi fructus (A. fructus) (Sharen) has been used therapeutically to treat gastrointestinal illnesses, including gastric ulcer (GU). The mechanism underlying this impact is still not fully known, though. Aim of the studyTo investigate the hidden mechanism by which A. fructus influences the pathogenesis of GU, we employed network pharmacology approaches and in vivo validated studies. Materials and methodsMultiple public databases were used to compile information on bioactive compounds, potential targets of A. fructus, and associated genes of GU. Then, the STRING database's protein-protein interaction (PPI) data of the drug-disease overlapping gene targets was obtained, and the core targets for A. fructus against GU were discovered. Additionally, molecular docking was done to examine the binding capabilities of the active substances and core targets. Then, the pathways of A. fructus that target GU were examined using the Annotation, Visualization and Integrated Discovery (DAVID)'s Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway studies. In a mouse model of acute stomach mucosal damage brought on by absolute ethanol, the findings of network pharmacology were finally validated. ResultsIn total, 610 targets derived from the 196 bioactive compounds in A. fructus, were discovered, and along with 115 A. fructus target genes for therapy of GU. Then, ten core targets associated with apoptosis and inflammation were determined based on node degree, and ALB, AKT1, TNF, EGFR, MAPK3, CASP3, MMP9, STAT3, SRC, and HRAS were identified as promising therapeutic targets of A. fructus against GU. The results of molecular docking also revealed that 65 active compounds had strong binding activity with the core targets, with volatile chemicals being the most significant active ingredients. So, for following in vivo tests, A. fructus volatile oils (AVO) were used. The KEGG analysis showed that the phosphoinositide-3-kinase/protein kinase B (PI3K/AKT) signaling pathway may be crucial for the therapeutic mechanism of GU. In experiments that were validated in vivo, AVO considerably decreased the ulcer area and enhanced the histological appearance of the gastric tissues. In addition, compared to the model group, up-regulated the expression of IGF-1, p-PI3K, and p-AKT and down-regulated the protein levels of TNF-α and Caspase 3 in the stomach tissues. ConclusionAccording to preliminary findings from this work, A. fructus may influence inflammatory response and apoptosis via regulating the PI3K/AKT signaling pathway and associated gene targets. Importantly, our research might offer a theoretical foundation for future research into the intricate anti-GU mechanism of A. fructus.

Luteolin alleviates depression-like behavior by modulating glycerophospholipid metabolism in the hippocampus and prefrontal cortex of LOD rats.

Late-onset depression (LOD) is defined as primary depression that first manifests after the age of 65. Luteolin (LUT) is a natural flavonoid that has shown promising antidepressant effects and improvement in neurological function in previous studies. In this study, we utilized UPLC-MS/MS non-targeted metabolomics techniques, along with molecular docking technology and experimental validation, to explore the mechanism of LUT in treating LOD from a metabolomics perspective. The behavioral results of our study demonstrate that LUT significantly ameliorated anxiety and depression-like behaviors while enhancing cognitive function in LOD rats. Metabolomic analysis revealed that the effects of LUT on LOD rats were primarily mediated through the glycerophospholipid metabolic pathway in the hippocampus and prefrontal cortex. The levels of key lipid metabolites, phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE), in the glycerophospholipid metabolic pathway were significantly altered by LUT treatment, with PC and PE showing significant correlations with behavioral indices. Molecular docking analysis indicated that LUT had strong binding activity with phosphatidylserine synthase 1 (PTDSS1), phosphatidylserine synthase 2 (PTDSS2), and phosphatidylserine decarboxylase (PISD), which are involved in the transformation and synthesis of PC, PE, and PS. Lastly, our study explored the reasons for the opposing trends of PC, PE, and PS in the hippocampus and prefrontal cortex from the perspective of autophagy, which may be attributable to the bidirectional regulation of autophagy in distinct brain regions. Our results revealed significant alterations in the glycerophospholipid metabolism pathways in both the hippocampus and prefrontal cortex of LOD rats. Moreover, LUT appears to regulate autophagy disorders by specifically modulating glycerophospholipid metabolism in different brain regions of LOD rats, consequently alleviating depression-like behavior in these animals.

Identifying potential therapeutic targets of mulberry leaf extract for the treatment of type 2 diabetes: a TMT-based quantitative proteomic analysis

BackgroundMulberry (Morus alba L.) leaf, as a medicinal and food homologous traditional Chinese medicine, has a clear therapeutic effect on type 2 diabetes mellitus (T2DM), yet its underlying mechanisms have not been totally clarified. The study aimed to explore the mechanism of mulberry leaf in the treatment of T2DM through tandem mass tag (TMT)—based quantitative proteomics analysis of skeletal muscle.MethodsThe anti-diabetic activity of mulberry leaf extract (MLE) was evaluated by using streptozotocin-induced diabetic rats at a dose of 4.0 g crude drug /kg p.o. daily for 8 weeks. Fasting blood glucose, body weight, food and water intake were monitored at specific intervals, and oral glucose tolerance test and insulin tolerance test were conducted at the 7th and 8th week respectively. At the end of the experiment, levels of glycated hemoglobin A1c, insulin, free fat acid, leptin, adiponectin, total cholesterol, triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were assessed and the pathological changes of rat skeletal muscle were observed by HE staining. TMT-based quantitative proteomic analysis of skeletal muscle and bioinformatics analysis were performed and differentially expressed proteins (DEPs) were validated by western blot. The interactions between the components of MLE and DEPs were further assessed using molecular docking.ResultsAfter 8 weeks of MLE intervention, the clinical indications of T2DM such as body weight, food and water intake of rats were improved to a certain extent, while insulin sensitivity was increased and glycemic control was improved. Serum lipid profiles were significantly reduced, and the skeletal muscle fiber gap and atrophy were alleviated. Proteomic analysis of skeletal muscle showed that MLE treatment reversed 19 DEPs in T2DM rats, regulated cholesterol metabolism, fat digestion and absorption, vitamin digestion and absorption and ferroptosis signaling pathways. Key differential proteins Apolipoprotein A-1 (ApoA1) and ApoA4 were successfully validated by western blot and exhibited strong binding activity to the MLE’s ingredients.ConclusionsThis study first provided skeletal muscle proteomic changes in T2DM rats before and after MLE treatment, which may help us understand the molecular mechanisms, and provide a foundation for developing potential therapeutic targets of anti-T2DM of MLE.Graphical

The potential pharmacological mechanism of prunetin against osteoporosis: transcriptome analysis, molecular docking, and experimental approaches

Background Prunetin is an O-methylated isoflavone, known for its beneficial properties. However, its specific pharmacological effects in the treatment of osteoporosis (OP) remain poorly understood. This study aims to elucidate the mechanisms underlying the antiosteoporotic effects of prunetin through a combination of bioinformatics analysis and cell experiments. Methods We gathered predicted targets of prunetin from various online platforms. Differential expression analysis of mRNAs in patients with OP was conducted using the Limma package, based on the GSE35959 dataset. A PPI network diagram was visualized and analyzed using Cytoscape 3.7.2 software. Molecular docking was employed to assess the binding affinity between ligands and receptors, and selected key genes were further validated through cell experiments. Results A total of 4062 differentially expressed genes (DEGs) were identified from the GSE35959 dataset. Among these, 58 genes were found to overlap with the targets of prunetin, indicating their potential as therapeutic targets. The enrichment analysis indicated these targets were mainly enriched in MAPK, FoxO, and mTOR signaling pathways. The molecular docking analysis demonstrated that prunetin exhibited strong binding activity with the core targets. Furthermore, cell experiments revealed that prunetin effectively reversed the expression levels of ALB, ESR1, PTGS2, and FGFR1 mRNA in MC3T3-E1 cells treated with dexamethasone (DEX). Conclusion Our research revealed the multi-pathway and multi-target features of prunetin in treating OP, shedding light on the potential mechanisms underlying the effectiveness of prunetin against OP. These findings serve as a theoretical foundation for future drug development in this field.

Venom Peptides of Crotalus atrox Against SARS-Cov-2 Spike Protein and Human ACE2 Receptor by Molecular Docking Analysis

Venoms are composed of about 100 to 500 pharmacologically active compounds. Less than 0.01% of these compounds have been identified and a significant majority of them act on unknown receptors. Here, the potential Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) activities of selected Crotalus atrox venom peptides (CVPs) including Atrolysin D (AD), vascular apoptosis-inducing protein-1 (VAIP-1), Catrocollastatin (CC), and Calcium-Free Phospholipase A2 (CFP) were investigated via molecular docking analysis. CVPs were docked against human angiotensin-converting enzyme-2 (ACE-2) and 3-chymotrypsin-like protease (3CLpro) viral spike protein. All CVPs had low binding energies to both 3CLpro and ACE2, suggesting that they interacted strongly with the active sites of enzymes, compared to the reference drugs lopinavir and ritonavir. The binding energy of 3CLpro was -139.517 kcal/mol, -96.239 kcal/mol, -121.590 kcal/mol, -259.424 kcal/mol with AD, VAIP-1, CC, and CFP, respectively. CFP showed a very strong binding activity with 3CLpro, suggesting that it could be a very effective compound in inhibiting the SARS-CoV-2 virus. The binding energy of ACE2 was -101.165 kcal/mol, -73.064 kcal/mol, -106.918 kcal/mol, -82.830 kcal/mol with AD, VAIP-1, CC, and CFP, respectively. AD made a much stronger bond with ACE2 than reference drugs, showing that it could be used as a virus-protective component in humans. The results suggest a potential drug candidate for the development of therapeutics against Coronavirus disease 2019 (COVID-19). In vitro and in vivo experiments are needed to confirm these compounds' potential preventive and therapeutic effects.

Three neutralizing mAbs induced by MPXV A29L protein recognizing different epitopes act synergistically against orthopoxvirus

ABSTRACT The worldwide outbreak of the monkeypox virus (MPXV) has become a “Public Health Emergency of International Concern” (PHEIC). Severe monkeypox virus infection can be fatal, however, effective therapeutic methods are yet to be developed. Mice were immunized with A35R protein and A29L protein of MPXV, and the binding and neutralizing activities of the immune sera against poxvirus-associated antigens and viruses were identified. A29L protein and A35R protein-specific monoclonal antibodies (mAbs) were generated and their antiviral activities of these mAbs were characterized in vitro and in vivo. Immunization with the MPXV A29L protein and A35R protein induced neutralizing antibodies against the orthopoxvirus in mice. None of the mAbs screened in this study against A35R could effectively neutralize the vaccinia virus (VACV), while three mAbs against A29L protein, 9F8, 3A1 and 2D1 were confirmed to have strong broad binding and neutralizing activities against orthopoxvirus, among which 9F8 showed the best neutralizing activity. 9F8, 3A1, and 2D1 recognized different epitopes on MPXV A29L protein, showing synergistic antiviral activity in vitro against the VACV Tian Tan and WR strains; the best activity was observed when the three antibodies were combined. In the vivo antiviral prophylactic and therapeutic experiments, 9F8 showed complete protective activity, whereas 3A1 and 2D1 showed partial protective activity. Similarly, the three antibodies showed synergistic antiviral protective activity against the two VACVs. In conclusion, three mAbs recognized different epitopes on MPXV A29L protein were developed and showed synergistic effects against orthopoxvirus.

Network pharmacology and bioinformatics analysis identifies potential therapeutic targets of Naringenin against COVID-19/LUSC.

Coronavirus disease 2019 (COVID-19) is a highly contagious respiratory disease that has posed a serious threat to people's daily lives and caused an unprecedented challenge to public health and people's health worldwide. Lung squamous cell carcinoma (LUSC) is a common type of lung malignancy with a highly aggressive nature and poor prognosis. Patients with LUSC could be at risk for COVID-19, We conducted this study to examine the potential for naringenin to develop into an ideal medicine and investigate the underlying action mechanisms of naringenin in COVID-19 and LUSC due to the anti-viral, anti-tumor, and anti-inflammatory activities of naringenin. LUSC related genes were obtained from TCGA, PharmGKB, TTD,GeneCards and NCBI, and then the transcriptome data for COVID-19 was downloaded from GEO, DisGeNET, CTD, DrugBank, PubChem, TTD, NCBI Gene, OMIM. The drug targets of Naringenin were revealed through CTD, BATMAN, TCMIP, SymMap, Chemical Association Networks, SwissTargetPrediction, PharmMapper, ECTM, and DGIdb. The genes related to susceptibility to COVID-19 in LUSC patients were obtained through differential analysis. The interaction of COVID-19/LUSC related genes was evaluated and demonstrated using STRING to develop a a COX risk regression model to screen and evaluate the association of genes with clinical characteristics. To investigate the related functional and pathway analysis of the common targets of COVID-19/LUSC and Naringenin, KEGG and GO enrichment analysis were employed to perform the functional analysis of the target genes. Finally, The Hub Gene was screened and visualized using Cytoscape, and molecular docking between the drug and the target was performed using Autodock. We discovered numerous COVID-19/LUSC target genes and examined their prognostic value in LUSC patients utilizing a variety of bioinformatics and network pharmacology methods. Furthermore, a risk score model with strong predictive performance was developed based on these target genes to assess the prognosis of LUSC patients with COVID-19. We intersected the therapeutic target genes of naringenin with the LUSC, COVID-19-related targets, and identified 354 common targets, which could be used as potential target genes for naringenin to treat COVID-19/LUSC. The treatment of COVID-19/LUSC with naringenin may involve oxidative stress, anti-inflammatory, antiviral, antiviral, apoptosis, immunological, and multiple pathways containing PI3K-Akt, HIF-1, and VEGF, according to the results of the GO and KEGG enrichment analysis of these 354 common targets. By constructing a PPI network, we ascertained AKT1, TP53, SRC, MAPK1, MAPK3, and HSP90AA1 as possible hub targets of naringenin for the treatment of COVID-19/LUSC. Last but not least, molecular docking investigations showed that naringenin has strong binding activity in COVID-19/LUSC. We revealed for the first time the pharmacological targets and potential molecular processes of naringenin for the treatment of COVID-19/LUSC. However, these results need to be confirmed by additional research and validation in real LUSC patients with COVID-19.

Hyperoside prevents doxorubicin-induced cardiotoxicity by inhibiting NOXs/ROS/NLRP3 inflammasome signaling pathway.

Clinical application of doxorubicin (Dox) in cancer chemotherapy is limited by its cardiotoxicity. Present study aimed to demonstrate the effect and mechanism of hyperoside in Dox-induced cardiotoxicity. C57BL/6 mice were injected with 12 mg/kg of Dox, and 1 μM Dox was exposed to primary cardiomyocytes. Cardiac function was evaluated by echocardiographic and myocardial enzyme levels. Cardiomyocyts apoptosis was analyzed by TUNEL staining and flow cytometry. Network pharmacology and molecular docking were utilized to explore potential targets of hyperoside. Protein expressions were detected by western blot and enzyme activities were determined by colorimetry. Cardiac dysfunction and cardiomyocyte apoptosis induced by Dox were attenuated by hyperoside. Mechanism of hyperoside was mainly related to "oxidative stress" pathway. Hyperoside exhibited strong binding activities with nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs, the main source of ROS in cardiomyocytes) and cyclooxygenases (COXs). Experiments proved that hyperoside suppressed the ROS generation and the elevated activities of NOXs and COXs induced by Dox. Dox also triggered the activation of NLRP3 inflammasome, which was reversed by hyperoside. Hyperoside bound to NOXs and COXs, which prevents Dox-induced cardiotoxicity by inhibiting NOXs/ROS/NLRP3 inflammasome signaling pathway. Hyperoside holds promise as a therapeutic strategy for Dox-induced cardiotoxicity.