Taohong Siwu Decoction (THSWD) was widely used for the treatment of varicocele-associated male infertility. However, the pharmacological mechanism of action is not completely clear. Therefore, network pharmacology and molecular docking were performed to explore potential mechanism of THSWD in the treatment of varicocele-associated male infertility. The Traditional Chinese Medicine Systems Pharmacology (TCMSP), Swiss Target Prediction, and GeneCards were used to retrieve candidate compounds, action targets, and disease-related targets. The construction of the protein–protein interaction (PPI) network and the screening of core genes were completed by the STRING and Cytoscape 3.9.1, respectively. The DAVID was used to obtain results of gene ontology function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The Mcule analysis platform was used to perform molecular docking. There were a total of 53 candidate compounds and 782 relevant targets in THSWD. There were 45 common targets between THSWD, varicocele, and male infertility, and 23 core genes were found in the PPI network. Biological processes involved response to hypoxia, regulation of blood pressure, cellular response to hypoxia, and regulation of the nitric oxide biosynthetic process. Furthermore, the KEGG pathway enrichment analysis showed that the common targets mainly regulated the disease of varicocele-associated male infertility through the HIF-1 signaling pathway, PI3K-Akt signaling pathway, Relaxin signaling pathway, and TNF signaling pathway. Finally, the molecular docking showed that luteolin, quercetin, and kaempferol had good intercalation with major targets. As predicted by network pharmacology, THSWD regulated varicocele-associated male infertility through multiple compounds and targets, and its mechanism was closely related to inflammatory response, reactive oxygen species damage, and function of blood vessels. Varicocele has been reported to be attributable to 35% to 40% of cases of male infertility. Surgery and medication are often used at this stage of clinical treatment, with medication being the main method. Taohong Siwu Decoction (THSWD) is composed of 6 Chinese herbs , which have the activity of removing blood stasis, clearing collaterals, and nourishing blood. It is often used in patients with varicocele, and can effectively relieve the symptoms of testicular swelling, perineal swelling and pain, and the bulging of testicular tendons. Nevertheless, the pathogenesis of THSWD treatment for varicocele-associated male infertility is not fully understood. Therefore, exploring the mechanism of action of THSWD in the treatment of varicocele-associated male infertility was beneficial to clinically rational drug use and the development of new drugs.Male infertility accounts for 60% of total infertility cases, and the common pathological causes of male infertility are varicocele and chronic reproductive system infections.,4 As we all know, molecular docking technology plays a key role in drug design, development, and research. By calculating the binding energy of different conformations , it is convenient to explore potential active compounds for the treatment of diseases. Therefore, we chose network pharmacology and molecular docking methods to study the mechanism of action of THSWD on varicocele-associated male infertility, excavate the potential active ingredients and key targets, and provide a theoretical basis for further research. The detailed technical flow of the study is shown in Figure In recent years, network pharmacology has been widely used to elucidate the mechanism of action of traditional Chinese medicine (TCM) in the treatment of diseases, and to screen the active ingredients and key targets of TCM by constructing a network of compounds and targets.https://old.tcmsp-e.com/tcmsp.php), respectively. The candidate compounds and related targets were screened according to the screening conditions . Compounds in the TCMSP database have no relevant targets, and Swiss Target Prediction (http://swisstargetprediction.ch/) was used for target prediction. The SMILES of the compound were retrieved in the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and then the SMILES were entered into the search column in the Swiss Target Prediction database (http://www.swisstargetprediction.ch/), the target information was predicted, and the target with a probability greater than 0 was selected as the compound-related target. Finally, the predicted targets were corrected with the UniProt database (https://www.uniprot.org/).The chemical compounds of Persicae Semen (PS), Carthami Flos (CF), Rehmanniae Radix Praeparata (RRP), Paeoniae Radix Alba (PRA), Chuanxiong Rhizoma (CR) and Angelicae Sinensis Radix (ASR) were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP)  was used for functional enrichment analysis of protein-protein interactions. The above common targets were uploaded to the STRING database, and “multiple proteins” was selected as the analysis module, “homo sapiens” as the species, and 0.4 as the minimum required interaction score. Other parameters were kept as defaults and searched. The obtained results were imported into Cytosape 3.9.1 software for visualization, and its built-in “Network Analyzer” tool module was used for topological network analysis.The STRING database (https://david.ncifcrf.gov/tools.jsp) is widely used for annotation of biological information, which helps researchers discover biological internal connections. The common targets were uploaded to “gene list” in DAVIA database, and “gene symbol” was selected as an identifier. The screening false discovery rate of less than 0.05 was used as the result of GO and KEGG enrichment. The GO-related results were mainly composed of biological process (BP), cell components (CC), and molecular function (MF).The DAVID database  stores a large amount of 3D structural data of biological macromolecules for research in basic biology, health, energy, education, and biotechnology. To obtain the PDB ID of target proteins, we inputted target proteins into the RCSB database. Mculle (https://mcule.com/) is an online drug discovery platform for drug research. 1-Click Docking (https://mcule.com/search/) is a functional module of Mculle for predicting ligand-target affinity. We only need to input the SMILES of the ligand and the PDBID of the receptor into the database to obtain 5 docking morphologies, and select the morphology with the lowest binding energy as the optimal docking. Finally, we visualized the molecular docking results using the PyMOL 2.5 (https://pymol.org/).The RCSB database . The higher the gene ratio of an item, the more likely it is that the item played a major role in the GO function. Therefore, the top 10 items of the BP, CC, and MM gene ratio were selected to make a stick chart , mainly involved pathways related to HIF-1 signaling pathway, PI3K-Akt signaling pathway, endocrine resistance, apoptosis, Relaxin signaling pathway, etc  that were closely related. Previous studies have reported that the closeness of protein binding to ligand correlates with the binding energy, with binding energy below −5.0 kcal/mol indicating good binding and binding energy below −7.0 kcal/mol indicating very firm binding. Docking results showed that quercetin, kaempferol and luteolin were tightly bound to target proteins , and binding energies to PTGS2, AKT1, ESR1, and CASP3 were lower than those of IL6, VEGFA, TNF, and TP53  from TCMSP. Among the candidate compounds, PS, CF, and PRA accounted for 39.6%, 37.7%, and 17%, respectively. PS maintained homeostasis through bidirectional regulation of plasma cAMP concentration and PKA protein in rats with hot and cold blood stasis. Tumor necrosis factor (TNF) is an important pro-inflammatory factor in the body, that plays an important role in the inflammatory response and is often used as a therapeutic target for rheumatoid arthritis, inflammatory bowel disease, and a variety of cancers. Serum albumin encodes the most abundant protein in human blood, which not only regulates plasma colloid osmotic pressure, but also serves as a carrier protein for a variety of endogenous molecules, including hormones, fatty acids, metabolites, and exogenous drugs. Vascular endothelial growth factor A (VEGFA) is a member of the platelet-derived growth factor/VEGF family, which can mediate increased vascular permeability, induce angiogenesis and endothelial cell growth, promote cell migration, and inhibit cell apoptosis. Zou et al found that emodin promoted the proliferation of seryl-trna synthetase, reduced VEGFA expression, and inhibited tumor angiogenesis in triple-negative breast cancer. As a marker of inflammation, interleukin 6 (IL6) is involved in a series of inflammatory responses in the body. Inhibition of its expression can effectively reduce the inflammatory response and reduce the damage caused by inflammation to the body. Statement of defense: PPI network analysis showed that the main functions of the common targets were the regulation of cell proliferation, apoptosis, inflammation, vasodilation, and angiogenesis. To further verify the regulatory roles of these common targets, GO biological process and KEGG pathway enrichment analyses were performed.782 targets related to the candidate components were obtained from the DrugBank and Swiss Target Prediction databases. Subsequently, the target genes related to varicocele and male infertility in the GeneCards database were intersected with the genes related to candidate compounds, and 45 common targets were obtained. The PPI network composed of 45 common targets showed that 21 targets had higher than average degrees, and the highest ranking targets were AKT1, TNF, ALB, TP53, VEGFA, and IL6, respectively. AKT serine/threonine kinase 1 is a serine/threonine protein kinase, a member of the AGC kinases family, which participates in a variety of biological processes, including cell proliferation, metabolism, survival, signal transduction, and angiogenesis. It has been found that Akt can promote cell survival by affecting the activities of caspase-9, YAP, Bcl-2, and Bcl-x. It also increases VEGF secretion and mediates eNOS phosphorylation, vasodilation, and angiogenesis. have shown that A plays a regulatory role under hypoxic conditions in tissues and organs by reducing oxygen consumption or increasing the release of NO or other vasodilators. The relaxin signaling pathway is involved in the regulation of vascular and smooth muscle contraction. In contrast, the PI3K-Akt signaling pathway and the TNF signaling pathway are involved in the regulation of the inflammatory response, which have been widely studied in cancer in recent years.,25Basically consistent with the above studies, BP was mainly involved in response to hypoxia, regulation of blood pressure, cellular response to hypoxia, and regulation of the nitric oxide biosynthetic process. KEGG pathway enrichment analysis showed that THSWD treatment of varicocele-associated male infertility disease was mainly through HIF-1 signaling pathway, PI3K-Akt signaling pathway, Relaxin signaling pathway, endocrine resistance, apoptosis, Estrogen signaling pathway, and TNF signaling pathway, etc. Previous studies Serban et al evaluated the effect of quercetin on blood pressure by systematic review and meta-analysis of randomized controlled trials, and found that quercetin had significant statistical significance in reducing blood pressure, and a daily dose of more than 500 mg was required. From the same source as quercetin, kaempferol is found in various vegetables, fruits, and plants and has anti-inflammatory properties. By regulating vascular endothelial inflammation, kaempferol has therapeutic effects on colitis, chronic hepatitis, lung injury, cancer, and diabetes. Feng et al found that different concentrations of kaempferol  induced G protein-coupled estrogen receptor activation, increased cell viability to nearly 10%, 19.8%, and 30%, respectively, and reduced the generation of cellular reactive oxygen species , thereby alleviating postmenopausal atherosclerosis. Yao et al found that kaempferol had the ability to protect the vascular endothelium in a mouse model of vascular injury by reducing oxidative stress mediated by H2O2 and paraquat (in vivo and in vitro). In addition, kaempferol inhibited the levels of TNF-α and IL-6 and the activation of NF-κB in aortic tissues and human umbilical vein endothelial cells. Luteolin is a kind of natural brass, and numerous studies have shown that it has anti-inflammatory, antioxidant, and anti-cancer biological activities.–33 Boeing et al found that luteolin could reduce the oxidative stress response caused by iritantan by reducing reactive oxygen species and enhancing endogenous antioxidants. It also alleviated inflammation by reducing TNF, IL-1β and IL-6 levels and increasing IL-4 and IL-10 levels. In addition, it was found that Luteolin could also reduce the LPS-induced inflammatory injury of cardiomyocytes and the excessive release of inflammatory cytokines.,36 Baicalein, a flavonoid originally extracted from the TCM scutellaria baicalensis, has a variety of pharmacological effects, such as inhibiting the oxidative stress response, inhibiting the cell apoptosis, inhibiting the inflammatory response, protecting mitochondria, and enhancing the expression of neuronal protective factors. Liu et al found that baicalein inhibited isoproterenol-induced cardiac hypertrophy in mice, and in vitro cell experiments found that Baicalin increased the expression of catalase and mitochondrial autophagy receptor FUN14 domain 1, promoted autophagy, cleared active oxygen species, and reduced isoproterenol-induced myocardial hypertrophy. Song et al found that baicalin could improve the cell viability of SH-SY5Y cells treated with MPP+ and reduce the accumulation of iron and lipid peroxides induced by MPTP, and the mechanism was closely related to inhibition of oxidative stress. In summary, the above studies showed that the main pharmacological effects of active ingredients were anti-oxidative stress, anti-inflammation, and improvement of blood circulation. In summary, the above studies showed that the main pharmacological effects of the active ingredients were anti-oxidative stress, anti-inflammation, and improvement of blood circulation, which was basically the same as our previous hypothesis.To figure out which active ingredients were involved in regulating the disease of varicocele-associated male infertility, we constructed the network of T-A-T-P. By analyzing the network, it was found that the degree of quercetin, kaempferol, luteolin, and baicalein in the network was higher, suggesting that these compounds play an important bridge role in the regulatory network, and indicating that this class of compounds plays a major role in the treatment of diseases. Quercetin is present in various fruits, vegetables, and Traditional Chinese Medicine. A large number of studies have shown that it has anti-oxidative, anti-inflammatory, anti-proliferative, anti-cancer, and anti-diabetic properties. In recent years, many researchers have found that it can lower blood pressure.Subsequently, to find out how closely the active ingredients bind to the key targets, we selected the top 3 compounds for molecular docking experiments. The results showed that luteolin, quercetin and kaempferol bind closely to PTGS2, ESR1, CASP3 and AKT1, respectively. These results indicate that these 3 compounds can be used as the main active ingredients of THSWD in the treatment of varicocele-associated male infertility.In this study, a total of 53 candidate compounds of THSWD were obtained. The active ingredients of THSWD including quercetin, kaempferol, luteolin, baicalein, and beta-sitosterol, and PTGS2, EGFR, AKT1, ESR1, CASP3, MMP9, IL6, VEGFA, TNF, TP53, and HSP90AA1 were predicted as major targets for the treatment of varicocele-associated male infertility. Molecular docking results showed that luteolin, quercetin and kaempferol had good binding effects on major targets. KEGG pathway enrichment analysis showed that THSWD may play a therapeutic role through HIF-1, PI3K-Akt, and Relaxin signaling pathways. In summary, our study further improves the mechanism of action of THSWD in the treatment of varicocele-associated male infertility, and provides scientific evidence for subsequent experimental research and clinical rational administration.We thank Director Ming Gao for her valuable comments and support for this study. Xiwei Song, Xingdong Wang, and Min Huang are not among the authors, but I would like to thank them for their assistance with data processing.Data curation: Xiaohong Lan, Qinyan Wu.Formal analysis: Yang Yang.Software: Xuqing Chen.Supervision: Xuqing Chen, Yuekun Wang.Validation: Yuekun Wang.Writing – original draft: Bo Wu.Writing – review & editing: Bo Wu, Yuekun Wang.


Candida genus, poses a significant threat to immunocompromised individuals. Natural compounds derived from medicinal plants have gained attention as potential sources of anti-fungal agents. Ajwa dates (Phoenix dactylifera L.) have been recognized for their diverse phytochemical composition and therapeutic potential. In this study, we employed a multi-faceted approach to explore the anti-candidiasis potential of Ajwa dates’ phytochemicals. Utilizing network pharmacology, we constructed an interaction network to elucidate the intricate relationships between Ajwa dates phytoconstituents and the Candida-associated molecular targets of humans. Our analysis revealed key nodes in the network , suggesting the potential modulation of several crucial processes  and fungal pathways . To validate these findings, molecular docking studies were conducted, revealing the binding affinities of the phytochemicals towards selected Candida protein targets of humans (ALB–rutin (−9.7 kJ/mol), STAT1–rutin (−9.2 kJ/mol), STAT3–isoquercetin (−8.7 kJ/mol), IL2–β-carotene (−8.5 kJ/mol), CASP1–β-carotene (−8.2 kJ/mol), TP53–isoquercetin (−8.8 kJ/mol), PPARG–luteolin (−8.3 kJ/mol), TNF–βcarotene (−7.7 kJ/mol), TLR4–rutin (−7.4 kJ/mol) and PTPRC–rutin (−7.0 kJ/mol)). Furthermore, molecular dynamics simulations of rutin–ALB and rutin-STAT1 complex were performed to gain insights into the stability and dynamics of the identified ligand–target complexes over time. Overall, the results not only contribute to the understanding of the molecular interactions underlying the anti-fungal potential of specific phytochemicals of Ajwa dates in humans but also provide a rational basis for the development of novel therapeutic strategies against candidiasis in humans. This study underscores the significance of network pharmacology, molecular docking and dynamics simulations in accelerating the discovery of natural products as effective anti-fungal agents. However, further experimental validation of the identified compounds is warranted to translate these findings into practical therapeutic applications.Candidiasis, caused by opportunistic fungal pathogens of the  Candida genus, has emerged as a prominent healthcare concern with increasing incidence and clinical significance .Several potential targets were identified in the study, including those that are predicted for phytochemical constituents of Ajwa dates as well as those connected with candidiasis in humans. FunRich tool version 3.1.3 was used to construct Venn diagrams for analyzing common targets ; the Swihttps://string-db.org/ (accessed on 20 February 2023)] was utilized to study protein–protein interactions (PPIs) of potential targets [The STRING database [ targets . Accordi targets . The nodhttps://david.ncifcrf.gov/ (accessed on 25 February 2023)]  . In ordeAn investigation of the interaction between phytochemical constituents of Ajwa dates and identified candidiasis targets of humans was conducted using AutoDock Vina . From th+ and Cl- counterions were added. A leap frog algorithm was used to equilibrate NVT and NPT for 100 ps steps. Following equilibration, the production MD was applied to the complex of the solvated protein and ligand for 100 nanoseconds. After removing periodic boundary conditions from the MD run, a trajectory file was further analyzed. Data were analyzed using the Chimera package. XMGRACE was used to generate the diagrams (https://plasma-gate.weizmann.ac.il/Grace/ (accessed on 1 March 2023)).A further study was undertaken to understand how ligands behave within the binding pocket of receptors as a result of their time-dependent conformational stability, which was based on the MD studies. The usefulness of this method has been demonstrated in several studies, including the identification of new inhibitors in a variety of applications ,49,50,51By using the Poisson-Boltzmann surface (PBSA) method of molecular mechanics (MM), binding free energy calculations were performed. The MM-PBSA method has been widely used in drug discovery to calculate the solvation-based score of protein-ligand interactions. Solvation free energy (polar and non-polar) and vacuum potential energy were used to determine the solvation-based binding free energy. Non-polar and polar solvation energy terms have been calculated using Poisson-Boltzmann equations and solvent accessible surface areas (SASA). In order to estimate the affinity between a ligand and a receptor, the Poisson–Boltzmann equation was used. By utilizing a van der Waals contact probe, the SASA method was used to predict which solvent was surrounding a protein surface. To calculate the MM-PBSA, a script in the G_MMPBSA module was used that uses the AMBERTOOLS  softwarebinding) was calculated using the equations given below:The solvation-based free energy of binding , molecular functions (MF), and cellular components (CC) A–C, alonmponents D. StatisAs one of the most popular computational methods for finding potential leads against predefined targets, virtual screening using molecular docking is an effective strategy. The application of this method resulted in the identification of compounds with high binding affinities and specific interactions with the target proteins. Molecular docking analysis was conducted in order to understand how phytochemical constituents of Ajwa dates interact with identified protein targets of humans that play a key role in the development of candidiasis. In a docking analysis, several compounds were found to significantly bind to the target proteins of humans . The hig2. SASA values showed that ALB and STAT1 was remained stable in the presence of rutin. Molecular stability can also be calculated from the compactness of protein molecules. The compactness measure in MD simulations is called Rg. The compactness of a protein structure is a useful parameter that can be used to examine the tertiary structure. Rg values were used to assess the compactness of ALB and STAT1 after daidzein binding. The ALB–rutin  [C. albicans with an inhibition zone diameter of 15 mm and a minimum inhibitory concentration of 7.5 mg/mL. Selim et al. (2021) [C. albicans, finding that gallic acid inhibited C. albicans with an inhibition zone diameter of 18 mm and a minimum inhibitory concentration of 5 mg/mL. In 2021, the authors of [C. albicans and A. niger with MIC values of less than 50 μg/mL by the polyphenol extract of Ajwa dates. In 2022, the authors of [C. albicans, where the former inhibited the latter’s growth and biofilm formation. We note that we have not found reports of interactions of the Ajwa date compounds with the target proteins we have identified. Nevertheless, some studies have shown that Ajwa dates and their seeds have anti-fungal properties against . (2019)  compared. (2021)  isolatedthors of  reportedthors of  also repCandida infections on mucosal surfaces. However, excessive STAT3 activation can also impair anti-fungal immunity by suppressing other immune cells in humans. IL-2 is a cytokine that stimulates the proliferation and activation of T cells and Tregs [Candida and promotes the differentiation and function of Th1 cells [Candida and stimulating the recruitment and extravasation of immune cells to the site of infection. TNF also regulates the differentiation and function of Th1 and Th17 cells. PPARG is a nuclear receptor that regulates various metabolic processes and has anti-inflammatory effects [Candida infection in humans [In this study, we applied an integrative network pharmacology approach to identify the potential targets of the phytochemical constituents of Ajwa dates and their relevance to candidiasis in humans. This comprehensive approach allows for the prediction of multiple human targets for candidiasis, such as STAT3, IL-2, PTPRC, STAT1, CASP1, ALB, TP53, TLR4, TNF and PPARG. STAT3 is a transcription factor that regulates the differentiation and function of Th17 cells, which are essential for the defense against nd Tregs ,69. IL-2nd Tregs . PTPRC ind Tregs ,72. STATh1 cells . CASP1 ih1 cells . ALB is h1 cells . TP53 ish1 cells . TLR4 ish1 cells . TNF is  effects . PPARG cn humans .+ T cells develop into Th17 cells, a subset of T helper cells that produce interleukin-17 (IL-17) and other pro-inflammatory cytokines. Th17 cells play an important role in the host defense against fungal infections, such as candidiasis, by stimulating the production of other cytokines, such as TNF-α, IL-6, and IL-1β, and enhancing the recruitment and activation of neutrophils, which are essential for killing Candida cells [Based on the GO analysis, possible targets of phytochemical constituents of Ajwa dates against candidiasis in humans are involved in multiple important GO processes, such as the regulation of the response to a cytokine stimulus, regulation of inflammatory responses, regulation of the cellular response to an external stimulus, etc, whereas according to the KEGG pathway analysis, potential targets of phytochemical constituents of Ajwa dates against candidiasis are significantly enriched in several important pathways, such as Th17 cell differentiation, the Toll-like receptor signaling pathway, the C-type lectin receptor signaling pathway and necroptosis. Th17 cell differentiation is a process by which naive CD4da cells . Th17 ceda cells .Candida species. TLRs are a family of transmembrane proteins that can recognize specific molecular patterns derived from microbes, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), flagellin, zymosan and nucleic acids. These patterns are called microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular patterns (PAMPs) [The Toll-like receptor (TLR) signaling pathway is a mechanism by which the innate immune system recognizes and responds to microbial pathogens, such as  (PAMPs) . The bin (PAMPs) . Another (PAMPs) .Candida species. CLRs are a group of PRRs that have a carbohydrate recognition domain that can bind to specific sugar moieties on the surface of microbes. Some of the CLRs involved in anti-fungal immunity are Dectin-1, Dectin-2, Mincle, DC-SIGN and MBL [Candida species can activate different TLRs and induce necroptosis in macrophages and dendritic cells [Candida cells [The C-type lectin receptor (CLR) signaling pathway is another mechanism by which the innate immune system recognizes and responds to fungal pathogens, such as  and MBL . The bin and MBL . Dectin- and MBL . Moreove and MBL . Necropt and MBL . Candidaic cells . Necroptda cells . TherefoMoreover, the results of molecular docking analysis confirmed that phytochemical constituents of Ajwa dates can bind stably to the active pockets of identified target proteins. Therefore, these compounds could be considered for use as a potential treatment for candidiasis in humans via modulating proteins such as, STAT3, IL-2, PTPRC, STAT1, CASP1, ALB, TP53, TLR4, TNF and PPARG. The further binding stability of the protein–ligand complex confirmed via molecular dynamics analysis revealed that these complexes display a stable conformation in solvation in water at a temperature of 300 K and at an atmosphere pressure of 1. This is in line with what was shown in the docking analysis. During the MD simulation, hydrogen bonds were found to be formed in protein–ligand complexes, which indicates that the interaction has a high level of affinity. As a result of this consideration of the importance of network pharmacology, the present study examines the phytochemical constituents of Ajwa dates, as well as their potential targets, pathways, and effects, as they relate to the treatment of candidiasis, thereby providing a theoretical foundation for further research in the future. Considering that network pharmacology has certain limitations, it is only through data mining that it is possible to identify the basic pharmacological mechanisms that are responsible for the treatment of candidiasis. The analysis of the bioactive properties of molecules in the context of network pharmacology is currently supported by a variety of databases. It is inevitable that there will be discrepancies between the information sources in a database since there are many different sources of information and experimental data. It is important to note that although we have presented some interesting results, the potential of Ajwa dates to serve as a preventive measure against candidiasis and other fungal diseases in humans still needs to be evaluated in further research and clinical trials.Phoenix dactylifera L.) in humans through the integration of network pharmacology, molecular docking and dynamics simulations. The findings collectively shed light on the promising attributes of Ajwa date phytoconstituents as potential anti-fungal agents against Candida infections in humans. Through network pharmacology analysis, a complex interaction between Ajwa dates’ phytochemicals and Candida-associated molecular targets was identified. This approach allowed us to identify key compounds that may exert significant modulatory effects on fungal pathways. Subsequent molecular docking studies reinforced these interactions, highlighting the binding affinities of specific phytochemicals towards selected Candida protein targets of humans. The dynamics simulations provided deeper insights into the stability and dynamics of the ligand–target complexes, offering a comprehensive understanding of the binding mechanisms and intermolecular interactions governing the efficacy of the identified compounds. Overall, the present study underscores the potential of Ajwa dates’ phytochemicals as a source of anti-fungal agents for candidiasis treatment. By integrating computational techniques, the present study accelerated the process of identifying and characterizing potential therapeutic candidates. While findings of the present study build a promising foundation, further experimental validation and in vitro studies are essential to confirm the efficacy and safety of these compounds.In the present study, a multifaceted approach was used to investigate the anti-candidiasis potential of phytochemicals derived from Ajwa dates (


Objective: Longkui Yinxiao Soup is a traditional Chinese medicine formula used to treat psoriasis for decades. Although Longkui Yinxiao Soup showed promising efficacy in clinical practice, the regulatory mechanisms of Longkui Yinxiao Soup remain elusive. This study aimed to explore the underlying mechanisms of Longkui Yinxiao Soup in a psoriasis-like mouse model.Methods: Longkui Yinxiao Soup was quality controlled by determining the contents of imperatorin and rhoifolin using high-performance liquid chromatography. The imiquimod-induced psoriasis-like mouse model was used to study the therapeutic effect and mechanism of Longkui Yinxiao Soup. The histopathological skin changes were observed by hematoxylin and eosin staining; the infiltration of proliferating proteins, proliferating cell nuclear antigen and Ki67, in skin tissues were observed by immunohistochemical analysis; and the inflammatory factors such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-23, and IL-17 in serum were detected using enzyme-linked immunosorbent assay. RNA sequencing and bioinformatic analysis were used to predict the mechanism of LYS against psoriasis. mRNA expressions of p38, extracellular regulated protein kinases (ERK), mitogen-activated protein kinase 3 (MEK3), mitogen-activated protein kinase 6 (MEK6), RAP1 GTPase activating protein (Rap1gap), and Rap1 were determined using real-time quantitative polymerase chain reaction. The expression levels of proteins related to Rap1–mitogen-activated protein kinase signaling pathways were measured by Western blotting.Results: A quality-control method for Longkui Yinxiao Soup was successfully established using imperatorin and rhoifolin as content determination indexes. Longkui Yinxiao Soup significantly ameliorated the psoriatic symptoms in mice. The serum levels of inflammatory cytokines such as IL-6, TNF-α, IL-23, and IL-17 were decreased, and the expression levels of antigen identified by monoclonal antibody Ki67 (Ki67) and PCNA in skin tissues were downregulated. Moreover, the inhibition of Rap1–MAPK signaling pathways by Longkui Yinxiao Soup was detected.Conclusion: This study confirmed the antipsoriatic activity of Longkui Yinxiao Soup in psoriasis-like mice. This might be due to the inhibition of inflammatory factor secretion, keratinocyte proliferation, and the Rap1–MAPK signal pathway. Psoriasis is a common, chronic papulosquamous skin disease characterized by the development of erythematous, indurated, scaly, pruritic, and often painful skin plaques . The etiExcessive proliferation and impaired differentiation of keratinocytes are typical histopathological features of psoriasis. The progression of psoriasis is triggered by the upregulation of pro-inflammatory cytokines resulting from the activation of keratinocytes and immune cells . DendritAlthough modern medicine makes remarkable progress in treating psoriasis by developing drugs such as glucocorticoids, tretinoin, methotrexate, and biological preparations, the long-term use of these drugs may cause various adverse effects . TherefoSolanum nigrum L. (Chinese name: Longkui), Pteris multifida Poir. (Chinese name: Fengweicao), Arnebia euchroma (Royle) Johnst. (Chinese name: Zicao), Angelica dahurica (Fisch.ex Hoffm.) Benth.et Hook.F. (Chinese name: Baizhi), and Dioscorea opposita Thunb. (Chinese name: Shanyao). In clinical observations of LYS treatment, the total effective rate of LYS and Avia capsule combination therapy was 87.50%, and the total effective rate of therapy using Avia capsule in psoriasis vulgaris was 70.00% (Longkui Yinxiao Soup (LYS) has been used as an in-hospital formulation for psoriasis vulgaris in the Shanghai Skin Disease Hospital for more than 30 years. LYS contains five botanical drugs , includis 70.00% . LYS hasIn this study, we performed the quality control of LYS and then evaluated its antipsoriatic effect in the imiquimod (IMQ)-induced mouse model of psoriasis by regulating keratinocyte proliferation and inflammatory factor secretion. Finally, RNA sequencing and molecular biology methods were adopted to predict the potential target of LYS. This study aimed to clarify the mechanism of the effective substances in LYS inducing efficacy and blocking the pathogenesis of psoriasis.LYS was obtained from Shanghai Baolong Pharmaceutical Co., Ltd. . IMQ cream was purchased from Sichuan Mingxin Pharmaceutical Co., Ltd. . Imperatorin (purity ≥98%) and rhoifolin (purity ≥98%) were purchased from the National Institutes for Food and Drug Control of China . All primers were synthesized by Sangon Biotech . Antibodies specific to MEK3 + MEK6 (ab200831) and p38 delta/mitogen-activated protein kinase (MAPK) 13 + p38 alpha/MAPK14 (ab31828) were obtained from Abcam Inc. , and Rap1gap (DF4383) was obtained from Affinity Biosciences Inc. . Ki67 rabbit monoclonal antibody (AF1738), proliferating cell nuclear antigen (PCNA) rabbit monoclonal antibody (AF1363), phospho-p38 MAPK (Thr180/Tyr182) (AM063) antibody, Horseradish Peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (H + L) (A0208), and HRP-labeled goat anti-mouse IgG (H + L) (A0216) were purchased from Beyotime Biotechnology . The enhanced chemiluminescence reagent was obtained from Yeasen .Male BALB/c mice (22 ± 2 g) aged 6–8 weeks, purchased from Shanghai JieSiJie Laboratory Animals Co., Ltd. , were used in the experiments. The mice were offered free access to food and water within a specific pathogen-free environment. The use of animal participants in this study was reviewed and approved by the Care and Use Committee of the Shanghai Skin Disease Hospital (ethics clearance number: 2022–60.)v/w) purified water for 2 h and then decocted in 8 times water (v/w) for 1 h. The decoction was then filtrated, and the filtrate was collected. The filtrate obtained after two times of filtration was mixed and concentrated at 80°C to a relative density of 1.27.The five botanical drugs included in LYS  were pur–1; and the sample volume was 10 μL.The analytical method was validated using a Waters Alliance high-performance liquid chromatography  system containing an e2695 separation module and a 2,998 photodiode array detector. Both imperatorin (active ingredient of Baizhi) and rhoifolin (active ingredient of Fengweicao) were analyzed using an Agilent Eclipse Plus C18 column . The other chromatographic conditions for HPLC were as follows: the eluent was acetonitrile–water (65:35) for imperatorin and acetonitrile–water (20:80) for rhoifolin; the monitoring wavelength was set to 300 nm for imperatorin and 345 nm for rhoifolin; the column oven temperature was 30°C; the flow rate of the eluent was 1.0 mL minw/w) daily on their back skins for five consecutive days. On day 13, the mice were sacrificed after being narcotized by inhalation of diethyl ether, and the blood samples and back skin tissues were collected.The mice were subjected to adaptive feeding for 7 days prior to the initiation of the experiments. Thirty-five mice were randomly categorized into five groups: the control group, the model group (IMQ group), the methotrexate -treated group, and two groups treated with LYS intragastric administration at the doses of 4.9 g/kg and 43.8 g/kg of crude drug (LYS-Low and LYS-High) from day 1 to day 12. The low dose of LYS was converted according to human equivalent dosages based on body surface area. The backs of all the mice were shaved and depilated using depilatory cream to expose the back skin. Four groups except the control group received the application of 62.5 mg IMQ cream (5% The body weight of the mice was recorded every day during the experiment. The psoriasis area and severity index (PASI) was used to evaluate the severity of the skin inflammation, and the spleen index was measured on day 13. The degrees of erythema, scaling, and thickening were scored independently on a scale of 0–4: 0 for none; one for slight; two for moderate; three for severe; four for very severe. The severity of the inflammation was measured using the cumulative score  on a scale of 0–12. The spleen index was calculated as follows:Spleen index (mg/g) = Spleen weight (mg)/Mouse weight (g) × 10.On day 13, the back skin tissues were collected and fixed in 10% buffered formalin and embedded in paraffin. After staining with hematoxylin and eosin (H and E), the samples were examined using an optical microscope . The epidermal thickness was measured at five randomly selected spots.g and 4°C for 20 min. Then, the levels of the cytokines IL-6 , TNF-α , IL-23  and IL-17 A  were determined using enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s protocols.The serum samples were separated by centrifugation from the blood samples at 12,000IHC analysis was performed on paraffin-embedded skin tissue sections. Antibodies anti-Ki67 (1:500) and anti-PCNA (1:500) were used. The images of sections were photographed using a light microscope.n = 3). Total RNAs were extracted to generate cDNA libraries for Illumina Hiseq4000 sequencing . Before further analyses, we used the relative logarithmic expression boxplots and principal component analysis (PCA) to control the quality of the data. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations with assembled differentially expressed genes (DEGs) were performed, and subsequently, LYS-induced DEGs and associated signaling pathways were generalized. Furthermore, quantitative polymerase chain reactions (qPCRs) and Western blotting (WB) were used to check the expression of some DEGs related to target signaling pathways to verify the results of RNA sequencing.Skin tissues were extracted randomly from the control, IMQ, and LYS-High groups, respectively . Then the RNAs were reverse transcribed into cDNA using a PrimeScript RT means Real time Master Mix . qPCR was performed using TB green is a fluorescent dyes Green Premix Ex Taq Mremix Ex Taqme (TaKaRa) on a LightCycler480 system. The PCR cycle parameters were as follows: denatured at 95 °C for 30 s, followed by 45 cycles of 95°C for 5 s and 60°C for 30 s, and then followed by one cycle of 95°C for 5 s and 60°C for 60 s (melting). The data were analyzed using the 2Approximately 100 mg of skin tissue was used to extract protein using 1 mL of RIPA buffer containing 10 μL of PMSF . The protein concentration was determined using BCA assay , and total proteins were separated using 6%–12% SDS-PAGE. The separated proteins were then transferred to PVDF membranes and blocked with 5% BSA in TBST for 2 h. After blocking, the membranes were washed three times with TBST and incubated with primary antibodies including PCNA, p38, P-p38, MEK3 and MEK6 , GAPDH (1:2000 diluted), and Ki67 (1:800 diluted) overnight at 4°C. The incubation with secondary HRP-conjugated antibody  was then performed at room temperature for 1 h to detect antibody binding. The protein bands were quantified and analyzed using an Image J system . Finally, the data were expressed as band intensity normalized to GAPDH or their respective total proteins.p-value of <0.05 indicated a statistically significant difference.The data were expressed as mean ± standard deviation (SD). A one-way analysis of variance was performed to analyze the data using SPSS Statistics 24.0 . A via selective phosphodiesterase four inhibition  (p < 0.001). After LYS treatment, the body weight decreased significantly compared with the model group (p < 0.01)    and S1.  < 0.01) . The splctively) .H and E staining revealed that the epidermal layer in the IMQ group was thickened, and numerous lymphocyte infiltrations were observed in the dermis and epidermis. After treatment with MTX and different doses of LYS, the thickness of the epidermal layer was effectively reduced, the stratum corneum became smooth, and the lymphocyte infiltration in the dermis layer was reduced. Furthermore, the higher the dose of LYS, the more obvious was the effect; notably, the high dose of LYS was equivalent to that of MTX .p < 0.01). The levels of these cytokines significantly decreased in the LYS and MTX groups compared with the IMQ group (p < 0.05). In the LYS group, the levels of IL-6, TNF-α, IL-23, and IL-17 decreased in a dose-dependent manner.The effects of LYS on serum cytokines were detected using ELISA. IL-6, TNF-α, IL-23, and IL-17 levels significantly increased in IMQ-induced mice with psoriasis  , cellular component (CC), and molecular function (MF). These DEGs were linked to the keratinocyte adhesion, migration, and dysfunction based on BP subgroups : “epithe55 DEGs) . In termThe KEGG enrichment analysis led to the identification of the top 10 pathways, including “AGE-RAGE signaling pathway in diabetic complications,” “Rap1 signaling pathway,” “Ras signaling pathway,” “MAPK signaling pathway,” and so forth . LYS significantly suppressed the expression of the aforementioned targets .We further detected the expression of critical molecules at the mRNA and protein levels to further verify the role of Rap1–MAPK signaling pathways in the proliferation of psoriatic keratinocytes. As shown in p < 0.001). These IMQ-induced enhancements in the expression were markedly prevented by LYS in a dose-dependent manner except p-JNK (p < 0.01 and p < 0.001). The expression levels of MEK3/6 and p-MEK3 were increased by IMQ treatment (p < 0.05 and p < 0.001), but were not suppressed by LYS treatment. IMQ treatment upregulated the expression of Rap1 and Rap1gap, while LYS significantly downregulated their expression (p < 0.001 and p < 0.01).As shown in Psoriasis is a complex disease caused by different factors, and its exact pathogenesis is unknown. Previous studies showed that keratinocyte hyperproliferation might play a critical role in the initiation and maintenance of psoriasis .Recent studies reported that the theory of the “IL-23/Th17 axis” could explain the pathogenesis of psoriasis to some extent. Activated by IL-23 and sourced from dendritic cells and macrophages in the psoriatic dermis, Th17 and T cells release inflammatory cytokines such as TNF-α, IL-6, IL-17A, and IL-22 . These iCell cycle–related antigens Ki67 and PCNA are the most common proliferation markers in the IMQ-induced psoriasis-like mouse model. This study showed that the expression of Ki67 and PCNA was significantly upregulated in the model group, accompanied by an increase in “IL-23/Th17 axis”–related inflammatory factors, compared with that in the control group. This was consistent with the previous findings . After LMAPK plays an important role in regulating most cellular processes, including proliferation, differentiation, and apoptosis and can be grouped into four subfamilies: ERK, p38, JNK, and BMK1  . P38, ERp38 MAPK can be activated by Rap1, which can regulate cell growth and death . Rap1 bein vivo by inducing the secretion of “IL-23/Th17 axis”–related inflammatory factors, thereby reducing the hyperproliferation of keratinocytes. The results of RNA sequencing and molecular biology experiments confirmed that the antipsoriatic effect of LYS might be related to the inhibition of Rap1–MAPK signaling pathway activation.The findings of this study demonstrated that LYS primarily targeted keratinocytes The results of this study revealed that LYS had obvious anti-psoriatic effects in the mice with IMQ-induced psoriasis. The anti-psoriasis effect of LYS was related to the inhibition of inflammatory factor secretion, keratinocyte proliferation, and the activation of the Rap1–MAPK signaling pathway. These findings confirmed the therapeutic effect and mechanism of LYS in treating psoriasis to some extent, thus providing important evidence for the clinical application of LYS.


Stk32a is expressed in hair cells on one side of the LPR in a pattern complementary to Emx2 expression in hair cells on the opposite side. Stk32a is necessary to align the intrinsic polarity of the bundle with the core planar cell polarity (PCP) proteins in EMX2-negative regions, and is sufficient to reorient bundles when ectopically expressed in neighboring EMX2-positive regions. We demonstrate that STK32A reinforces LPR formation by regulating the apical localization of GPR156. These observations support a model in which bundle orientation is determined through separate mechanisms in hair cells on opposite sides of the maculae, with EMX2-mediated repression of Stk32a determining the final position of the LPR.The vestibular maculae of the inner ear contain sensory receptor hair cells that detect linear acceleration and contribute to equilibrioception to coordinate posture and ambulatory movements. These hair cells are divided between two groups, separated by a line of polarity reversal (LPR), with oppositely oriented planar-polarized stereociliary bundles that detect motion in opposite directions. The transcription factor EMX2 is known to establish this planar polarized organization in mouse by regulating the distribution of the transmembrane receptor GPR156 at hair cell boundaries in one group of cells. However, the genes regulated by EMX2 in this context were previously not known. Using mouse as a model, we have identified the serine threonine kinase STK32A as a downstream effector negatively regulated by EMX2.  Planar polarity is an organizational feature of epithelia in which the polarity of cells and tissues is coordinated along a plane perpendicular to the apical-basal cell polarity axis. Planar polarity is seen across diverse species and tissues, and this organization is essential for early developmental events including left/right asymmetry in the primitive streak and coordinated cellular movements such as convergent extension and neural tube closure . SimilarThe hair cell stereociliary bundle is comprised of rows of stereocilia of increasing length with the tallest row positioned alongside the kinocilium, a true tubulin-based cilium located at one side of the hair cell apical surface . When thEmx2 and Gpr156 mutants, intrinsic polarization of the bundle remains intact but these bundles are uniformly oriented and fail to form an LPR. However, despite this functional relationship, GPR156 is not transcriptionally regulated by EMX2. Instead GPR156 function is regulated post-translationally, and is only present at the apical cell boundaries in hair cells expressing EMX2 where it acts to reorient the intrinsic polarity relative to the PCP axis  mice  mice .Emx2Cre/Cre utricles that included the downregulation of genes that are presumed to be direct or indirect transcriptional targets of EMX2  (Stk32a mRNA was detected in clusters that overlapped with mRNA transcribed from the hair cell gene Gfi1 (Genes that show increased expression in  the LPR . One sucn (fISH) . STK32A n (fISH) . Within ene Gfi1 . A similene Gfi1 . The haiene Gfi1 .Stk32a mRNA detected by RNAseq is due to an expansion of the Stk32a expression domain and in the absence of Emx2, Stk32a mRNA can be detected throughout the entire sensory epithelia  Emx2 overexpression mice (LSL-Emx2Rosa26 ), Stk32a expression is reduced wherever Emx2 is induced  vector to overexpress Stk32a mRNA in lateral hair cells of the utricle where Stk32a expression is normally repressed. For these experiments, utricles were micro-dissected and grown as explants prior to AAV transduction. Since the AAV vector does not contain the genomic regulatory elements surrounding Stk32a it is not subject to EMX2 associated repression. Transduced cells were identified based upon EGFP expression from a bicstronic Stk32a-P2A-EGFP mRNA . Therefore, in order to determine the sub-cellular distribution of STK32A, ir cells . Since Sir cells . STK32A:ir cells . Motif-by bundle . Moreoves affect . TogetheStk32a was also necessary for the planar polarized development of medial hair cells  mouse was generated using CRISPR-mediated mutagenesis to determine if ir cells . This tatermates –3. SinceStk32a-/- utricle was severely disrupted , but not in the rest of the organs (Emx2-negative domains), as reported previously  also had an inverted intrinsic planar polarity that was revealed by changes in the distribution of aPKC  to regulate Stk32a expression. Prior transcriptional profiling has shown that EMX2 contributes to both transcriptional activation and repression in the developing telencephalon prior to cortical arealization . The Stk32a KO mouse line was generated by targeting the second exon of the Stk32a gene, which contains the translational start, using CRISPR technology. Two gRNAs (5’-UAGAAAUGACCAUGGUGCAU-3’ and 5’-UACAGUGUAUCUAAGGAUAG-3’) that are complementary to intronic DNA sequences located on either side of Stk32a exon 2 were designed by the University of Utah Mutation Generation and Detection Core. These were combined with recombinant Cas9 protein for pronuclear injections into B6CBAF1/J mouse zygotes (Jax strain#100011) that were subsequently implanted into pseudo-pregnant females by University of Utah Transgenic and Gene Targeting Core. One male founder with an appropriate deletion generated by NHEJ and identified by PCR amplification followed by Sanger sequencing was selected for line expansion through backcross with B6129SF1/J females. PCR primers used for mutant genotyping are: Stk32a primer A (5’-AGTTTTCTTGATACCTGGGGATGT-‘3’) and Stk32a primer B (5’-GGATGGCAGTGATTATGAAAGTTG-3’). The Stk32a-/- phenotype was initially evaluated by intercrossing N2 heterozygous mice and the mutant phenotype was consistently observed when N3 and later generations were experimentally intercrossed. Experimental crosses consisted of -/-Stk32a  males crossed with +/-Stk32afemales, and heterozygous littermates generated from these crosses were used as controls. CD1 mice were purchased from Charles River and intercrossed for utricle explant experiments. Mice were genotyped by PCR using allele specific primers. All animal work was reviewed for compliance and approved by the Animal Care and Use Committee of The University of Utah (IACUC protocol #00001498) and the Animal Care and Use Committees of The Jackson Laboratory ./wt mice  were proInner ear tissues were fixed using 4% paraformaldehyde in 67 mM Sorensons’ phosphate buffer (pH 7.4) for 2 hr on ice, or using 10% trichloroacetic acid (TCA) for 10 min on ice depending upon primary antibody selections (see below). For wholemount immunofluorescent labeling, utricles and saccules were micro-dissected and detergent permeabilized using a blocking solution , PBS) supplemented with 0.5% Triton-X100. Primary antibodies and Phalloidin Alexa Fluor 488 (Invitrogen A12379) were diluted in blocking solution supplemented with 0.1% Tween-20 and incubated overnight at 4 °C. Tissue was washed thoroughly using PBS with 0.05% Tween-20 and incubated with species-specific Alexa-conjugated or Cy3 conjugated secondary antibodies , before mounting for fluorescence microscopy using Prolong Gold . Fluorescent images were acquired by structured illumination microscopy using the Zeiss Axio Imager M.2 with ApoTome.2 attachment and Axiocam 506 mono camera. Images in AB_609894), Mouse anti-aPKC , Mouse anti-βII Spectrin , Guinea Pig anti-CELSR1 ), Rabbit anti-GFP , Goat anti-GPR156 , Rabbit anti-MST4/STK26 , Goat anti-Oncomodulin/OCN , Rabbit anti-pericentrin/PCNT , Rabbit anti-PK2 .Antibodies and fixatives used in this study are: Mouse anti-Acetylated Tubulin , Rat anCre/wtEmx2  intercross, under nuclease free conditions. The bony labyrinth was lightly perforated and ears were stored in RNAlater (ThermoFisher AM7020) at 4 C for 1 week to allow for subsequent collections and genotyping. Cre/CreEmx2  and wild type samples were dissected under nuclease free conditions to isolated the utricle from surrounding mesenchyme and adjacent cristae. Four to 6 utricles were combined per genotype to constitute one biological replicate, and 4 replicates were collected for each. Tissue was transferred to 350 µl of RNeasy Lysis buffer (Qiagen 79216) Buffer RLT and lysed using the Qiagen Tissue Lyser LT at 20 Hz with one stainless steel bead for 4 min. Following centrifugation, supernatant was transferred to a clean tube and RNA isolated using the RNeasy Micro Kit (Qiagen 74004). DNA and RNA concentrations were determined using Qubit broad range DNA and RNA assay kits (Invitrogen Q32853 Q10211) and RNA quality evaluated using the Agilent 2200 TapeStation which returned RNA integrity numbers ranging from 8.3 to 9.1. Libraries were generated at the Huntsman Cancer Institute Genomic Core facility using TruSeq Stranded Total RNA Library Prep with Ribo-Zero Gold kits (Illumina 20020598) and sequenced using the Illumina NovaSeq 6000 for 25 M 2x50 bp reads per biological replicate. Significant unmapped reads were found in one WT sample which was excluded from subsequent analysis after principal component analysis revealed it to be a significant outlier. Remaining samples were compared using the Bioconductor DESeq package as well as by post hoc-analysis using Swish. Benjamini-Hirshberg false discover rate was used to normalize data across all transcripts and produce adjusted p-values for significantly downregulated or upregulated genes based on log2-fold changes compared to WT baseline transcript levels.Whole inner ear capsules were dissected from E18.5 mouse pups, generated by fISH inner ears were fixed overnight at 4 °C using 4% paraformaldehyde prepared in phosphate buffered saline  and stored as needed at –20 °C in 100% MeOH. Prior to hybridization tissue was dissected to expose sensory epithelia and rehydrated through a decreasing MeOH gradient  ending in PBST (PBS with 0.1% Tween-20). fISH was completed using Hairpin Chain Reaction protocols (For wholemount rotocols . In briet-test.For quantification of mRNA detected by fISH (HCR v3.0) images were captured in three rectangle regions of interest (ROIs) of identical sizes were imaged with identical Z-plane depth from the lateral and medial regions of the utricle  and procTo quantify GPR156 in cochlear explants, we defined a 2x0.5 µm window in Fiji and positioned it on an medial or lateral outer hair cell junctional region where GPR156 signals were the most intense in the cell. The mean grey value was measured for each electroporated cell along with two adjcent un-electroporated control cells. GPR156 enrichment was calculated as a ratio of averaged values for control cells. Statistical significance was determined using 1-way ANOVA . Purified virus was aliquoted and stored at –80 °C. Virus titer was determined by qPCR using SYBR Green and StepOnePlus (AppliedBiosystems) quantitative PCR machine as described  cells were used for AAV preparation based upon an established protocol . Cells wescribed . Variant7 virions per µl) and incubated in a cell culture incubator at 37 °C for 4 hr. After the incubation, 2–3 ml fresh culture medium was added to each well and the explant was cultured for 3 days, with fresh media added after 48 hr. Following culture, explants were washed with PBS, then fixed for 30 min with 4% PFA/PBS on ice. After fixation and brief PBS wash, coverslips were superglued onto glass microscope slides with the explant facing up and processed for wholemount immunolabeling as described previously. Following labeling explants were coverslipped using Prolong Gold Antifade mounting media with the explant was sandwiched between the two glass coverslips and the bottom most coverslip superglued to the glass slide.Utricles were dissected from the inner ears of P0 CD1 mouse pups in pre-chilled dissection solution  and 10 µg/ml Ciprofloxacin (SigmaAldrich 17850), transferred to 100 µl culture media (DMEM/F12 1:1 (ThermoFisher Cat 11330032) with 10% FBS, 0.1X N2 Supplement and 10 µg/ml Ciprofloxacin), and attached to a sterilized coverslip using Cell-Tak Cell and Tissue Adhesive (Corning 354240). Coverslips were placed into individual wells of a six-well plate and transduced with AAV premixed in culture media with AAV at  were collected at E13.5 in HBSS +5 mM Hepes buffer . Plasmid DNA was mixed with Fast Green FCF  and injected at 2 µg/µl into the cochlear duct using the Wiretroll plunger system . Next, the whole inner ear was electroporated , and the membranous labyrinth was dissected away from the condensed mesenchyme and embedded in Matrigel . The explants were cultured for 6 days in DMEM with 10% fetal bovine serum and 10 µg/ml ciprofloxacin , and then fixed in 10% TCA for 10 min on ice before being processed for immunolabeling.To evaluate bundle orientation, utricles, saccules, and cristae were immunolabeled with antibodies against βII-SPECTRIN  and Oncomodulin/OCN  as described above. Orientation was determined based on the polarized position of the fonticulus and measurements aided with a customized Python script  that allPou4f3 since hair cell number would not vary with the size of the cropped vestibule. For each primer set, three biological replicates and two technical replicates were used. PCR amplifications used the following primers: Stk32a primer C (5’- GACTTGACCATGGGAGCCAA –3’), Stk32a primer D (5’-TGGTGTCATTCTTCCGCACA –3’) Stk32a primer E (5’-GGCCCTGGACTACCTACAGA-3’), Stk32a primer F (5’-TAAGGCTTGGTGCCAGCTAC-3’), GAPDH for (5’-TGGAGCCAAAAGGGTCA-3’), GAPDH rev (5’-CTTCTGGGTGGCAGTGA-3’), Pou4f3 for (5’-CCAGACTCCCGAAGATGATGG-3’), Pou4f3 rev (5’-GCCAGCAGGCTCTCATCAAA-3’).Inner ears were dissected from individual mice and coarsely cropped to separate the cochlea and semi-circular canals from the utricle and saccule, and this vestibular portion was transferred to individual Eppendorf tubes and flash frozen in liquid nitrogen. Following PCR genotyping, RNA was extracted from three knockout and three littermate control samples using the QIAGEN RNeasy Micro Kit (QIAGEN 74004) according to the manufacturers protocol. RNA concentration was determined by Nanodrop spectrophotometry and 2.5 µg of RNA was used to prepare cDNA using the Invitrogen SuperScript III Reverse Transcriptase (ThermoFisher Scientific 18080093). Quantitative RT-PCR was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems) and PCR reactions contained 0.6 µM forward and reverse primer, 0.2 μL of cDNA and 2 X Power SYBR Green master mix. Following an incubation at 95 °C for 10 min, the reactions cycled between 95 °C denaturation for 15 s, and 58 °C extension for 1 min 40 times, and a melting curve was generated after the final cycle to verify the amplification specificity. Relative fold changes of gene expression were normalized by against the hair cell gene t-test was used to analyze the comparison and a p-value <0.05 was considered to be statistically significant. For measures of GPR156 medial and lateral localization, ne-way ANOVA and unpaired t-test were respectively used to analyze the comparison and p-values <0.05 was considered to be significant. Gpr156 data is graphed as 25–75% boxplots where exterior lines show minimum and maximum values, the middle line the median and +the mean. For analysis of RNAseq datasets, Benjamini-Hirshberg false discover rate was used to normalize data across all transcripts and produce adjusted p-values for significantly downregulated or upregulated genes based on log2-fold changes compared to WT baseline transcript levels. Unless otherwise stated, fluorescent imaging was completed on a minimum of three biological replicates with higher quality representative images selected for publication.All data is graphed as mean +/- SEM or SD as indicated in figure legends and statistical analysis was conducted using GraphPad Prism 9. The student’s  This important study provides a significant advance in the understanding of the molecular mechanisms that establish planar cell polarity in hair cells of the mammalian inner ear. The conclusions, which are supported by compelling evidence, will be of interest to those studying the development and function of the vestibular system, and planar cell polarity. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Hernán López-Schier (Reviewer #1); Brian McDermott (Reviewer #3).Thank you for submitting your article "The dark kinase STK32A regulates hair cell planar polarity opposite of EMX2 in the developing mouse inner ear" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Demonstrate whether or not Stk32a is a direct transcriptional target of Emx2, or tone down this conclusion.2) Provide further analysis of the nature of the Stk32a mutation, including whether or not any protein is produced.3) Test whether the kinase activity of Stk32a is required for its function.4) Either test whether Stk32a directly interacts with Gpr156, or discuss whether Stk32a regulates Gpr156 via direct or indirect mechanisms.Reviewer #1 (Recommendations for the authors):Figures are truncated in my version of the manuscript.I do not understand how the authors can claim that the Stk32a-/- mice have massively disrupted vestibular hair cells, but have no discernible behavioural differences from controls.Please revise the idea that Stk32a is a direct target of Emx2.Reviewer #2 (Recommendations for the authors):Specific comments:1. Since mutant mRNA perdures in the Stk32a mutant utricle , it would be important to more clearly define the nature of the mutation and determine whether any Stk32a protein is produced in the mutant utricle by Western blot. Multiple WB-validated Stk32a C-terminal antibodies are commercially available and can be tested (e.g. Σ SAB1405383). In addition, it should be clarified whether Stk32a heterozygous maculae had any PCP defects, to rule out potential dominant-negative effects.2. How might Emx2 repress Stk32a transcription? Is this regulation direct or indirect? Was there a precedent for Emx2 as a repressor? This should at least be discussed if not experimentally addressed. Of note, ectopic expression of Emx2 in the MES did not appear to repress Stk32a expression as effectively as in the lateral region , suggesting that additional factors may be involved to fully repress Stk32a.3. Several outstanding questions remain regarding the mechanism by which Stk32a regulates GPR156 surface expression, some of which are well within the scope of the current study. For example, is the kinase activity of Stk32a required for its function? It should be entirely feasible to test whether a Stk32a kinase-dead construct can reduce GPR156 levels in cochlear explants, as shown in Figure 7 . The kinase-dead construct would also help distinguish whether Stk32a overexpression reorients PCP via dominant-active or dominant-negative effects.4. Related to the above, the data and model really beg the question of whether Stk32a directly interacts with Gpr156. It should be feasible to test this in heterologous cells transfected with Gpr156 and GFP-tagged Stk32a wt or kinase-dead constructs using co-IP assays. Essential revisions:1) Demonstrate whether or not Stk32a is a direct transcriptional target of Emx2, or tone down this conclusion.Stk32a transcription. As recommended, we have made text edits throughout the manuscript that are meant to specifically tone done the conclusion that Stk32a is a direct target of Emx2. Notable changes include:We appreciate this concern and recognize that our experiments do not distinguish between a direct or indirect relationship between EMX2 and 2) Provide further analysis of the nature of the Stk32a mutation, including whether or not any protein is produced.We appreciate this recommendation as well as the underlying concern that the KO phenotype reported here may not be null, and that possible expression of mutant protein may provide residual function or even a dominant phenotype. We have tested multiple commercial antibodies , and have contracted the production of our own custom antibodies yet remain unable to identify a reagent with suitable specificity or affinity to detect STK32A by Western Blot or Immunofluorescence assays.Stk32a mutation that support our interpretation that the Stk32a KO is a null, and that any mutant protein, if produced, is not functional. We feel that these analyses should be sufficient because in the event that we had detected mutant protein via Western blot, these are the experiments we would have conducted to determine whether or not that mutant protein had an effect.In the absence of a suitable reagent we have provided the following additional analyses of the Stk32a+/- littermates. Thus, targeted deletion of Stk32a exon2 does not result in a dominant phenotype. Please see Figure 4 —figure supplement 3 in the resubmitted manuscript. Moreover, this addition allows us to clarify an experimental detail that was not clearly stated during the first submission; that the ‘Control’ mice used for the KO analysis were all heterozygous for the Stk32a exon2 deletion.We have prepared a new figure supplement demonstrating that no phenotype emerges in Stk32a heterozygotes and that the organization of vestibular hair cells in the utricle is not changed between wild type and Stk32a mutant allele is not functional. In brief, we demonstrate that a predicted myristoylation site at the second amino acid (Glycine) of STK32A is necessary for GFP-tagged protein localization to the apical surface of hair cells. Furthermore, deletion of this Glycine prevents stereociliary bundle reorientation following AAV-mediated expression of GFP-tagged Stk32aΔ2G mutant protein in Emx2-positive hair cells demonstrating that myristoylation is essential for this STK32A function.We have added additional experimentation demonstrating that any mutant protein that might be produced from the Stk32a KO we anticipate that any mutant proteins translated from alternative start codons will also be non-functional.Since this amino acid is encoded by exon2 and is deleted in the 3) Test whether the kinase activity of Stk32a is required for its function.Stk32a construct that is predicted to disrupt kinase function. This mutation replaces an essential Lysine at amino acid position 52 with Arginine (K52RStk32a ). Based upon sequence similarities to related kinases this Lysine is necessary for STK32A to bind ATP prior to substrate phosphorylation  and substitution with Arginine disrupts kinase activity. This mutant has been used to demonstrate that the kinase activity of STK32A is required for function in the following experiments:We have improved the resubmitted manuscript as recommended by the reviewers by conducting additional experiments using a mutant K52RStk32a  to electroporate hair cells in the developing cochlea. These experiments have been added to Figure 7. In brief, kinase-dead Stk32a K52R does not impact GPR156 delivery or maintenance at the apical cell surface, indicating the kinase activity is necessary for this aspect of STK32A function.We have used plasmids expressing Based upon these experiments we conclude that the kinase activity of Stk32a is required for its function and appreciate the recommendation to incorporate these additional experiments.4) Either test whether Stk32a directly interacts with Gpr156, or discuss whether Stk32a regulates Gpr156 via direct or indirect mechanisms.The potential for physical interactions between STK32A and GPR156 has been evaluated in heterologous cell lines by co-transfecting GST and GFP tagged constructs. This approach has not revealed a direct physical interaction, however negative results using these approaches have little meaning since we cannot exclude the possibility that intermediaries or cofactors present in hair cells, but not in cultured cells, facilitate the proposed interactions. As such we have decided not included those studies in this manuscript. Instead, as recommended by the reviewers, we have discussed the possibility that STK32A regulation of GPR156 may be indirect.Reviewer #1 (Recommendations for the authors):Figures are truncated in my version of the manuscript.This is unfortunate and, if given the opportunity, we will ensure that each figure is appropriately formatted prior to publication.I do not understand how the authors can claim that the Stk32a-/- mice have massively disrupted vestibular hair cells, but have no discernible behavioural differences from controls.We were also surprised that changes in planar polarity that are so readily detected based upon vestibular hair cell anatomy did not impact the mouse’s behavior in its home cage. It is possible that deficits will be revealed by more advanced behavioral testing but that is beyond the scope of this developmental study.Please revise the idea that Stk32a is a direct target of Emx2.Stk32a repression. These changes were described in detail in response to Essential Revision #1.We have made changes to revise this idea and discuss the possibility of an intermediary between EMX2 and Reviewer #2 (Recommendations for the authors):Specific comments:1. Since mutant mRNA perdures in the Stk32a mutant utricle , it would be important to more clearly define the nature of the mutation and determine whether any Stk32a protein is produced in the mutant utricle by Western blot. Multiple WB-validated Stk32a C-terminal antibodies are commercially available and can be tested (e.g. Σ SAB1405383). In addition, it should be clarified whether Stk32a heterozygous maculae had any PCP defects, to rule out potential dominant-negative effects.Despite the availability of several commercially available antibodies we have not identified one that can detect STK32A protein from mouse tissues by either Western blot or immunofluorescent labeling.Nonetheless, we have taken two steps to address these important concerns as presented in greater detail in Essential Revision #2.Stk32a KO mouse we do not expect that any mutant protein, if produced, to be functional in the KO.We demonstrate that an N-myristoyl Glycine at amino acid position 2 is necessary for STK32A localization and function. Since this amino acid is encoded by exon2 which is deleted in the We demonstrate by comparing wild type and Stk32a heterozygous hair cells that there are no PCP defects and thus no dominant-negative effects of any mutant protein that may be produced.2. How might Emx2 repress Stk32a transcription? Is this regulation direct or indirect? Was there a precedent for Emx2 as a repressor? This should at least be discussed if not experimentally addressed. Of note, ectopic expression of Emx2 in the MES did not appear to repress Stk32a expression as effectively as in the lateral region , suggesting that additional factors may be involved to fully repress Stk32a.Emx2 mutation or overexpression (similar to our own study). Notable exceptions occur in the regulation of Sox2 expression where EMX2 binds POU sites in the Sox2 enhancer which has been proposed to repress gene expression in a dose dependent manner , and in the regulation of Gsx2 expression where EMX2 functions as a repressor in the presence of cofactors . We have added a paragraph to the Discussion section that presents these examples, while clearly stating that our own study does not distinguish between direct or indirect modes of regulation.There is a well-established precedent for EMX2 acting as a repressor during the course of telencephalic development and cortical arealization. However, much of this work is based upon changes in gene following 3. Several outstanding questions remain regarding the mechanism by which Stk32a regulates GPR156 surface expression, some of which are well within the scope of the current study. For example, is the kinase activity of Stk32a required for its function? It should be entirely feasible to test whether a Stk32a kinase-dead construct can reduce GPR156 levels in cochlear explants, as shown in Figure 7 . The kinase-dead construct would also help distinguish whether Stk32a overexpression reorients PCP via dominant-active or dominant-negative effects.We have conducted additional experiments using a kinase-dead STK32A construct that test its function in both cochlear and utricle explants. These changes were described in detail in response to Essential Revision #3.4. Related to the above, the data and model really beg the question of whether Stk32a directly interacts with Gpr156. It should be feasible to test this in heterologous cells transfected with Gpr156 and GFP-tagged Stk32a wt or kinase-dead constructs using co-IP assays.We have conducted experiments to test the potential for STK32A and GPR156 to physically interact or for STK32A to phosphorylate GPR156 following co-transfection of heterologous cell lines. To date these efforts have only yielded negative results. Unfortunately, an unavoidable flaw in this approach is that it is not known whether or not the cell lines we employed express cofactors or intermediaries needed for these interactions to occur  which makes interpretation of a negative result impossible. For these reasons those results have not been included in the current manuscript.


The aim of the study is to verify the active ingredients of peach blossom and to explore the molecular mechanisms of their therapeutic effects against constipation through network pharmacology and molecular docking analysis.  The potential active ingredients of peach blossom were identified from published literature and the BAT-TCM database, and their potential targets were predicted using the SwissTargetPrediction and PharmMapper platforms. In addition, targets related to constipation were retrieved using OMIM, DrugBank, GeneCards, TTD, and DisGeNET databases. The intersection of drug targets and disease targets was considered as the potential targets of peach blossom in the treatment of constipation. The STRING platform was used to construct a protein interaction network. Gene ontology (GO) functional analysis and KEGG pathway enrichment analysis were performed on key targets using the DAVID database. Molecular docking verification between the active ingredients of peach blossom and the targets was conducted using AutoDock software.  A total of 33 active ingredients of peach blossom and 185 corresponding targets were identified, and 88 intersection targets were obtained after Venny mapping. These 33 active ingredients  in peach blossom may play a role in the treatment of constipation by regulating signaling pathways through targets such as EGFR, VEGFA, ESR1, GSTP1, and PTGS2.  A variety of active ingredients of peach blossom regulate multiple signaling pathways by acting on targets, which reflects the characteristic of “multiple ingredients-multiple targets-multiple pathways,” thereby playing a role in the treatment of constipation. Constipation is a common gastrointestinal disease characterized by a prolonged or shortened defecation cycle, difficult defecation, and dry stool. In some conditions, the stool is not hard, but the process of defecation is not smooth though there is a desire to defecate . Constip, Fructus aurantii, Magnolia officinalis, Atractylodes, Fructus fructus, Cistanche, Astragalus, peach kernels, and glycyrrhiza. Both alone and in combination with other TCM applications, obvious efficacy without toxic side effects has been observed. In addition, it has been reported that the therapeutic effect of TCM on antipsychogenic constipation was more effective than that of western medicines, such as phenolphthalein and glycerin enema [Currently, the main strategy for the treatment of constipation is the use of laxatives, including bulk laxatives , osmotiin enema .Rosaceae Prunus persica (L.) Batsch or Prunus davidiana (Carr.) Franch. The blooming of flowers precedes the growth of leaves, and the flowers are harvested between March and April [Peach blossoms are the flowers of rosaceous plants nd April . Peach bnd April . Its usend April  used “Shnd April . In addind April . In clinnd April .In this study, an “active ingredient-target-signaling pathway” network was constructed based on the network pharmacology analysis, and the potential molecular mechanisms of the therapeutic effect of peach blossom against constipation were explored by molecular docking analysis and verification. Our findings may provide a theoretical basis for the clinical application of peach blossom in the treatment of constipation, and provide novel insights for further research on the mechanisms of peach blossom.https://bionet.ncpsb.org/batman-tcm/) [https://pubchem.ncbi.nlm.nih.gov). The SwissADME database (https://www.swissadme.ch/) [The chemical ingredients of peach blossom were retrieved from the BATMAN-TCM database (an-tcm/) , and thean-tcm/) –22 to sudme.ch/)  was seardme.ch/)  and highdme.ch/)  and Phardme.ch/)  platformhttps://www.omim.org/) [https://www.drugbank.ca/) [https://www.genecards.org/) [https://db.idrblab.net/ttd/) [https://www.disgenet.org/) [Homo sapiens,” and the search was conducted using “constipation” as the keyword, and the targets closely related to constipation were screened. The obtained targets were standardized using the UniProt database [The OMIM  , DrugBanank.ca/) , GeneCards.org/) , TTD (Thet/ttd/) , and Diset.org/)  databasedatabase .https://bioinfogp.cnb.csic.es/tools/venny/index.html). A Venn diagram was constructed to obtain the intersection targets, which were the potential targets of peach blossom in the treatment of constipation.The screened targets of active ingredients and disease were uploaded to the Venny 2.1 online tool website (Cytoscape 3.8.0 software  was usedhttps://string-db.org/) database [Homo sapiens,” and “Medium confidence = 0.400” was set to obtain the PPI network. The “.tsv” format file was downloaded and imported into the Cytoscape 3.8.0 software. The plug-in CytoNCA [The drug-disease intersection targets were uploaded to the STRING (database  to const CytoNCA  was usedhttps://david.ncifcrf.gov/) [P < 0.05 was set as the threshold for screening. The GO and KEGG enrichment results were plotted into histograms and bubble charts using the bioinformatics online platform (https://www.bioinformatics.com.cn/).Using the DAVID database (rf.gov/) , Gene OnThe potential active ingredients of peach blossom, their molecular targets, and the potential signaling pathways mediating the therapeutic effect of peach blossom against constipation were imported into Cytoscape 3.8.0 software to construct a visual network. The Network Analyzer plug-in was used for network topology analysis to screen important active ingredients and action targets.https://www.rcsb.org/), and the SDF structures of the active ingredients of peach blossom were converted into “mol2” format files using OpenBabel software. The AutoDock Tools-1.5.6 software was used to dewater and hydrogenate the proteins, and save it as “PDBQT” format. Then, the processed protein was combined with its corresponding active ingredient by AutoDock Tools-1.5.6 software to obtain the affinity. Affinity < 0 indicated that the ligand and the receptor can spontaneously bind, and the conformation with the lowest affinity was the optimal conformation. The PyMOL software was used to conduct a visualization analysis of the molecular docking results.The 3D structures of MAPK1, EGFR, VEGFA, PTGS2, and ESR1 proteins were downloaded from the RCSB PDB database , cellular component (CC), and molecular function (MF) of GO, as well as the KEGG pathway. With istogram . A bubbl-catechin, were found to act on the important signaling pathways mediating the therapeutic effect of peach blossom against constipation by binding to targets such as EGFR, VEGFA, and ESR1. Among these, the top 5 targets with a degree ≥2 times of the median were EGFR, VEGFA, ESR1, GSTP1, and PTGS2. These findings suggested that these proteins are potential core targets of peach blossom in the treatment of constipation. The active ingredients of peach blossom act on multiple targets and different pathways interact with each other through multiple common targets, thereby playing a synergistic role in the treatment of constipation.Cytoscape 3.8.0 was used to draw a network diagram for the top 10 pathways in KEGG analysis and their corresponding active ingredients and targets, and visualization analysis was performed . The netMolecular docking was performed for the five core targets  and their corresponding compounds. It is generally believed that the lower the affinity, the greater is the possibility of the target protein binding to the compound, and the more stable is the binding conformation. The results of molecular docking between the active ingredients of peach blossom and the core targets are shown in Peach blossom can not only be used as a laxative, as recorded by Shizhen Li in “Compendium of Materia Medica (本草纲目),” but also for dietary therapy to treat dry feces and intestinal obstruction ever since . As recoESR1 gene encodes the estrogen receptor ERα. Studies have demonstrated upregulation of the expression of ERα in the intestinal mucosa of patients with irritable bowel syndrome, resulting in the dysregulation of estrogen-mediated local immune responses, which may be related to the pathogenesis of irritable bowel syndrome [A search conducted on the BATMAN-TCM database identified 79 potential active ingredients of peach blossom. After the literature review and excluding the ingredients with low gastrointestinal absorption rate, a total of 33 active ingredients were included in the study; most of these were flavonoids and alkaloids, including naringenin, hesperetin, aromadendrin, and persicogenin. A previous study  found thsyndrome . Vasculasyndrome . VEGFA isyndrome . VEGFA isyndrome .In this study, GO enrichment analysis found that the main molecular functions regulated by peach blossom in the treatment of constipation were nitric-oxide synthase (NOS) regulatory activity, heme binding, and efflux transmembrane transporter activity. NO plays an important role in regulating gastrointestinal motility and is the main inhibitory neurotransmitter affecting intestinal motility. NOS is a key enzyme for the production of endogenous NO and exists in the gastrointestinal tissues from the esophagus to the internal anal sphincter . Fan et KEGG pathway enrichment analysis revealed the potential involvement of several pathways in mediating the therapeutic effect of peach blossom against constipation including cancer signaling pathways, metabolism of cytochrome P450 to exogenous substances, and tryptophan metabolism. Cytochrome P450 (CYP450) is a superfamily of enzymes that exists widely in the body. CYP3A is a subenzyme with the highest content in the CYP450 enzyme system, which mainly exists in the intestine and liver of animals. Approximately, 60% of drugs are catalyzed by the CYP3A enzyme system to complete the metabolic process . Wang etStudies have shown that Chaihu Shugan powder can significantly inhibit the apoptosis of gastric Cajal interstitial cells and effectively improve the gastrointestinal function . SimilarIn this study, we performed network pharmacology analysis to systematically analyze the potential active ingredients, effect targets, and signaling pathways mediating the therapeutic effect of peach blossom against constipation. A total of 33 active ingredients  in peach blossoms were identified, which might play a role in the treatment of constipation by regulating signaling pathways through targeted genes such as EGFR, VEGFA, ESR1, GSTP1 and PTGS2, which reflects the characteristic of “multi-ingredient-multi-target-multi-pathway” therapy. This study lays a foundation and provides a theoretical basis for further research on the mechanism of the laxative effect of peach blossom.


Polycystic Ovary Syndrome (PCOS) is associated with a 3 to 4-fold increased risk of endometrial cancer (EC), but molecular mechanisms are unclear. Upregulation of the IGF1 gene in PCOS endometrium may increase EC risk, but this is uncertain. We aimed to investigate links between EC and PCOS, by analysing publicly available transcriptomic data. The NCBI Gene Expression Omnibus was used to identify relevant studies. Differentially expressed genes (DEGs) were identified and analysed using Metascape to identify pathways of interest. PCOS DEGs that encode proteins secreted into blood were identified using the Human Protein Atlas blood protein database. EC DEGs that are cellular receptors were identified using EcoTyper. These were intersected to identify which EC receptors interact with PCOS secreted proteins. Seven receptors were identified in EC but only PTPRF, ITGA2, ITGA3 and ITGB4 genes were expressed on epithelial cells. Pathway enrichment of these genes showed that the major and common pathway involved was that of the PI3K-AKT signalling pathway which was consistent with a link between PCOS and EC. However, IGF1 was down regulated in PCOS and EC. These findings hold significant promise for improving our understanding of mechanistic pathways leading to EC in PCOS. Endometrial cancer (EC) is the second most diagnosed cancer in women globally, with 417,000 new cases and 97,000 deaths in 2020 .The most frequently altered mutation in type 1 EC, is the PI3K/AKT/mammalian target of the rapamycin (mTOR) (P1K3CA) pathway . ElevatePolycystic Ovary Syndrome (PCOS), the most common female endocrinopathy (3.1–20% of women of reproductive age), is associated with obesity, infrequent menstrual periods, infertility, hirsutism, enlarged ovaries with many small follicles on ultrasound imaging, increased risks of type-2 diabetes in later life and a 3 to 4-fold increased risk of EC . MechaniIGF1 may therefore increase EC risk in PCOS. However, this requires validation, and it is important to investigate potential confounders which affect insulin resistance . In a pilot-study  in Nottiin-vivo studies. The original aim of this study was to investigate the links between EC and PCOS, by analysing publicly available transcriptomic data and investigate IGF-1 and IGFBP gene expression in the endometrium of women with PCOS and EC compared with normal endometrium. Our conclusions at this stage do not support a link between IGF-1 and IGFBP genes in PCOS and EC. However, we have identified four novel receptors which may underpin the risk of EC in PCOS, and we believe our findings provide sufficient evidence to form the basis for a transcriptomic atlas to underpin future research into the links between PCOS and EC and the molecular mechanisms underpinning both diseases.There is thankfully now an increasing amount of publicly available transcriptomic data from studies carried out in women with PCOS and endometrial cancer. Bioinformatic analyses of these data offer the potential to investigate research questions, without the need for At the time of search, NCBI GEO search yielded 813 results for the keyword ‘polycystic ovary’ and 3021 results for ‘endometrial cancer’ in Homo sapiens datasets. Of the polycystic ovary results, 37 were expression profiling by array and 18 were by high throughout sequencing, while in endometrial cancer there were 90 expression profiling by array and 58 by high throughput sequencing. Furthermore, of the 127-expression profiling by array studies identified, only 11 were of patients and healthy controls without or prior to any drug treatment.The cell sources and location of DEGs in polycystic ovary syndrome and endometrial cancer are different. For PCOS DEGs to exert their effect on endometrial cancer, these cells must secrete proteins into the blood that leaves the ovary and goes to the epithelial lining of the uterus. The identified DEGs of PCOS were checked against the protein atlas blood secretome database and protein coding genes were identified (Supplementary Table 1) as well as the corresponding epithelial receptors for the DEGs from endometrial cancer (Supplementary Table 2).The protein encoding DEGs identified in PCOS were intersected with EC receptor DEGs to identify interacting ligands . There wMost of the ligands in PCOS were down regulated in patients, with receptors being either up regulated or down regulated in EC . PathwayGSE48301  looked ain-silico analysis and laboratory validation is required to support any hypothesis that can be generated from this analysis. Both IGF1 and INSR were however down regulated in PCOS and EC patients, when we looked at the DEGs that were common between all PCOS datasets and all EC datasets, except for one dataset (GSE178671) where it was up regulated in recurrent endometrial cancer stage IB in comparison to non-recurrent endometrial cancer stage IB. This finding goes against the findings from the pilot study in the UK [The aim of this study was to investigate the possible molecular associations between PCOS and EC by analysing data from publicly available transcriptomic data sets. We set out to further investigate findings from previous research showing that IGF-1 and IGFBP gene expression in the endometrium of women with PCOS and EC was increased compared with normal endometrium and identify other novel genes that may be commonly expressed. We were unable to confirm these specific findings from our data analysis at this stage, especially as this was an n the UK  where IGOn the other hand, several of the DEGs were related to insulin and insulin-related pathways such as IGFALS, IGFB5, SYNAP5, IGF2BP2-AS1, and IGFBP4. Pathways affected by or involving insulin include GO:1990314 cellular response to insulin-like growth factor stimulus, hsa04910 Insulin signalling pathway, hsa04931 insulin resistance. It is of interest that in the study GSE178671, where recurrent and non-recurrent endometrial cancer were compared, the DEGs were involved in the IGFR1 cascade signalling, signalling by type 1 insulin-like growth factor-1 receptor, IRS-related events triggered by IGF1R, all of which are within the MAPK signalling pathway (Supplementary File 11 and Table 3 within Supplementary File 11). The MAPK signalling pathway when exposed to growth factors stimulates the cells to proliferate and grow and is a known tumour suppressor pathway. Changes in the insulin signalling pathway may therefore underpin the association between PCOS and EC as insulin resistance is a key feature of PCOS, though the precise molecular mechanisms which underpin this association remains unclear.Despite this uncertainty, we believe our work has identified some novel findings and provide sufficient data to form the basis for a transcriptomic atlas to improve our understanding of the underlying mechanisms as well as underpin future research into PCOS and EC. Similar transcriptomic atlases have been published for other disease states e.g., COVID-19 . For exaFrom our transcriptomic atlas, the focus was on extracellular proteins. Using the human protein atlas, we successfully identified which DEGs encoded proteins secreted in the blood, and their receptors on the epithelial lining of the uterus were identified using the online tool, EcoTyper. Four receptors  were identified as differentially expressed in EC with ligands or gene products secreted by PCOS. While these receptors appeared to be either up or down regulated in endometrial cancer, their ligands appear to be mostly down regulated by PCOS.Next, it was important to determine which of these molecules were known to be signalling molecules and what their effects were in terms of altering gene expression or enzyme activity . Following this, it was important to determine which gene expression changes in EC were linked to a cancerous state. In terms of the identified receptors, three – ITGA2, ITGA3 and ITGB4 – appeared to be involved in the PI3K-AKT signalling pathway, which is a known oncogenic pathway that promotes various cell functions such as proliferation, cell survival and angiogenesis. PTPRF, the fourth gene, was an up regulated receptor gene in EC and involved in various pathways linked to cellular adhesion and organization. Its ligand, NID1, a gene that plays a role in cellular adhesion and extracellular matrix organization, appeared to be down regulated in PCOS. Subsequent research to decipher the precise mechanisms by which these receptors might play a role in the association between EC and PCOS should ideally find out from the literature what molecules cause these gene expression changes, and in a pathway analysis, these can be linked with their target affected genes.The results of this study also provided data to form the basis for a transcriptomic atlas to underpin future research into the molecular mechanisms underpinning PCOS and/or EC. For example, in PCOS, we found that in the study GSE106724  that looOther findings were the association between DEGs involved in activation of GTPase activity receptor and pathways, such as response to growth factor stimulus and lutein granulosa cells in PCOS (GSE98595) ; pathwayFurthermore, when we investigated the pathways associated with the 54 DEGs in skeletal muscle of PCOS patients from two different studies (GSE8157  and GSE6Findings which form the basis for a transcriptomic atlas to underpin future research into EC included the association between cellular adhesion and grades 2 and 3 EC (GSE115810) , microtuThe use of gene-enrichment analysis (GEA) to identify potential pathways was a strength and helpful step to enable us to investigate our research question. We are also unaware of a similar approach to decipher the possible molecular links between PCOS and EC. A potential limitation however is that results from GEA can sometimes be misleading. The differences identified may reflect the fact that each disease is caused by different factors  and DEG changes in the same direction might be coincidental or due to some other factor exerting the same change in both diseases. The same change in gene expression in both diseases might not have anything to do with one (PCOS) causing a change in the other (EC). Having identified a potential pathway, it is then crucial to look at what is happening to the expression levels of all the components. Several members of a pathway might have elevated transcript levels while others might have reduced levels or have elevated levels of inhibitory factors which nullify the effectiveness of the pathway. Further interrogation of the data presented in our transcriptomic atlas is therefore required.Our transcriptomic atlas allows research groups globally to independently undertake further data analyses to investigate the molecular links between PCOS and EC. We identified four novel receptors which may underpin the risk of EC in PCOS, but there may well be others. Finally, both IGF1 and INSR were down regulated in PCOS and EC patients, which was not consistent with previous research. Therefore, more studies are required to decipher the exact role of the insulin signalling pathway in EC risk in PCOS.Institutional review board approval was not required for this study as we have used publicly available data, and the study was conducted between February and May 2022. The Gene Expression Omnibus (GEO) database from NCBI was used to identify transcriptomic studies containing the keywords ‘polycystic ovary’ and ‘Endometrial cancer’ . Of the P value of ≤ 0.05 were considered as statistically significant. All DEGs were analysed using the Metascape database, a gene enrichment and analysis resource, for identification of pathways [Differentially expressed genes (DEGs) were identified using the built-in GEO2R program with the limma  R package and applying forced normalization on all data. Comparisons used for analysis were between healthy controls and cases, or different grades of tumours . Genes with an adjusted pathways , 30.PCOS DEGs encoding proteins that are secreted in the blood were identified using the Human Protein Atlas blood protein database . The end