The study aimed to explore the active composition and mechanism of the Chaiqin Qingning capsule (CQQN) against pharyngitis based on the network pharmacology and through using a pharyngitis rat model.  The active ingredients and targets of CQQN were queried using the TCMSP database. Disease-related target genes were queried in the DrugBank, GeneCards, OMIM, and DisGeNEt databases using “pharyngitis” as the search term. The STRING database was used to establish a protein-protein interaction (PPI) network. GO function enrichment and KEGG pathway enrichment analyses were performed to identify active components and key targets. Cytoscape software (version 3.7.2) was used to construct an active component/target gene/enrichment pathway network. AutoDock software was used to select the best binding target for molecular docking. The effect of CQQN was verified in the pharyngitis rats. β. Signaling pathways included virus infection, TNF, IL-17, and cancer pathways. The molecular docking results showed that the critical components in CQQN had good potential for binding to key target genes. Animal experiments showed that CQQN could significantly reduce the expression of TNF-α, IL-1β, IL-6, and IL-17 in the serum of rats with pharyngitis (P < 0.05). Hematoxylin and eosin staining showed that the inflammatory state of pharyngeal tissue in rats was significantly reduced compared to that in the model group.  Network pharmacology showed 30 active compounds in CQQN with 240 targets, including 54 for the treatment of pharyngitis. Potential active ingredients included quercetin, kaempferol, stigmasterol, saikosaponin D, and isorhamnetin. The key targets involved were AKT1, TNF, IL-6, and IL-1 CQQN can improve pharyngitis by regulating the TNF and IL-17 signaling pathways. The study makes a positive exploration and provides a new idea for a more comprehensive and in-depth excavation of CQQN with an intervention effect on pharyngitis and other upper respiratory diseases in the future. Pharyngitis is usually caused by various viral or bacterial infections. The virus is the most common cause, accounting for about 40%-60% of cases. The second cause is a bacterial infection, accounting for about one-third of children's pharyngitis, but only about 5%-10% of that in adults [Pharyngitis is inflammation of pharyngeal mucosa and surrounding lymphoid tissue, characterized by congestion and edema . The main adults , 3. SignChaiqin Qingning capsule (CQQN) is a Chinese patent medicine preparation composed of Radix Bupleuri, Baicalin, and Artificial Calculus Bovis. Radix Bupleuri has antipyretic, anti-inflammatory, immune enhancement, antibacterial, and antitumor pharmacological effects. Baicalin has antipyretic, antibacterial, anti-inflammatory, antiviral, antiendotoxin, antioxidant, and antitumor effects. Artificial Calculus Bovis has the pharmacological effects of clearing away heat, detoxifying, and regulating immunity . CQQN isThe clinical effect of traditional Chinese medicine (TCM) results from the comprehensive action between its active component group and the complex biological network of the body. Integration analysis between the active component group and the target group of the body is a challenge for studying the pharmacological mechanism of TCM , 11. ThrThis study aimed to explore the intervention mechanism of CQQN in pharyngitis by network pharmacology and to verify the mechanism in a rat pharyngitis model. The results can provide references for follow-up research.CQQN capsules were purchased from Yangzijiang Pharmaceutical Group Co., Ltd. (China), batch number 21022312. Ammonia water was purchased from Nanjing Chemical Reagent Co., Ltd. (China), batch number 200303196X. The enzyme-linked immunosorbent assay (ELISA) kit was purchased from Jiangsu Enzyme Immunoassay Industry Co., Ltd. (China), batch number 202106.The rats were purchased from Hangzhou Medical College, animal license number SCXK (Zhejiang) 2019-0002 and certificate number 20210628Aazz0100000604. The rats were raised under controlled humidity (55-65%), temperature (22 ± 2°C), and daily light intensity (12-h/12-h light/dark cycles). The rats had free access to water and standard diets. The experimental protocols were approved by the Committee on Laboratory Animal Care of China Pharmaceutical University, and all animals were treated with humane care according to the National Institutes of Health (USA) guidelines.http://lsp.nwu.edu.cn/tcmsp.php/), SwissTargetPrediction (http://www.swisstargetprediction.ch/), DrugBank database (https://go.drugbank.com/), GeneCards database (https://www.genecards.org/), OMIM database (https://www.omim.org/), DisGent database (https://www.disgenet.org/home/), UniProt database (http://www.uniprot.org/), PubChem database (http://pubchem.ncbi.nlm.nih.gov/), STRING database (https://string-db.org/), protein structure database (http://www.rscb.org/), Cytoscape (version3.7.2), ChemBioDraw (version 14.0), Venny plot production platform (http://bioinformatics.psb.ugent.be/webtools/Venn/), PyMOL software, AutoDockTools 1.5.6 software, and R 4.1.0 software were used.TCMSP platform  ≥30% and drug-likeness (DL) ≥0.18 were used as screening criteria for active compounds, and related target genes were collected . The staThe relevant pharyngitis targets were queried in the DisGeNET, GeneCards, OMIM, and the DrugBank databases, and duplicate targets were deleted after collection. The remaining targets were used as pharyngitis disease targets.The potential targets of CQQN on pharyngitis were obtained by matching the target genes of the active ingredients with the target genes related to pharyngitis. The Venny map was used to draw the intersection map of the active drug component and disease targets.The protein-protein interaction (PPI) network between targets was created by selecting “Multiple proteins” in the STRING database interface, copying the intersection targets into “List of Names,” and selecting “Homo sapiens” in “Organism.” Other parameters remain as default. The obtained PPI network was imported into the Cytoscape software and visualized using the network analyzer function.http://org.Hs.eg.db) that includes “DOSE,” “clusterProfiler,” and “pathview” was used to perform the Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on the common target of CQQN and pharyngitis. According to P < 0.05, the top ten biological processes (BP), molecular functions (MF), cellular components (CC), and the top 20 KEGG pathways were selected. Subsequently, experimental data such as active ingredients, targets, and the top 20 KEGG pathways were imported into the Cytoscape software to construct a network of “active ingredient/target/enrichment pathways.”R packages including “colorspace” and “string” were installed. A Bioconductor package , the medium-dose , and the large-dose  CQQN groups. Each CQQN group was administered CQQN once a day for five days. The blank and model groups received an equal volume of 0.9% sodium chloride (NS) solution. After the last administration of CQQN or 0.9% NS, the pharyngeal mucosa of the animals was observed for quantitative rating evaluation. The pharyngeal scoring standard was based on the color of the pharynx tissue, secretion, degree of hyperemia, and swelling. The following four grades were assigned: grade (–): the rat's pharyngeal tissue was light red, moist, smooth with no congestion; grade (+): the rat's pharyngeal tissue was close to grade (-), but there were visible mild chronic congestion, poor mucosal gloss, and pharyngeal secretions; grade (++): the pharyngeal tissue of rats showed chronic congestion, dark red, and mild swelling with a small amount of secretion; and grade (+++): the pharyngeal tissue was in a state of chronic hyperemia, dark red, increased mucus secretion, and noticeable swelling .β, IL-6, TNF-α, and IL-17 were measured by ELISA according to the kit instructions.Upon the completion of CQQN or 0.9% NS administration, the serum of rats was taken, and the blood samples were placed at 4°C for 30 min. Serum was separated by 3000 rpm centrifugation for 10 min. Serum levels of IL-1Pharyngeal mucosa and submucosa were extracted and fixed with 10% formalin solution. After hematoxylin and eosin (HE) staining, the histomorphological changes were observed under a light microscope.P < 0.05 was considered statistically significant, and P < 0.01 indicated that the difference was extremely significant.Excel was used for data entry, SPSS 25.0 software for data analysis, and GraphPad Prism 9.0 was used for mapping. Measurement data were expressed as mean ± standard deviation (mean ± SD). One-way analysis of variance was used for pairwise comparison between groups. A total of 374 ingredients were obtained from the TCMSP database . The active compounds were then screened with DL ≥0.18 and OB ≥30% as the condition. Saikosaponins A, B, C, and D were reported to be the main active components of Radix Bupleuri [With the keywords “pharyngitis,” 46 pharyngitis-related targets were found in the DrugBank database, 536 in the GeneCards database, 38 in the OMIM database, and 30 in the DisGent database. After removing duplicate targets, 618 pharyngitis targets were obtained , including 1,506 BP items, 14 CC items, and 70 MF items. BP is mainly related to the response to reactive oxygen species (ROS) in the metabolic process, the response to drugs, the regulation of ROS, and the T cell activation. CC is related to the membrane raft, the side of the membrane, and the outer membrane. MF is primarily related to binding cytokines, protein phosphatase, kinase, and tumor necrosis factor (TNF) receptor superfamily . KEGG analysis showed that the relevant CQQN signaling pathways in the treatment of pharyngitis included virus infection, TNF, IL-17, and cancer pathways  were imported into the Cytoscape software to construct a network diagram of active components/target/enrichment pathways. There were 93 nodes  and 347 interacting edges in the network, as shown in The active ingredients of CQQN, the potential targets for treating pharyngitis, and the top 20 KEGG-enriched pathways  chromone), three saponins , one sterol component (stigmasterol), one lignan component (cubebin), and a steroid compound (chenodeoxycholic acid). The results show that these compounds may be the key components of CQQN in the treatment of pharyngitis, as shown in β, CASP3, PTGS2, VEGFA, CXCL8 STAT3, MMP9, HIF1A, IL10, and MYC  for each pair of molecular docking. It is generally believed that binding energy of less than -4.25 kcal/mol indicates that the ligand has a specific binding activity to the receptor, less than -5.0 kcal/mol has better binding activity, and less than -7.0 kcal/mol has strong binding activity.β, and AKT1) were selected for the analysis of the binding mode. As shown in The ten active pharmaceutical ingredients were bound to 14 target proteins (TP50 was deleted because the ligand structure could not be found), and the data obtained were analyzed by a heat map, as shown in All rats in the blank group were in good physical condition, had bright fur color, and had normal eating and drinking patterns. After modeling, all rats had swelling and congestion of the pharynx, and oral secretions gradually increased. Some rats had local ulceration of the mouth and lips, and superficial ulcers were formed in the pharynx. After continuous administration of CQQN for five days, the redness and swelling of the small-, medium-, and large-dose groups of rats in the CQQN were reduced to varying degrees. The amount of secretion was significantly reduced compared to that of the model group .In the structure of the blank group, the rat pharynx tissue and the submucosal glands were normal, and there were no inflammatory cells. In the structure of the model group, the rat pharynx tissue changed with the congested mucosal layer of blood vessels. The submucosal layer of connective tissue proliferated, and many inflammatory cells infiltrated. The pharyngeal mucosa of rats in the high-dose CQQN group was basically restored to normal, and the submucosal inflammatory cells and mucosal glands were significantly reduced. In the medium-dose CQQN group, a small number of inflammatory cells were observed in the mucosa and submucosa in the pharyngeal tissue. In the small-dose CQQN group, the pharyngeal mucosa of rats became thinner. Inflammatory cells were significantly reduced, and the submucosal mucosal glands were basically normal, as shown in P < 0.01). Compared to the model group, the pharyngeal tissues of the CQQN small-, medium-, and large-dose groups improved significantly (P < 0.01), as shown in The histopathological grade of the pharynx in the model group was significantly higher than that of the blank group . Compared to the model group, the CQQN large-dose group significantly reduced the levels of IL-6, TNF-α, and IL-1β (P < 0.01) and significantly reduced the level of IL-17 (P < 0.05). The medium-dose CQQN group significantly reduced the levels of IL-1β and IL-6 (P < 0.01) and significantly reduced the TNF-α level (P < 0.05), as shown in Compared to the blank group, the levels of TNF-Pharyngitis is an inflammation of the upper respiratory tract. Sore throat and dysphagia in the disease process will seriously affect patients' daily lives and even cause serious complications, such as rheumatic heart disease, glomerulonephritis after streptococcal infection, sepsis, or autoimmune diseases . CQQN isα and NO [α production in macrophages [α in bronchoalveolar lavage fluid, and reduce the reaction of oxidative stress [κB-α and p65 and inhibit inflammation in vitro and in vivo by regulating the MAPK and NF-κB pathways. NF-κB, which is involved in the immune response and the inflammatory response, is a familiar molecular target for the treatment of pharyngitis [α, IL-6, IL-1β, iNOS, and COX-2) and increase the expression of anti-inflammatory cytokines (IL-10) through downregulating the expression of NF-kBp65 (inhibiting p-IKB-α activation) and p38MAPK [κB pathways induced by TNF-α [Network pharmacology results show that quercetin, kaempferol, stigmasterol, saikosaponin A, saikosaponin C, saikosaponin D, isorhamnetin, and other ingredients play a vital role in the treatment of pharyngitis by CQQN. Quercetin is a flavonoid compound. It is considered the most effective ROS scavenger and inhibits the production of several proinflammatory factors, such as TNF-α and NO . In vitrα and NO . Severalrophages , 26 and rophages . Kaempfee stress . Kaempferyngitis . Saikosaryngitis . Stigmas p38MAPK . Isorhamby TNF-α . The aboα is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation and is responsible for a wide range of signaling events within cells, leading to necrosis or apoptosis [The GO analysis result shows that the cytokine receptor binding and TNF receptor binding are at the top. It indicates that these receptors may be the primary drug targets of CQQN in the treatment of pharyngitis. TNF is a multifunctional cytokine. It plays an essential role in the pathophysiology of several diseases . TNF-α ipoptosis .α is the most important cytokine in this pathway by upregulating MAPK, ERK, NF-κB, and other signaling pathways to induce apoptosis and plays a proinflammatory role [β, IL-6, TNF-α, and other inflammatory mediators [The results of the KEGG pathway enrichment analysis show that the targets of CQQN are mainly concentrated in virus infection and TNF, IL-17, and cancer pathways. Studies have found that the TNF signaling pathway plays a key role in the inflammatory response. TNF-ory role . MAPK isediators . IL-17 iediators . IL-17 iediators . In addiediators , 40. Conβ are the predicted targets of CQQN. The molecular docking results showed that the active components of CQQN had a strong affinity with the key targets, a stable hydrogen bond at the binding site, and a stable conformation and good binding activity. It can provide a theoretical basis for CQQN treating pharyngitis at the molecular level.The PPI topological analysis shows 16 strongly associated proteins, among which AKT1, TNF, IL-6, and IL-1Through the animal experiment, we found that the pharyngeal mucosa of pharyngitis rats in each dose group improved to various degrees after CQQN was administered for five days. Under a light microscope, it was found that the pathological changes of the pharyngeal tissue in CQQN were less than that in the model group. Inflammatory cells in the submucosa were significantly reduced, and mucosal glands were also decreased.β, TNF-α, and IL-17 in the medium and large-dose CQQN groups were significantly lower than those in the model group. It is suggested that CQQN inhibits the development of inflammation by reducing the production of proinflammatory factors and mediators and blocking the transmission of proinflammatory signals and playing a role in the treatment of pharyngitis.IL-6, IL-1α, and IL1β after anorectal surgery in rats [α and IL-6 in mouse nasal mucosa tissue [α, IL-1β, and IL-6) [Saikosaponin A has been reported to reduce serum IL-6, TNF- in rats , and quea tissue . Stigmasnd IL-6) . This shα, IL-6, IL-1β, and IL-17) to inhabit the TNF and IL-17 signaling pathways. However, this study only conducted a single efficacy test through the pharyngitis rat model; more studies of CQQN need to be conducted. This study provides ideas and a basis for a more comprehensive and in-depth exploration of TCM with intervention effects on pharyngitis in the future.This study screened potential active ingredients, key targets, and CQQN pathways of CQQN in the treatment of pharyngitis through network pharmacology. The results show that CQQN can intervene in the expression of inflammatory factors through multiple components, targets, and pathways. The results of animal experiments indicate that CQQN exerts a therapeutic effect by downregulating proinflammatory cytokines (TNF-


Aidi injection (ADI) is a compound preparation injection of Chinese herbs used to treat patients of nonsmall cell lung cancer (NSCLC) in China. This study aimed to reveal the mechanism of ADI in the treatment of NSCLC by using network pharmacology and molecular docking.  The related targets of ADI and NSCLC were obtained from multiple databases. The network diagram of disease-drug-components-targets (DDCT) and protein-protein interaction (PPI) was constructed to screen key targets. Then, the key targets and main signaling pathways were screened by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Next, in order to validate the results of network pharmacology, expression analysis and survival analysis of key genes were performed. Finally, we carried out the technology of molecular docking to further validate the accuracy of the above results. p < 0.001). In lung adenocarcinoma (LUAD), the expression of TP53, CASP3, MMP9, AKT1, and PTGS2 had statistical differences compared with normal tissue (p < 0.001), while the expression of MAPK1 had no statistical difference (p > 0.05). The results of survival analysis of key genes showed that AKT1, MAPK1, CASP3, MMP9, TP53, and PTGS2 had statistical differences in the OS or RFS of NSCLC patients (p < 0.05). In addition, the results of molecular docking indicated that the key genes and the main components have good docking activity.  A total of 207 targets of ADI and 5282 targets of NSCLC were obtained finally. Through the construction of DDCT and PPI network diagrams, 28 key targets were finally obtained. The results of the KEGG enrichment analysis indicated that multiple signaling pathways were associated with NSCLC, which included the MAPK signaling pathway, the IL-17 signaling pathway, and the PI3K/AKT signaling pathway. The key genes in the signaling pathway mainly include TP53, CASP3, MMP9, AKT1, PTGS2, and MAPK1. The results of differently expressed analysis of key genes showed that TP53, CASP3, MMP9, AKT1, PTGS2, and MAPK1 had statistical differences in lung squamous cell carcinoma (LUSC) compared with normal tissue ( This study revealed the potential mechanism of ADI in the treatment of NSCLC with multipathways and multitargets and provided a scientific basis for the in-depth study of ADI in the treatment of NSCLC. Lung cancer is the leading cause of cancer-related deaths worldwide, with the 5-year survival less than 21% , easy drAidi injection (ADI) is a compound preparation injection of Chinese herbs Z52020236, CFDA), which is extracted from Mylabris (Banmao), Panax Ginseng (Renshen), Acanthopanax Senticosus (Ciwujia), and Astragali Radix (Huangqi), four traditional Chinese herbs . In rece, CFDA, wEmphasizing the holistic concept when treating diseases with TCM, as a TCM injection preparation, the function of ADI naturally depends on the mutual coordination and complementation of each Chinese herb. In order to comprehensive inquiry the effect of ADI and reveal the mechanism of ADI in the treatment of NSCLC, we employed a network pharmacology strategy. Network pharmacology is a novel discipline based on the theory of systems biology, analyzing the network of biological systems and selecting specific signaling nodes for multitargets drug molecular design . Its reshttps://tcmspw.com/tcmsp.php) database. The active ingredients of senticosus were obtained from the TCMID (https://www.megabionet.org/tcmid/) database and then imported back into the TCMSP database one by one to obtain the related targets of senticosus. Screening conditions are oral bioavailability (OB) ≥30% and drug likeness (DL) ≥0.18.The active ingredients and related targets of Panax Ginseng, Astragali Radix, and Mylabris were obtained from the TCMSP (https://omim.org/) and GeneCards Suite (https://www.genecards.org) databases, using nonsmall cell lung cancer or NSCLC as the search term. Merge all targets from the two databases and delete duplicate targets as the final target for NSCLC.The NSCLC-related targets were obtained from the OMIM (https://bioinfogp.cnb.csic.es/tools/venny/index.html).In order to obtain the intersecting targets and make a Venn diagram of ADI and NSCLC, the related targets of ADI and NSCLC obtained above were inputted into the Venny 2.1.0 (https://string-db.org). We imported the PPI linkage file into Cytoscape software to create a PPI network diagram and screened out targets with a degree and betweenness centrality (BC) greater than their mean values as the key targets for the study.Running the prepared Cyto.pl script through the Perl program to obtain the linkage network file and classification file of nodes, the active ingredient file and the file of the intersecting targets of ADI and NSCLC, and he above files were imported into Cytoscape software (version 3.7.2) to build the DDCT network diagram. The PPI linkage file was obtained by using the intersecting targets of ADI and NSCLC in the STRING database (https://david.ncifcrf.gov/) for GO and KEGG enrichment analysis, and the species selected was “Homo sapiens.” The cluster profiler package and the ggplot2 data package [p adjust <0.05. Genes in major signaling pathways were regarded as key genes for further study.The selected key targets were entered into the DAVID database  database. Then, the RNAseq data were converted in FPKM format to TPM format and log2 conversion. The R (3.6.3) software and ggplot2 package were used for statistical analysis and visualization [In order to explore the expression differences of the screened key genes in tumor tissue and normal tissue, we downloaded the Level 3 RNAseq data in HTSeq-FPKM format of key genes in LUSC and LUAD from the TCGA  was used for this study, which is a powerful online tool for assessing the correlation between the expression and survival of 30,000 genes in more than 25,000 samples from 21 tumor types [p < 0.05 was considered statistically significant.To validate the results of network pharmacology, we used a survival analysis method to investigate whether the expression of key genes had an effect on survival rate with OS  and RFS  as study indicators. The Kaplan–Meier plotter (or types . We splihttps://pubchem.ncbi.nlm.nih.gov) and the RSCB PDB database (https://www.rcsb.org/), respectively. AutoDock Vina software (https://vina.scripps.edu/) was used to perform molecular docking of key proteins and active ingredients, and the affinity value represents the binding energy of both. It is generally believed that the lower the binding energy, the more stable the binding of active ingredients and proteins [To further verify the above results, we performed molecular docking on key genes. To further verify the above results, we selected the top 10 active compounds in the DDCT network and key genes in the main signaling pathway for molecular docking. The structures of the related active compounds and key genes were obtained from PubChem  were used to perform statistical analysis on the results of enrichment analysis and gene expression differences. To compare survival curves, we used the log-rank test to calculate the HR and log-rankIn this study, network pharmacology was used to analyze the potential targets of ADI and NSCLC, and the Venn diagram was used to obtain intersecting targets for ADI and NSCLC at the same time. Next, to identify key targets, the intersecting targets of ADI and NSCLC were used to construct a network diagram of disease-drugcomponents-targets (DDCT) and protein-protein interactions (PPI). Then, we obtained the main biological functions and signaling pathways involved in the ADI treatment of NSCLC through key targets. In order to verify the targets and signaling pathways obtained in previous, we performed differential expression analysis and survival analysis of key genes in mainly signaling pathways. Finally, the molecular docking technique was used to further verify the above results. The workflow chart of this study is shown in Through screening, a total of 37 active compounds  and 207 By searching each database, combining the results of each database and removing duplicates, a total of 5282 targets of NSCLC , cellular component (CC), and molecular function (MF). A total of 1850 entries with p < 0.001)    . Compare > 0.05) . Similar > 0.05) . Convers > 0.05) . These rp < 0.05)  . Patient < 0.05) , and MAP < 0.05) . Patient < 0.05) , and sim < 0.05) . Convers < 0.05) . The resTo further verify the accuracy of the results, we performed molecular docking technique on the top 10 compounds in terms of degree in the DDCT network diagram and the top 5 key genes . As shown in NSCLC has no obvious clinical symptoms, and most NSCLC patients are in the advanced stage once diagnosed, with poor prognosis, which seriously threatens human health. In recent years, there have been more and more clinical trials and basic researches on ADI in the treatment of NSCLC, providing a new method for the treatment of NSCLC. Several studies have shown that ADI combined with conventional western medicine treatment can significantly improve clinical efficacy and reduce the occurrence of adverse reactions –16. In oWe found that the main components of ADI were quercetin, formononetin, kaempferol, beta-sitosterol, and stigmasterol by using the method of network pharmacology. Basic research showed that quercetin could trigger BCL2/BAX-mediated apoptosis, necrosis, and mitotic mutation, and inhibit the migration potential of A549 cells, showing good anti-NSCLC tumor activity . FormonoThrough the PPI network map, we found that there were 28 key targets of ADI in the treatment of NSCLC, including 6 key genes that existed in the main signaling pathways, which were AKT1, TP53, CASP3, MAPK1, MMP9, and PTGS2. AKT1 was a member of the AKT kinase family, and research showed that AKT1 plays an antimetastatic role in the NSCLC cells with KRAS or EGFR mutations . In addiα/NF-κB1 signaling pathway, and blocking CD47 could enhance anti-tumor effects [κB was widely used by eukaryotic cells as a gene regulator to control cell proliferation and cell survival, which can regulate anti-apoptotic genes and play an indispensable role in the process of apoptosis. Research showed that miR-449a suppresses tumor growth, migration, and invasion in NSCLC by targeting the HMGB1-mediated NF-κB signaling pathway [The results of KEGG enrichment analysis indicated that multiple signaling pathways were involved in the ADI treatment of NSCLC, which included the MAPK signaling pathway, the IL-17 signaling pathway, and PI3K/AKT signaling pathway, etc. Mitogen-activated protein kinase (MAPK) is an important transmitter of signals from the cell surface to the nucleus, which is a group that can be activated by different extracellular stimuli, such as cytokines, neurotransmitters, hormones, cell stress, and cell adhesion. The basic component of the MAPK pathway is a tertiary kinase pattern that is conserved from yeast to humans, including MAP kinase kinase kinase (MKKK), MAP kinase kinase (MKK), and MAPK, which can be activated in turn to jointly regulate cells growth, differentiation and response to inflammation and other important cellular physiological or pathological processes . MAPK si effects . Several effects . Therefo effects . Dieckol effects . It was  effects , 49. The pathway . VEGF wa pathway , 52. TheIt is necessary to use the method of survival analysis for the analysis of the occurrence, development, and prognosis of chronic diseases, especially malignant tumors. OS and RFS are two important reference indicators to describe the survival rate. In our study, the results of survival analysis showed that the expression of key genes in the signaling pathway had a significant impact on the OS or RFS of NSCLC patients, which verified the accuracy of the network pharmacology results, and indicated that the antitumor effect of Aidi injection was by upregulating or downregulating the expression of these key genes in tumor tissue. Molecular docking results showed that the main components of ADI and the key genes in the signaling pathway had good binding activity. It is well known that AKT1 plays an indispensable role in regulating tumor cell proliferation and apoptosis . InteresOur results revealed the mechanism of ADI in the treatment of NSCLC may be through the targets of AKT1, TP53, CASP3, MAPK1, and PTGS2, regulating the MAPK signaling pathway, PI3K-AKT signaling pathway, and TNF signaling pathway to exert its therapeutic effect. In conclusion, this study revealed the mechanism of ADI against NSCLC through multipathways and multitargets, which provided new insights for clarifying the molecular mechanism of ADI and also provided a reference for the research and development of anti-NSCLC drugs.


The targets of the active ingredients of DSY related to EndMT and atherosclerosis were obtained and used to construct a protein-protein interaction (PPI) network followed by network topology and functional enrichment analysis. Network pharmacology analysis revealed that the PI3K/AKT pathway was the principal signaling pathway of DSY against EndMT in atherosclerosis. Molecular docking simulations indicated strong binding capabilities of DSY’s bioactive ingredients toward PI3K/AKT pathway molecules. Experimentally, DSY could efficiently modify expression of signature EndMT genes and decrease expression of PI3K/AKT pathway signals including integrin αV, integrin β1, PI3K, and AKT1 in TGF-β2-treated HUVECs. LASP1, which is upstream of the PI3K/AKT pathway, had strong binding affinity to the majority of DSY’s bioactive ingredients, was induced by EndMT-promoting stimuli involving IL-1β, TGF-β2, and hypoxia, and was downregulated by DSY. Knock-down of LASP1 attenuated the expression of integrin αV, integrin β1, PI3K, AKT1 and EndMT-related genes induced by TGF-β2, and minimized the effect of DSY. Thus, our study showed that DSY potentially exerted anti-EndMT activity through the LASP1/PI3K/AKT pathway, providing a possible new therapeutic intervention for atherosclerosis.Atherosclerosis is a chronic inflammatory disease characterized by the formation of plaque and endothelial dysfunction. Under pro-inflammatory conditions, endothelial cells adopt a mesenchymal phenotype by a process called endothelial-to-mesenchymal transition (EndMT) which plays an important role in the pathogenesis of atherosclerosis. Dan-Shen-Yin (DSY) is a well-known traditional Chinese medicine used in the treatment of cardiovascular disease. However, the molecular mechanism whereby DSY mitigates atherosclerosis remains unknown. Therefore, we employed a network pharmacology-based strategy in this study to determine the therapeutic targets of DSY, and  Atherosclerosis (AS) is a progressive inflammatory disease triggered by numerous systemic risk factors, including smoking, diabetes, hyperlipidemia, hyperglycemia, hypertension and disturbed blood flow in local areas. These stimuli promote hardening and thickening of artery walls as well as the formation of plaques. Upon formation, plaques can result in intravascular thrombosis and interruption of blood flow, potentially leading to morbidity or death. Studies have shown that plaque formation is associated with the accumulation of mesenchymal cells in the arterial intima, which secrete pro-inflammatory molecules and synthesize extracellular matrix and metalloproteinases . Althougin vivo lineage tracing experiment, the endothelial-specific Cre-lox system VE-cadherin-CreERT2 or SCL-CreERT2 in combination with ApoE−/− or LDLR−/− mouse AS models were used to study the deep relationship between EndMT and AS , in which endothelial markers continue to be expressed and new mesenchymal markers are acquired. EndMT was first described during heart development and was characterized morphologically as representing a shift from a cobblestone-like shape to a spindle morphology accompanied by alterations in gene expression profiles . EndMT hT and AS . ApproxiT and AS  and FAP T and AS  after 4 T and AS , indicatT and AS , and theT and AS , suggestSalvia miltiorrhiza), sandalwood and fructus amomi (amomi fruit). As a famous traditional Chinese formula, DSY has been considered to improve blood circulation in Chinese theory and has been prescribed for centuries to treat coronary artery disease. Studies have indicated that DSY exerted protective effects against inflammation and reduced oxidative stress in a rat model of diabetic AS , sandalwood, Chuanxiong rhizoma, carthami flos, Rehmanniae radix preparata, and Angelica sinensis radix showed beneficial therapeutic effects against AS in an ApoE−/− mouse model  comes from the Shi Fang Ge Kuo and is comprised of red sage  in treating complex diseases. In this work, we used network pharmacology and experimental strategies to investigate and elucidate the mechanism of DSY’s effects on EndMT to support the rationale of using DSY in treating AS. We obtained the ingredients of DSY with the help of several databases and screened them for their EndMT and AS targets, which were acquired with multi-source databases and literature. We then set up protein-protein interaction (PPI) networks to determine the interactions among these targets and in turn performed enrichment analysis using gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). This analysis resulted in the identification of PI3K/AKT signaling as a potential target. We validated the involvement of PI3K/AKT signaling in DSY against EndMT in treating AS and found that PI3K/AKT was the downstream signaling pathway of LASP1.www.symmap.org), TCMSP(tcmsp-e.com), TCMID (http://47.100.169.139/), HERB (http://www.tcmip.cn/ETCM/). The SMILES structures of the compounds were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The parameters of oral bioavailability (OB) ≥20% in TCMSP, quantitative estimate scores ≥0.49 in ETCM (https://fafdrugs4.rpbs.univ-paris-diderot.fr/), and ADME with ‘high’ GI in SwissADME (http://www.swissadme.ch/) were designated as thresholds to screen for potential bioactive compounds. In addition, we included the ingredients not screened by the databases, but mentioned in the literature.The compounds in DSY were acquired using several databases including SymMap  (www.symg), TCMSP (tcmsp-e), TCMID  (http:///), HERB  (herb.ac/), HERB   and PubMed. Because we were unable to obtain the targets of some of the compounds from the databases or the literature, we then used SwissTargetPrediction (http://swisstargetprediction.ch/) to predict targets based on structurally similar molecules. Targets with probability ≥0.7 were retained. EndMT and AS target genes were searched from DisGeNET database (https://www.disgenet.org/), GeneCards database (https://www.genecards.org/) and DrugBank database (https://go.drugbank.com/). The overlapping genes among targets of DSY, EndMT and AS were visualized by Jvenn (http://jvenn.toulouse.inra.fr/app/example.html).The potential targets of DSY compounds were collected by manually searching the HERB database, TCMSP database, TCMID database, SymMap database, as well as from CNKI (database  (https:/database  (https:/database  (https:/by Jvenn  (http://The component-target network was constructed by Cytoscape (v3.9.1) and the degree value of each node, which reflects the importance of components or targets, was calculated by Network Analyzer. PPI data were acquired from Cytoscape plug-in BisoGenet  which inIn order to study PPIs at the system level, we then merged the PPI networks of DSY, EndMT, and AS by Cytoscape, and the Cytoscape plugin, cytoNCA , was usep value < 0.05, and the associated GO terms and KEGG pathways were ranked by adjusted p value.GO term enrichment analysis was performed using the R package clusterProfiler. Pathway enrichment gene symbols were converted to entrez ID with the R package org.Hs.eg.db (Version3.10.0). All of the targets in the networks were also subjected to KEGG pathway enrichment analysis. The significant terms and pathways were selected according to adjusted http://www.rcsb.org/pdb/). The 3D structures of the bioactive ingredients in DSY were downloaded from PubChem database and ZINC database (https://zinc12.docking.org/) (Autodock vina (v1.1.2) in Autodock tools (v1.5.7)  was usedng.org/) . The proSalvia miltiorrhiza was bought from Kangmei Pharmaceutical Co.,Ltd., (#210800301), Fructus amomi was purchased from Sinopharm Holding Shenzhen Medicinal Materials Co., Ltd.(#210502), sandalwood was obtained from Lingnan Traditional Chinese Herbal Medicine Co., Ltd. (# 2006001). In total, 200 g of Salvia miltiorrhiza, 30 g fructus amomi and 30 g of sandalwood  were extracted in 500 ml of ethyl acetate for 1 h with heating and refluxing. The extract was collected into a clean beaker and filtered. Afterwards, the extract was placed in an ultra-clean tube and evaporated to dryness. The dried powder was dissolved in DMSO and stored at -20°C for later usage. The stability of the product was verified by high performance liquid chromatography (HPLC) (y (HPLC) .2 at 37°C. Human umbilical vein smooth muscle cells (HVSMCs) were obtained from Otwo Biotech (#HTX2305) and maintained in DMEM supplemented with 10% FBS and 1% P/S. To establish the EndMT model, HUVECs were stimulated with 10 ng/ml TGF-β2  in conditioned medium supplemented with 2.5% FBS for four consecutive days. For DSY application, HUVECs pretreated with TGF-β2 for 2 days were incubated with different doses of DSY for a further 2 days.Human umbilical vein endothelial cells (HUVECs) were purchased from Cyagen (#HUVEC-2001). The HUVECs were cultured in a 1% gelatin coated plate in complete endothelial cell medium  containing 5% FBS, and endothelial growth factor and were maintained in a humidified chamber with 5% COLASP1 siRNAs  and non-targeting siRNA (siNT: UUCUCCGAACGUGUCACGU) were obtained from Sangon Biotech. A total of 25–100 nM of specific or siNT was introduced into the cells using RNATransMate  according to the manufacturer’s recommendations.Salvia miltiorrhiza, sandalwood and fructus amomi were added for an additional 2 days. For IC50 analysis, cells were treated with different doses of DSY for 1 day, then 10 μl of CCK-8 assay reagent  was added to each well. The absorbance at 450 nm was measured using a spectrophotometer. Linear-regression analysis in Graphpad Prism 7 was performed to calculate IC50 value.HUVECs were plated in 96-well plates in complete ECM medium and cultured overnight. The following day, cells were changed to ECM medium containing 2.5% FBS and were supplemented with 10 ng/ml TGF-β2 for 1 day. Then different doses of DSY as well as −ΔΔCT.HUVECs were homogenized in Trizol reagent (Thermo-Fisher Scientific) and total RNA was prepared using the EZ-press RNA purification Kit  according to the manufacturer’s procedure. Reverse transcription was performed using the Color Reverse transcription kit  following the manufacture’s instructions. RT-qPCR was conducted using 2xColor SYBR green qPCR mix  on a C1000 thermocycler (Bio Rad), and measurements were normalized to the expression of the housekeeping gene GAPDH. The relevant primers are shown in Beyotime) containing 1x proteinase inhibitor cocktail and 1x phosphatase inhibitor cocktail. The determination of protein concentration was performed by Bradford protein assay. Equal amounts of protein were loaded and separated by 10% polyacrylamide gel electrophoresis, and proteins were transferred to a PVDF membrane by wet electroblotting. The membrane was incubated with anti-SM22α , anti-LASP1 , anti-AKT1 , anti-phospho-AKT (Ser473) , and anti-β-actin  at 4°C overnight, followed by washing and incubation with secondary anti-mouse, -goat, and -rabbit antibodies . Immunoreactive bands were detected.Cells were rinsed with cold PBS and lysed in SDS buffer  for 10 min at 4°C. After three washes, cells were incubated with rabbit anti-SM22α (1:50), rabbit anti LASP1 (1:50) and goat anti-VE-cadherin  overnight at 4°C. After four washes, samples were further incubated with appropriate AlexaFluor-coupled secondary antibodies  for 1 h at RT. Cells were then washed and mounted in mounting medium .p value less than 0.05 was considered to be significant. Data are shown as the mean ± SEM from at least three independent experiments.Statistical analyses were carried out using GraphPad Prism 7 software . The differences among more than two groups were compared using one-way ANOVA followed by Tukey’s post-hoc test. A Salvia miltiorrhiza, 97 compounds from fructus amomi and 38 compounds from sandalwood were extracted from SymMap, TCMSP, HERB, TCMID and ETCM databases. In addition, another four components from Salvia miltiorrhiza including caryophyllene oxide, rosmarinic acid, ailanthoidol, salvianolic acid L as well as three ingredients in fructus amomi including α-copaene, 4-methoxycinnamic acid, and 4-hydroxycinnamic acid were retrieved from the literature and included for subsequent analysis. A total of 227 ingredients were identified after merging and removing the duplicates. A detailed list of DSY ingredients is given in A total of 113 compounds from Salvia miltiorrhiza, fructus amomi, and sandalwood, 3478, 3968, and 353 targets were identified, respectively , protein complexes with DNA , and lumen organization   . KEGG ene lumen) .As shown in Salvia miltiorrhiza, 30 g of sandalwood, and 30 g of fructus amomi were extracted with ethyl acetate, yielding 1.4043 g of extract. The stability of the DSY extract was determined by HPLC  is a specific focal adhesion protein that is induced under inflammatory conditions and in a variety of tumors . It has ds LASP1 . Furtherds LASP1 . DSY decds LASP1 . As expeds LASP1  and downds LASP1 . But therivation . Taken tAs the most common cardiovascular disease, AS gives rise to high morbidity and mortality worldwide. The endothelial lining on the inner surface of blood vessels has recently been determined to be transformed into mesenchymal cells through EndMT in response to various stimuli including growth factors, cytokines, hypoxia, and oscillatory flow, which contributes to the hardening and thickening of the arterial wall and plaque formation . A substSalvia miltiorrhiza, fructus amomi, and sandalwood, is a famous traditional Chinese formula for treating cardiovascular disease. Pharmacological studies revealed that DSY possessed protective effects against acute ischemic myocardial injury, and diabetic AS by its anti-inflammatory and antioxidant activities (−/− mouse model (DSY, which is composed of tivities ; but, whtivities . The PI3tivities , and thetivities . Blockinse model . Therefose model . During se model . In the The remaining question was how the PI3K/AKT pathway was being regulated. Many papers had reported that the PI3K/AKT pathway was activated by LASP1 in many types of cancer cells . LASP1 iSalvia miltiorrhiza was the main active herb in DSY that exhibited inhibitory activity against EndMT, in agreement with the results from network pharmacology analysis that the majority of bioactive ingredients were from Salvia miltiorrhiza. Our work highlights the importance of Salvia miltiorrhizain in treating AS, which has been suggested in other works (Salvia miltiorrhiza regulated EndMT. Also, the effects and mechanisms of the bioactive ingredients in Salvia miltiorrhiza have to be characterized. Furthermore, we found that DSY could strongly decrease mesenchymal specific genes while moderately upregulating endothelial genes, suggesting their strong ability to inhibit the transition to the mesenchymal state, while preserving a relatively refined role in recovering the endothelial function. In future, more work has to be conducted to study the anti-AS effect of DSY in combination with other compounds with pro-endothelial activity.TCM is a promising method for the treatment of cardiovascular disease due to its use of multiple compounds with multiple targets. We provide network pharmacology and molecular biology evidence that DSY regulates EndMT in AS mainly through the LASP1/PI3K/AKT pathway. In this work, experimental analysis uncovered that er works , but mor


PXR-CYP3A4 expression of cluster 2 caused DILI through inflammation-drug interaction and drug-drug interactions among pharmaco-immunomodulatory agents, including tocilizumab, glucocorticoids , and ritonavir. NRF2 of cluster 3 and HLA targets of cluster four promoted DILI, being related to ritonavir/glucocorticoids and clavulanate/vancomycin. This study showed the pivotal role of PXR associated with inflammation-drug and drug-drug interactions on DILI and highlighted the cautious clinical decision-making for pharmacotherapy to avoid DILI in the treatment of COVID-19 patients.Of the patients infected with coronavirus disease 2019 (COVID-19), approximately 14–53% developed liver injury resulting in poor outcomes. Drug-induced liver injury (DILI) is the primary cause of liver injury in COVID-19 patients. In this study, we elucidated liver injury mechanism induced by drugs of pharmacologic treatments against SARS-CoV-2 (DPTS) using bioinformatics and systems biology. Totally, 1209 genes directly related to 216 DPTS (DPTSGs) were genes encoding pharmacokinetics and therapeutic targets of DPTS and enriched in the pathways related to drug metabolism of CYP450s, pregnane X receptor (PXR), and COVID-19 adverse outcome. A network, constructed by 110 candidate targets which were the shared part of DPTSGs and 445 DILI targets, identified 49 key targets and four Molecular Complex Detection clusters. Enrichment results revealed that the 4 clusters were related to inflammatory responses, CYP450s regulated by PXR, NRF2-regualted oxidative stress, and HLA-related adaptive immunity respectively. In  Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuing to spread globally. This disease resulted in thousands of hospitalizations and deaths . As per In addition, drugs of pharmacologic treatments against SARS-CoV-2 (DPTS) are repurposed and off-label used in combination due to the lack of standard guideline for COVID-19 management . Pharmacp < 0.01) (Accumulating evidences  have sho < 0.01) . Moreove < 0.01) . Current < 0.01) . HoweverIn this present study, we used publicly available data to comprehensively screen DPTS and genes encoding their therapeutic and PK targets (DMEs and drug transporters) named as genes that were directly related to DPTS (DPTSGs). DPTSGs were analyzed by functional enrichment and matched with DILI targets to identify candidate targets of DPTS-induced liver injury to establish regulatory network. Further MOCDE clusters analysis of candidate targets was conducted to elucidate the molecular mechanism underlying the DILI in COVID-19 patients .www.covid19-druginteractions.org) (The off-label drugs against SARS-CoV-2 were extracted from the list of COVID-19 agent section on the University of Liverpool COVID-19 drug interaction website (ons.org) , the guihttps://go.drugbank.com/) (https://www.pharmgkb.org/) (https://pubchem.ncbi.nlm.nih.gov/) (http://ctdbase.org/)  , “Variankb.org/) , and “Biih.gov/) . The DILse.org/) .https://biit.cs.ut.ee/gprofiler/gost)  were identified. The enrichments were visualized using a dot plot using the ggplot2 package in R software.The DPTSGs were functionally enriched in Gene Ontology (GO) , Kyoto Encyclopedia of Genes and Genomes (KEGG), and Wikipathway (WP) enrichment pathways using go: profiler (er/gost)  to analyer/gost) . The sighttps://cn.string-db.org/)  (clusters) with the high clustering scores, we conducted Molecular Complex Detection (MCODE) cluster analysis of candidate targets using the Cytoscape plugin MCODE clustering algorithm  (The candidate targets underlying DPTS-induced liver injury were obtained by matching DPTSGs and DILI targets. The protein-protein interaction (PPI) network of candidate targets was constructed. The PPI data with medium confidence interaction score >0.4 were downloaded from the STRING database (db.org/) , and then 3.7.2) . The node = 100) . The higclusters have a higher risk of DILI. Moreover, drugs with log p ≥ 3 and a daily dose ≥100 mg were associated with a significantly increased risk of DILI (p < 0.05)  were extracted from the “Chemical and Physical Properties” section in the Pubchem database (https://pubchem.ncbi.nlm.nih.gov/) (https://go.drugbank.com/) (https://www.ncbi.nlm.nih.gov/books/NBK548710/) (Drugs that relate to the key targets and significant pathways in each MOCDE  < 0.05) . Informaih.gov/) . The metnk.com/)  and Live548710/) , respectA total of 216 drugs, including antiviral drugs for SARS-CoV-2, pharmaco-immunomodulatory drugs, anticoagulants, anti-platelet drugs, and antibiotics against bacterial co-infections with SARS-CoV-2 in community and ward, were DPTS and listed in 10 p values were shown in p = 2.16 × 10–218) confirmed that DPTSGs mainly included the PK targets of DPTS . As per the main KEGG pathway - “Drug metabolism - cytochrome P450” (p = 1.60 × 10–14), CYP450s were the main DMEs of DPTS. Furthermore, the primary WP pathway of “Nuclear Receptors Meta-Pathway” (p = 1.21 × 10–23) suggested that PK targets of DPTS were regulated by nuclear receptors, including PXR/NR1I2 (WP2876: Pregnane X Receptor pathway with p = 6.38 × 10–11). Above enrichment analysis results of DPTSGs indicated the DILI of DPTS associated with PXR-mediated CYP450s DM and the necessity of further analysis for DPTS-induced liver injury. ACE2, AGT, CCL2, CCL3, CSF3, CXCL10, CXCL8, IL10, IL1B, IL2, IL2RA, IL6, TMPRSS2, and TNF were the genes related to the “COVID-19 adverse outcome pathway” [WP4891 with p = 3.27 × 10–11 and genes ratio of 93.3% (14/15)]. The genes, including IL6, IL1B, and TNF, encode inflammatory cytokines of CRS in COVID-19. They were also the therapeutic targets of pharmaco-immunomodulatory drugs.We obtained 1209 DPTSGs after deleting the duplicate genes . GO, KEGcluster 1 with the highest MCODE score of 22.08 contained the top ten key targets with high connectivity in the PPI network (cluster 2 sub-network (MCODE score = 8.421), nuclear receptors of NR1I2 (PXR) and PPARA and their downstream CYP3A4 and ABCB1 were included . The PPI network had 104 nodes (after removing six isolated candidate targets) and 1914 edges, with an average degree of 18.4 . A total network . In the included . The essincluded . The tra-network . Identifeactions .cluster 1 revealed that this primary sub-network was associated with “cytokine activity” (p = 9.12 × 10–21), “Cytokines and Inflammatory Response” (p = 5.78 × 10–13), and “COVID-19 adverse outcome pathway” , “Pregnane X Receptor pathway” (p = 2.10 × 10–15), “Nuclear Receptors in Lipid Metabolism and Toxicity” (p = 2.10 × 10–15), and “Drug metabolism - cytochrome P450”  metabolism. HLA-B, HLA-DRB1, HLA-A, and HLA-DQB1 of cluster four were enriched in the MHC protein complex and involved in the regulation of adaptive immune response.Enrichment results of cluster 1. These drugs included the specific IL-6 antagonist of tocilizumab, non-specific agents (corticosteroids and interferons), macrolides, stains, and hydroxychloroquine/chloroquine. Additionally, immune-suppressive agents and some antibiotics also elicited anti-inflammatory properties  suppressed Nrf2 which regulated cellular antioxidant activity. Colchicine, vitamin D, simvastatin, estradiol, and resveratrol could activate Nrf2 and have antioxidant effects  and high daily doses (800/200 mg). These findings highlight their potential risk of DILI. Likewise, other anti-SARS-CoV-2-Virus drugs with log p ≥ 3 and a high daily dose ≥100 mg, e.g., hydroxychloroquine/chloroquine, may be also involved in DILI.Pharmaco-immunomodulatory agents such as tocilizumab and glucocorticoids  and ritonavir affect PXR regulation and cause DILI. Corticosteroids have a high likelihood score of DILI A with hepatic enlargement and steatosis or glycogenosis . Log cluster 1, MCODE score = 22.08), PXR-regulated drug metabolic sub-network , NRF2-regulated oxidative sub-network (cluster 3), and HLA-related adaptive immune sub-network (cluster 4). In the primary DILI sub-network of cluster 1, IL6, IL1B, TNF, and CCL2 were among the top ten key targets and related to the COVID-19 adverse outcome pathwaya inflammation of CRS exacerbated DILI. Pharmaco-immunomodulatory agents, including tocilizumab that targets at IL-6 of CRS and glucocorticoids , and ritonavir affected PXR regulation. DDIs between these above drugs resulted in DPTS-induced liver injury. Moreover, ritonavir and glucocorticoids  suppressed antioxidant Nrf2, promoting DILI. Additionally, clavulanate and vancomycin, associated with HLA targets of cluster 4, had high risk of idiosyncratic DILI. Our results identified the key regulatory effects of PXR in DILI associated with CRS of COVID-19 and DDIs between some medications in the treatment of COVID-19 patients.In the present study, 1209 DPTSGs, including genes encoding PK and therapeutic targets of DPTS, were found to be associated with drug metabolism of CYP450s, nuclear receptor of PXR, and COVID-19 adverse outcome. Regulatory network of DPTS-induced liver injury contained inflammatory sub-network  in the patients suffering SARS-CoV-2 infection. CRP is an acute inflammatory protein synthesized by hepatocytes, and its production is mainly regulated by IL-6 (trough) was reached an extremely high level >20,000 ng/ml (six times the upper therapeutic limit), leading to the increased liver injury, in approximately 75% of the ICU patients receiving the halving dose (400/100 mg q24 h) with plasma CRP >200 mg/L (trough levels (median of 13600 ng/ml with the standard 400/100 mg q12 h) were significantly correlated with CRP (Spearman correlation coefficient = 0.81) and approximately 2-fold higher in the hospitalized COVID-19 patients than in the HIV patients (7100 ng/ml) (trough was significantly lower when the IL-6 antagonist of tocilizumab was pre-administered (p < 0.001). These findings demonstrated that tocilizumab activated PXR with up-regulated CYP 3A4 expression by inhibiting IL-6 signaling.This PXR inhibition mediated inflammation-drug interactions via the decreased CYP450s activity such as CYP3A4 and increased substrate drugs to the supratherapeutic levels that might result in DILI. In the study of Lenoir, et al. , they ob by IL-6 . Lopinav200 mg/L . Consist0 ng/ml) . Additio0 ng/ml) . In this0 ng/ml) . Further0 ng/ml) . The loc0 ng/ml) . In cont0 ng/ml)  found thp < 0.01) and immunosuppressants  was observed in COVID-19 patients during their hospitalization , ritonavir, and glucocorticoids , caused DILI. Increased CYP3A4 expression and abnormal lipid metabolism also contributed to DILI. PXR was activated as a result of the anti-inflammatory properties of pharmaco-immunomodulatory agents administrated to severe COVID-19 patients (the inflammatory phase) . Tocilizlization .Furthermore, PXR regulation was accompanied by the suppression of Nrf2, an antioxidant response regulator, by glucocorticoids and lopinavir/ritonavir, aggravating DILI. Nrf2 regulates GSH production and the expression of GSTs and participates in ROS and xenobiotic (drugs) detoxification . KratschIn vitro studies of Ambrus, et al.,  and valproic acid (antiepileptic). In the complex pharmacotherapy, most of drugs were metabolized by CYP3A4 in the liver (. The reduced CYP3A4 activity increased the risk of severe adverse drug events and affected clinical outcomes. Whereas, oleoylethanolamide and simvastatin increased the CYP3A4 expression via PPARA (This study has some limitations. Although bioinformatics analysis has been widely used in the advanced researches in biomedical sciences and drug design, there are the gaps between bioinformatics and system biology simulations and clinical practice. Firstlyhe liver . The redia PPARA . Secondlia PPARA . Future In conclusion, we provided in-depth insights to understand the mechanism underlying DPTS-induced liver injury : PXR reg


Mutations can occur in Gα proteins which prevent GTP hydrolysis; this allows the G proteins to signal independently of G protein–coupled receptors and can result in various cancers, such as uveal melanoma (UM). Most UM cases harbor Q209L, Q209P, or R183C mutations in Gαq/11 proteins, rendering the proteins constitutively active (CA). Although it is generally thought that active, GTP-bound Gα subunits are dissociated from and signal independently of Gβγ, accumulating evidence indicates that some CA Gα mutants, such as Gαq/11, retain binding to Gβγ, and this interaction is necessary for signaling. Here, we demonstrate that disrupting the interaction between Gβγ and Gαq is sufficient to inhibit aberrant signaling driven by CA Gαq. Introduction of the I25A point mutation in the N-terminal α helical domain of CA Gαq to inhibit Gβγ binding, overexpression of the G protein Gαo to sequester Gβγ, and siRNA depletion of Gβ subunits inhibited or abolished CA Gαq signaling to the MAPK and YAP pathways. Moreover, in HEK 293 cells and in UM cell lines, we show that Gαq-Q209P and Gαq-R183C are more sensitive to the loss of Gβγ interaction than Gαq-Q209L. Our study challenges the idea that CA Gαq/11 signals independently of Gβγ and demonstrates differential sensitivity between the Gαq-Q209L, Gαq-Q209P, and Gαq-R183C mutants.Heterotrimeric G protein stimulation  Q209L or Q209P mutations in Gαq or Gα11 occur in 90% of UM patients, and mutations at R183 occur in about 5% of patients (Heterotrimeric G proteins (Gαβγ) are canonically activated through G protein–coupled receptors (GPCRs), which allows for GDP release from the Gα subunit and further GTP binding. It is generally accepted that GTP binding on the Gα subunit promotes an active conformation and allows for the dissociation from Gβγ. Active GTP-bound Gα proteins can further bind and stimulate downstream effectors , 2. Hydrc tumors , 6. Mutapatients . Approxipatients .q/11 in UM, driven by the activating Q209L/P and R183 mutations, promotes the stimulation of two major signaling pathways: 1) the mitogen-activated protein kinase (MAPK) pathway and 2) the Yes-associated protein and Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) pathway. The MAPK cascade is stimulated by the direct binding of CA Gαq/11 to phospholipase C–β (PLC-β), which hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol further activates PKC, which phosphorylates and activates RasGRP3. The further activation of Ras promotes the stimulation of the MAPK cascade and results in the phosphorylation and activation of ERK. Phospho-ERK (pERK) dimerizes and translocates into the nucleus to bind transcription factors and promote the transcription of proliferative genes  transcription factor in the nucleus to promote the transcription of genes involved in proliferation and cell survival  and FR900359 (FR) have been shown to inhibit both WT and CA Gαq/11 and have shown promising results in cancer cell models , Q209P (QP), and R183C (RC)—is sufficient to inhibit oncogenic signaling of these mutationally active Gαq. We provide evidence that disrupting the interaction between CA Gαq and Gβγ significantly inhibits Gαq-mediated activation of the MAPK and YAP/TAZ pathways. Surprisingly, we show differential sensitivity between Gαq-QL and the Gαq-QP/RC mutants in that oncogenic signaling by Gαq-QP and Gαq-RC is more sensitive to the disruption of Gβγ binding than Gαq-QL.Here, we tested the hypothesis that disrupting the interaction between Gβγ and the CA Gαq, such as PLC-β, requires the binding of Gαq to Gβγ , Q209P (QP), and R183C (RC) mutants. To monitor the activity of the YAP/TAZ pathway, we transfected the CA mutants, with and without the I25A mutation, into HEK 293 Gαq/11 CRISPR-Cas9 KO cells and monitored YAP activity using the TEAD luciferase reporter assay, which is stimulated in response to YAP activation and nuclear translocation. Gαq-QL, Gαq-QP, and Gαq-RC all showed strong activation in the assay. Cells expressing the pcDNA3 vector and WT Gαq were used as negative controls and indicated no significant change in TEAD luciferase reporter activity (A). The Gαq-QL-C9,10S palmitoylation-deficient mutant was used as a negative control for Gαq-QL. The C9,10S mutation in WT Gαq has been previously established to prevent plasma membrane localization and consequently prevent GPCR-dependent activation . It has been previously established that activation of the Rho-dependent YAP pathway results in stabilization and dephosphorylation of YAP and its subsequent translocation into the nucleus  as a control resulted in the loss of nuclear YAP and its accumulation in the cytoplasm. Gαq-I25A-QL also promoted nuclear localization of YAP, consistent with its retention of signaling in the TEAD-luciferase assay (A). Cells expressing CA Gαq-QP and Gαq-RC had robust nuclear YAP localization, indicating stimulation of this pathway. However, Gαq-I25A-QP and Gαq-I25A-RC failed to stimulate the nuclear localization of YAP, as indicated by the cytoplasmic localization of YAP. Similar to the TEAD-luciferase reporter assay results, this data suggests that Gαq-I25A-QL is more resistant to disruption of signaling than the Gαq-I25A-QP and Gαq-I25A-RC mutants. In immunofluorescence microscopy studies, we also examined the subcellular localization of the CA Gαq mutants with and without the I25A mutation . Expression of CA Gαq-QL resulted in pERK stimulation >4-fold compared to cells transfected with pcDNA3 or WT Gαq. The Gαq-I25A-QL mutant also activated pERK at a slightly decreased level compared to Gαq-QL, although the difference was not statistically significant. Interestingly, the Gαq-QP and Gαq-RC mutants stimulated pERK, but pERK activity was significantly decreased in cells expressing the Gαq-I25A-QP and Gαq-I25A-RC mutants (C and D). The effects of the I25A mutants on MAPK activity were further validated with the serum response element (SRE) luciferase reporter assay, which monitors both Rho and MAPK-dependent activity (E). As indicated, the CA Gαq-QL mutant robustly stimulated SRE-dependent luciferase activity. Introduction of the I25A mutation into Gαq-QL (Gαq-I25A-QL) strongly inhibited the ability to stimulate SRE luciferase activity, similar to the lack of signaling by the Gαq-QL-C9,10S palmitoylation-deficient mutant and the loss of signaling upon treatment of cells expressing Gαq-QL with YM. As expected, the CA Gαq-QP and Gαq-RC mutants also robustly stimulated SRE luciferase activity, while the Gαq-I25A-QP and Gαq-I25A-RC mutants completely failed to stimulate luciferase activity, similar to cells treated with YM (E). The decreased signaling by Gαq-I25A-QL in comparison to Gαq-QL is much more drastic in the SRE luciferase assay than the pERK immunoblot assay (C and D). Although this is somewhat surprising since the SRE luciferase readout is downstream of the MAPK pathway, we note that the SRE luciferase assay provides a very high signal-to-noise readout for Gαq-QL signaling  and is consistently very sensitive to disruption of Gαq-QL signaling . Taken together, these results indicate that the introduction of the I25A mutation to disrupt binding to Gβγ inhibited oncogenic signaling of the CA Gαq mutants through the YAP and MAPK pathways, and the Gαq-QP and Gαq-RC mutants are more sensitive to the I25A mutation than Gαq-QL.Along with stimulation of the YAP pathway, CA Gα mutants , C and D mutants , C and Dactivity E. As ind with YM E. The deot assay , C and Dq-QL has an increased association with Gβγ compared to Gαq-RC  and was further quantified (B). As expected, WT Gαq displayed a strong pull down with Gβ1γ2, but the introduction of the I25A mutation into WT Gαq strongly decreased this interaction, indicating that the I25A mutation disrupts binding between Gαq and Gβγ. Interestingly, Gαq-QL had a similar binding association to Gβ1γ2 with that of WT Gαq, and Gαq-QL bound significantly stronger to Gβ1γ2 than Gαq-QP and Gαq-RC (A and B). Furthermore, Gαq-I25A-QL was more strongly bound to Gβ1γ2 than Gαq-I25A-QP and Gαq-I25A-RC (A and B). We did not see a statistically significant difference in association with Gβγ between Gαq-QP and Gαq-RC and their corresponding I25A mutants. This is likely due to the low initial binding to Gβγ with both Gαq-QP and Gαq-RC. Although we previously demonstrated the surprising ability of Gαq-QL to interact strongly with Gβγ and the poor interaction of Gαq-RC with Gβγ, we now show a dramatically decreased association of Gαq-QP with Gβγ compared to Gαq-QL, even though both have a mutation of Q209. This differential binding to Gβγ between the CA Gαq mutants likely contributes to the varying sensitivity in oncogenic signal disruption between the I25A mutants . Interestingly, cotransfection with 200 ng of the Gαo DNA was required to decrease Gαq-QL stimulation of TEAD luciferase activity by 63%. In comparison, cotransfection with 100 ng of Gαo plasmid DNA achieved 83% and 84% reduction in signaling by Gαq-QP and Gαq-RC, respectively. Transfection with the control Gαo G2A constructs did not reduce the luciferase reporter activity in response to the CA Gαq mutants (A). The ability of the expression of Gαo to reduce CA Gαq-stimulated YAP activity was also monitored through nuclear localization of YAP by immunofluorescence microscopy. Gαo localized strongly at cellular membranes, while the Gαo G2A mutant was primarily cytoplasmic, indicating the expected localization of the proteins. Strong nuclear YAP was detected with the CA Gαq-QL, Gαq-QP, and Gαq-RC mutants, indicating stimulation of the YAP pathway (B). As expected, expression of Gαo G2A with the CA Gαq mutants did not disrupt nuclear localization of YAP. Overexpression of Gαo inhibited YAP nuclear localization stimulated by Gαq-QP and Gαq-RC (B). Conversely, cotransfection of Gαo with Gαq-QL failed to prevent strong YAP nuclear localization (B), further suggesting that the Gαq-QP and Gαq-RC mutants are more sensitive to Gβγ binding disruption than Gαq-QL.Considering that inhibiting the association between the CA Gαin cells , 42, 43.embranes . YAP actplasmids A. Intere mutants A. The ab pathway B. As expd Gαq-RC B. Converlization B, furtheo expression and subsequent Gβγ sequestration on CA Gαq-promoted MAPK activity, we immunoblotted for pERK levels (A and B). The expression of the CA Gαq mutants resulted in strong stimulation of pERK. Expression of Gαo resulted in no significant decrease in pERK activity for Gαq-QL. However, cotransfection of Gαq-QP or Gαq-RC with 50 ng and 100 ng of Gαo plasmid resulted in a strong decrease in pERK activity (A and B). There was no significant change in pERK levels with the Gαo G2A control (A and B). These results were further validated using the SRE luciferase reporter assay (C). Expression of Gαq-QL, Gαq-QP, and Gαq-RC robustly activated SRE-dependent luciferase activity, while treatment with YM abolished luciferase activity. Consistent with results from the other signaling assays (A and B), cotransfection with 50 ng Gαo plasmid was sufficient to completely abolish Gαq-QP– and Gαq-RC–stimulated SRE luciferase activity, while up to 250 ng of Gαo plasmid was required for near complete inhibition of Gαq-QL–stimulated SRE luciferase activity (C). These findings indicate that the expression of Gαo to sequester endogenous Gβγ inhibits the signaling activity of Gαq-QL/P and Gαq-RC. The results also further validate that the Gαq-QL CA mutant is less sensitive to signaling disruption than the Gαq-QP and Gαq-RC mutants.To monitor the effects of GαK levels , A and Bactivity , A and B control , A and Ber assay C. Expresg assays , A and Bactivity C. These o could sequester Gβγ and inhibit the binding of Gβγ to Gαq-QL/P and Gαq-RC. To examine this, we coexpressed Gαo or Gαo G2A with the CA Gαq mutants in 6x-His-Gβ1y2 HEK 293 stable cells and pulled down Gβ1γ2 with Ni-NTA beads. Immunoblots were used to detect Gαq and Gαo bound to Gβ1 (A and B). As indicated in q-QL had a stronger binding association with Gβ1 than Gαq-QP and Gαq-RC. Gαo was bound to Gβ1 in the pull-down extract, but Gαo G2A had minimal to no interaction with Gβ1 as expected. The expression of Gαo resulted in significantly decreased binding of Gαq-QL to Gβ1 (A and B). Expression of Gαo consistently resulted in a noticeable decrease in the pull down of Gαq-QP and Gαq-RC with Gβγ, but this did not reach statistical significance due to the already low level of Gαq-QP and Gαq-RC association with Gβγ in the absence of Gαo expression. These findings confirm that overexpression of Gαo sequesters Gβγ and prevents binding of Gαq-QL/P and Gαq-RC to Gβγ.Next, we wanted to confirm that the expression of Gαd to Gβ1 , A and BL to Gβ1 , A and Bq through the N-terminal I25A mutation or expression of Gαo is sufficient to inhibit overactive Gαq signaling in HEK 293 Gαq/11 KO cells. We next wanted to determine if inhibiting Gβγ binding to CA Gαq would be sufficient to disrupt oncogenic signaling in UM cells containing the Gαq-QL or Gαq-QP mutants. To study this, we depleted Gβ1 and Gβ2, two predominant Gβ subunits, through siRNA transfections. Sufficient knockdown of Gβ1 and Gβ2 was first validated in HEK 293 Gαq/11 KO cells (A and B). siRNA molecules specific for Gβ1, Gβ2, and both Gβ1 and Gβ2 were transfected into the HEK 293 Gαq/11 KO cells, and WT Gαq, Gαq-QL, or Gαq-QP constructs were transfected after 24 h. pERK levels were detected and quantified through immunoblotting (A and B). The results indicate that siRNA depletion of Gβ1/2 significantly disrupted pERK activity driven by both Gαq-QL and Gαq-QP (A and B). Gβ1 or Gβ2 siRNA knockdown alone was insufficient to significantly inhibit pERK activation (A and B). Considering the effectiveness of Gβ1/2 depletion on pERK activity in the HEK 293 Gαq/11 KO cells, we decided to knock down Gβ1/2 in four UM cell lines: 1) Mel202 (Gαq-Q209L mutation), 2) 92.1 (Gαq-Q209L mutation), 3) OMM1.3 (Gαq-Q209P mutation), and 4) UM001 (Gαq-Q209P mutation). We also used the OCM-3 cell line as a control. The oncogenic activity of OCM-3 cells is driven by mutant BRAF (V600E) as opposed to mutant Gαq/11; thus, we would expect Gβ1/2 depletion to have minimal effect on pERK activity  and quantified (D). Interestingly, the OMM1.3 and UM001 cell lines which contain the Gαq-Q209P mutation had significantly decreased pERK levels with Gβ1/2 knockdown, similar to that of YM treatment (C and D). There was no significant difference in pERK levels with Gβ1/2 depletion in the Mel202 and 92.1 cells which contain the Gαq-Q209L mutation (C and D). This data further suggests that there is differential sensitivity to the inhibition of oncogenic signaling between cell lines with the Gαq-Q209L and Gαq-Q209P mutations.Our studies demonstrate that disrupting the interaction between Gβγ and CA GαKO cells , A and Bblotting , A and Bd Gαq-QP , A and Btivation , A and Bactivity . All celoblotted C and quaantified D. Interereatment , C and Dmutation , C and Dq and show a surprising differential sensitivity of CA Gαq mutants to the inhibition of signaling upon disruption of their interaction with Gβγ. We used two methods to disrupt Gβγ interaction with CA Gαq—introduction of a Gβγ-binding disrupting mutation, I25A, into CA Gαq and sequestration of endogenous Gβγ by expression of Gαo—to show that inhibiting interaction with Gβγ can drastically reduce CA Gαq-stimulated signaling to both the MAPK and YAP/TAZ pathways. Moreover, signaling by CA Gαq-Q209P, as well as CA Gαq-R183C, was much more effectively prevented by Gβγ binding disruption than signaling by CA Gαq-Q209L. Lastly, we extended the analysis to UM cell lines containing either the oncogenic driver mutant Gαq-Q209L or Gαq-Q209P and showed that depletion of Gβ1/2 strongly inhibited activation of the MAPK pathway in cells with the Gαq-Q209P mutant but not in cells with the Gαq-Q209L. Our results suggest that disrupting the interaction between Gβγ and CA Gαq may be a novel approach for inhibiting aberrant Gαq/11 signaling.The results presented in this study demonstrate the importance of Gβγ for signaling by CA mutants of Gαq mutants studied herein, it would be expected that CA Gα mutants would not retain binding to Gβγ. However, several studies have challenged the classical heterotrimer dissociation model, indicating that at least some Gα subunits maintain association with Gβγ when they are in the active, GTP-bound state due to the presence of a GTPase-inhibiting mutation or in response to GPCR activation  a central switch region, which undergoes a conformational change upon GTP binding and 2) an N-terminal α helical domain . Thus, one reason why CA Gαq mutants in this study showed decreased or loss of signaling to the MAPK and YAP pathways when interaction with Gβγ is disrupted is a failure to be efficiently palmitoylated and membrane-bound. Indeed, Gαq-I25A-QL retains stronger plasma membrane localization than Gαq-I25A-QP and Gαq-I25A-RC , although only partial loss of signaling was observed in when measuring pERK (C and D). Expression of the increasing amounts of Gαo to sequester Gβγ provided a powerful tool to demonstrate titratable differences in sensitivity to disruption of Gβγ binding to the CA Gαq mutants . Using the SRE-luciferase assay, transfection with 50 ng of Gαo expression plasmid was sufficient to completely abolish signaling of Gαq-QP and Gαq-RC, while transfection with 250 ng of Gαo plasmid was required to observe a similar almost-complete inhibition of Gαq-QL signaling (C). Likewise, transfection with 50 or 100 ng of Gαo plasmid failed to inhibit Gαq-QL–stimulated pERK levels, but transfection with 50 or 100 ng of Gαo plasmid provided significant inhibition of Gαq-QP– and Gαq-RC–stimulated pERK (A and B). Gβγ pull-down experiments showed the interaction of Gαq-QL with Gβγ, similar to the levels of WT Gαq interaction with Gβγ, and, importantly, the I25A mutation and expression of Gαo only partially disrupted the interaction . The Gαq-I25A, Gαq-I25A-QL, and Gαq-I25A-RC constructs were previously described . YFP-tagged Gαq was provided by Catherine Berlot. Gαo and Gαo-G2A plasmids were described previously (q and Gαo transient transfections was obtained from Invitrogen (Cat # 11668-019). siRNA targeting Gβ1 (GGAUAACAUUUGCUCCAUU), Gβ2 (ACUGGGUACCUGUCGUGUU), and both Gβ1 and Gβ2 (Gβ1/2) (ACGACGACUUCAACUGCAA) were previously described  (for immunoblots with HEK 293 Gαq/11 KO lysates), GAPDH (Cat # 60004-1-Ig), and Gαo (Cat # 12635-1-AP) were from Proteintech. The Gαq (Cat # ab199533) (for immunofluorescence experiments), Gβ1 (Cat # ab137635), and Gβ2 (Cat # ab108504) antibodies were obtained from Abcam. The YAP antibody (Cat # sc-101199) was purchased from Santa Cruz. The Gαq (Cat # 14373S) , ERK (Cat # 4696S), pERK (Cat # 9101S), and Myc-tag (Cat # 2272S) antibodies were obtained from Cell Signaling Technologies. The HA-tag antibody 12CA5 was from Covance. The GRK4-6 antibody (Cat # 05-466) was obtained from Sigma-Aldrich. For immunofluorescence microscopy, the secondary antibodies Alexa Fluor 488 (goat anti-rabbit) (Cat # A-11034), Alexa Fluor 594 (goat anti-mouse) (Cat # A-11032), Alexa Fluor 594 (goat anti-rabbit) (Cat # A-11037), and Alexa Fluor 647 (goat anti-mouse) (Cat # A-32728) were purchased from Invitrogen. The secondary antibodies IRDye 680RD goat anti-rabbit IgG (H + L) (Cat # 92568071) and IRDye 800CW donkey anti-mouse IgG (H + L) (Cat # 92532212) were obtained from LI-COR and were used to visualize protein from all of the immunoblots.The antibodies for Gαq/11 KO cells were generously provided by Dr Asuka Inoue and were described previously  with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin . HEK 293 Gβ1γ2 stable cells were described previously . The SRE luciferase reporter plasmid was described previously  with 1% Triton X-100. For the Gαq-I25A experiments, the cells were incubated with the anti-rabbit Gαq antibody (Abcam) and the anti-mouse YAP antibody in 2.5% milk/TBS-Triton X-100. For the Gαo experiments, the cells were incubated with the anti-rabbit Gαo antibody and the anti-mouse YAP antibody in 2.5% milk/TBS-Triton X-100. The primary antibodies were incubated for 60 min. The cells were washed 5 times with 2.5% milk/TBS-Triton X-100. For the Gαq-I25A experiments, the cells were incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 antibodies in 2.5% milk/TBS-Triton X-100. For the Gαo experiments, the cells were incubated with goat anti-rabbit Alexa Fluor 594 and goat anti-mouse Alexa Fluor 647 antibodies in 2.5% milk/TBS-Triton X-100. The secondary antibodies were incubated for 30 min. The cells were washed 5 times in TBS/1% Triton X-100. The cells were incubated in DAPI  diluted in warmed PBS for 5 min. The coverslips were rinsed in distilled water and mounted onto glass slides with ProLong Diamond Anti-fade Mountant . The images were acquired using the Olympus IX83 microscope with a 60× oil immersion objective and an ORCA Fusion sCMOS camera (Hamamatsu) controlled by Olympus cellSens software.HEK 293 Gα1γ2 pull-down assay was done as previously described . The cell lysates were incubated for 1 h on ice and were then centrifuged at 13,000 rpm  to pellet the nuclei and insoluble material. Forty microliters of the lysate were reserved separately for the input fraction. The remaining lysate was added to 30 μl of Ni-NTA beads  and rotated in an end-over-end rotator for 2 h at 4 °C. The tubes were placed in a magnetic rack, and the remaining supernatant was aspirated. The beads were washed 3 times with lysis buffer C. Fifty microliters of elution buffer (lysis buffer C with 0.25 M Imidazole) were added to elute Gβ1 and any bound proteins from the Ni-NTA beads. Forty microliters of the pull-down eluate were transferred to a new tube. Ten microliters of 5× SDS-PAGE sample buffer with 3.5% β-mercaptoethanol were added to the input fraction and the pull-down fraction. The input and pull-down lysates were separated on a 10% SDS-PAGE gel and protein bound was detected via immunoblotting. The blots were probed with the HA-tag and Myc-tag antibody . The blots for the Gαo experiments were also probed with Gαo and GAPDH. The blots were imaged on LI-COR, and the bands were quantified on the ImageJ software (https://imagej.nih.gov/ij/). The pull-down samples in Gαq-I25A experiments were normalized to its respective input fraction. The pull-down samples in the Gαo experiments were normalized to WT Gαq.The Gβescribed . Brieflyq  for the HEK 293 Gαq/11 KO cells or Cell Signaling Gαq  for the UM cells, along with GAPDH  as a loading control. The ERK and pERK bands were quantified by densitometry using the ImageJ software, and the relative pERK signal was divided by ERK. The quantified signals were normalized to WT Gαq (D), the individual CA Gαq mutants (B), or control siRNA between the CA Gαq mutants (B) or the UM cell lines (D).Cells were lysed in SDS-PAGE sample buffer. Lysates were run on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked in 2.5% bovine serum albumin (BSA)  in 1× TBS supplemented with 0.05% Tween 20. The blots were incubated with both pERK  and ERK  antibodies overnight. A duplicate immunoblot was performed and was probed with either Proteintech Gαo WT Gαq D, the in mutants B, or con mutants B or the ll lines D.q/11 KO cells and 12-well plates for the UM cells. For the HEK 293 Gαq/11 KO experiments, 30 pmol of control, Gβ1, Gβ2, or Gβ1/2 siRNA were transfected into cells using Lipofectamine RNAiMax , according to manufacturer's instructions. The media was changed after 5 h. After 24 h, the corresponding WT Gαq, Gαq-QL, or Gαq-QP constructs were transfected into the cells using Lipofectamine 2000 . The media was changed to serum-free media after 24 h and corresponding cells were treated with 1 μM of YM. The cells were lysed in 1× SDS-PAGE sample buffer with 0.7% β-mercaptoethanol after 16 h. For experiments with the UM cells, 15pmol of control or Gβ1/2 siRNA were transfected into cells using Lipofectamine RNAiMax . The media was changed after 5 h. After 72 h, the media was changed to serum-free media, and the corresponding cells were treated with 1 μM of YM. The cells were further lysed after approximately 16 h in 1× SDS-PAGE sample buffer with 0.7% β-mercaptoethanol. The lysates were run on 10% SDS-PAGE gels and were immunoblotted for pERK , ERK , Gβ1 , Gβ2 , GAPDH , and Proteintech Gαq (Cat #13927-1-AP) for the HEK 293 Gαq/11 KO cells or Cell Signaling Gαq (Cat # 14373S) for the UM cells.Cells were plated in 6-well plates for the HEK 293 GαProtein lysates were run on 10% SDS-PAGE gels and transferred to LI-COR nitrocellulose membranes (Cat # nc9680617). The membranes were blocked in either 2.5% BSA or 2.5% milk in 1× TBS/0.05% Tween 20 at room temperature for 60 min. The blots were incubated in corresponding primary antibodies in 2.5% BSA or milk in 1× TBS/0.05% Tween 20 at 4 °C overnight. The blots were washed three times in 1× TBS/0.05% Tween 20 and incubated in LI-COR anti-rabbit (Cat # 92568071) and anti-mouse (Cat # 92532212) secondary antibodies for 60 min at room temperature. The immunoblots were further washed three times in 1× TBS/0.05% Tween 20 and once with PBS. The blots were then imaged on the LI-COR Odyssey imager.B and D) multiple comparison test were used to calculate significance. Error bars in all experiments indicate mean ± SD with significant differences indicated as ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005; ∗∗∗∗p < 0.0001.GraphPad Prism was used to analyze the data for all of the figures. A two-way ANOVA followed by Šidák's or Tukey’s , B and DAll data is contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.