NCT02848131) is currently underway. This study aimed to investigate the mechanism of QUE protecting against podocyte injury to attenuate DN through network pharmacology, microarray data analysis, and molecular docking. QUE-associated targets, genes related to both DN, and podocyte injury were obtained from different comprehensive databases and were intersected and analyzed to obtain mapping targets. Candidate targets were identified by constructing network of protein-protein interaction (PPI) of mapping targets and ranked to obtain key targets. The major pathways were obtained from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) term enrichment analysis of candidate targets via ClueGO plug-in and R project software, respectively. Potential receptor-ligand interactions between QUE and key targets were evaluated via Autodocktools-1.5.6. 41. Candidate targets, of which three key targets , and the major AGE-RAGE signaling pathway in diabetic complications were ascertained and associated with QUE against podocyte injury in DN. Molecular docking models showed that QUE could closely bind to the key targets. This study revealed that QUE could protect against podocyte injury in DN through the following mechanisms: downregulating inflammatory cytokine of TNF, reducing VEGF-induced vascular permeability, inhibiting apoptosis by stimulating AKT1 phosphorylation, and suppressing the AGE-induced oxidative stress via the AGE-RAGE signaling pathway.Quercetin (QUE), a health supplement, can improve renal function in diabetic nephropathy (DN) rats by ameliorating podocyte injury. Its clinical trial for renal insufficiency in advanced diabetes ( Podocyte injury is a critical event resulting in the eventual podocyte loss in the development and progression of diabetic nephropathy (DN), accounting for 40–45% of patients with diabetes mellitus –3. MoreoRecently, podocyte injury has been regarded as a novel early mechanism involved in DN . In the NCT02848131) are currently underway   (diabetic nephropathy) and [all] (Homo sapiens).”Genes associated with DN were retrieved from five comprehensive databases, including the Online Mendelian Inheritance in Man database  , GeneCards database with the keyword “[all] (podocyte injury) and [all] (Homo sapiens),” and Gene Expression Omnibus (GEO) database [P < 0.05) using the Limma package [Genes related to podocyte injury were searched from four databases: OMIM and DigSee database  with thedatabase  with thedatabase  titled “ package  of the Rhttps://string-db.org) using Cytoscape 3.71 [QUE-associated targets, podocyte injury-related genes, and DN-associated genes were subjected to intersection analysis to identify the mapping targets that were considered to be highly relevant to QUE protecting against podocyte injury in DN. The “protein-protein interaction (PPI)” topological network of the mapping targets was constructed using the STRING database   was considered to be the major pathway for QUE protecting against podocyte injury in DN.The candidate targets were imported into the ClueGO plug-in  of CytosGO terms describe the biological function of genes through three semantic terms, namely, biological process (BP), cellular component (CC), and molecular function (MF) . KEGG cohttps://pubchem.ncbi.nlm.nih.gov/) and the Protein Data Bank database (PDB) (https://www.rcsb.org/pages/contactus), respectively.The interaction between QUE (ligand) and key targets (receptors) were evaluated using Autodocktools-1.5.6  and visuA total of 355 targets of QUE  both was the AGE-RAGE signaling pathway in diabetic complications that was also identified as the major signaling pathway of QUE protecting against podocyte injury in DN. Oxidoreductase activity, antioxidant activity, and peroxidase activity from R project were consistent with the regulation of reactive oxygen metabolism (36.92%) from ClueGO [The top pathway obtained in ClueGO (accounting for 94.44%) and R project  could easily enter and bind to the key target TNF (2JG9), VEGFA (1MKK), and AKT1 (1UNR) with several interactions, hydrogen bonds, and amino acid residues, shown in The present study shows that QUE would protect against podocyte injury in DN mainly by regulating the major AGE-RAGE signaling pathway and three key targets: TNF mediating the proinflammatory, VEGF promoting vascular permeability and proliferation, and AKT1 participating in apoptosis. Furthermore, QUE, having the five hydroxy groups , should have suitable binding sites with three key targets and interacts with amino acid residues of targets through multiple hydrogen bonding and Van der Waals using molecular docking analysis.QUE regulates the oxidative stress-associated AGE-RAGE signaling pathway to protect against podocyte injury in DN. It is known that the binding of AGEs to the receptor RAGE can induce oxidative stress and inflammation, eliciting podocyte injuries , 50. Encα, a member of the TNF receptors superfamily, is an intermediate factor for excessive ROS-induced podocyte injury and apoptosis [α plays a predictive role in DN, attributing to its involvement in the onset and progression of DN [α and ROS synthesis [QUE inhibits key targets of TNF mediating the proinflammatory and regulates VEGF promoting vascular permeability and AKT mediating apoptosis to protect podocytes from injury in DN. Experiments in the streptozocin-induced diabetic rat have demonstrated that the inflammatory cytokine of TNF-poptosis –60. Moreon of DN . Encouraynthesis  induced ynthesis  which isynthesis . High Hcynthesis , 65, andynthesis , 67. Whiynthesis . Interesynthesis , 70. Metynthesis . Treatmeynthesis . Furtherynthesis . VEGF isynthesis . It is rynthesis , 75. Butynthesis , 77. So,ynthesis . Interesynthesis . Hence, ynthesis . Howeverynthesis . Noteworynthesis . Thus, iynthesis , 81, andynthesis , 83.P=0.0048 from the R project). Autophagy, which can accelerate the metabolism of ROS induced by HG, significantly accelerates the metabolism of ROS and inhibits the activation of VEGF, showing its importance to maintain the postmitotic podocytes cells [α and AKT and ameliorating doxorubicin-induced podocyte injury in rats [Moreover, this study also indicates that QUE protects against podocytes injury having relation with autophagy from enrichment analysis , inhibit endothelial cell proliferation (VEGFA) and apoptosis of podocytes (AKT1 and TP53), and suppress the AGE-induced oxidative stress by regulating the AGE-RAGE signaling pathway activated by HG to protect against podocytes injury in DN . This st


Yikunyin is an empirical prescription that exhibits good efficacy in the clinical treatment of menopausal syndrome; however, its underlying mechanism remains unclear. This study investigates the mechanism implicated in the therapeutic effect of Yikunyin by identifying its hub genes, central pathways, and key active ingredients.  The active ingredients and targets of Yikunyin were obtained from the Traditional Chinese Medicine Systems Pharmacology database, whereas the targets related to menopausal syndrome were obtained from GeneCards, PharmGKB, Therapeutic Target Database (TTD), and Comparative Toxicogenomics Database (CTD). To reveal the pharmacological mechanism, the component-target and the intersecting protein-protein interaction (PPI) networks were constructed, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Finally, molecular docking was carried out to assess the strength of binding between the key active ingredients and key targets.  A total of 418 targets and 121 active ingredients were identified in Yikunyin. The intersection of Yikunyin's 418 targets with the 2822 targets related to menopausal syndrome shows that there are 247 common targets that can be considered potential targets of Yikunyin in the treatment of menopausal syndrome. The topology analysis of the constructed PPI network conducted using the Cytoscape software shows that there are 15 hub genes implicated in the therapeutic effect of Yikunyin: AKT1, PRKCA, TLR9, CXCL10, PRKCD, PARP1, ABCB1, TP53, CAV1, MAPK8, PPARA, GRB2, EGFR, IL-6, and JAK2. Moreover, the key active components acting on these genes are paeoniflorin, luteolin, quercetin, beta-sitosterol, and kaempferol. GO and KEGG analyses indicate that Yikunyin can treat menopausal syndrome by regulating cellular response to chemical stress (GO:0062197), cellular response to oxidative stress (GO:0034599), phosphatase binding (GO:0019902), cytokine receptor binding (GO:0005126), PI3K-Akt signaling (hsa04151), lipid and atherosclerosis (hsa05417), and hepatitis B (hsa05161). Finally, the results of molecular docking suggest that the key active ingredients and key targets can bind well, with binding energies of less than −5 kJ/mol.  The research conducted herein reveals that Yikunyin treats menopausal syndrome by targeting AKT1 and IL-6 and by regulating the PI3K-Akt signaling pathway. Moreover, it provides a new idea for understanding the therapeutic effects of traditional Chinese medicines. Menopause is a physiological stage characterized by the permanent cessation of a woman's menstrual period due to the loss of ovarian follicle function. According to a global project comprising 36 international studies conducted in 24 countries across 6 continents, the mean age of natural menopause onset ranges between 46 and 52 years, with an overall average of 48.8 years . During Yikunyin is a Gui Shen Pill-based formula composed of nine herbs, namely Gou Qi Zi (Lycii Fructus), Fu Ling (Poria), Bai Shao (Paeoniae Radix Alba), Xian Ling Pi (Epimedii Folium), Huang Qi , Shu Di Huang (Rehmanniae Radix Praeparata), Gou Teng (Ramulus Uncariae Cum Uncis), Muli (oysters), and He Huan Pi (Albizia Peel). These herbs are rich in flavonoids, which are known for their anti-inflammatory, antioxidant, anticancer, and antidiabetic effects , 8. ClinUnlike Western medicines, TCM prescriptions, such as Yikunyin, comprise a variety of herbs. Considering their complex composition, the effects of these prescriptions cannot be readily studied using simple methods. In 2008, Hopkins proposed network pharmacology as an effective method that can be used to study the molecular mechanisms of complex systems  by establishing the relationships between multiple compounds and targets . In thishttps://tcmspw.com/tcmsp.php) [https://202.127.145.134/scdb/), and a high-throughput experiment- and reference-guided database of traditional Chinese medicine (HERB) database (http://herb.ac.cn/). The oral bioavailability (OB) and drug-likeness (DL) of each component were obtained from the TCMSP database. Since Yikunyin is administered orally, OB, a measure of the drug's ability to enter systemic circulation, is an important indicator [The chemical composition of Yikunyin was obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (msp.php) , the Shahttps://www.swissadme.ch/) provides pharmacokinetic and drug-likeness data of compounds not included in the TCMSP database. In this research, Swiss ADME was used to identify the active compounds in Yikunyin based on GI absorption and drug-likeness. In particular, the compounds with “High” GI absorption and more than three “Yes” drug-likeness criteria were considered to be active.The Swiss Absorption, Distribution, Metabolism, and Elimination (ADME) system (https://www.swisstargetprediction.ch/) were used to predict the relevant targets of Yikunyin. The compound SDF files required for SwissTargetPrediction were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The UniPort database (https://www.uniprot.org/) was used to convert protein targets into genetic targets, species restricted to humans, and reviewed targets.The TCMSP database and SwissTargetPrediction (https://www.genecards.org/), PharmGKB (https://www.pharmgkb.org/), the Therapeutic Target Database(TTD) (https://db.idrblab.net/ttd/), and Comparative Toxicogenomics Database(CTD) (https://ctdbase.org/), using the keyword “menopausal syndrome.” The identified menopausal syndrome targets were intersected with the drug targets using the online website https://bioinformatics.psb.ugent.be/webtools/Venn/. The resulting Venn diagram was visually processed, and the intersection targets were considered to be the targets of Yikunyin in the treatment of menopausal syndrome.As for the targets of menopausal syndrome, they were collected from four databases, namely GeneCards (https://string-db.org). The organism criterion was set to “Homo sapiens,” and the minimum required interaction score was fixed at 0.7. For visualization and topology analysis, the resulting TSV files were downloaded from the STRING database and uploaded into Cytoscape 3.7.2. The important targets in the network were identified based on their degree value (large degree) [To construct the intersecting protein-protein interaction (PPI) network, the identified targets were entered into the STRING database ( degree) .CytoHubba is a Cytoscape plug-in that uses 12 algorithms  to characterize the nodes in the PPI network and to identify the central elements based on their importance . After cR package. P values below 0.01 were considered statistically significant [Gene Ontology (GO) analysis was used to identify the genes implicated in biological process (BP), cell composition (CC), and molecular function (MF) related to menopausal syndrome, whereas the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to identify the corresponding pathways. The analyses were carried out using the clusterProfiler nificant .https://pubchem.ncbi.nlm.nih.gov/), whereas the structures of proteins were obtained from the Protein Data Bank . The PDB files of proteins and drugs were uploaded into AutoDockTools 1.5.6 for pretreatment , and then, molecular docking was conducted to determine the binding energy. The obtained PDBQT files were converted to PDB files and imported into PyMOL 2.2.0 for visual processing.To predict whether the key active compounds can bind well to the key protein targets, molecular docking analysis was carried out using AutoDockTools 1.5.6. The molecular structures of the key active compounds were taken from PubChem , eight of which are in Bai Shao, six in Fu Ling, 32 in Gou Teng, 35 in Gou Qi Zi, 16 in Huang Qi, two in Shu Di, 21 in Xian Ling Pi, 10 in He Huang Pi, and seven in Muli. These components are listed in Supplementary In total, 418 targets corresponding to the 121 active ingredients in Yikunyin were identified using the TCMSP database and SwissTargetPrediction. Based on searches conducted in the GeneCards, CTD, PharmGKB, and TTD databases, a total of 2,822 targets are associated with menopausal syndrome. Of these targets, 247 are also associated with Yikunyin, as shown in the Venn diagram presented in To better understand the relationship between TCM components and targets, a common target-component network was constructed, as shown in The PPI network was constructed by inputting all 247 common targets into the STRING database, and it was visualized using Cytoscape . TopologThe key compounds corresponding to the 15 hub genes identified herein were determined by a reverse search, and they are paeoniflorin, luteolin, octadecatrienoic acid, quercetin, beta-sitosterol, kaempferol, acacic acid lactone, julibroside j24, julibrotriterpenoidal lactone A, norarmepavine, S-(2-carboxyethyl)-L-cysteine, and machaerinic acid lactone. P value). The obtained results indicate that the mechanism of menopausal syndrome treatment by Yikunyin is mainly related to cellular response to chemical stress (GO:0062197), cellular response to oxidative stress (GO:0034599), phosphatase binding (GO:0019902), cytokine receptor binding (GO:0005126), PI3K-Akt signaling (hsa04151), lipid and atherosclerosis (hsa05417), and hepatitis B (hsa05161).Since the 15 hub genes play important roles in the treatment of menopausal syndrome by Yikunyin, it is necessary to identify the pathways in which these genes are enriched. Based on GO and KEGG analyses, the hub genes are implicated in 644 GOs (624 BPs and 20 MFs) and 91 KEGGs. The strength of binding between hub genes and key active compounds was assessed by molecular docking. With binding energies less than −5 kJ/mol, the proteins in the hub genes are well connected to the key compounds. The 3D representation and binding energies of AKT1 and IL-6 docking are shown in Gou Qi Zi (Lycii Fructus), Fu Ling (Poria), Bai Shao (Paeoniae Radix Alba), Xian Ling Pi (Epimedii Folium), Huang Qi , Shu Di Huang (Rehmanniae Radix Praeparata), Gou Teng (Ramulus Uncariae cum Uncis), Muli (oysters), and He Huan Pi (Albizia Peel) are the nine TCM components of Yikunyin. According to Jae Hyun Kim et al., the water extract of Gou Qi Zi can inhibit RANKL-induced osteoclast differentiation and improve menopause osteoporosis . SimilarThe key active ingredients identified herein are paeoniflorin, luteolin, octadecatrienoic acid, beta-sitosterol, kaempferol, quercetin, acacic acid lactone, julibroside J24, julibrotriterpenoidal lactone A, norarmepavine, S-(2-carboxyethyl)-L-cysteine, and machaerinic acid lactone. Among these ingredients, paeoniflorin, luteolin, kaempferol, beta-sitosterol, and quercetin are noteworthy, as they can provide a reference for the future development of new drugs. Based on previous studies, luteolin inhibits insulin resistance, a condition caused by diseases (diabetes and metabolic syndrome) associated with the onset of menopause, by promoting the PI3K-Akt signaling pathway . MeanwhiThe 15 hub genes identified herein are as follows: AKT1, PRKCA, TLR9, CXCL10, PRKCD, PARP1, ABCB1, TP53, CAV1, MAPK8, PPARA, GRB2, EGFR, IL-6, and JAK2. MCODE analysis shows that AKT1 and IL-6 played the most important roles. IL-6 was used to develop tocilizumab, a drug that clinically treats arthritis and alleviates the symptoms of COVID-19 by targeting interleukin-6 receptors . AccordiTo further assess the complex relationships existing between different component, targets, and pathways implicated in the treatment of menopausal syndrome by Yikunyin, GO and KEGG analyses of the 15 key targets were conducted. The results of GO analysis show that the 15 targets are mainly enriched in phosphatase binding, cytokine receptor binding, cellular response to oxidative stress, and cellular response to chemical stress, with the former three biological processes being the most important. After entering menopause, the level of alkaline phosphatase in women increases significantly, and it keeps on increasing with time. This indicates that the bone turnover level, a marker of menopausal osteoporosis, also increases . As mentP < 0.001), as manifested by the recovery of liver fibrosis. Moreover, curcumin, a compound whose structure is similar to those of quercetin and luteolin, has a hepatoprotective effect [The KEGG analysis results demonstrate that most of the 15 hub genes are involved in PI3K-Akt signaling, lipid and atherosclerosis, and hepatitis B, and the former seems to be the most critical pathway implicated in the therapeutic effect of Yikunyin on menopausal syndrome. Unlike Lee et al. , we belie effect , and quee effect . TherefoThis study highlights the importance of Yikunyin in improving menopausal syndrome by upregulating the PI3K-Akt signaling pathway, improving inflammatory response, promoting antioxidant, and adjusting lipid metabolism to prevent atherosclerosis. The most important active ingredients in Yikunyin are paeoniflorin, luteolin, kaempferol, beta-sitosterol, and quercetin. In addition, our research provides some new targets for the future development of new drugs.


Effective treatments for patients experiencing temperature-related symptoms are limited. The hot and cold effects of traditional herbal medicines have been utilized to treat and manage these symptoms, but their molecular mechanisms are not fully understood. Previous studies with arbitrarily selected herbs and ingredients may have produced biased results. Here, we aim to systematically elucidate the molecular mechanisms of the hot and cold properties of herbal medicines through an unbiased large-scale investigation of herbal ingredients, their target genes, and the transcriptome signatures induced by them. Using data regarding 243 herbs retrieved from two herbal medicine databases, we statistically identify (R)-Linalool, (-)-alpha-pinene, peruviol, (L)-alpha-terpineol, and cymol as five new hot-specific ingredients that share a common target, a norepinephrine transporter. However, no significant ingredients are cold-specific. We also statistically identify 14 hot- and 8 cold-specific new target genes. Pathway enrichment analysis of hot-specific target genes reveals the associated pathways including neurotransmitter reuptake, cold-induced thermogenesis, blood pressure regulation, adrenergic receptor signaling, and cation symporter activity. Cold-specific target genes are associated with the steroid pathway. Transcriptome analysis also shows that hot herbs are more strongly associated with coagulation and synaptic transmission than cold herbs. Our results, obtained from novel connections between herbal ingredients, target genes, and pathways, may contribute to the development of pharmacological treatment strategies for temperature-related pain using medicinal plants. Approximately one-fifth of the world’s population suffers from chronic pain . Among tTraditional herbal medicines utilize the properties of herbs with hot or cold temperament to treat and manage temperature-related pain . SeveralTo avoid biased results due to analysis of only a few specific herbs or ingredients, we aim to systematically elucidate the molecular mechanisms of the hot and cold properties of herbal medicines through an unbiased large-scale investigation of herbal ingredients, their target genes, and the transcriptome signatures induced by them. Using the data of 243 herbs obtained from herbal medicine databases, we perform statistical analysis to identify the enriched ingredients and target genes specific to herbs with hot or cold effects. The transcriptome signatures related to hot or cold effects are also analyzed from the gene expression data to reveal associated pathways. Our study provides a comprehensive understanding of the cold and hot properties of herbal medicine and possible therapeutic interventions for patients with temperature-related pain.Information regarding herbal medicines was obtained from two recent databases: The Integrative database of Traditional Chinese Medicine enhanced by Symptom Mapping   and the Due to a parsing problem in ETCM, where commas in ingredient names are ambiguous because they are used for both synonym and chemical nomenclature punctuation, ingredient information for each herb was extracted from SymMap only. Data linked to the duplicate synonyms of each ingredient were integrated based on alias names, PubChem IDs, and CAS IDs, and then target gene information of the corresponding ingredient was extracted.dhyper function in R. First, we collated ingredient information of all herbs in each category, and then calculated the frequency of occurrence of each ingredient in each category. Next, we also collated target gene information of all the ingredients of all herbs in each category, and then calculated the frequency of occurrence of each target gene in each category. The hypergeometric test was used to statistically compare the frequency of a particular ingredient or target gene in one category compared to another considering the overall size of the two categories. Adjusted p values, Padj, were obtained using Benjamini–Hochberg multiple testing correction after the hypergeometric tests were performed.Hypergeometric tests were performed between the herb categories to identify the enriched ingredients or target genes associated with the hot or cold properties using the Using the identified specific target genes, we performed pathway enrichment analysis and network visualization . Pathwaylimma package [fgsea package [To identify specific transcriptomic alterations associated with hot or cold properties, we obtained microarray data  containi package  after mu package  to analyWe integrated the information from databases to systematically investigate the molecular mechanisms underlying the cold and hot properties of herbal medicines. We obtained the annotated cold and hot properties of 322 herbs from SymMap  and 402 The properties of the 243 matched herbs were categorized into one of nine temperament levels ranging from great/extreme cold to great/extreme hot b. They wpadj > 0.05). The most significant differentially enriched CG ingredient was apigenin. However, 21 ingredients were significantly enriched in the HG compared with those in both CG and NHG (padj < 0.01)  a. We ide < 0.01) b. In par(SLC6A2), which encodes a norepinephrine (NE) transporter, as a common target gene of all hot-specific ingredients  were found to be commonly enriched across the HG. We defined the genes that were significantly more enriched in the HG than in either the CG or CGE (padj < 0.01) as hot-specific target genes, which were CHRM1, SLC6A2, SLC6A3, SLC6A4, ADRA1A, PTGS2, RXRA, ADRB2, MAOA, MAOB, BCHE, ADRA2A, PLB1, and ADRB1. Similarly, the cold-specific target genes, which were NOS2, PRSS1, GSK3B, CALM3, CKD2, AR, CASP3, and ESR2, were defined as the genes that were significantly more enriched in the CG than in either the HG or HGE (padj < 0.01). The full name of each target gene is provided in Next, we investigated the enriched target genes of the herb ingredients in the HG and CG. We performed the enrichment analysis of the top 50 target genes using the hypergeometric test and Benjamini–Hochberg multiple testing correction . Each grTo demonstrate the hot and cold properties of the herbs at the level of biological pathways, we performed a pathway enrichment analysis of the hot- and cold-specific target genes using g:Profiler  with theTo examine the effects of the hot and cold properties at the transcriptome level, we analyzed the gene expression data obtained after treating MCF7 breast cancer cells with 102 herbal ingredients . We calcTo reveal the pathways differentially active between the HCs and CCs, we performed GSEA using the Hallmark and GO BP gene sets. Consistent with our previous findings, the upregulated genes after treatment with HCs were found to be associated with the downregulation of coagulation, which is related to vasodilation, blood pressure, and upregulation of synaptic transmission b. We alsWe systematically identified novel molecular mechanisms involved in the hot and cold properties of herbs from two herbal medicine databases (SymMap and ETCM) by investigating the key ingredients, target genes, and transcriptomic signatures. We found that 21 key ingredients were significantly enriched in the HG, with the top five ingredients defined as hot-specific ingredients. We also found that 14 hot-specific and 8 cold-specific target genes were significantly enriched in the HG and CG, respectively. In addition, consensus pathways related to synaptic transmission and blood pressure were identified from both pathway enrichment analysis of the target genes and transcriptome analysis of the gene expression data for herbal ingredients. Network analysis further suggested that adrenergic signaling and associated synaptic transport could be core pathways for the hot properties of herbal medicines.SLC6A2 have been reported to be related to pain sensitivity [(R)-Linalool, (-)-alpha-pinene, peruviol, (L)-alpha-terpineol, and cymol were identified as the top five hot-specific ingredients. (R)-Linalool is a natural monoterpene with anti-inflammatory and analgesic effects . (-)-Alpsitivity , suggestHeat production is a function of adipose tissues induced by sympathetic neuron innervation from secreted catecholamines, such as NE. Exposure to low temperatures increases NE release from neurons, inducing lipid metabolism and thermogenesis in adipose tissue. Recent studies have shown that sympathetic neuron-associated macrophages regulate NE concentrations in adipose tissue by causing the reuptake of extracellular NE mediated by SLC6A2, the identified common target of hot-specific ingredients ,30. ThesSLC6A2 and MAOA, may be involved in these effects of herbs with hot properties. In addition, the hot-specific target genes were also associated with cation symporter activity, consistent with the findings of a previous study, which showed that herbs with hot properties induced the Na+-K+-ATPase and Ca2+-Mg2+-ATPase pump activities [Heart rate and blood pressure are regulated by NE release. Previously, herbs with hot properties have been shown to stimulate NE release in PC12 cells, whereas herbs with cold properties had no effect or suppressed NE release . This retivities . Modulattivities .We found that the cold-specific ingredients, their target genes, and the associated pathways were less in number than their hot-specific counterparts. Similarly, a previous study examining 10 cold and 10 hot herbs identified 27 target protein molecules associated with hot properties and only 2 target protein molecules associated with cold properties . These dThe molecular response of a cell to treatment with specific ingredients or drugs can be systematically assessed by its transcriptomic signature, which refers to the changes induced in gene expression after treatment. Therefore, we analyzed the gene expression data for the molecular responses against 102 herbal ingredients . This daPaeoniae Radix can reveal novel mechanisms of action [A previous study also analyzed the same dataset under the assumption that the major ingredients of herbs are representative of their effects . Howeverf action . TherefoWe conclude that the novel molecular mechanisms underlying the effects of herbs with hot properties are associated with multiple pathways including NE synaptic transmission, adipose tissue thermogenesis, and blood pressure regulation. Their pharmacological activities may act on major protein targets such as SLC6A2 and MAOA. Although the molecular mechanisms underlying the effects of herbs with cold properties appear heterogeneous, the steroid hormone signaling pathway was found to be a common mechanism among them. Our findings, based on the comprehensive systematic approach, may contribute to the development of treatment strategies for temperature-related pain.


To investigate microRNA-320a-related differentially expressed genes (DEGs) and pathways in osteoarthritis (OA) by bioinformatic analysis.The target genes of microRNA-320a were searched and collected from MiRTarBase microRNA Targets dataset, the TargetScan Predicted Nonconserved microRNA Targets dataset and the TargetScan Predicted Conserved microRNA Targets dataset. OA-related microRNAs and OA-related target genes were collected from GeneCards databases. The pathway enrichment analysis of miRNAs ware performed by Funrich analysis tool. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was obtained from Database for Annotation, Visualization and Integrated Discovery (DAVID). GeneMANIA and STRING are used for protein-protein interaction (PPI) network analysis. Module analysis was performed by Cytoscape.A total of 176 OA related miRNAs were searched and collected for enrichment analysis, and microRNA-320a was one of OA related miRNAs. Enrichment pathway and analysis of 1721 miRNA-320a-related target genes from MiRTarBase and TargetScan were performed using the online tools Metascape. And results shown that the biological processes were remarkably enriched in chromatin organization, cellular response to DNA damage stimuli, mRNA metabolic process, protein ubiquitination, and regulation of cell adhesion. And then we analysed miRNA-320a-targeted OA genes via KEGG, GO enrichment and PPI Network. Our results showed that miRNA-320a played a role in OA through FoxO signaling pathway, PI3K-Akt signaling pathway, focal adhesion, MAPK signaling pathway, HIF-1 signaling pathway and cellular senescence. And we speculate that MAPK signaling pathway plays a key role in the effect of miRNA-320a on OA.This study implied microRNA-320a-related DEGs and dysregulated pathways in OA. The aim is to screen miRNA-320a-related genes and pathways in OA and, eventually, to improve the understanding of underlying mechanisms of miRNA-320a in OA. As one of the most common chronic degenerative articular cartilage diseases, osteoarthritis (OA) has a positive correlation with age, and its incidence is increasing, seriously affecting people's quality of life and physical and mental health, and causing a heavy burden to families, society and the country. The main pathological features of OA include wear and tear of articular cartilage, degeneration and excessive apoptosis of chondrocytes, degeneration of articular chondrocytes –3.MiRNA (MicroRNA) is a class of endogenous small RNAs with about 20–24 nucleotides in length, and plays important roles in cell differentiation, biological development and disease occurrence and development. Bastami et al. developed a bioinformatics method to identify miRNAs target sites polymorphisms related to obesity or colorectal cancer (CRC), which could provide potential biomarkers for diagnoses, prognoses and treatment for CRC or obesity . The patIn this study, MiRTarBase microRNA Targets dataset, TargetScan Predicted Nonconserved microRNA Targets dataset and the TargetScan Predicted Conserved microRNA Targets dataset were analyzed to identify the target genes of miRNA-320a. Subsequently, GO and KEGG pathways were enriched, PPI network was established, and modules were analyzed. OA related miRNAs and target genes were collected from GeneCards. Enrichment pathway and analysis were performed using the online tools Metascape. This work aims to screen miRNA-320a-related genes and pathways in OA and, eventually, to improve the understanding of underlying mechanisms of miRNA-320a in OA.http://mirtarbase.mbc.nctu.edu.tw/php/index.php), the TargetScan Predicted Nonconserved microRNA Targets dataset and the TargetScan Predicted Conserved microRNA Targets dataset (https://www.targetscan.org/vert_80/). OA-related microRNAs and OA-related target genes were collected from GeneCards databases (https://www.genecards.org/).The target genes of the microRNA-320a were searched and collected from MiRTarBase microRNA Targets dataset . Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was obtained from DAVID (https://david.ncifcrf.gov/), and the visualization of the signaling pathways was drawn by bioinformatics (http://www.bioinformatics.com.cn/).The pathway enrichment analysis of miRNAs ware performed by Funrich analysis tool. The difference was statistically significant at k-core = 2, and max.depth = 100), so as to facilitate downstream analysis.In order to predict protein-protein interaction (PPI), GeneMANIA in Cytoscape is used for PPI network analysis. Nodes represent genes and edges represent connections between genes. Molecular complex detection (MCODE) plug-in component in Cytoscape v3.8.2 software is used to find key sub-networks and genes in a huge network according to the relationship between edges and nodes  pathway . The bioBetween these OA related miRNAs, we focus on the role and mechanism of miRNA-320a in OA. We searched and collected target genes of miRNA-320a from MiRTarBase and TargetScan. 480 target genes of the miRNA-320a were obtained from the MiRTarBase microRNA Targets dataset, 788 target genes of the miRNA-320a predicted using conserved miRNA seed sequences and 535 target genes of the miRNA-320a predicted using nonconserved miRNA seed sequences were obtained from the TargetScan microRNA Targets dataset. By integrating these data, we get 1,721 target genes of the miRNA-320a. And then Pathway enrichment analyses of miRNA-320a-targeted genes were performed using the online tools Metascape. It was shown that the biological processes were remarkably enriched in chromatin organization, cellular response to DNA damage stimuli, mRNA metabolic process, protein ubiquitination, and regulation of cell adhesion. The biological pathway was significantly enriched in cellular responses to stimuli, cell cycle, transcriptional regulation by RUNX1, signaling by Rho GTPases, Miro GTPases and RHOBTB3 . KEGG fu3,183 OA-related target genes were collected from GeneCards. And then Pathway enrichment analyses of OA-related target genes were performed using the online tools Metascape. It was shown that the biological processes were remarkably enriched in inflammatory response, positive regulation of cytokine production, cell adhesion, Mitogen-activated protein kinases (MAPK) cascade and cell population proliferation. The biological pathway was significantly activated in extracellular matrix organization, cytokine-cytokine receptor interaction, and signaling by interleukins .We intersected miRNA-320a-targeted genes with OA-related genes. Based on overlapping results in the Venn diagrams , 259 genWe obtained the PPI network from by GeneMANIA. Our study showed that physical interactions account for 56.93%, co-expression accounts for 25.69%, genetic interactions account for 7.76%, predicted accounts for 4.18%, co-localization accounts for 3.78%, pathway accounts for 1.63, and shared protein domains account for 0.04% . To furtOA is the most common joint disease in the elderly population. Affected by age, gender, obesity, genetics, mechanical stress and other pathogenic factors, the incidence of OA is getting higher and higher. With the increase of age, the disease gradually worsens, resulting in joint deformity and seriously affecting the quality of life of patients , 13. At A total of 176 OA related miRNAs were searched and collected from the GeneCards. Biological pathways, biological processes and cell components were enriched and analyzed. Pathway analysis demonstrated that Btra1 integrin cell surface interactions, proteoglycan syndecan-mediated signaling events, glypican pathway, TRAIL signaling pathway, integrin family cell surface interactions, and sphingosine 1-phosphate (S1P) pathway play important roles. OA is associated with extracellular matrix (ECM) changes and tissue degeneration. The integrin protein family connects structural information in the extracellular matrix of OA to the complex response mechanisms inside the cell . Fibrone1,721 target genes of miRNA-320a from MiRTarBase and TargetScan were performed using the online tools Metascape. It was shown that the biological processes were remarkably enriched in chromatin organization, cellular response to DNA damage stimuli, mRNA metabolic process, protein ubiquitination, and regulation of cell adhesion. The biological pathway was significantly enriched in cellular responses to stimuli, cell cycle, transcriptional regulation by RUNX1, signaling by Rho GTPases, Miro GTPases and RHOBTB3. MiRNA silences gene expression through transcription, post-transcription, and chromatin dependent pathways , 19. AgiκB signaling pathway, JNK signaling pathway, MAPK signaling pathway, Wnt signaling pathway, PI3K/AKT/mTOR signaling pathway, and so on. Among them, NF-κB signaling pathway is reported to be involved in chondrocyte catabolism, chondrocyte survival and synovial inflammation (Many signaling pathways are involved in the progression of OA –33, inclammation . And MAPammation . Researcammation , 35. Foxammation . A largeammation . PI3K/AKammation , 39. Comammation . Evidencammation , 42.PPI Network Analysis of miRNA-320a-targeted ORG also showed that MAPK signaling pathway plays a key role in the effect of miRNA-320a on OA. MAPK serine/threonine protein kinase family is widely present in eukaryotes and regulates a variety of cellular processes including proliferation, differentiation, survival and apoptosis , 44. LANMiRNA-320a is a very important miRNA that can regulate the expression of multiple target genes. This study implied microRNA-320a-related DEGs and dysregulated pathways in OA. The aim is to screen miRNA-320a-related genes and pathways in OA and, eventually, to improve the understanding of underlying mechanisms of miRNA-320a in OA.


Tumor budding (TB) is a small cluster of malignant cells at the invasive front of a tumor. Despite being an adverse prognosis marker, little research has been conducted on the tumor immune microenvironment of tumor buddings, especially in cervical cancer. Therefore, RNA sequencing was performed using 21 formalin-fixed, paraffin-embedded slides of cervical tissues, and differentially expressed genes (DEGs) were analyzed. Immune Pathway and Gene Database (IMPAGT) was generated for immune profiling. “Pathway in Cancer” was identified as the most enriched pathway for both up- and downregulated DEGs. Kyoto Encyclopedia of Genes and Genomes Mapper and Gene Ontology further revealed the activation of the PI3K/Akt signaling pathway. An IMPAGT analysis revealed immune dysregulation even at the tumor budding stage, especially in the PI3K/Akt/mTOR axis, with a high efficiency and integrity. These findings emphasized the clinical significance of tumor buddings and the necessity of blocking the overactivation of the PI3K/Akt/mTOR pathway to improve targeted therapy in cervical cancer. Tumor budding (TB) is the formation of a cluster of fewer than five malignant cells at the invasive edge of a tumor ,2,3. FirDespite its clinical significance, the tumor immune microenvironment (TIME) of TB has not been extensively studied, especially in cervical cancer. To date, immune gene databases such as ImSig (Immune Cell Gene Signatures)  or platfTherefore, we used a slightly different approach than traditional analysis methods. Instead of using diverse databases to infer biological implications and deduce potential clinically meaningful biomarkers in cancer etiology, we generated a new immune database, namely, Immune Pathway and Gene Database (IMPAGT), based on the gene set of CIBERSORT. Using IMPAGT, the tumor immune microenvironment of tumor buddings in cervical cancer was investigated.Following approval by the Institutional Review Board of Kyungpook National University Chilgok Hospital , 21 patients with cervical cancer were selected for the study. Cervical cancer tissue from each patient was collected after radical hysterectomy with pelvic and/or para-aortic lymphadenectomy to treat early-stage and locally advanced cervical cancer. All patients were clinically staged according to the revised 2009 International Federation of Gynecologic Obstetrics staging system for cervical cancer .All cervical tissue specimens were collected after biopsy, and formalin-fixed, paraffin-embedded (FFPE) slides were generated. The FFPE slides were stained with hematoxylin and eosin (H&E), and multiple sections of H&E-stained slides were examined by pathologists at Kyungpook National University Chilgok Hospital. Based on the criterion used in a previous study , among 21 cervical cancer samples, 15 had high tumor budding, whereas the remaining samples had low tumor budding . The medp < 0.05. The DEGs were plotted in a volcano plot graph using the “ggplot” package in R.RNA was extracted from FFPE slides of 21 patients using the ReliaPrep™ FFPE Total RNA Miniprep kit . Extracted RNA was then synthesized into cDNA using the TruSeq RNA Exome Kit , and an RNA sequencing dataset was generated using the Nova 6000 platform (Illumina). The raw data were quantified using the STAR  and RSEMCIBERSORT is a computational platform designed by the Alizadeh Laboratory at Stanford University  that uses the machine learning technique, support vector regression, to deconvolute and characterize diverse cell types from bulk sequencing data . The plaFor immune-related analysis, LM22 was used as a signature matrix. According to Alizadeh et al., LM22 contains 547 genes that distinguish 22 mature human hematopoietic populations isolated from peripheral blood or in vitro culture conditions, including seven T cell types, naïve and memory B cells, plasma cells, NK cells, and myeloid subsets. CIBERSORT analysis using this LM22 matrix provides proportional gene expression measures of the input GEP file corresponding to 22 distinct immune cell types .The 547 gene symbols of LM22 are listed in https://www.genome.jp/kegg/pathway.html (accessed on 13 June 2022)), and all corresponding genes were collected using the R package “KEGGREST”. An LM22-based IMPAGT was generated, with the KEGG pathways of LM22 presented in columns and the corresponding gene symbols presented in rows.STRINGdb  is a datDEGs overlapping in tumor budding and IMPAGT were identified using the basic R function “intersect”. Then, the intersecting DEGs were further searched using IMPAGT, and upregulated (orange) and downregulated (blue) DEGs were highlighted. Subsequently, the number of highlighted DEGs in each pathway was counted.Pathways with more than 10 DEGs were considered significantly enriched in the TB environment of cervical cancer. Among these pathways, the most enriched pathway was visualized using several pathway visualization tools, such as KEGG Mapper Color , DAVID , and Shihttps://www.genome.jp/kegg/mapper/color.html (accessed on 20 June 2022)), “hsa” was selected as the search mode to identify human-specific pathways. The lists of DEGs were uploaded, with upregulated DEGs colored red and downregulated DEGs colored cyan.From the KEGG Mapper Color website (https://david.ncifcrf.gov/tools.jsp (accessed on 20 July 2022)) [GO analysis was performed using the DAVID database (y 2022)) ,27. In tThe RNA sequencing revealed 1253 DEGs between patients with high tumor budding and those with low tumor budding. Among these DEGs, 903 genes were upregulated and 349 were downregulated. A volcano plot of the top 30 up- and downregulated DEGs is presented in −16.When the 547 genes of LM22 were analyzed in STRINGdb, the resultant PPI network was closely interrelated, forming an intricate ball cluster . There wKEGGREST. When overlapping genes were filtered, 2534 genes were left. This list of 2534 genes was considered the “IMPAGT gene set” which could be used for immune analysis. IMPAGT and the IMPAGT gene set are presented in STRINGdb also identified the KEGG pathways associated with genes in the LM22 dataset. In total, 51 pathways from the KEGG database were analyzed, all of which were related to immune reactions of the human body. From these pathways, those related to direct viral infection, such as influenza A pathways, or those irrelevant to tumor budding, such as asthma pathways, were excluded, leaving 26 immune pathways. Meanwhile, 4131 genes were collected from the 26 pathways using In total, 133 upregulated DEGs overlapped with the IMPAGT gene set, and they were engaged in all 26 immune pathways. Considering pathways with more than 10 DEGs as significantly enriched pathways, seven significantly enriched pathways were associated with upregulated DEGs: “Pathway in Cancer (Hsa 05200)”, “Antigen processing and presentation (Hsa 04612)”, “Transcriptional misregulation in Cancer (Hsa 05202)”, “MAPK signaling pathway (Hsa 04010)”, “NOD-like receptor signaling pathway (Hsa 04621)”, “Cell adhesion molecules (Hsa 04514)”, and “Cytokine-cytokine receptor interaction (Hsa 04060)”.Among 31 downregulated DEGs, there were 31 overlapping DEGs, which were involved in 17 different immune pathways. The only significantly enriched pathway was “Pathway in Cancer”, and the remaining pathways contained fewer than five DEGs.To summarize, “Pathway in Cancer” was the most strongly enriched pathway, indicating that it was the most strongly activated and dysregulated pathway in our sample. It was considered to be the most significantly enriched pathway in the TB environment of cervical cancer.The significantly enriched pathways for both upregulated and downregulated DEGs, along with the number of DEGs involved in each pathway, are listed in “Pathway in Cancer” was selected as the most significantly enriched pathway. Subsequently, it was visualized using KEGG Mapper Color. To clarify the overall involvement of TB DEGs in the pathway, all 1253 DEGs were inserted into KEGG Mapper.The DEG-mapped “Pathway in Cancer” revealed the points at which the upregulated and downregulated DEGs engaged the pathway . Most DEp-values of <0.05, ”Signal transduction (GO: 0007165)” and “Immune response (GO: 0006955)” were the top two BP terms. Other significantly enriched BP terms included “Positive regulation of cell proliferation (GO: 0008284)”, “Positive regulation of apoptotic process (GO: 0043065)”, “Regulation of apoptotic process (GO: 0042981)”, “Regulation of cell proliferation (GO: 0042127)”, and “Positive regulation of protein phosphorylation (GO: 0001934)” ” .Meanwhile, the 133 overlapping DEGs were further visualized using STRINGdb, and the PPI network revealed a tendency to cluster into three groups, which were termed “Apoptotic process”, “Protein Phosphorylation”, and ”Cell proliferation” .This study aimed to investigate the tumor immune microenvironment (TIME) of TB in cervical cancer using the newly generated immune database IMPAGT. For immune profiling analyses, researchers mainly use immune gene databases, such as ImSig  or ImmuCConsidering these limitations, our study holds significance in terms of several aspects. First, IMPAGT allows researchers to interpret their data with high efficiency and integrity and a low risk of bias. IMPAGT, the “two in one” immune database, provides immune pathways and the corresponding genes of each pathway simultaneously. The integrated database enables users to investigate their genes of interest in a more rapid and efficient manner than individually searching and merging separate results from multiple databases. Furthermore, instead of obtaining “one gene–one pathway” associations based on the researcher’s interpretation, IMPAGT provides several immune pathways in which certain genes of interest are involved. In particular, it proposes “one gene–many immune pathways” interconnections. As it offers diverse immune pathways in which specific genes are engaged, IPMAGT encourages researchers to view and interpret the tumor immune microenvironment (TIME) from a more holistic, comprehensive, and integrative perspective.Second, IMPAGT presents the dysregulation of the immune system even at the tumor budding stage in cervical cancer. “Pathway in Cancer” was the most strongly enriched and activated pathway for both up- and downregulated DEGs. This highlighted the presence of abnormal shifts in gene expression in TB, indicating the activation of carcinogenesis dynamics even from a small cluster of tumor cells. This process supported the initiation of cancer from tumor budding and further revalidated their clinical significance.In addition, DEG-mapped pathway visualization of “Pathway in Cancer” using KEGG Mapper revealed overactivation of “Signal transduction pathways”, such as the PI3K/Akt, JAK/STAT, and Notch signaling pathways. These signaling pathways eventually pointed toward “Sustained angiogenesis”, “Evading apoptosis”, and “Proliferation”. A detailed investigation of DEG-mapped pathways revealed that up- and downregulated DEGs were mainly involved in the PI3K/Akt signaling pathway. Furthermore, the GO analysis of 133 IMPAGT-overlapped upregulated DEGs revealed that the immune-related genes were directly associated with the characteristics of the PI3K/Akt pathway.The PI3K/Akt/mTOR signaling pathway is one of the most common aberrantly stimulated pathways in solid tumors . The patThe oncogenic stimulation of this pathway is caused by several mechanisms: inappropriate activation of RTK; somatic mutations in PI3KCA, which encodes p110α of PI3K; and the loss of the tumor suppressor PTEN and other molecular signals ,36,40,41The PI3K/Akt/mTOR axis is related to antiapoptosis and proliferation in cancer cells. Apoptosis is a programmed cell death pathway that is activated by both the extrinsic death receptor and intrinsic mitochondrial pathways. Peng et al. argued that PI3K/Akt/mTOR signaling leads to the binding of extrinsic death complexes, such as FasL and Fas, which activate caspase 8 and trigger apoptosis with other caspases . AdditioThe PI3K/Akt/mTOR pathway is also critical in sustaining angiogenesis by serving as a master controller of angiogenic signaling in the endothelium . Tumor aAlthough it was not specifically identified in the DEG-mapped pathway, PI3K/Akt/mTOR signaling is also closely associated with the EMT ,53. As mDespite the necessity and continuous trials of PI3K inhibitors, only a few treatments have been approved for clinical use for several reasons . First, On that note, a recently developed technology, termed proteolysis-targeting chimeras (PROTACs), can be used to solve issues related to small-molecule inhibitors. PROTACs are bivalent molecules consisting of a ligand of the protein of interest (POI) and a covalently linked ligand of an E3 ubiquitin ligase. PROTACs recruit E3 ligase in proximity. After binding and forming the complex, the POI is transported to the 26S proteasome for degradation ,68. PROTThe limitations of our study include the small number of samples with pathway-level results. Because the current data focused on the most enriched pathway related to the TIME of TB in cervical cancer, specific details, such as gene-level biomarkers or specific aspects of molecular reaction in the PI3K/Akt/mTOR pathway, have not been elucidated. However, these issues can be resolved by planned follow-up research on PI3K PROTACs with more clinical samples and validation studies in cervical cancer cell lines. Moreover, protein-level analyses, such as Immunohistochemistry and Western blotting, are planned in the next follow-up study as well. Despite these limitations, our study validated the presence of immune dysregulation in TB of cervical cancer, especially the dysregulation of the PI3K/Akt/mTOR signaling pathway. In addition, the findings emphasized the clinical significance of tumor budding in relation to tumorigenesis. Furthermore, the study asserted the necessity of in-depth investigations on the PI3K/Akt/mTOR axis to develop more specifically targeted patient-tailored treatments in cervical cancer.The newly generated database IMPAGT revealed the existence of immune dysregulation dynamics and the specific enrichment of the PI3K/Akt/mTOR signaling pathway in the tumor immune microenvironment of cervical cancer with high tumor budding. Further research focused on the PI3K/Akt/mTOR axis and selective therapeutic approaches targeting this pathway could help develop better targeted therapy strategies for cervical cancer.


The endometrium receptivity was impaired by controlled ovarian hyperstimulation (COH), which would then lead to fertility issues and increased abortion clinically. In the present study, to explore the effectiveness of Tiaojing Zhuyun Formula (TJZYF) in improving endometrial receptivity of COH rats and the possible active ingredients and mechanisms, an approach of network pharmacology was performed and a COH animal model was established. As analyzed, stigmasterol and quercetin may be the active ingredients of TJZYF on improving endometrial receptivity and positive regulation of ion transport, the cytokine-mediated signaling pathway, and endocrine process, and vascular endothelial growth factor receptor signaling pathway may be involved. Eighty female rats were divided into four groups randomly: control, model, TJZYF, and TJZYF+si-VEGFA. COH rat models were constructed by injecting with human menopausal gonadotropin (HMG) and human chorionic gonadotropin (HCG). We found that both endometrial thickness and number of embryo implantations in model were substantially reduced vs. control. The gene and protein expressions of VEGF, PI3K, and p-Akt in the uterus were significantly reduced. TJZYF could increase the endometrial thickness and number of embryo implantations and enhance the expressions of VEGF, PI3K, and p-Akt in the uterus. In the TJZYF+si-VEGFA group, the effect of TJZYF was impaired. Generally, TJZYF could improve the endometrium receptivity and facilitate embryo implantation of COH rats by upregulating VEGF and enhancing the PI3K/Akt signaling pathway. An estimated 48 million couples and 186 million individuals have developed infertility, which has a negative impact on the sexual behavior and psychology of infertile couples and has a profound impact on the global quality of life [The World Health Organization (WHO) has reported that the reproductive health of populations such as infertility is attracting global health concerns , surgery, or reproductive tract infection , 10. AmoTraditional Chinese medicine (TCM) is currently attracting more and more international attention for reproductive and disease prevention, including the treatment of female infertility. Studies have shown that TCM can benefit gonadotropin hormone release, ovulation induction, improvement of uterine blood flow, and menstruation in the endometrium –18. OtheThe present work firstly used a network pharmacological technical to explore potential active components and key targets of TJZYF which might act on enhancing endometrial receptivity. And through the establishment of a COH animal model, the experimental research on TJZYF improving the uterine membrane receptivity of the model animals was carried out. The mechanism of TJZYF in improving endometrial receptivity was preliminarily discussed, and a certain scientific basis was provided for the research and development of new Chinese patent of TJZYF in the later period.Rehmanniae Radix Praeparata , Cuscutae Semen , Morindae Officinalis Radix , Cistanchs Herba , Cornus Officinalis Sieb. Et Zucc , lycii fructus , Carapax testudines , Angelicae Sinensis Radix , Chuanxiong Rhizoma , Atractylodes macrocephala Koidz , Radix Salviae , Cyperi Rhizoma , and Paeoniae Radix Alba . 80 female SD rats  were purchased. The approval for animal experiments was obtained from the Guizhou University of Chinese Medicine (20210018). The rats were fed adaptively for 7 days; the female rats were subjected to vaginal exfoliated cell smear observation. Then, the animals were grouped as control, model, TJZYF, and TJZYF+si-VEGFA, with 20 rats in each group. Ten days before model establishment, rats were given 4.5 mL TJZYF by gavage (0.38 g/mL) twice a day. Intraperitoneal injection was given to model at 9:00 in the late estrus of rats for continuous 9 days. The dosage was 40 μg/100 g of GnRHa and 40 IU/100 g of HMG at the same time. 100 IU/100 g of HCG was injected 48 h later. Equal volume of saline was given to rats of normal group. Apparent increase of vaginal secretion in rats marks the successful establishment of the rat model. Meanwhile, abundant nuclear-free keratinocytes can be observed using a microscope.TJZYF consists of https://tcmspw.com/tcmsp.php) based on principles of network pharmacology. The active ingredients of TJZYF was composed of Shudihuang (SDH), Tusizi (TSZ), Bajitian (BJT), Roucongrong (RCR), Shanzhuyu (SZY), Gouqi (GQ), Guiban (GB), Danggui (DG), Chuanxiong (CX), Baizhu (BZ), Danshen (DS), Xiangfu (XF), and Baishao (BS). Targets of the active ingredients were collected from SwissTargetPrediction (http://www.swisstargetprediction.ch/) subsequently. The disease genes ware retrieved from Genecards (https://auth.lifemapsc.com/) by using “decreased endometrial receptivity” as a search term. After the disease genes relevant to decreased endometrial receptivity were sorted out, we constructed a protein-protein interaction (PPI) network based on an online tool String and established a drug-component-target diagram based on Cytoscape 3.6.1 software. Data exported from the Cytoscape software were used for Gene Oncology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis.We firstly obtained active ingredients of TJZYF using TCMSP website (http://www.rcsb.org/pdb) and PubChem database, respectively. A series of processes including hydrogenation was performed using AutoDock 4.2.6 software. Following discharge calculation, molecular docking was conducted for the receptor protein and the ligand using AutoDock Vina 1.1.2. The binding energy with the highest score was selected for subsequent analysis, and the results were processed for visualization using PyMOL software.Firstly, 3D structures of both proteins, namely, AKT and VEGFA, and ligands, namely, quercetin and stigmasterol, were obtained via the Protein Data Bank  and images collected.HE staining was performed to observe the sample tissue characteristics of rats. Firstly, the sample was fixed using a fresh solution of 4% paraformaldehyde and embedded using paraffin under routine steps. Sample slices were prepared into 4.5 The concentrations of estrogen (E2) and progesterone (PROG) in plasma were determined by the use of quantitative sandwich ELISA kits  that is commercially available. A microplate reader was applied to measure the produced products, and a standard curve was plotted for analysis.The uteruses of rats in each group were taken by laparotomy, rinsed with PBS, longitudinally dissected, and fixed in 2.5% glutaraldehyde solution, and the development of endometrial pinopodes could be visualized using a scanning electron microscope. Its developmental process of the pinopodes undergoes 3 phases: development, maturation, and decline. During development, microvilli become slender, dense, and erect. There will be a protrusion growing at the top and developing the entire top cell. After that, microvilli shortens and merges. During maturation, the microvilli disappeared completely with a smooth surface. Meanwhile, the protrusion grows bigger and higher than ciliate cells. Finally, during recession, it gradually shrinks, and microvilli reappear on the surface.−∆∆CT was employed for calculating relative expression of genes.Total RNA from the sample was extracted using Trizol reagent  as per manufacturer's protocol. cDNA synthesis was performed using PrimeScript TMR T kit . The qRT-PCR reactions were carried out as per TB Green Premix Ex Taq II  instructions. The platform applied CFX96 real-time system . The algorithm of 2The primers used were listed: VEGF-F: 5′-CTGCCGTCCGATTGAGACC-3′, VEGF-R 5′-CCCCTCCTTGTACCACTGTC-3′; PI3K-F: 5′-ACACCACGGTTTGGACTATGG-3′, PI3K-R 5′-GGCTACAGTAGTGGGCTTGG-3′; AKT-F: 5′-ATGAACGACGTAGCCATTGTG-3′, AKT-R 5′-TTGTAGCCAATAAAGGTGCCAT-3′.The protein extracted from frozen endometrial samples was measured via Western blot. The protein lysis buffer was added to the tissue; the homogenate was kept cold on ice for 30 min before supernatant collection via centrifugation. Bicinchoninic acid (BCA) was used for protein quantification and followed by denaturation at 100°C for 8 min. Following protein isolation, membrane transference and blocking were performed. The primary antibody (1 : 1000) was added for incubation overnight. After washing with TBST, secondary antibody  was provided for culture. One hour later, optical density (OD) value of the band was analyzed and target band of which was selected as its corresponding relative expression. The antibodies used were presented below: anti-VEGF (A12303), anti-PI3K (A0265), anti-AKT (A17909), anti-p-AKT (AP0637), and GAPDH , and all were ABclonal.The obtained tissues were processed as previously described including fixation, dehydration, and slice preparation. Following the supplement of primary antibodies, the samples were cultured for IHC staining and incubated with HRP-Polymer-conjugated secondary antibody. One hour later, chromogenic reagent diaminobenzidine  was added for staining three minutes; hematoxylin  was applied for counterstaining. Subsequently, a Pannoramic Scan 250 Flash was employed for scanning.P < 0.05.The results were processed and analyzed using GraphPad Prism version 9.0  and presented as mean value ± standard deviation. Statistical significance among three or more groups were determined by one-way analysis of variance (ANOVA) with Tukey post hoc testing. The difference was statistically significant when The main components of each medicinal material were found in the TCMSP database, and the compounds with the conditions of OB ≥ 30% and DL ≥ 0.18 were considered as the main active components, and a total of 135 compounds were screened. The corresponding targets for the reduction of endometrial receptivity were collected from the Genecards database, with a total of 621 targets, and a total of 1033 targets for the components of TJZYF. The two were processed for intersection, and there were a total of 130 intersection genes as shown in Thirty potential targets screened by PPI analysis were used for GO and KEGG pathway annotation analysis. Through GO analysis, enriched terms of biological process (BP), cellular component (CC), and molecular function (MF) were obtained, respectively, and the functional distribution of the target was predicted. As shown in The results of KEGG pathway annotation are shown in The docking studies of small molecules (quercetin and stigmasterol) and proteins (AKT1 and VEGFA) were performed using the Autodock software. The conformation of quercetin and stigmasterol binding to AKT1 was displayed in Figures The emotional cycle of rats was detected by HE staining and observed under optical microscope. The HE-stained sections of the rat uterine tissues in each group were observed, as shown in P < 0.01) whereas that of the TJZYF and TJZYF+si-VEGFA groups were increased as compared with model (P < 0.01). Additionally, the number of embryo implantation in TJZYF+si-VEGFA group was lower than TJZYF group (P < 0.01).Pinocytosis is generated through endometrial epithelium. The development of pinocytosis is shown in P < 0.01), while TJZYF and TJZYF+si-VEGFA could increase VEGF, PI3K, and p-AKT expression in endometrial tissue (P < 0.01). Moreover, there were significant protein expression differences between TJZYF and TJZYF+si-VEGFA, and the expression in TJZYF+si-VEGFA was lower than that in TJZYF (P < 0.01). The mRNA detection indicated that the expression trend of VEGF and PI3K in each group in the endometrial tissue were consistent with protein expression in In order to verify the potential target of TJZYF to enhance endometrial receptivity obtained in the network pharmacology analysis, we designed WB and immunohistochemistry to detect the protein expression and mRNA level of VEGF, AKT, PI3K, and p-AKT in rats' endometrial tissue. Protein detection results are shown in In vitro ART is an extracorporeal intervention technique for people suffering from infertility and includes assisting conception and improving pregnancy rates . In vitrThe COH method effectively promotes the development of IVF-ET technology by inhibiting the early appearance of luteinizing hormone peak, improving egg quality, and increasing ovulation rate and high-quality embryo rate; however, the problem of low pregnancy rate is still remained . It is cWith the aim to investigate TJZYF's action on endometrial receptivity improvement in COH rat models, we first explored active ingredients in TJZYF and key targets and possible mechanisms for improving endometrial receptivity by using a network pharmacology technique. The experimental results show that the active ingredients of TJZYF may be A1 (stigmasterol) and G1 (quercetin). Collecting the intersection genes of TCM and decreased endometrial receptivity and then conducting GO and KEGG enrichment analysis, we found that cytokine-mediated signaling pathway, endocrine process, and vascular endothelial growth factor receptor signaling pathway show great impact. And the molecule functions are closely related to activation of protein kinase B activity and growth factor receptor binding. According to the results, the binding effect of stigmasterol, quercetin, and protein VEGFA and AKT was further studied by molecular docking, implying that quercetin had strong binding to VEGFA and AKT, followed by stigmasterol.Subsequently, the COH animal model was established to investigate relevant effects and mechanisms of TJZYF on endometrial receptivity. Endometrial thickness and tissue morphology are considered important factors affecting IVF and ET , 33. EndTJZYF could increase endometrium receptivity and the number of embryo implantations in COH rats. Our results suggested that the TJZYF might mainly regulated the receptivity of endometrium by cytokine-mediated signaling pathway, endocrine process, and vascular endothelial growth factor receptor signaling pathway. AKT1 and VEGF are considered as key target genes of TJZYF for endometrial receptivity management. Stigmasterol and quercetin may be the important active ingredients of TJZYF. Furthermore, it also demonstrated that TJZYF may improve the endometrial receptivity via mediating VEGF and PI3K/AKT signaling pathway.


To investigate the mechanism of action of Datura metel in the treatment of sinus bradycardia based on network pharmacology and molecular docking.The active ingredients and targets of Datura metel were collected by TCMSP database, and the Cytoscape software was used to map to show the interrelationship. Use 5 databases: GeneCards, PharmGKB, OMIM, DisGeNET, and Drugbank to obtain targets related to sinus bradycardia; establish a protein-to-protein interaction network with the help of the STRING platform; GO and Kyoto Encyclopedia of Genes and Genomes analysis of the selected core targets using the Metascape platform; Finally, the AutoDock platform was used for molecular docking and the results were displayed through Pymol.27 kinds of active ingredients of the drug were screened, including 10 kinds of main ingredients; 198 drug targets and 1059 disease targets. There are 54 targets of action in the treatment of sinus bradycardia, of which 19 targets such as AKT1, IL6, TNF, and VEGFA are the core targets of Datura metel in the treatment of sinus bradycardia. Kyoto Encyclopedia of Genes and Genomes obtained 18 results suggesting that AGE-RAGE, hepatitis C, relaxin, and JAK-STAT may be key signaling pathways. Molecular docking shows that most components of the drug have good docking ability with the core target, indicating that the prediction results have certain reliability.This study preliminarily explores the potential active ingredients and possible mechanisms of action of Datura metel in the treatment of sinus bradycardia and provides a basis for in-depth investigation of its medicinal material basis and mechanism of action. Asymptomatic sinus bradycardia often requires no treatment, but in modern medicine, western drugs such as atropine have short-lived and unstable effects, as well as the high cost of pacemakers.Sinus bradycardia (SB or DH) refers to a sinus rhythm with a frequency of fewer than 60 beats/min, Modern pharmacological results have confirmed that scopolamine, the active ingredient of Datura metel (DM or YJH), can increase heart rate better at higher doses,,4 and clinical reports of formulas using Datura metel as one of the key drugs to increase sinus rhythm have been increasing year by year. The clinical observation found that the combination of Heart Treasure Pill with Datura metel as the main ingredient and the basic treatment of western medicine can increase the improvement rate of sinus bradycardia with ventricular anterior contraction by more than 15%. In addition, it has been clinically found that the combination of Heart Treasure Pill and Irbesartan can better increase heart rate while improving cardiac function. Although Datura metel is effective in increasing sinus rhythm, few studies use big data systems to deeply explore the mechanisms of its components. From the idea of the holistic concept of traditional Chinese medicine, the author has used modern network pharmacology and molecular docking methods to reveal the main chemical components, targets, and mechanisms of Datura metel to increase heart rate, to provide a more reliable theoretical basis for the use of Datura metel in the treatment of sinus bradycardia. The corresponding workflow is shown in Fig. Chinese medicine is a unique treasure trove of medicine in China and has significant and irreplaceable advantages in the prevention and treatment of many diseases such as sinus bradycardia.Web-based pharmacology and molecular docking studies require the use of web-based databases and software platforms, and the URLs of the databases used in this paper are listed in Table  and then the active ingredients and related targets of Datura metel were screened with the conditions of oral bioavailability ≥ 30%, and drug-like properties ≥ 0.18. The active ingredients and the corresponding targets were compiled in Excel and entered into the UniProt database one by one, with “Reviewed” and “Human” as constraints, to obtain the gene abbreviations of all targets. After de-duplication, the gene results of the targets of the active ingredients of Datura metel were obtained.All the chemical components of Datura metel were firstly obtained from the TCM Systematic Pharmacology (TCMSP) database, Online Human Mendelian Genetic, DrugBank, DisGeNET, Pharmacogenomics Knowledge Base, and the results were collated in Excel format and deduplicated for the desired The results are then deduplicated to the desired disease target. GeneCards were used to retain results for genes greater than the median. The corresponding targets of the active ingredient and the disease target were imported into Venny2.1 to obtain the intersecting targets.Using “sinus bradycardia” as a keyword, searches were conducted in the 5 classic databases GeneCards, database. The intersecting targets from 2.2.2 were imported into the string database, species were selected as “Homo sapiens” and free nodes were hidden, other parameters were set to default values, PPI networks were constructed, and the “tsv” file was exported. The built-in plug-ins Bisogenet and CytoNCA of Cytoscape 3.7.1 were used to filter for degree centrality, proximity centrality, and intermediate centrality to derive core targets.Protein interactions were analyzed using the String The biological process (BP), cellular component (CC), molecular functions (MF), and KEGG pathways were obtained and collated in Excel format. The results of the data were plotted using the Bioinformatics online mapping website as GO triple bar graphs and KEGG bubble plots.GO functional annotation and KEGG pathway enrichment analysis were performed for the intersection targets of Homo sapiens and sinus bradycardia based on the Metascape database.To visualize the modern pharmacological rationale for the treatment of sinus bradycardia with Datura metel, the Cytoscape 3.7.1 software was used to construct a “drug-key component-pathway-intersection target-disease” network diagram. were obtained in PDB format, and the water molecules and ligands were removed in Pymol 2.4.0 software; the mol2 structures of the top 4 core targets in the PubChem database were obtained in mol2 format; molecular docking was performed by AutoDockTools 1.5.6 software. Docking was performed by Autodock software to obtain the highest binding energy sites; visualization was performed by Pymol software.The 3D structures of the top 4 core targets (AKT serine/threonine kinase 1 (AKT1), IL6, vascular endothelial growth factor (VEGFA), tumor necrosis factor (TNF) in the PDB databaseA total of 27 chemical components of Datura metel were obtained in TCMSP Table .Retrieved from 5 major databases, GeneCards, Online Human Mendelian Genetic, DrugBank, DisGeNET, and Pharmacogenomics Knowledge Base, respectively, “sinus bradycardia “277, 20, 9, 900, and 114 corresponding targets were obtained, and 1059 targets remained after all targets were combined and deweighted. The intersecting targets were obtained by importing the drug targets and disease targets together into Venny2.1, suggesting that the 54 intersecting targets  were identified by importing 54 intersecting targets into the Metascape database, with the species selected as “Homo sapiens.” Of these, 3700 entries were for BP, mainly related to the cellular response to organic cyclic compound, negative regulation of cell population proliferation, blood circulation, response to cytokine, and regulation of system process. There are 285 entries in CC, mainly dealing with the extracellular matrix, synaptic membranes, plasma membrane protein complex, membrane raft, transcription regulator complex, and other CCs. MF has 458 entries, mainly related to cytokine receptor binding, protein domain specific binding, endopeptidase activity, protein kinase binding, G protein-coupled neurotransmitter receptor activity, and other MFs. The top 10 of BP, the top 9 of CC, and the top 10 of MF were taken for display , of which the first 18 were significant pathways as shown in Table The 54 intersecting targets were imported into the Metascape database, and the species selected was “Homo sapiens,” and KEGG enrichment analysis was performed. 209 key signaling pathways were identified (The interrelationship data files and attribute files of drug and key components, key components and intersecting targets, intersecting targets and pathways, and intersecting targets and diseases were prepared in Excel, and both were imported into Cytoscape 3.7.1 line optimization to obtain the final visual network Fig. , where tSince kaempferol and quercetin are the 2 most important components of Datura metel, and AKT1, TNF, IL6, and VEGFA are the top 4 most important ranked core targets screened by degree value among the intersection targets of drug disease, so the molecular docking validation of the most important components and the most important targets has better credibility. Molecular docking of the top 4 Degree values of the core targets and their corresponding components was carried out by AutoDock software and the binding energy is shown in Table  mostly increased excitability of the vagus nerve, impaired function of the sinus node itself, numerous organic heart diseases, etc. It is also related to the long-term use of certain drugs. The pathogenesis is usually related to abnormalities in myocardial ion channel function and autonomic dysfunction. The development of sinus bradycardia is complex, with multiple cells and pathways of action interacting with each other, thus forming a complex network system of interactions. Single-target, single-pathway therapy is not effective, while Chinese medicine has the natural qualities of multiple components, multiple targets, and multiple pathways of action, so it can regulate the body systematically. It has been used for a long time to treat illnesses, with the famous doctor Hua Tuo using it as the main ingredient to make mafei san in the late Eastern Han Dynasty. Abroad, it has a wider range of uses,–25 including heart disease, convulsions, diarrhea, hemorrhoids, dysmenorrhea, eczema, and toothache. Although the Chinese Pharmacopoeia 2020 edition does not state that the drug can increase heart rate, several studies have shown that important chemical components of the flower, such as quercetin, have a significant effect on improving heart function, and call for quercetin to be taken seriously in the treatment of many cardiovascular diseases. As research into the chemical composition of the drug has become more advanced, clinical formulations using it as a major component to increase sinus heart rate have been frequently reported, with the Heart Treasure Pill–31 being widely used clinically. Although there are many reports, the specific molecular regulatory mechanism of the effect of Datura metel on sinus bradycardia remains unclear. In this study, by searching the TCMSP database, we found that quercetin, apohyoscine, kaempferol, datumetine, atropine, and other active ingredients have more target genes and are the main active ingredients of Datura metel. We obtained the interrelationship network between the above components and sinus bradycardia-related targets through network pharmacology and validated quercetin and kaempferol among them by molecular docking.Sinus bradycardia belongs to the category of “palpitation,” or “chest stuffiness and pains” in Chinese medicine, The causes, mechanisms, and treatment of this disease have been described in ancient works such as Yellow Emperor’s Classic of Internal Medicine, Treatise on Febrile Diseases. Modern medicine has confirmed that the causes of this disease are Chen Qiuhong and others suggest that they may protect against ischemia-reperfusion injury by increasing Na+/K+-ATPase activity and thus improve arrhythmias. The compounds have been shown to improve cardiac arrhythmias by increasing Na+/K+-ATPase activity. These compounds have been shown to exert anti-arrhythmic effects on several ion channels, including potassium, sodium, and calcium, in the myocardial cell membrane. Animal studies have reported that flavonoids have a strong effect on the reduction of inward rectifier potassium currents caused by aconitine. Pei et al found that quercetin had a protective effect on myocardial damage caused by adriamycin in mice. This shows that quercetin and kaempferol, as flavonoids in Chinese medicine, play a “multi-component, multi-target” role in the treatment of sinus bradycardia. In addition, alkaloids also have a role in the improvement of sinus bradycardia. In particular, it is widely known that higher doses of atropine can increase heart rate by relieving vagal inhibition. Studies have shown that scopolamine and scopolamine, which are contained in Hypericum, can improve blood microcirculation by blocking α1 and M receptors, increasing cell membrane fluidity, reducing blood viscosity, promoting tissue healing, increasing heart rate, enhancing myocardial contractility, and inhibiting ectopic excitability of the heart.–40After screening by oral bioavailability and drug-like properties parameters, 27 active ingredients were obtained, and a total of 10 active ingredients were identified after eliminating those that could not be found as targets. From the “drug-component-pathway-key target-disease network diagram,” it can be seen that there are 6 major active ingredients directly connected to the key targets, accounting for 60% of the total number of corresponding target ingredients, which are flavonoids and alkaloids. Flavonoids are mostly found in the flowers and leaves of the woody herb and are effective in a variety of diseases of the cardiovascular system. In addition to effectively improving the number of episodes of arrhythmias due to myocardial ischemia, they also have multiple effects such as antiatherosclerosis, anticoagulation, antilipid, and vasodilatation, IL-6 and VEGFA are closely related to the progression of cardiac hypertrophy and coronary artery disease, while VEGFA is widely distributed in the brain, VEGFA is widely distributed in the brain, heart, and other tissue cells, and has an important regulatory role in angiogenesis and atherosclerosis. Studies have shown that MSCs can reduce inflammation in the heart and lungs through TNF signaling. Mathematical modeling of TNF-α overexpression in transgenic mouse hearts has revealed an effect on cardiac arrhythmias. All of the above suggests a strong association between these targets and the development of arrhythmias.The results of the network screening of core targets showed that AKT1, IL6, VEGFA, and TNF were the top 4 core target proteins in terms of degree value, and maybe the most core targets for the treatment of sinus bradycardia with Yohimbe. A study showed that resveratrol targeting AKT1 inhibited inflammatory vesicle activation in cardiomyocytes under sympathetic stress, inhibited β-adrenergic receptor-mediated NLRP3 inflammatory vesicle activation in cardiomyocytes and mouse hearts, and the resulting cardiac inflammation.C. elegans revealed that C. elegans was highly correlated with the BPs of blood circulation, regulation of smooth muscle cell proliferation, and response to peptides in the cardiovascular system, which were also reflected in the key targets of C. elegans for the treatment of cardiac arrhythmias. The KEGG pathway enrichment results showed that Hepatitis C, AGE-RAGE signaling pathway, relaxin signaling pathway, JAK-STAT signaling pathway, T cell receptor signaling pathway, calcium signaling pathway, p53 signaling pathway, and adrenergic signaling in cardiomyocytes as the main regulatory pathways. This suggests that the same regulatory mechanisms may exist between different diseases. Evidence suggests a relationship between viral invasion and the development of arrhythmias, such as hepatitis B and C.,46 Numerous previous studies have shown that relaxin can hinder fibrosis through many cell targets and signaling pathways. The AGE-RAGE signaling pathway was found to affect the migration and differentiation of cardiac fibroblasts, which in turn affected ventricular reconstruction and cardiac function. In addition, the JAK-STAT signaling pathway plays an important role in myocardial ischemia, infarction, myocarditis, and other cardiac injuries, and its blockade is effective in reducing the inflammatory response in the myocardium and preventing the formation of myocardial fibrosis. Some components, such as arginine pressors, can regulate current by affecting the T-type calcium signaling pathway, increasing intracellular calcium ion concentrations, and triggering prolonged action potential duration and differences in the spatial distribution of action potentials, leading to arrhythmias.The analysis of the BPs of the key core targets of In summary, through the use of network pharmacology combined with molecular docking, and the comprehensive discussion and analysis of various networks and databases, the composition targets and mechanisms of action of Datura metel. in the treatment of sinus bradycardia through “multi-component, multi-target, and multi-pathway” have been more fully explored. The results of this study should be investigated in cellular or animal studies to provide further ideas and references for basic and clinical research.Conceptualization: Feifei Yang.Data curation: Pihong Liu, Xiaosi Zhang.Formal analysis: Feifei Yang, Pihong Liu, Xiaosi Zhang.Investigation: Zhe Zhang.Methodology: Feifei Yang, Hao Lu.Project administration: Naizhi Geng.Resources: Naizhi Geng.Software: Feifei Yang.Supervision: Xiaosi Zhang, Zhe Zhang, Hao Lu.Validation: Naizhi Geng.Visualization: Feifei Yang, Xiaosi Zhang, Zhe Zhang, Hao Lu.Writing—original draft: Feifei Yang.Writing—review and editing: Naizhi Geng, Xiaosi Zhang.The article type has been changed from Narrative Review to Observational Study. “A Review” has been removed as the subtitle of the article title.