Objective: To analyze the key targets and potential mechanisms underlying the volatile components of Scutellaria baicalensis Georgi acting on gliomas through network pharmacology combined with biological experiments.Methods: We have extracted the volatile components of Scutellaria baicalensis by gas chromatography-mass spectrometry (GC-MS) and determined the active components related to the onset and development of gliomas by combining the results with the data from the Traditional Chinese Medicine Systems Pharmacology database. We screened the same targets for the extracted active components and gliomas through network pharmacology and then constructed a protein-protein interaction network. Using a Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, we analyzed the protein effects and regulatory pathways of the common targets. Lastly, we employed ELISA and Western blot in verifying the key targets in the regulatory pathway.Results: We ultimately determined that the active component in S. baicalensis Georgi related to the onset and development of gliomas was Wogonin. The results of the network pharmacology revealed 85 targets for glioma and Wogonin. We used gene ontology to analyze these target genes and found that they involved 30 functions, such as phosphatidylinositol phosphokinase activation, while the KEGG analysis showed that there were 10 regulatory pathways involved. Through the following analysis, we found that most of the key target genes are distributed in the PI3K-Akt and interleukin 17 signaling pathways. We then cultured U251 glioma cells for the experiments. Compared with the control group, no significant change was noted in the caspase-3 expression; however, cleaved caspase-3 expression increased significantly and was dose-dependent on Wogonin. The expression of Bad and Bcl-2 with 25 μM of Wogonin has remained unchanged, but when the Wogonin dose was increased to 100 μM, the expression of Bad and Bcl-2 was noted to change significantly  and was dose-dependent on Wogonin. The ELISA results showed that, compared with the control group, the secretion of tumor necrosis factor alpha, IL-1β, and IL-6 decreased as the Wogonin concentration increased. Tumor necrosis factor alpha downregulation had no significant dose-dependent effect on Wogonin, the inhibitory effect of 25 μM of Wogonin on IL-6 was not significant, and IL-1β downregulation had a significant dose-dependent effect on Wogonin.Conclusion: Wogonin might promote the apoptosis of glioma cells by upregulating proapoptotic factors, downregulating antiapoptotic factors, and inhibiting the inflammatory response, thereby inhibiting glioma progression. Glioblastoma multiforme, also called glioma, is a relatively common malignant brain tumor in adults, which originates from glial cells, is a highly invasive and fatal tumor of the central nervous system , and it S. baicalensis Georgi (Chinese skullcap) plant, has been determined to have numerous pharmacological activities in TCM, including anti-inflammatory effects, which are particularly important, in addition, This TCM has an antiapoptotic effect and is currently employed in treating gliomas, although more research is needed. One of the active ingredients of S. baicalensis Georgi that plays a role in TCM is the flavonoid baicalin, which has a significant effect in the treatment of gliomas  in order to assist in the treatment of glioma has raised interest. TCM can improve the clinical symptoms of various types of glioma, reduce the adverse reactions after chemotherapy, enhance immunity, and prolong survival times . Current gliomas ; howeverS. baicalensis on gliomas, we extracted the volatile components of S. baicalensis by gas chromatography-mass spectrometry (GC-MS) and then searched and matched the active components related to the onset and development of glioma from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Employing the network pharmacology method, we screened the possible pathways of the involvement of S. baicalensis Georgi in the onset and development of glioma and further verified and analyzed the key signal nodes through biological experiments to reveal the potential mechanisms of the volatile components of this plant in the treatment and prevention of gliomas.Therefore, to examine the effects of the volatile components of Scutellaria and divided the result into three equal parts (10 g each), which we later immersed in hot water for 4 h with the ethanol(81.5°C), methanol(64.7°C), and benzenol (71C), respectively. After passing the resulting liquid through a 0.22-µm ultrafiltration membrane, the filtrate was concentrated to 5 ml using a rotary evaporator and filtered through a 0.22-µm filter membrane into a sample injection bottle, which we then analyzed using GC-MS as detailed below.We crushed and sipped 30 g of We heated an RTX-5MS (30 m × 0.25 mm × 0.25 µm) elastic quartz capillary column using a heating program. The column temperature was raised to 210°C at a rate of 10°C/min, then to 220°C at 1°C/min, and then to 280 °C at 10°C/min. The temperature was then kept steady for 1 min. The total determination time was 20 min, the shunt ratio was 20:1, the inlet temperature was 280°C, the column front pressure was 50 kPa, the injection volume was 1 μL, the carrier gas was high purity nitrogen (99.999%), and the flow rate was 1.1 ml/min.The settings were as follows: electron ionizationion source; ion source temperature of 200°C; electron energy of 70 eV; connector temperature of 250°C; solvent delay of 3.5 min; scanning range: 50–600 amu; and electron multiplier voltage of 1200 V.For the screening, we generated the corresponding compound ion flow chart, based on the mass spectrum of each chromatographic peak fragment figure, the literature review, and the database data. We also checked the mass spectrum data on the base peak’s mass-to-charge ratio and the relative abundance condition as the intuitive comparison. Finally, we determined the crude drug’s chemical composition and its physical and chemical properties and pharmacological action.S. baicalensis extracts were screened using the TCMSP database under conditions of oral bioavailability of 30%, druggability of 0.18, and blood-brain barrier (BBB) permeability index of −0.3. We downloaded the chemical structure files corresponding to the active components.The active components of the GC-MS S. baicalensis) using the PubChem database. We predicted the target genes using BATMAN-TCM combined with the web-based SwissTargetPrediction tool. Lastly, we selected the target protein name, gene name, UniProt ID, and other information.We then determined the specific chemical information for Wogonin  and Kyoto Encyclopedia of Genes and Genomes (KEGG) in the ClueGo plug-in for the biological process analysis and pathway analysis of the TCM volatile components acting on glioma-related targets. The parameters were set as ClueGo:Function, GO and KEGG, and Use GO Term Fusion (p ≤ 0.05).We entered the retrieved target genes into the Cytoscape software and employed and Gene Ontology As per the established Wogonin and disease-related targets, we screened out the common targets of drugs and diseases and constructed the disease-target regulation network of volatile component action targets and glioma-related diseases of Genuine medicinal materials in Henan using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. We then inserted the acquired volatile components, key targets of compounds, and main pathways for TCM into Cytoscape software, constructing the glioma-related component-target-main disease pathways using Cytoscape’s Merge and Union functions.We cultured human U251 cells and treated them with 0, 25, 100, and 200 μM of Wogonin  for 24 h, WOG concentration was based on reference . we used2. These cells are obtained from the laboratory. We then grouped the cells as follows: control group (0 μM), 25 μM, 100 μM, and 200 μM.We cultured U251 cells with 10% FBS supplemented DMEM in an incubator set to 375% COWe used ELISA kit  to detect the expression of TNF-α, IL-1β and IL-6 according to the instructions of the kit.1) After the cells were lyzed, the protein in it was extracted, and the concentration of the above-mentioned protein was determined using a measurement kit .2) We used polyacrylamide gel to extract the protein of each lysate (20 μg), and then transferred it to a polyvinylidene fluoride membrane .3) The above membrane was washed with 0.5% Tween 20 (TBST) in Tris buffered saline for 5 min, and then blocked in 5% skim milk for 1 h.4) The membranes were incubated with antibodies against caspase3/cleavedcaspase-3, BAD, and Bcl-2  overnight at 4°C.5) Successively thereafter, the membranes were washed three times with TBST for 5 min each, incubated for 1 h at 37°C with rabbit HRP-conjugated antimouse antibody  , and washed three times with TBST for 5 min each.β-actin was used to normalize the band density.6) We used electrochemiluminescence reagent  to detect the protein bands. p < 0.05 indicating statistically significant differences.The statistical analysis was performed using the SPSS v. 23.0 software package, with the measurement data expressed as (x S). We then calculated the differences between groups using a one-way ANOVA, with S. baicalensis by GC-MS. We used the methanol-ethanol-cresol method to extract the volatile active ingredients of S. baicalensis, obtaining information on 117 common targets, with 85 combined targets for glioma and Wogonin  combined with the SwissTargetPrediction tool was able to predict the target genes of the volatile components of  Wogonin . We empl Wogonin .l-amino acid translocation, amino acid translocation, and reverse translocation  and IL-17 signaling pathway (an important inflammatory pathway) . We thenThe secretion of TNF-α, IL-1β, and IL-6 was detected via ELISA. The groups with Wogonin have showed a decreasing trend in TNF-α, IL-1β, and IL-6 secretion with increased Wogonin concentrations compared with the control group . FurtherWe detected the expression of apoptotic regulation genes such as caspase-3/cleaved caspase-3, Bad, and Bcl-2 by Western blotting . The resS. baicalensis Georgi  and analyzed the components with GC-MS. We then searched and matched those components related to the onset and development of glioma using the TCMSP database. Lastly, we identified the volatile active component Wogonin by analyzing the results of the GC-MS.In recent years, extracting effective antitumor components with low toxic and adverse effects from TCM and natural plants has become a new line of research. TCM currently employs mostly boiling water processing in extracting the components of hydrothermally soluble substances in TCM materials. The pharmacological effects of non-hydrothermally soluble substances contained in TCM have gradually gained the attention of researchers . Other vAlthough TCM has been gaining attention worldwide, understanding the scientific basis of TCM from the molecular and systemic level is still a major challenge for evidence-based medicine . Unlike S. baicalensis Georgi in gliomas by means of network pharmacology. The results revealed 85 targets of Wogonin acting with glioma. The results of the GO annotation and KEGG pathway analysis showed that these target genes were involved in the binding of phosphatidylinositol phosphokinase (to activate chemokines) and l-amino acid translocation (to activate amino acid translocation and thus activate and reverse the transport. The regulatory pathway involves the PI3K-Akt signaling pathway, the Rap1 signaling pathway, and the IL-17 signaling pathway. These results suggest that Wogonin could affect gliomas through the above regulatory pathways. Further research found that important target genes are concentrated in PI3K-Akt and IL-17 signaling pathways. We selected apoptotic factors such as caspase-3, Bad, and Bcl-2 and inflammatory factors such as IL-1β, TNF-α, and IL-6 for further experimental verification.This study has analyzed the molecular mechanism of Glioma is a common and destructive brain tumor that usually occurs in the glial cells of the brain or spine. Immunological studies have established the importance of the tumor microenvironment as a driver of tumor development. Inflammatory mediators such as IL-1β released by monocytes regulate the transcription network needed for malignant cell growth . The uncS. baicalensis Georgi, is a flavonoid with a relative molecular weight of 284.26 and a molecular formula of C16H12O5. In vivo and in vitro analysis found that Wogonin can reduce the growth and metastasis of leukemia, liver cancer, colon cancer, breast cancer, and other malignant tumors (S. baicalensis and found that Wogonin can inhibit the proliferation of glioma cells and induce G0/G1 stagnation in a dose-dependent manner. The expression of G1-phase cyclin D1 and cyclin-dependent kinases 2 and 4 was significantly decreased, and the cyclin-inhibiting protein P27 was overexpressed. The mechanism of promoting glioma differentiation might be the inhibition of glycogen synthase kinase-3/-light-chain protein pathway (Wogonin, a monomer component extracted from t tumors . Wogonint tumors . (Wang et tumors  extracte pathway . suggest pathway . Bcl-2 i pathway . The dow pathway . In thisIn conclusion, we speculate that Wogonin might promote glioma cell apoptosis by upregulating Bad gene expression and cleaved caspase-3 gene activation and by downregulating Bcl-2 expression. Wogonin also inhibits the progression of gliomas by downregulating the secretion of the inflammatory cytokines TNF-α, IL-6, and IL-1β. To provide a theoretical basis for clinical treatment, further in-depth experimental studies are needed to explore the specific molecular mechanisms of Wogonin’s action on gliomas.


There is evidence that schizophrenia is characterized by activation of the immune-inflammatory response (IRS) and compensatory immune-regulatory systems (CIRS) and lowered neuroprotection. Studies performed on antipsychotic-naïve first episode psychosis (AN-FEP) and schizophrenia (FES) patients are important as they may disclose the pathogenesis of FES. However, the protein–protein interaction (PPI) network of FEP/FES is not established. The aim of the current study was to delineate a) the characteristics of the PPI network of AN-FEP and its transition to FES; and b) the biological functions, pathways, and molecular patterns, which are over-represented in FEP/FES. Toward this end, we used PPI network, enrichment, and annotation analyses. FEP and FEP/FES are strongly associated with a response to a bacterium, alterations in Toll-Like Receptor-4 and nuclear factor-κB signaling, and the Janus kinases/signal transducer and activator of the transcription proteins pathway. Specific molecular complexes of the peripheral immune response are associated with microglial activation, neuroinflammation, and gliogenesis. FEP/FES is accompanied by lowered protection against inflammation, in part attributable to dysfunctional miRNA maturation, deficits in neurotrophin and Wnt/catenin signaling, and adherens junction organization. Multiple interactions between reduced brain derived neurotrophic factor, E-cadherin, and β-catenin and disrupted schizophrenia-1 (DISC1) expression increase the vulnerability to the neurotoxic effects of immune molecules, including cytokines and complement factors. In summary: FEP and FES are systemic neuro-immune disorders that are probably triggered by a bacterial stimulus which induces neuro-immune toxicity cascades that are overexpressed in people with reduced anti-inflammatory and miRNA protections, cell–cell junction organization, and neurotrophin and Wnt/catenin signaling. In 1995, Smith and Maes  showed iThere is now evidence that schizophrenia phenotypes are accompanied by activation of the immune-inflammatory response system (IRS), including first episode psychosis (FEP), first episode schizophrenia (FES), multiple episode schizophrenia (MES), MES with worsening, the acute phase of schizophrenia, chronic schizophrenia, treatment resistant schizophrenia, deficit schizophrenia, and schizophrenia with comorbid mood symptoms and chronic fatigue-like symptoms ,7. In thStudies on antipsychotic-naïve (AN)-FEP and FES patients are extremely important as the results may disclose the pathogenesis of the disease, and because the results are not affected by the effects of multiple episodes . MoreoveFES, which is the consequence of FEP, is characterized by (a) activated IRS/CIRS with increased levels of CCL11 , TRYCATs, oxidative stress indicants, and IL-10; (b) increased IgA C1q circulating immune complexes (CICs); (c) disorders in the expression of β-catenin, E-cadherin, plasmalemma vesicle associated protein (PLVAP), indicating dysfunctions in cell–cell, adherens, and vascular junctions; (d) increased IgA/IgM responses to Gram-negative bacteria, including Klebsiella pneumoniae ; and (e)As such, schizophrenia is an incredibly complex disease that involves the interaction of genetic factors, immune and intracellular signaling, metabolic pathways, and alterations in proteins/enzymes. Until now, the majority of genetic, pathway, and metabolic studies have concentrated on identifying and characterizing the individual genes, proteins, and metabolics involved in schizophrenia susceptibility. Nevertheless, genes/proteins with small to moderate effects interact to influence susceptibility and outcome in complex diseases such as schizophrenia. Network, pathway enrichment, and annotation analysis may provide insight into how genes and proteins interact to influence disease susceptibility . AdditioHence, the aims of the current study were to delineate (a) the interactome or PPI network of FEP/FES and possible sub-networks; (b) the most influential core genes in the interactome, either hubs (disease causing genes) or bottlenecks (controlling the network); (c) the biological functions, pathways, molecular patterns, and cellular components that characterize FEP/FES; (d) the possible trigger factors of the FEP/FES interactome; and (e) the possible pathway similarities with other medical illness. This study is a secondary data analysis on existing data using open, deidentified, and non-coded datasets and, therefore, this is non-human subject research which is not subject to IRB approval. In our previous case-control studies conducted on Brazil and Thai patients with AN-FEP and FES, we identified significant biomarkers or differently expressed genes/proteins (DEPs) as compared with normal controls ,10,15,16https://string-db.org, as accessed 19 September 2021). The STRING database was employed to assess the PPIs (minimum required interaction score was 0.400) and to construct zero-order (seed proteins only) and first-order  PPI networks among the genes (set organism is homo sapiens). We examined the network characteristics  of the zero and first-order networks as well as further expanded networks using STRING and Cytoscape  plugins including NetworkAnalyzer . Hub nodes were authenticated as the top five nodes with the highest degree, and top non-hub bottlenecks as nodes with the highest betweenness centrality. Together they shape the backbone of the network.Construction of the PPI network with network expansion was conducted using STRING version 11.0 , which robustly identifies annotated complexes, and Cytoscape plugins, namely ClusterMaker. Molecular Complex Detection (MCODE) was performed using Metascape to detect smaller components of densely connected nodes which represent molecular complexes .https://www.ebi.ac.uk/intact/, as accessed 19 September 2021), GOnet , Metascape , inBio Discover , Enrichr , or the R package ClusterProfiler 4.0 and examined for their pathway, function, or disease enrichment scores. In this study, we use the false discovery rate (FDR) corrected p-values. We searched the networks against GO biological processes (assemblies of molecular functions in pathways), GO molecular functions (protein activities at the molecular level), GO cellular component (location of the proteins) , Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways , REACTOME (the European Bio-informatics Institute pathway database) , Translational Regulatory Relationships (TTRUST) , and DOID human disease phenotypes . Moreover, Metascape was used to delineate and visualize the GO biological pathways and molecular processes, and PANTHER , as accessed 19 September 2021), REACTOME, KEGG, and Wiki  pathways, which are over-represented in the gene sets. Metascape automatically clusters, hierarchically, the significant terms into a tree based on Kappa-statistical similarities among their gene memberships. This is useful because GO terms heavily overlap and, therefore, output GO terms show a large degree of redundancy. The top 10 performing terms obtained with Enrichr are displayed as bar graphs produced with Appyters.The list of seed and first-order genes  were extended with known protein interactions from STRING, IntAct . inBio Discover was used to show the network with the top 4 selected DOID annotations. R package ClusterProfiler was employed to make dot plots of annotated GO term leaves, with size of the dots indicating gene number and color of the dots indicating the n = 88) exceeded the expected number of edges (n = 24), with p-enrichment value of 1.0 × 10−16, and average node degree = 7.65 and average local clustering coefficient = 0.709. There were three singletons, namely PLVAP, DROSHA, and C1QA. n = 1063) exceeded the expected number of edges (n = 273) with p-enrichment value of 1.0 × 10−16, average node degree = 23.1, average local clustering coefficient = 0.678, average number of neighbors = 23.109, network diameter = 4 and radius = 2, characteristic path length = 1.919, network density = 0.254, and heterogeneity = 0.586.All genes/proteins were used to construct an undirected network representing the protein interactions of FEP/FES. The zero-order network consisted of 23 nodes, and the number of edges , TNF (58), IL10 (46), IL4 (42), and CSF2 (39) were the top five hubs, while CTNNB1 , BDNF (0.0285) and CDH1 (0.014) were the top three non-hub bottlenecks. Top rank hubs and bottlenecks (computed as z degree + z betweenness) computed for the selected 23 seed proteins in a further enlarged network showed that the most influential proteins were in descending order of importance: CTNNB1, IL6, TNF, CDH1, IL4, IL10, and BDNF.n = 811) exceeded the expected number of edges (n = 193) with p-enrichment value of 1.0 × 10−16, with average node degree = 25, average local clustering coefficient = 0.75, average number of neighbors = 24.954, network diameter = 4 and radius = 2, characteristic path length = 1.780, network density = 0.390, and heterogeneity = 0.549. The top five hubs are, in descending order of importance, IL6 (87), TNF (85), IL10 (71), IL4 (71), and IFNG (68). BDNF was the most important bottleneck (0.0563), followed by IL6 (0.0473) and TNF (0.046).The seed proteins were used to construct a PPI network representing the protein interactions in FEP only. The first-order network (first shell) shows 65 nodes, the number of edges . p = −38) and NFKB1 .−7.13) was accumulated in the seed gene list selection. It is interesting to note that behavior  and learning and memory  accumulated in the FEP/FES genes.−10), type-2 immune response , positive regulation of T cell activation , positive regulation of B cell proliferation , positive regulation of immunoglobulin production , and positive regulation of myeloid leukocyte differentiation . Moreover, other important GO terms were: cellular response to LPS , microglial cell activation , positive regulation of gliogenesis (pFDR = 7.962 × 10−4), and positive regulation of tyrosine phosphorylation of STAT protein .−2), entry of bacterium into the host cell , adherens junctions organization , modulation of chemical synaptic transmission , synapse assembly , neuron projection development , positive regulation of axonogenesis , positive regulation of neuroblast proliferation , cerebral cortex radial glia guided migration , and cellular response to indole-3-methanol .Consequently, we explored the network of interrelated genes using inBio Discover to delineate which diseases (DOID) are over-represented in the query gene list. The first major finding of this study is that we were able to construct zero and first-order PPI networks of FES/FEP which show high connectivity and some unexpected interactions, including the central role of BDNF. We found that the backbone of the FEP network is shaped by six genes  and the backbone of FEP/FES by IL6, TNF, IL10, CCTNB1, CDH1, and BDNF. Moreover, our network analysis revealed two major protein communalities, namely one centered around immune-inflammatory genes and BDNF, and a second cluster around cell–cell junction genes (CTNNB1 and CDH1). Most importantly, BDNF appears to function as a switch between both communalities, as it shows interconnections with immune genes and CTNNB1 and the high-affinity receptor of BDNF, namely neurotrophic tyrosine kinase receptor 2 (NTRK2), which was allocated to the cell–cell junction cluster. Another, smaller, bridge between both communalities is CDH1, which is interconnected with CTNNB1 and with CSF2, IL4, TNF, IL10, and IL6.Therefore, it appears that an immune response, as observed in FEP/FES, comprises not only IRS and CIRS components , but alsGO annotation and MCODE analysis revealed that the upregulated genes in FEP and the cluster-1 genes in FEP/FES were highly significantly associated with a response to a molecule of bacterial origin and a response to LPS. In fact, a response to LPS was the second most important GO term overexpressed in the upregulated proteins in FEP. Moreover, annotation analysis using a custom-made GO list with possible “responses to…” or “cellular responses to …” or a “defense response to…” showed that the most significant paths enriched in the network were responses to an organic substance (determined as cytokine or lipid), an external stimulus, response to other organisms, or an external biotic stimulus. Further analysis showed that a response to a bacterium and LPS were the most significant paths enriched in the FEP/FES network, whereas a response to a virus was less significant and did not appear in the GO and MCODE enrichment analysis. Moreover, an abiotic stimulus and fungal or parasite stimuli were not significantly enriched.All in all, it appears that FEP and FEP/FES may be triggered or maintained by a response to LPS of Gram-negative bacteria. These findings corroborate a recent study which reported increased IgA/IgM responses to LPS of Gram-negative bacteria in FES and deficit schizophrenia, and a significant association between these indicants of bacterial load and the G-CoDe  and symptom profiles, especially psychosis ,18. SuchThe third major finding of this study is that the upregulated genes in FEP were enriched in key pathways and cellular functions that play a key role in immune-inflammatory signaling, namely the receptor signaling pathway via Janus kinases/signal transducer and activator of transcription proteins (JAK-STAT) pathway, the TNFR1-induced NFκB signaling pathway, and TNF- and death receptor signaling. Moreover, STAT3 and STAT6 were prominent nodes in the first order network, with STAT3 occupying a central position. Many cytokines  signal via the JAK-STAT pathway, thereby transactivating Janus kinases leading to translocation of STATs to the nucleus and upregulation of cytokine-modifiable genes . The JAKRELA gene and has a significant effect on pre-pulse inhibition [TNF2(A) allele, which affects plasma TNF levels, is significantly increased in schizophrenia and TNF2 homozygotes are detected in schizophrenia only [Our enrichment analyses show that TNF plays an important role in FEP and suggests that TNF-induced IκB kinase (IKK) activation with consequent NFκB translocation to the nucleus is one of the key processes in FEP. Moreover, we found that both NFκB1 and RELA (NFκB p65 unit or transcription factor p65) are the most prominent transcription factors in the FEP network. RELA plays a role in NFκB activation and translocation of the released NFκB complex to the nucleoplasm and contributes to DNA binding in the NFκB complex . This NFhibition . Importahibition . Previouhibition . Moreovep < 0.004), and that these effects are not influenced by the drug state of the patients [BDNF and NTRK2 gene polymorphisms may increase susceptibility to paranoid schizophrenia [The fourth major finding is that the downregulated genes were enriched in the receptor protein tyrosine kinase (RTK) and neurotrophin/Trk receptor signaling pathways, cellular component morphogenesis, and production of miRNAs involved in gene silencing. RTK is a family of high-affinity cell surface receptors, including Trk, which is activated by neurotrophins such as BDNF . Interespatients . Moreoveophrenia .We found that the downregulated DISC1 gene is enriched in various GO biological terms indicating neurogenesis, axonogenesis, and axon extension. DISC1 is now established as a risk factor for schizophrenia and other major psychiatric illness and is involved in aspects of adult progenitor proliferation, neurogenesis, neurite outgrowth, cytoskeletal modulation, signal transduction, and CTNNB1 abundance ,36. It iAnother highly significant GO path enriched in the downregulated network was the “production of miRNAs involved in gene silencing by miRNA”, which may, at least in part, be ascribed to downregulation of DROSHA, which is a ribonuclease (RNase) III family enzyme and plays a key role in miRNA maturation . In mammThe enrichment and annotation analysis revealed other important drug targets in FEP/FES. Firstly, we found that a neuroinflammatory response was enriched in the seed gene FEP/FES list, whilst MCODE showed that a cytokine/chemokine complex of IFN-γ, IL-6, IL-12A, CCL3, IL-4, and IL-13 was strongly associated with microglial cell activation and tyrosine phosphorylation of STAT proteins. These results extend the findings that schizophrenia is accompanied by microglial activation . MoreoveSecondly, WikiPathway and PANTHER enrichment analysis revealed that the upregulated genes were strongly associated with the TLR signaling  and tolerance pathways. These findings extend those of previous publications indicating activation of the TLR4 proinflammatory pathway in schizophrenia .Third, GO annotation analysis revealed that the cluster 2 genes are enriched in the Wnt/catenin pathway and cell–cell junction organization. Moreover, different combinations of the downregulated genes were associated with the Wnt/catenin pathway (DISC1 and CTNNB1), adherens junctions organization (CDH1 and CTNNB1), synapse assembly (CDH1 and BDNF), neuron projection development , neuroblast proliferation (DISC1 and CTNNB1), cerebral cortex radial glia guided migration (DISC1 and CTNNB1), positive regulation of axonogenesis (BDNF and DISC1), and modulation of chemical synaptic transmission . BDNF polymorphisms are associated with changes in the Wnt/β-catenin pathway [CTNNB1 is a component of the Wnt/β-catenin signaling pathway and the E-cadherin-catenin adhesion complex, which play a key role in epithelial integrity and tissue architecture maintenance ,51. The  pathway . In hipp pathway .Hp2 allele and the Hp2.2 genotype (prehaptoglobin-2 or zonulin) [CTNNB1 gene (c.1943 A>G) are associated with schizophrenia [CTNNB1 KO mice display anxiety behaviors and CTNNB1 KO in paraventricular interneurons accompanied by impairments in social interactions, repetitive behaviors, and object recognition [Disorders in the Wnt/catenin pathway were previously described in FES and schizophrenia ,61,62 anzonulin)  and incrzonulin) . Moreoveophrenia , whilst ognition . Associaognition ,68.Fourth, exploration of transcriptional regulation showed that SP1, NFκB1, and RELA were the most prominent transcription factors in the FEP/FES network. SP1 or specificity protein 1 (or transcription factor Sp1) is a ubiquitously expressed transcription factor, which regulates the expression of a variety of house-keeping and tissue-restricted genes frequently involved in immune responses, response to DNA damage, and apoptosis . This exFifth, biological GO term classifications showed that complement factors were enriched not only in microglial activation and humoral immune responses, but also in synapse pruning and organization. The involvement of complement in FEP/FES agrees with previous findings showing increased plasma C3C and C4 and CSF and brain C4 complement factors in schizophrenia ,75,76. MBy inference, individuals with reduced CIRS , includiInterestingly, almost all pathways or molecular patterns enriched in the interactome of FEP/FES are directly  or indirectly  affected by LPS. As such, the increased LPS levels in FES may maintain peripheral and central immune activation resulting in neurotoxic effects on central neuronal circuitry, neurogenesis, and synapse functions . DisordeFuture research in FEP should examine the microbiome, gut dysbiosis, and consequent bacterial translocation due to leaky gut as well as alterations in the microbiota–gut–brain axis , which m


Astragalus membranaceus  exerts significant therapeutic effect on gastric ulcer (GU); however, there are scarce studies on its molecular mechanism against GU. This study aims to explore the key ingredients, key targets, and potential mechanisms of AM in the treatment of GU by utilizing network pharmacology and molecular docking.  Several public databases were used to predict the targets of AM and GU, respectively, and the drug and disease targets were intersected to obtain the common targets. Next, the key ingredients and key targets were identified by constructing ingredient-target network and protein-protein-interaction (PPI) network. Gene Ontology biological processes (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out on the common targets in order to ascertain the biological processes and signaling pathways involved. Finally, molecular docking was conducted to verify the binding affinity between the key ingredients and key targets.  A total of 552 predicted targets were obtained from 23 screened active ingredients, of which 203 targets were the common targets with GU. Quercetin, kaempferol, and isorhamnetin were identified as the key ingredients by constructing ingredient-target network, and TP53, AKT1, VEGFA, IL6, TNF, CASP3, and EGFR were selected as the key targets by constructing PPI network. GOBP and KEGG pathway enrichment analysis suggested that the therapeutic effect of AM on GU involved multiple biological processes and signaling pathways related to inflammation, oxidative stress, apoptosis, cell proliferation, and angiogenesis. Molecular docking validation demonstrated that all key ingredients had good binding affinity with the key targets.  This study revealed the key ingredients, key targets, and potential mechanisms of AM against GU, and these data may provide some crucial references for subsequent research and development of drugs for treating GU. Helicobacter pylori (H. pylori) infection, overuse of nonsteroidal anti-inflammatory drugs, excessive drinking, smoking, and stress [H. pylori eradication and gastric acid secretion inhibition has improved the clinical cure rate of GU, but their therapeutic effect is limited and the recurrence rate of GU is still high [Gastric ulcer (GU) is a common disease in which gastric acid and pepsin self-digest the gastric mucosa due to the imbalance of defense mechanism and injury factors of gastric mucosa caused by multiple aggressive factors, including d stress , 2. Ulced stress , 4. At td stress , 6. The ill high . Furtherill high , 8.Gastrointestinal diseases are the preponderant diseases in herbal therapy. A large number of studies in human and animal models have shown that the efficacy of herbal medicines in the treatment of GU is comparable or superior to that of omeprazole and cimetidine, and herbs display fewer adverse reactions. Besides, the cost of herbal therapy in the treatment of GU is only about one-sixth of that of western medicine . TherefoAstragalus membranaceus (AM), also known as “Huangqi” in China, is one of the most important Chinese herbal medicines and has a medicinal history of two thousand years [in vivo and in vitro, including immunomodulatory [nd years . More thnd years . These idulatory , anti-indulatory , 14, antdulatory , antifatdulatory , antiagidulatory , and hypdulatory  effects.Herbs are multi-ingredient and multitarget in treating diseases, and their pharmacodynamic ingredients and molecular mechanisms are often difficult to identify. With the advancement of network technology and bioinformatics, computer-aided identification methods of drug-target interactions represented by network pharmacology and molecular docking have been developed to provide support for the research of herbs with complex action mechanism. They greatly reduce the initial time and cost of experimental determination of drug-target interaction and improve the chances of finding ideal drug candidates –20. Althhttps://tcmsp-e.com/) by using “Huangqi” as the keyword. The chemical ingredients meeting both oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18 were screened out as the bioactive ingredients. Besides, chemical substances that do not meet the above screening criteria but have potential activity in the treatment of GU were also included by searching and reading relevant literature on PubMed (https://pubmed.ncbi.nlm.nih.gov/).The chemical ingredients of AM were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  [https://pubchem.ncbi.nlm.nih.gov/) and then were used to predict potential gene targets in SwissTargetPrediction platform (http://www.swisstargetprediction.ch/) [Protein targets associated with each bioactive ingredients were acquired from TCMSP and transformed into corresponding gene symbols via Uniprot database (ot.org/) . In addiion.ch/) , 23. Thehttps://www.genecards.org/) [https://www.disgenet.org/) [https://omim.org/) [http://db.idrblab.net/ttd/) [With “gastric ulcer” as the keyword, GeneCards Database(ds.org/) , DisGeNEet.org/) , Online im.org/) , and Theet/ttd/)  were useThe common targets of ingredient targets and disease targets were obtained by intersection and a Venn diagram was drawn to display visually. Furthermore, a network based on the interactions between drug, ingredients, common targets, and disease was constructed and visualized by Cytoscape v3.7.1 .https://www.string-db.org/) to build PPI network, and the result was exported in tab-separated value (TSV) format after hiding the unconnected nodes [The common targets were imported into STRING database (ed nodes . Subsequed nodes .https://metascape.org/) for GOBP and KEGG pathway enrichment analysis [p value from small to large, the top 25 GOBP items and the top 25 KEGG pathways were selected to draw a bar diagram and a bubble diagram, respectively.The previously obtained common targets were input into Metascape  and converted into PDB format by PyMOL v2.1.0 after removing water and original ligands [In order to confirm the accuracy of the network pharmacology prediction, the key ingredients were used as small molecular ligands to perform molecular docking with the key target proteins. The 2D structures of ligands were obtained from PubChem database and structurally optimized and transformed into PDB format by Chem3D v19.0 and PyMOL v2.1.0. The 3D structures of receptor proteins were downloaded from RSCB PDB database , astragaloside III , and ononin  were included together for their reported antiulcer potential in several studies though they did not meet the inclusion criteria –37. Fina1226 known disease targets of GU were collected from GeneCards Database, 136 from DisGeNET, 79 from OMIM, and3 from TTD. A total of 1320 disease targets were collected by merging the disease targets obtained from these databases and removing the duplicates.203 common targets of AM and GU were identified  and an iThe PPI network exported from STRING database had 201 nodes (two free nodes were hidden) and 5149 edges. The nodes with degree ≥84 were selected to build a new network, but there were still too many targets. Therefore, a core network composed of 14 nodes and 91 edges was further extracted by using the median of degree, BC, and CC. The extraction process is shown in p values, the top 25 GOBP items and the top 25 KEGG pathways were selected to draw a bar diagram  were predicted to be the key ingredients of AM in the treatment of GU, and TP53, AKT1, VEGFA, IL6, TNF, CASP3, and EGFR were identified as the key targets. Molecular docking also verified that all key ingredients had good binding affinity with the key targets. GOBP and KEGG pathway enrichment analysis suggested that the therapeutic effect of AM on GU involved multiple signaling pathways and biological processes related to the occurrence and healing of GU, such as inflammation, oxidative stress, apoptosis, cell proliferation, and angiogenesis.H. pylori of the screened key ingredients have been reported previously. Quercetin was found to exert excellent inhibitory activity on urease, an enzyme which enables H. pylori to survive in acidic gastric juice and infect gastric mucosa [H. pylori colonies in a dose-dependent manner both in vitro and in vivo [H. pylori. Furthermore, KEGG pathway enrichment analysis showed that EBV infection was associated with the antiulcer effect of AM, and Epstein-Barr virus (EBV) was proved to promote gastrointestinal mucosal inflammation, indicating that AM may be considered for the treatment of GU caused by EBV [H. pylori is the leading cause of GU. The bioc mucosa . Kaempfe in vivo . GOBP end by EBV , 42.α and IL-6 are regarded as the major contributors to gastric inflammation and mucosal injury, and their serum levels are positively correlated with the severity of GU [Gastric mucosal inflammation caused by increased expression of proinflammatory factors is an important reason for the occurrence, aggravation, and long-term nonhealing of GU , 44. TNFty of GU –47. Our ty of GU –50. Furtty of GU , 52.Oxidative stress (OS) is a state of imbalance between oxidation and antioxidation in the body, under which excessive reactive oxygen (ROS) produces oxidative damage to lipids, proteins, and DNA of cells . GastroiH. pylori infection [The balance of proliferation and apoptosis of gastric epithelial cells maintains the integrity of gastric mucosa. When this balance is destroyed by various pathogenic factors and apoptosis is dominant, gastric ulcers can be induced . Studiesnfection , 60. Amonfection . GOBP anAngiogenesis is essential for the healing of gastric mucosal injury because it contributes to delivering oxygen and nutrients to the healing site. Angiogenesis is initiated and regulated by angiogenic factors, among which VEGF is the most effective and basic regulator . The facH. pylori and virus, anti-inflammation, antioxidation, regulation of gastric mucosal cell proliferation and apoptosis, and promotion of angiogenesis. These data can provide some crucial references for the research and development of new anti-GU drugs in the future.To sum up, our study has revealed that the therapeutic effect of AM on GU is multi-ingredient, multitarget, and multimechanism. The key ingredients of the curative effect are mainly flavonoids, and the molecular mechanism is closely related to inhibition of


On the other hand, gabapentin led to a complete regression of the hiccup probably by reducing the nerve impulse transmission and modulating the diaphragmatic activity. The present case series suggests the use of low doses of gabapentin as an effective treatment for aripiprazole-induced hiccups. However, our knowledge of the neurotransmitter functioning of the hiccup reflex arc is still limited, and further research is needed to characterize the neurotransmitters involved in hiccups for potential novel therapeutic targets.Hiccups are sudden, repeated, and involuntary contractions of the diaphragm muscle (myoclonic contraction). It involves a reflex arc that, once activated, causes a strong contraction of the diaphragm immediately followed by the closure of the glottis translating into the classic “hic” sound. Hiccups can be short, persistent, and intractable depending on the duration. The most disabling hiccups often represent the epiphenomenon of a medical condition such as gastrointestinal and cardiovascular disorders; central nervous system (CNS) abnormalities; ear, nose, and throat (ENT) conditions or pneumological problems; metabolic/endocrine disorders; infections; and psychogenic disorders. Some drugs, such as aripiprazole, a second-generation antipsychotic, can induce the onset of variable hiccups. We describe herein the cases of three hospitalized patients who developed insistent hiccups after taking aripiprazole and who positively responded to low doses of gabapentin. It is probable that aripiprazole, prescribed at a low dosage (<7.5 mg/day), would act as a dopamine agonist by stimulating D Hiccup is the sudden onset of erratic diaphragmatic and intercostal muscular myoclonus with early glottis closure terminating inspiration; hence, the abrupt air rush into lungs induces the vocal cords to close leading to a characteristic “hic” sound –3. Hiccuacute hiccups or “hiccup bouts” are of less than 48-hour duration, persistent lasts over 2 days, and intractable lasts over a month [An individual's hiccup rate is usually consistent for each hiccup episode, occurring at a frequency of 4 to 60 hiccups per minute , 8. The  a month . The inc a month , 11. Psy a month , 13.Brief episodes of hiccups are common in healthy individuals, and they are believed to be induced by the rapid stomach distension and irritation in terms of overeating, eating too fast, ingesting spicy food, drinking carbonated drinks, aerophagia, sudden change in ingested food temperature , or sudden excitement , 15.Persistent and intractable hiccups may signify a more serious underlying etiology such as gastrointestinal and cardiovascular disorders; central nervous system (CNS) abnormalities; ear, nose, and throat (ENT) conditions or pneumological problems; metabolic/endocrine disorders; infections; and psychogenic disorders including excitation, hyperventilation, malingering, somatization, and stress , 16–21.γ-aminobutyric acid (GABA) pathways in the brainstem and medulla [Finally, some drugs, such as chemotherapeutic agents and some glucocorticoids, showed a strong association with hiccups. Nearly 42% of patients taking both cisplatin and dexamethasone may develop hiccups . Interes medulla , 24.Benzodiazepines show a dose-dependent and inverse relationship with hiccups. At low doses, benzodiazepines correlate with the development of hiccups, while at higher doses, they may be useful in their treatment. Other medications associated with hiccups include alpha-methyldopa, donepezil, levodopa, inhaled anesthetics, ethanol, methohexital, morphine, sulfonamides, tramadol, and azithromycin , 25–27.Although formally known as one of the elective hiccup treatments, recent case reports suggest that antipsychotics (As) may trigger hiccups in some cases –31. TherAnticonvulsants, such as valproic acid, phenytoin, carbamazepine, and gabapentin enhancing central and peripheral GABA transmission, seem to be helpful in blocking the hiccup stimulus. Specifically, gabapentin produces a blockade of neural calcium channels and increases the release of GABA, which might modulate diaphragmatic excitability. Unfortunately, however, only a few data are available to confirm its effectiveness. One study, involving 43 patients with advanced cancer, noted a reduction of hiccups in 95.3% of patients by taking high daily doses of gabapentin (900-1200 mg) , with noOther drugs proposed in the treatment of protracted hiccups include defoaming and prokinetic agents (domperidone and metoclopramide), peppermint, proton pump inhibitors, baclofen, nifedipine, methylphenidate, midazolam, lidocaine, and sertraline , 35–39.In drug-induced hiccups, aripiprazole, a second-generation A (SGA), is very often incriminated for transient and persistent hiccups amongst both adult and adolescent patients , 40, 41.2 and D3) partial agonist (as opposed to a full DA antagonist like a SGA), type 1A serotonin (5-HT) receptor (5-HTlA) partial agonist, and type 2A serotonin (5-HT) receptor (5-HT2A) antagonist. As a partial agonist, aripiprazole acts as a functional antagonist in conditions of high DA concentrations and as a functional agonist in conditions of low DA concentration. This partial agonism might reduce the D2 hyperactivation in the mesolimbic pathway, thus alleviating positive symptoms of schizophrenia, but providing enough D2-receptor stimulation in the mesocortical pathway and the nigrostriatal pathway, thus preventing, respectively, negative symptoms and extrapyramidal side effects [Aripiprazole shows a unique mechanism of action as a type 2 and 3 dopamine receptor  according to DSM-5 criteria , admitteThe patient, a healthy carrier of beta-thalassemia, did not suffer from any other current or past medical conditions and had no family history of psychiatric disorders.At admission, on psychic examination, the patient presented a slight stutter and logorrheic language with hyperphonic tone. Mimicry was slightly overexpressed with verbal and motor stereotypes. He was euphoric with mild motor restlessness and with reduced need for sleep. Slight acceleration of thoughts with poorly structured paranoid and persecutory ideas.His longitudinal evaluation revealed that he was born preterm (34th week of gestation due to liver dysfunction from preeclampsia) and acquired walking after the 15th month. Since childhood, the patient has showed some language (expressive and receptive), learning , and behavioral problems (impulse-control), and since adolescence, he has experienced mood swings, obsessive thoughts , compulsive accumulation, stereotyped language, aggressive attitudes (self-injurious tics), and specific phobias  with serious impairment of personal autonomy and school performance.In those years, the patient regularly performed several personalized multidisciplinary therapeutic programs in different neuropsychiatric institutes with partial clinical benefit.In the last three years, Mr. A reported a period of euthymia, achieving the high school diploma and occasionally working as a waiter. However, four months before our consultation, the patient showed mood swings with irritability, nervousness, verbal aggression, and paranoid and persecutory ideas towards parents and colleagues, and he underwent two hospitalizations in other clinics with poor clinical benefit. When he was admitted to our inward, he was taking zuclopenthixol (20 drops/day) and diazepam (15 mg/day).Lithium sulfate was introduced on the first day, zuclopenthixol and benzodiazepines were gradually suspended until the third day, and aripiprazole (5 mg/day) was given on the fifth day. Approximately 8 hours after aripiprazole, the patient began to experience protracted and bothersome hiccups that lasted for the following 8 hours. The doctor in charge was called by the nursing staff and decided to give him gabapentin (100 mg) that led to a complete hiccup resolution in about 60 minutes.Since hiccups were considered as an adverse event of aripiprazole, this drug was immediately stopped and replaced by risperidone and clozapine. The patient remained in our hospital for the next 18 days, and he experienced no more hiccups.Mr. G was a 20-year-old man, working in the family business, admitted to our department for the onset of a major depressive episode (according to DSM-5 criteria ) with suThe family history was positive for anxious spectrum disorders in both maternal and paternal lineages. He referred previous cannabis and alcohol misuse. The patient did not suffer from any medical conditions.On psychic examination, he was not very talkative, and if questioned, he used short and concise sentences reporting experiences of self-devaluation and inadequacy. Mr. G described a sense of apathy, anhedonia, and a not-better-specified “emptiness.” He referred low energy levels, hypersomnia, difficulties in attention and memorization, poor prospects for the future, suicidal thoughts, and anxiety fluctuations with recent cardiorespiratory and neurovegetative critical episodes. The psychomotricity was slightly slowed. He denies recent alcohol and substance use.Neurological physical examination did not detect any signs of stiffness or tremor with regular peripheral-induced reflexes.His longitudinal evaluation revealed no complications at birth, pregnancy, and delivery with regular neuropsychomotor development.The onset of the psychopathological picture seemed to date back to the age of 14 when the patient experienced a drop in mood with anhedonia, loneliness, feelings of self-devaluation and inadequacy, irritability, anguish, thoughts of death, fluctuations of anxiety levels, and self-cutting. For this reason, he decided to begin a psychotherapeutic treatment with clinical benefit. At the age of 16 years, Mr. G started taking alcohol and cannabinoid for recreational-disinhibitory purposes.μg/mL), perphenazine (4 mg/day), and sertraline (50 mg/day).When he was 18, the patient reexperienced depressive symptoms with structured anticonservative ideation and progressive impairment of social and scholastic functioning (he quit the school). He underwent psychiatric evaluations, and several psychopharmacological associations were prescribed including antidepressants, antipsychotics, benzodiazepines, and mood stabilizers with partial clinical benefit. Considering the persistence of the symptomatology, he consulted us, and he was hospitalized in our clinic. At the admission, he was taking valproic acid . In this case, two doses of 100 mg gabapentin were administered to stop hiccups at a distance of about 2 hours due to the ineffectiveness of the first dose. About 35-40 minutes after the second dose, a complete regression of hiccups was obtained. The medical team stopped aripiprazole, and the hiccup never showed up again.Mr. R was a 41-year-old unemployed man with a diagnosis of BDI and a previous polysubstance use disorder (SUD) (DSM-5) . The patThe patient does not suffer from any other medical illness.At the admission, the patient was partially accessible to intrapsychic experiences. High reactivity, irritability, and nervousness emerged. He expressed apathy with low interest for work and for social relationships. There are nuanced self-reference and paranoid ideas. He presented an inversion of the sleep-wake rhythm. He described anxiety infradian fluctuations which worsened at evening.The neurological examination, the routine blood tests, and the toxicological urine test were normal and showed no recent substance use.The family psychiatric history was positive for mood disorders in the paternal lineage.The parents described him as extremely vulnerable to life events with high interpersonal sensitivity. Since adolescence, he alternated periods of dynamism, extroversion, and productivity with others of work and social withdrawal.The onset of the psychopathological picture was traced back to the age of 25, when Mr. R experienced depressive mood with a tendency to isolation and paranoid and persecutory ideas. With family aid, he underwent a first psychiatric consultation, and he took antidepressants (n.s.) with significative clinical improvement.The following years, the patient achieved a degree in agricultural science and, shortly thereafter, received a teaching job. When he was 34, after the end of a romantic relationship, he experienced a depressive episode again with death thoughts, paranoid ideas, and social and work impairment.He decided to consult a psychiatrist who prescribed various psychopharmacological associations and progressively regained mood stability and global functioning.About 6 months before the hospitalization, he presented a reexacerbation of depressive symptoms with paranoid and persecutory ideations toward colleagues, and thus, he decided to contact our clinic.At the admission, he was taking valproic acid (1000 mg/day), olanzapine (20 mg/day), and biperiden hydrochloride (2 mg/day). Olanzapine was gradually stopped and carbolithium (600 mg/day) was introduced. Aripiprazole (5 mg/day) was added on the third day and increased up to 7.5 mg/day the next day.Approximately 8 hours after the second aripiprazole, the patient reported the onset of a persistent hiccup. He tried different physical countermeasures, such as holding breath and drinking water while pinching the nose, but none were effective to stop it. Therefore, we decided to administer him 100 mg of gabapentin, and considering the lack of response after about two hours later, an additional 100 mg of gabapentin was given. In less than 60 minutes from the second dose, hiccup regression was observed.The aripiprazole was discontinued, and the hiccup did not recur again.The comprehension of hiccups is still being clarified. Current theories include an amorphous neural network coordinating various afferent and efferent pathways, interconnected, coordinated, and managed by a so-called “hiccup center.” The “hiccup reflex arc” consists of three components, the afferent limb including the glossopharyngeal nerve , vagus nerve , nuclei of the solitary tract, nucleus ambiguus, phrenic nerve, and sympathetic nerves to convey somatic and visceral sensory signals; the “processing core unit” in the midbrain; and the efferent limb traveling in motor fibers of phrenic nerves to the diaphragm and accessory nerves to the intercostal muscles , 46–51. 2 receptor, it is possible to hypothesize that a hyperdopaminergic state at the central level involves an activation of the hiccup center. This would explain both how other antipsychotics  can be effective in the treatment and how drugs having dopamine-agonist activity can trigger the appearance of simple or protracted hiccups. In fact, many Parkinson disease (PD) patients receiving dopamine agonists have frequent hiccups [3 receptors that may be involved in the hiccup reflex arc [1A and antagonizing 5-HT2A receptors [Considering that the only treatment approved by the Food and Drug Administration is chlorpromazine, which has a receptor profile characterized mainly by an antidopaminergic activity acting as a functional antagonist of the D hiccups –56. A re hiccups . Dopaminflex arc . Howeverflex arc . Even ineceptors . Therefo2 and D3 partial agonist, a serotonin 5-HTlA partial agonist, and a serotonin 5-HT2A antagonist, aripiprazole could potentially induce hiccups both through the regulation of the serotonergic and dopaminergic systems. At the serotonergic system level, aripiprazole could enhance the phrenic nerve activity through agonizing 5-HT1A and antagonizing 5-HT2A receptor and consequently trigger the hiccup reflex arc.Being a dopamine D2 and D3 receptors. Our cases presented protracted hiccups when they were still taking low doses of aripiprazole, specifically when the antidopaminergic activity had not yet started [Aripiprazole at low doses (approximately up to 7.5 mg) acts as a dopaminergic agonist, stimulating D started .1A receptor and antagonizing the 5-HT2A receptor.Thus, in our case, aripiprazole potentially could have triggered the hiccups through the stimulation of the dopaminergic system by acting as a dopamine agonist and through the serotonergic system by activating the 5-HTOn the other hand, gabapentin is an alpha-2-delta ligand, structurally similar to GABA, with the ability to block voltage-operated calcium channels, to reduce the release of several neurotransmitters (glutamate and substance P) and to modulate the diaphragmatic activity .Gabapentin, an alpha-2-delta ligand, structurally similar to GABA, has the ability to block voltage-operated calcium channels, to reduce the release of several neurotransmitters , and to modulate the diaphragmatic activity , 60, 61.In the present paper, we described the cases of three men hospitalized in our psychiatric department who experienced the onset of protracted and invalidating hiccups following the intake of a low dose of aripiprazole (<7.5 mg). The hiccups completely disappeared following the administration of low doses of gabapentin and the prompt suspension of aripiprazole. Furthermore, in each case, the hiccups did not recur with drug changes.This case series would suggest gabapentin as an ideal medication for the acute treatment of aripiprazole-induced hiccups. Given the great use of aripiprazole and considering that hiccups are a common side effect in clinical practice, the administration of low doses of gabapentin might represent an additional tool in the psychiatrist's armamentarium. Gabapentin is a promising and safe drug to treat prolonged, frequent, and persistent hiccups having reported high response rates and few side effects. Furthermore, gabapentin might be considered the first choice in the treatment of hiccups in some psychiatric patients. Indeed, it could be useful in those who are already taking antipsychotic therapy, so any treatment with an additional antipsychotic could lead to a worsening of clinical conditions or in subjects who could be negatively affected by treatment with an antipsychotic from a point of psychomotor and mood view.However, the exact roles of neurotransmitters within the hiccup reflex arc and the reason why there is a high interindividual variability in response to treatment are still not known. Further research is needed to characterize the neurotransmitters involved in hiccups for potential new therapeutic targets.


Diabetes mellitus, especially type 2 (T2DM), is a major public health problem globally. DM is characterized by high levels of glycemia and insulinemia due to impaired insulin secretion and insulin sensitivity of the cells, known as insulin resistance. T2DM causes multiple and severe complications such as nephropathy, neuropathy, and retinopathy causing cell oxidative damages in different internal tissues, particularly the pancreas, heart, adipose tissue, liver, and kidneys. Plant extracts and their bioactive phytochemicals are gaining interest as new therapeutic and preventive alternatives for T2DM and its associated complications. In this regard, isorhamnetin, a plant flavonoid, has long been studied for its potential anti-diabetic effects. This review describes its impact on reducing diabetes-related disorders by decreasing glucose levels, ameliorating the oxidative status, alleviating inflammation, and modulating lipid metabolism and adipocyte differentiation by regulating involved signaling pathways reported in the in vitro and in vivo studies. Additionally, we include a post hoc whole-genome transcriptome analysis of biological activities of isorhamnetin using a stem cell-based tool. Calendula officinalis L. ..6-C3 bas7H9O5). This later presents the first ring, which characterizes phenolics with two hydroxyl groups. The shikimate undergoes six reactions which end in the first amino acid: phenylalanine. Thanks to two key enzymes, phenylalanine ammonia-lyase (PAL) and cinnamate-4-hydroxylase (C4H), phenylalanine successively forms cinnamate and p-coumarate.. In this step, the level of PAL activity could quantitatively regulate the accumulation of phenolic compounds. The p-coumarate molecule is at the origin of coumarin derivatives. The pathway for the synthesis of phenylpropanoid molecules is characterized by the presence of a key enzyme called 4-coumarate CoA ligase (4CL), which catalyzes in the presence of the thiol function of the coenzyme A (CoA) the p-coumaric acid into 4-coumaroyl CoA (C30H42N7O18P3S). The aromatic A-ring of flavonoids provides from the condensation of three molecules of malonyl-CoA (-C6).More precisely, shikimic acid is at the base of several reactions, representing the skeleton of aromatic amino acids, which are the initiators of phenolic compounds. The first step consists of a combination of two molecules: phosphoenolpyruvate and erythrose 4-phosphate, which after four reactions, lead to forming the first skeleton of phenolics: the shikimate or shikimic acid . Chalcone is a key element in this topic because it is a precursor of all flavonoids based upon a fifteen-carbon skeleton consisting of two benzene rings. Afterward, chalcone is converted to naringenin  by the action of chalcone isomerase (CHI). On the one hand, flavones such as apigenin, acacetin, chrysin or luteolin are synthesized from naringenin in the presence of flavone synthase (FNS). On the other hand, different flavonols and succinic acid compounds are produced from naringenin in the presence of two enzymes: flavanone 3-dioxygenase (F3D) and flavonol synthase (FS). Apart from flavonol derivatives, other compounds, such as kaempferol, myricetin, and quercetin, are also produced. By a methyl group transfer from the S-adenosyl-L-methionine, the isorhamnetin is produced in the presence of the flavone 3′-O-methyltransferase (FMT) ,48. Thennalis L. .In fact, flavonoids are considered one of the most important groups of the polyphenol family. They have a structure based on a diphenyl propane type with two benzene rings . Firstly, fractionation can be used to simplify the extraction by removing all oil and lipophilic pigments from lipid-containing samples. The defatted sample is also sonicated before maceration in a mixture of methanol and water. Chromatography can then be used for analyses of phenolic compounds, especially flavonoids [Calligonum azel Maire plant fractions, collected from the Tunisian desert, have been assessed and an ultra-high performance liquid chromatography coupled to quadruple time of flight mass spectrometry UHPLC-ESI-QTOF was used to identify phenolics among them flavones and flavanols, which were the most abundant phenolic compounds identified [Mesembryanthemum edule using LC/ESI-MS/MS technology [Arthrocnemum indicum [Tamarix gallica [Glaucium flavum [Salsola kali [Artemisia herba-alba was performed to identify phenolics, such as flavones, flavonols and flavonoid alkaloids [Pancratium maritimum and the analysis by HPLC-DAD-ESI/MS revealed the presence of flavonoids including flavonol as isorhamnetin with their pentoside and hexoside conjugates such as isorhamnetin di-hexoside [Limoniastrum guyonianum using HPLC showed the presence of many phenolics, among them isorhamnetin-3-O-rutinoside [The distribution of isorhamnetin in medicinal plants is very wide and the methods of extraction and analyses are varied. Isorhamnetin derivatives are particularly wanted. The hydroxyl and methyl groups help in their characterization. Some methods are used to extract isorhamnetin, among them based on fractionation, using chemometric approaches, enzyme, and supercritical fluid extraction  seeds obtained with a green solvent such as 2-methyl tetrahydrofuran (MeTHF) as an alternative to petroleum or hexane-based solvent to extract oil-enriched phenolic compounds [Peucedanum aucheri Boiss collected in Marivan city, Kurdistan province, Iran [Calendula officinalis. Linear, quadratic, full cubic and special cubic models were analyzed. At the final full cubic was the most appropriate one, allowing greater efficiency in the extraction of isorhamnetin-3-O-rutinoside by 60%. For this study, the enzymes, Rapidase Maxi Fruit and Viscozyme, were used under some important factors affecting the enzyme activity under supercritical CO2 conditions such as pressure, temperature, pH, time and aqueous ethanol solution. Finally, the supercritical fluid extraction method was used to extract phenolic compounds such as flavonols. Many years ago, the supercritical fluid extraction procedure was used on the Eucalyptus globulus bark for the first time, using pure and modified CO2 with water, ethyl acetate and ethanol [2 combined with ethanol could extract significant amounts of phenolic compounds, including isorhamnetin. HPLC-MS quantification determined some flavonols, such as isorhamnetin-hexoside (0.26 g·g−1 of extract) and simple isorhamnetin (14.29 mg·g−1 of extract). Ethanol was used as a co-solvent. The quantification was carried out with high-pressure liquid chromatography equipped with a photodiode array detector. Isorhamnetin 3-O-glucosyl-rhamnosyl-rhamnoside, isorhamnetin 3-O-glucosyl-rhamnosyl-pentoside, isorhamnetin 3-O-glucosyl-rhamnoside, and isorhamnetin 3-O-glucosyl-pentoside were the most abundant flavonols extracted from O. ficus-indica supercritical extracts. As mentioned before, high-pressure liquid chromatography or HPLC is a commonly used method for analyzing phenolic compounds. In apple juice and pear juice, isorhamnetin 3-O-glucoside was detected. In fact, the separation of the flavonol glycosides in a “Brettacher” apple extract by HPLC and mass spectrometry revealed the presence of two glycoside forms such as isorhamnetin 3-O-glucose and isorhamnetin 3-O-galactoside [Far from phenolic extracts, phenolic compounds are also found in oils such as olive oil . These compounds . The prece, Iran . The NMRce, Iran . A multi ethanol . Authorsactoside .2/M stage by the activation of the ATM-Chk2 pathway. Li and colleagues revealed that isorhamnetin could also inhibit the growth of the gefitinib resistant PC9 cells (PC9-IR) by downregulating BCL-2 gene expression and PCNA protein expression, inhibiting DNA synthesis and upregulating P53, BAX and CASP3 gene expression [Casp3 activity (a biomarker of apoptosis) and improving neurological function repossession. Likewise, the mice treated with this flavonol showed decreased cerebral edema, enhanced blood–brain barrier function, and upregulated gene expression of tight junction proteins, including Ocln, Zo-1, and Cldn-5 [Cox-2 expression in male BALB/c mice [Oxidative stress is an endogenous and exogenous process that plays a role in aging. Oxidative stress occurs due to dysregulation in the antioxidant defences resulting from the imbalanced production of ROS and nitrogen species . Severalpression . Along wpression  demonstrd Cldn-5 . Ishola d Cldn-5 . Likewisd Cldn-5 . Yang etd Cldn-5  demonstrB/c mice . SimilarB/c mice  showed tB/c mice .Furthermore, isorhamnetin has been reported to prevent pulmonary tuberculosis . AdditioIsorhamnetin has been reported to alleviate shared metabolic complications in type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), but until the present, it is more aligned with T2DM. For example, Matboli et al. showed that isorhamnetin used at three different doses  possessed anti-diabetic action by regulating the insulin pathway at the microscopic, molecular and protein levels in the streptozotocin/high fat diet-induced T2DM rat models . AuthorsMetabolic syndrome is a constellation of cardiometabolic risk factors first described 90 years ago . It inclSeveral in vitro and in vivo studies showed that isorhamnetin might modulate carbohydrate metabolism and exhibit anti-diabetic activities by involving different mechanisms. Researchers have suggested that isorhamnetin promotes carbohydrate metabolism from the stages of digestion and intestinal absorption , improveVaccinium vitis berries, could enhance muscle cell glucose uptake [Glucose transporters (GLUT) are a large group of membrane proteins that transport glucose from epithelial cells to blood and from blood to cells passing the intestinal barrier in the gradient direction by passive transport. Some of them are insulin-dependent, notably GLUT4. It is a major insulin-regulated glucose transporter and coordinator of insulin action in adipose. In fact, it is well established that insulin, which enhances glucose uptake, also stimulates the recruitment of GLUT4 to the cell membrane of insulin-sensitive tissue. In a study underlying the anti-diabetic properties of quercetin and isorhamnetin, the authors proved that physiological concentrations of isorhamnetin promoted GLUT4 translocation to the plasma membrane in L6 myotubes through different mechanisms without altering GLUT4 expression . In facte uptake . Guided Nitraria retusa isorhamnetin-rich extract could reduce the fat accumulation in 3T3-L1 adipocytes in a dose-dependent manner [Nitraria retusa extract could regulate PPARγ and lipogenic enzymes target genes LPL and FAS [PPARs belong to the transcription nuclear receptor factors class II family with an important action in the glucose and lipid metabolism regulation . They art manner . The sin and FAS . Importa and FAS .The liver is an organ that plays a key role in carbohydrate homeostasis . Beyond Fatty liver disease or non-alcoholic steatohepatitis (NASH) represents a potential hepatic complication. It is characterized as an abnormal accumulation of TGs inside liver cells. In type 2 diabetes, insulin resistance leads to the accumulation of toxic fatty acids. When this accumulation occurs in liver cells it causes non-alcoholic fatty liver disease (NAFLD) and in severe forms steatosis or NASH ,114. Ganα-SMA, PAI-1, and COL1A1 [Oenanthe javanica, could reduce fibrosis in rat HSCs by blocking the extracellular signal regulated kinase (ERK) signaling pathway and inhibiting the proliferation and collagen synthesis of HSC-T6 cells [Hepatic fibrosis is excessive scarring resulting from the build-up of connective tissue in the liver. It is caused by excessive production and/or insufficient degradation of extracellular matrix. There are many causes of liver fibrosis. Among them is NASH origin. When there is resistance to insulin, fatty acids build up in the liver, causing toxicity and inflammation. This is accompanied by the production of cytokines and reactive oxygen species. In fibrosis, cytokines are responsible for the activation of hepatic stellate cells (HSCs) and will activate an excessive production of collagen fibers ,116. Thed COL1A1 . In factd COL1A1 . LikewisT6 cells . In factHippophae rhamnoides L., showed potent anti-tumor activity in hepatocellular carcinoma BEL-7402 cells, exerting strong cytotoxicity with IC50 at the concentration of 74.4 ± 1.13 µg/mL and 72 h incubation [2/M phase via activating the ataxia-telangiectasia mutated Chk2 pathway [Cleome droserifolia, reduced HepG2, a liver hepatocellular carcinoma cell, cell viability and anchorage-independent cell growth in a dose- and time-dependent manner but with a highest effect with the quercetin derivative. Furthermore, they exhibited a reduced migration capacity of HepG2 cells [Hepatocellular carcinoma is often discovered late at the metastasis stage (in 64% of cases); the most frequent secondary locations are the lung, lymph nodes, kidneys, and adrenal glands . As mentcubation . Cytotox pathway . In anotG2 cells .Vernonia anthelmintica herb, could suppress the proliferation of pancreatic cancer cells  via arresting the cell cycle in the S phase, which may be an alternative way to prevent pancreatic carcinoma and its metabolic complications [Salicornia herbacea plant) could promote glucose-stimulated insulin secretion in insulin-secreting rat insulinoma (INS-1) pancreatic β-cells without affecting cell viability [Insulin is a hormone naturally secreted by the body that helps our cells, particularly muscle cells, liver cells, adipose cells, and brain cells, absorb glucose from food to be converted into energy for all biological processes and functions ,126. In ications . Consistiability . Moreoveiability . The enhCastanea sativa plant species has a protective effect on streptozotocin-induced oxidative damage and β-cell apoptotic death [Furthermore, Grdović et al. reported that isorhamnetin originated from the ic death . This beic death . In the ic death . It coulic death .NF-κB is a protein of the superfamily of transcription factors involved in the immune and cellular stress responses and is considered as the major mediator of the secretion of inflammatory markers such as cytokines, chemokines, and different key enzymes ,134,135.We have established a stem cell-based tool using a perinatal stem cell, human amniotic epithelial cells (hAECs), to evaluate the bioactivities of natural compounds employing whole-genome microarray analysis ,143,144.It is worth noting that upon appropriate differentiation protocol, hAECs can be differentiated into hepatocyte-like cells ,154,155,n = 3) and isorhamnetin-treated (n = 3) hAECs. The cells were grown on 3D cell culture. Control cells were maintained in placental basal epithelial cell medium  in absence of any differentiation medium or growth factors, whereas treatment cells were supplemented with 20 mM of isorhamnetin  for 10 days. Differentially expressed genes (DEGs) are referred to as genes with a linear fold change > 2 and p-value < 0.05 (one-way between-subjects ANOVA). A total of 303 DEGs were identified; among them, 60 were upregulated and 243 were downregulated. Details of methodology have been explained elsewhere [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE153149, accessed on 24 November 2021).Considering the complex pathophysiology of DM, hAEC may not be an ideal in vitro model to study the anti-diabetic effects of compounds. However, because of its stem cell-like properties, it can be used for the initial screening of target compounds. We have previously explored antifibrotic  and hepalsewhere ,170. AllIn our previous studies on hAECs, we found that different types of compounds could direct the differentiation of hAECs towards different cell lineages, such as a caffeic acid ester, rosmarinic acid  and a cahttps://software.broadinstitute.org/gsea/index.jsp; accessed on 26 November 2021) [We examined the significantly enriched cell type signature data sets using the Molecular Signatures Database (MSigDB) ver. 7.4 of GSEA online software (er 2021) . These g+fibro-adipogenic progenitor (FBN1+FAP) cells, fibro-adipogenic progenitor (FAP) cells, and skeletal muscle pericytes [p-value after Benjamini and Hochberg correction for multiple hypothesis testing .We found that the most significantly enriched cell type gene set was pancreatic mesenchymal stromal cells  Figure A. Additiericytes . BiologiNext, we examined the significantly enriched hallmark gene sets on MSigDB (retrieved on 26 November 2021). Hallmark gene sets represent specific, well-defined biological states or processes generated based on identifying gene set overlaps and retaining genes that display coherent expression. The hallmarks have a collection of 50 gene sets condensed from over 4000 overlapping gene sets and thus have reduced noise and redundancy .Significantly enriched hallmark gene sets include genes defining epithelial-mesenchymal transition, genes upregulated in response to hypoxia, genes regulated by NF-kB in response to TNF, genes up and downregulated by KRAS activation, genes mediating apoptosis by activation of caspases, genes involved in myogenesis, genes upregulated in response to TGF-β1, genes upregulated by STAT5 in response to IL-2 stimulation, genes defining inflammatory response, genes involved in p53 pathways and networks, and genes encoding proteins involved in glycolysis and gluconeogenesis B. SignifMMP9, TSPAN1, and ITGBL1. Hyperglycemia triggers genomic instability leading to KRAS mutations in pancreatic cells [An interesting finding is enrichment of KRAS activation by the DEGs of the isorhamnetin-treated hAECs. Several KRAS-induced gene expressions were found to be significantly downregulated by isorhamnetin, such as ic cells  and has ic cells  and coloic cells . Wang etic cells . Therefohttp://ctdbase.org/; accessed on 29 November 2021) [Further pathway analysis of the DEGs was conducted using the Comparative Toxicogenomics Database (CTD) (er 2021) . CTD repCOL1A1, COL1A2, COL4A6, FN1, MMP2, and SERPINE1. As discussed in the earlier section, isorhamnetin’s antifibrotic effects have been well documented [Advanced glycation end products (AGEs) are produced through the non-enzymatic glycation and oxidation of proteins, lipids, and nucleic acids. The receptors for advanced glycation end products (RAGE) belong to the immunoglobulin superfamily. AGE/RAGE signaling is a complex and intricate cascade that activates multiple intracellular signal pathways involving protein kinase C, NADPH oxidase, and MAPKs, resulting in NF-κB -induced expression of IL-1, IL-6, TNF-α, VCAM-1, and VEGF. Particularly, AGE/RAGE signaling has been implicated in diabetes-mediated vascular calcification through activation of TGF-β mediated fibrosis, NFκB, and ERK1/2 pathways ,183,184.cumented ,99,119, Curated gene–disease association data were retrieved from the CTD (retrieved on 29 November 2021). We curated only significantly enriched metabolic diseases. The significance of enrichment was calculated by the hypergeometric distribution adjusted by the Bonferroni method. Significantly enriched metabolic diseases included DM, glucose and lipid metabolism disorders, hyperglycemia, and obesity D. HeatmaOur targeted microarray data analysis of isorhamnetin-treated hAECs also confirmed the potential of isorhamnetin in regulating biological functions related to DM and its associated complications.With the presence of various categories of flavonoids in nature, it is interesting to analyze the presence of these compounds in the body. When we talk about the metabolism of isorhamnetin and its availability in the human body after consumption, we are talking about the origin, the metabolism and the transport. This is based on an observation made by chromatography of some flavonoids such as flavonols in human serum. Liquid chromatography-mass spectrometry provides insight into the bioavailability of certain flavonoids in their aglycone and glycoside forms in the body . Mass spSeveral factors play a role in the entry of nutrients through the digestive tract. For example, enzymes from the intestinal microbiota affect the entry of phenolic compounds. A study carried out on the ginkgo leaf extracts in a mice model showed the importance of gut microbiota on the bioavailability and absorption from the gastrointestinal tract of some bioactive molecules, especially isorhamnetin . In thisOn the other hand, different membrane transporters control the transport of flavonoids, such as the sodium-dependent glucose transporter 1 (SGLT1) and the multidrug resistance-associated proteins 2 and 3 (MRP2 and MRP3) . In thisIsorhamnetin is a phenolic compound of the flavonoid family, more precisely of the flavonols. Originally it is a quercetin molecule but has undergone methylation. Isorhamnetin is distributed in the plant kingdom in many wild and cosmetic plants. In addition, several medicinal plants produce this molecule and several studies have attested to its anti-diabetic effect among other biological activities. All these data thus show the interest of isorhamnetin in the therapeutic industry. From this perspective, it would be very interesting to explore the effect of isorhamnetin and its derivatives, isolated especially from natural resources, on metabolic disorders. It is also necessary to highlight and review the clinical studies performed in this context using flavonoid-rich fractions and natural products in order to avoid the impact of side effects caused by synthetic and chemical drugs.