In China, the sale of freshly slaughtered chickens is becoming increasingly popular in comparison with that of live chickens, and due to this emerging trend, the skin and feather follicle traits of yellow-feathered broilers have attracted a great deal of research attention. The feather follicle originates from the interaction between the epidermis and dermis in the early embryonic stage. Feather follicle morphogenesis is regulated by the Wnt, ectodysplasin (Eda), epidermal growth factor (EGF), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), sonic hedgehog (Shh), Notch, and other signaling pathways that exist in epithelial and mesenchymal cells. The Wnt pathway is essential for feather follicle and feather morphogenesis. Eda interacts with Wnt to induce FGF expression, which attracts mesenchymal cell movement and aggregates to form feather follicle primordia. BMP acts as an inhibitor of the above signaling pathways to limit the size of the feather tract and distance between neighboring feather primordia in a dose-dependent manner. The Notch/Delta pathway can interact with the FGF pathway to promote feather bud formation. While not a part of the early morphogenesis of feather follicles, Shh and BMP signaling are involved in late feather branching. This review summarizes the roles of miRNAs/lncRNA in the regulation of feather follicle and feather growth and development and suggests topics that need to be solved in a future study. This review focuses on the regulatory mechanisms involved in feather follicle morphogenesis and analyzes the impact of SNP sites on feather follicle traits in poultry. This work may help us to understand the molecular regulatory networks influencing feather follicle growth and provide basic data for poultry carcass quality. The yellow-feathered broiler, also known as the quality broiler, encompasses local chicken breeds in China or new crossbreeds from local chicken varieties. China ranks second in chicken meat production in the world . In compFeathers are derived from feather follicles, which can produce feathers of diverse colors and shapes with different functions during different physiological periods. The basic structure of a feather follicle is similar to that of a hair follicle in mammals . HoweverThe initiation of feather follicle primordium results from the condensation of dermal mesenchymal cells below the epidermal placode. First, the dermal precursor cells migrate to the subectoderm region and form different skin regions (macro-patterning). Then, feather follicle primordia are induced to form epidermal placodes and dermal condensations within the skin regions. The feather follicle primordium positioning process in particular areas during embryonic development is also called micro-patterning. The arrangement of feather tracts begins on embryonic day 6 (E6), resulting in bare spaces between the tracts of the chicken embryo. These bare spaces remain unfeathered throughout a chicken’s life cycle; however, in geese, these bare spaces are filled with small fluffy feathers. We can observe the obvious feather follicle primordium emerging in the dorsal midline of the chicken embryo on embryonic day 8 (E8). The dorsal skin displays clear protrusions across the feather tract, which emerge on embryonic day 9 (E9) (short buds).Histological measurements have demonstrated that feather follicle development mainly occurs between embryonic days 7 and 15 (E7–E15) in chicken embryos  Figure . The skiThe interaction between epidermal and dermal mesenchymal cells is essential for feather follicular morphogenesis. Isolation and recombination experiments have demonstrated that dermal mesenchymal cells determine the direction of skin appendage development . The speThe Wnt signal expressions change in dynamic patterns and induce the formation of feathers at various stages. The Wnt1 class  functions through the canonical Wnt/β-catenin signaling pathway. Wnt1 and 3a, which activate the classical Wnt signaling pathway expressed throughout the feather tract (E6), are restricted to the placode epithelium (E8) and are related to the size of the spinal tract size . Wnt8a a2+ pathways, which act independently of the Ctnnb1 pathway [Non-canonical Wnt signaling is initiated by the Wnt5a type , defined as Wnt/planar cell polarity (PCP) or Wnt/Ca pathway . Wnt5a a pathway . The ove pathway . Wnt11 i pathway . The seq pathway . The hig pathway . HoweverThe epidermal growth factor (EGFs) family acts by binding to its tyrosine kinase receptor (EGFR), causing the phosphorylation of tyrosine residues and activating downstream-signaling pathways, such as the Jak, SRC kinase, and ERK pathways, etc. . EGF sigThe fibroblast growth factor (FGF) family is composed of eighteen secreted proteins (FGFRs) and four tyrosine kinase FGF receptors (FGFRs). The activated FGFRs phosphorylate specific tyrosine residues that are coupled with downstream signals, which include the RAS-MAPK, PI3K-AKT, PLCγ, and STAT pathways . FGFs arThe ectodysplasin pathway, including ectodysplasin, a secreted signaling molecule of the TNF superfamily, its receptor (EDAR), and the downstream death domain adaptor (EDARADD), plays an important role in the formation of epidermal appendages in mammals ,34. The Bone morphogenetic proteins (BMPs) are the members of the secreted signaling molecules transforming growth factor (TGF)-β superfamily and function by binding to specific BMP receptors (BMPRIA and BMPRIB). The canonical Smad and non-canonical Smad signaling pathways  are the down-signal transduction pathways of BMPs ,41. BMPsBMPs also repress various signaling pathways, including Wnt, Eda, and FGF, in order to control the formation of feather follicles. In the initial stages of feather development, the expression wave of Ctnnb1/EDA induces FGF20 expression, thereby triggering the mesenchymal cell condensation and feather patterning formation. The treatment of BMPs reduces the amount of feather primordium in a dose-dependent manner through the suppression of FGF20 expression but cannot eliminate cell aggregation . FGF4 an2+ channels, and coordinate mesenchymal cell movements to enhance feather bud elongation [The sonic hedgehog (Shh) is a secreted protein that belongs to the highly conserved hedgehog (Hh) family. In birds, Shh signals play crucial roles in feather follicle formation from initiating dermal condensation to feather filament shaping. Shh binds its receptor Patched (Ptc), leading to the activation of the seven-pass smoothened transmembrane protein (Smo) . Then, tongation . Fgf4, Songation . Shh is ongation . The effongation . During ongation ,51. Notch signaling is evolutionarily conserved and affects the development process of differentiation, proliferation, and apoptosis in many different tissues of vertebrates and invertebrates ,53,54. TNon-coding RNAs are a variety of RNAs that do not translate into proteins and were originally considered by-products of transcription with fewer biological functions. However, recent studies have suggested some of them, such as micro-RNAs (miRNAs), long-noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), act as key regulators in diverse aspects of biological functions, such as chromosome silencing, genomic imprinting, chromatin modification, transcriptional activation, and interference. NcRNAs exist widely in animal tissues and organs and regulate target gene expression via epigenetic regulation, transcriptional regulation, and post-transcriptional levels ,61. A grBao et al.  detectedA study on chicken feather regeneration found that lnc1589, lnc3500, and lnc7831 are able to regulate feather regeneration and axis formation through the inhibition of the Wnt signaling pathway . RNA seqIt has been found that ncRNAs play significant modulatory roles in hair follicle development through the regulation of Wnt/β-catenin, Shh, BMP, and Notch signaling pathways . As showMost miRNAs/LncRNAs target genes identified belong to feather follicle- or feather formation-related signaling pathways, suggesting that complex regulatory networks involving genes and ncRNAs exist in feather follicle development. We should pay more addition to the process of experimentally validating the specific regulatory mechanisms of the discovered miRNAs/LncRNAs in chicken feather follicle formation.Single nucleotide polymorphisms (SNPs) are the simplest forms of genetic markers and are caused by a single nucleotide mutation. They account for more than 90% of all known polymorphisms . SNPs arIn poultry, Feng et al. reported that a C to G transversion significantly reduced PDSS2 expression during feather development, resulting in a silky feather phenotype . It was 2 area in different sections. More research is needed to determine the suitability of detecting the feather follicle density in chickens. Compared with the known SNP molecular markers identified in hair follicles, how the SNPs control, interact with, or regulate gene expression and the different degrees of correlation with the core genes identified in the feather follicle development process remain unclear. Further studies need to be carried out to confirm the relationships between SNP markers and feather follicle traits in chickens.Overall, there are only a few studies related to feather follicle density and diameter size in chickens in comparison with those on hair follicles. A large number of SNP sites that are associated with feather follicles or feather traits were found, but the function of the candidate genes is still unclear. There are few relevant works that report on the differences in feather follicle density between chicken breeds. The measurement methods of feather follicle density or diameter can be divided into two categories: histopathologic section or direct count within a 2 × 2 cmFeather follicle morphogenesis is completed during the embryonic stage. The Wnt, EGF, Eda, FGF, Shh, and Notch/Delta pathways are positive pathways, and the BMP pathway has negative roles and is involved in feather follicle development. The crosstalk between these factors in the feather follicle formation process still needs to be investigated further. The ncRNAs collaborate with genes of the Wnt, Shh, BMP, and Notch pathways to form a complex regulatory network, which regulates feather follicle growth and development. Most studies focus on the temporal changes in the feather follicle growth and development processes. There is little research on the differences in feather follicle traits and gene expression among the different chicken breeds. Few SNP sites have been found to be closely related to feather follicle traits. It is necessary to further study the molecular regulatory mechanisms of feather follicle traits to provide a theoretical basis for novel interventions that can enhance the carcass appearance for the commercial sale of yellow-feathered broilers.


Background/aim: Hypertensive nephropathy (HN) is a common complication of hypertension. Traditional Chinese medicine has long been used in the clinical treatment of Hypertensive nephropathy. However, botanical drug prescriptions have not been summarized. The purpose of this study is to develop a prescription for improving hypertensive nephropathy, explore the evidence related to clinical application of the prescription, and verify its molecular mechanism of action.Methods: In this study, based on the electronic medical record data on Hypertensive nephropathy, the core botanical drugs and patients’ symptoms were mined using the hierarchical network extraction and fast unfolding algorithm, and the protein interaction network between botanical drugs and Hypertensive nephropathy was established. The K-nearest neighbors (KNN) model was used to analyze the clinical and biological characteristics of botanical drug compounds to determine the effective compounds. Hierarchical clustering was used to screen for effective botanical drugs. The clinical efficacy of botanical drugs was verified by a retrospective cohort. Animal experiments were performed at the target and pathway levels to analyze the mechanism.Results: A total of 14 botanical drugs and five symptom communities were obtained from real-world clinical data. In total, 76 effective compounds were obtained using the K-nearest neighbors model, and seven botanical drugs were identified as Gao Shen Formula by hierarchical clustering. Compared with the classical model, the Area under the curve (AUC) value of the K-nearest neighbors model was the best; retrospective cohort verification showed that Gao Shen Formula reduced serum creatinine levels and Chronic kidney disease (CKD) stage . With respect to target and pathway enrichment, Gao Shen Formula acts on inflammatory factors such as TNF-α, IL-1β, and IL-6 and regulates the NF-κB signaling pathway and downstream glucose and lipid metabolic pathways.Conclusion: In the retrospective cohort, we observed that the clinical application of Gao Shen Formula alleviates the decrease in renal function in patients with hypertensive nephropathy. It is speculated that Gao Shen Formula acts by reducing inflammatory reactions, inhibiting renal damage caused by excessive activation of the renin-angiotensin-aldosterone system, and regulating energy metabolism. Hypertensive nephropathy (HN) is one of the common complications of hypertension and can significantly increase the prevalence of chronic kidney disease (CKD) . AccordiBotanical drugs have advantages in improving HN symptoms. In the theory of traditional Chinese medicine (TCM), the use of botanical drugs emphasizes the symptom characteristics of the population. According to existing research , compareBotanical drugs are rich in compounds that act on multiple targets of the disease and improve the imbalance of the disease biological networks . HN invoThis study was based on data derived from the EMRs of patients with HN. The hierarchical network extraction algorithm and the fast unfolding algorithm were used to summarize the network information on patients' prescriptions and disease symptoms. A machine learning model was used to comprehensively analyze the clinical information and biological characteristics and obtain the core prescription Gao Shen Formula (GSF). Spontaneously hypertensive rats were used to establish a model for the verification of the mechanism of action of GSF .Chinese Pharmacopoeia.A total of 30,695 electronic medical records of patients diagnosed with hypertension were collected from the Affiliated Hospital of Shandong University of Traditional Chinese Medicine from 1 July 2014 to 31 May 2017. A total of 2,055 patients with HN were extracted. The diagnosis, demographic information (such as age and sex), chief complaint, medication prescriptions, and results of auxiliary examinations were collected from the patients’ EMRs. The patients’ symptoms were described using standard terminology, and the database was standardized with reference to the Medical Subject Headings (MeSH) and the In this study, prescriptions were extracted from the EMRs of HN patients. Setting botanical drugs as the node and the frequency of occurrence of two botanical drugs in different prescriptions as weights, a weighted prescription network was established. The hierarchical network extraction algorithm  was usedGephi (0.9.4) was used to implement the fast unfolding algorithm.The fast unfolding algorithm  has beenThe compounds and targets of core botanical drugs were collected from public databases, including the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) , SymMap HN-related genes were collected from the GEO database. The data were analyzed online using the GEO2R tool and supplemented using data from several mature databases, including Online Mendelian Inheritance in Man (OMIM) , DisGeNEHomo sapiens database to establish the protein‒protein interaction network (PPIN). In this study, shortest path analysis was used to identify the botanical drugs that are closely related to HN in the PPIN. The Dijkstra algorithm is a classical network node shortest-distance algorithm that is used to calculate the shortest distance between nodes in the PPIN. Nodes with a shortest distance of less than three were defined as strongly connected nodes  in R 3.6.3 was used to evaluate the compactness of compounds in each module in the matrix.To evaluate the regulation of each HN symptom by different compounds, this study summarized the number of genes regulated by different compounds in different symptom communities and established a frequency matrix. The Manhattan distance (Man) implemented in the Oral availability reflects the extent to which compounds are absorbed by the gastrointestinal tract and metabolized by the liver and is used as an important indicator of a compound’s effectiveness . The OB The Jaccard similarity coefficient (Jac) is used to measure the similarity between datasets. In this study, a Jac value was calculated for each compound target. The average Jac value was used to quantify the similarity of the botanical drug targets.BioConductor, implemented in the GOSemSim package (version 2.12.1) in R 3.6.3, was used to provide annotation data (The semantic similarity of Gene Ontology (GO) provides a method for calculating similarities among genes. In this study, GO biological process semantic similarity (GoSim) was used to measure the similarity between regulatory genes affected by compounds and biological processes related to HN. ion data .K-nearest neighbors (KNN) is a supervised machine learning algorithm that can generate a variety of feature information about compounds, match the input features with the expected output to create a learning function, and complete the classifier after cross-learning to adjust the parameters. The KNN model is sensitive to local information on interactions. KNN can be used to reflect the connection characteristics of input compounds and is more reliable for analysis of the targets of botanical drugs . In thispyrwr package (version 1.0.0) in Python 3.7.5.To determine the screening criteria to be used in the model, the random walk with restart (RWR) algorithm was used to find the key compounds. The HN-related gene was set in the PPIN as the seed node, and the restart probability was 0.75 . The lowkknn package (version 1.3.1) in R 3.6.3. To avoid data waste, the training uses 10 cross-validations to complete the model performance evaluation  were used as the input information for the KNN model and implemented in the aluation .To evaluate the performance of the KNN model, three other KNN control models were designed; they included input only OB (Comparison 2), input only Jac (Comparison 3), and input only gene regulatory information including GoSim and Man (Comparison 4).Receiver operating characteristic (ROC) curves of the four models were drawn, and the area under the curve (AUC) values were calculated to determine the effectiveness of the original model. After determining the best model, the point closest to the upper left corner of the ROC curve was used as the threshold point. Machine learning models such as support vector machines (SVMs), gradient boosting decision trees (GBDTs), and Bayesian networks (BNs), which are widely used in botanical drug bioinformation network analysis , were coThe KNN model can perform the screening of effective compounds. To effectively evaluate the effectiveness of botanical drugs, this study calculated the number of effective compounds contained in each botanical drug and used the hierarchical clustering algorithm (HCT) to classify and screen botanical drugs. In this study, Euclidean distance is used, and the clustering method of the org.Hs.e.g.db package (version 3.10.0) in R 3.6.3 was used to annotate the regulatory genes affected by GSF, the clusterProfiler package (version 3.14.3)   in R 3.6 3.14.3)  was usedMatching package (version 4.911) in R 3.6.3, was used to screen for matching patients, stabilize the tendency score between groups, and achieve a baseline balance among the patients.To evaluate the reliability of the KNN model and the HCT screening results, a retrospective cohort study was designed and used to evaluate the efficacy of GSF treatment based on real-world data. The retrospective cohort was established by analyzing the EMRs of 2,055 HN patients, screening both patients whose prescriptions included botanical drugs composed of GSF and patients who were not treated with botanical drugs, and evaluating changes in their clinical indices after six months of treatment. To ensure a balance among the selected patients, the tendency score was used to comprehensively quantify baseline factors such as age, sex, hospitalization time, and renal function, and the inverse probability processing weighted model was used. Inverse propensity of treatment weighting (IPTW) , implemeThe inclusion criteria were as follows: 1) a history of hypertension for more than five years; 2) persistent microalbuminuria or mild to moderate albuminuria during the course of the disease; 3) renal dysfunction caused by primary renal disease with hypertension and exposure to nephrotoxic substances, congenital or hereditary kidney disease, or other diseases; 4) a clear diagnosis in the medical record; and 5) the patient’s data in the medical record are complete.The exclusion criteria were as follows: 1) secondary hypertension; 2) severe cardiovascular or cerebrovascular disease, malignant tumors, severe hematopoietic system, respiratory system, digestive system, or infectious disease; 3) incomplete auxiliary examination of renal function during hospitalization; and 4) hospitalization time less than 1 week.The data of each eligible patient began with the electronic medical record recorded during hospitalization. The items collected include 1) the patient’s demographic characteristics ; 2) clinical features including length of hospital stay, serum creatinine, uric acid, urinary protein, blood lipids, and other parameters; and 3) details of the patient’s treatment, including the composition of any traditional Chinese medicine prescriptions used. The estimated glomerular filtration rate (eGFR) was estimated using the CKD-EPI formula . The CKDThe sample size was estimated based on reports in the clinical literature and on the previous research performed by the research group. The effective rate of modern medicine for symptomatic treatment is approximately 71% and that of combined traditional Chinese medicine is approximately 88%. The formula used to calculate the sample size is as follows:The traditional Chinese medicine prescriptions taken by patients in the GSF group included the constituent drugs of GSF. During the 6-month study period, all patients had records of traditional Chinese medicine prescriptions during outpatient visits or hospitalization in the Affiliated Hospital of Shandong University of Traditional Chinese Medicine and blood or urine test results related to their treatment with traditional Chinese medicine. During this period, all patients took antihypertensive drugs and patients with a GFR greater than 60 Thirty 14-week-old male spontaneously hypertensive (SHR) rats with body weights of 280–300 g were purchased from SPF (Beijing) Biotechnology Co., Ltd., China . Ten age-matched Wistar Kyoto (WKY) rats with body weights of 280–300 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., China . Chinese granule herbal extracts of GSF were purchased from Tiangjiang Pharmaceutical Co., Ltd, China. Irbesartan tablets [lot No. 0000009987] were purchased from Zhejiang Huahai Pharmaceutical Co., Ltd. China. The SHR and WKY rats were housed in standard plastic cages with a 12-h light/dark cycle at 23°C ± 1 °C and given free access to food and water. After a 10-week acclimation period, the 24-week-old SHR rats were randomly divided into three groups: 1) a GSF group in which each animal was treated with Chinese granule herbal extract at 4.5 g/kg body weight per day (i.g.); 2) a positive group in which each animal received irbesartan tablets 13.5 mg/kg body weight per day (i.g.); and 3) an SHR group in which each animal received 2 mL of saline per day (i.g.). The WKY group consisted of 10 rats, each of which received 2 mL of saline per day (i.g.). The intervention lasted for 8 weeks. The study was approved by the Animal Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine and the Institutional Animal Care and Use Committee (IACUC) of Shandong University of Traditional Chinese Medicine.To evaluate histological changes in the kidney, the animals were euthanized, and their kidneys were removed and quickly immersed in 4% paraformaldehyde for 24 h. The kidney tissue was dehydrated, embedded in paraffin, and sectioned at a thickness of 3 μm. Finally, hematoxylin-eosin staining was performed, and the pathological changes in the animals’ kidney tissue were observed under a light microscope at 400-fold magnification.The kidney tissue was ground in liquid nitrogen and cleaved with a strong RIPA buffer . The protein concentration was determined using a bicinchoninic acid (BCA) protein quantitation kit . Primary antibodies targeting NF-κB p65 , NF-κB p65 (phospho-S536) , and beta-actin  were incubated overnight with the target protein at 4 °C. The samples were then incubated with HRP-conjugated secondary antibodies . Protein expression was measured using an enhanced chemiluminescence reagent (ECL) kit .p < 0.05 were retained as highly specific botanical drugs, they were Dianthus superbus L.  (qumai) 3–10 g/d, Plantago asiatica L.  (cheqianzi) 10–20 g/d, Curcuma phaeocaulis Valeton  (ezhu) 10–20 g/d, Euryale ferox Salisb.  (qianshi) 10–20 g/d, and Achyranthes bidentata Blume  (niuxi) 15–30 g/d. The botanical drugs in the EMR data were boiled for 30 min and filtered. The combination of two parts with a total of 14 botanical drugs produced 961 compounds and 1,328 regulatory targets from public databases.Information on 2,055 patients with HN was collected; the information included 1,499 prescriptions, including 425 botanical drugs that were used 35,734 times. The hierarchical network extraction algorithm identified the core botanical drugs, they included Astragalus mongholicus Bunge  (huanqi) 15–25 g/d, Salvia miltiorrhiza Bunge  (danshen) 10–20 g/d, Codonopsis pilosula (Franch.) Nannf.  (dangshen) 10–30 g/d, Poria cocos  (fuling) 15–30 g/d, Atractylodes macrocephala Koidz.  (baizhu) 10–20 g/d, Angelica sinensis (Oliv.) Diels  (danggui) 15–30 g/d, Citrus × aurantium f. deliciosa Pericarpium  (chenpi) 10–20 g/d, Pinellia ternata (Thunb.) Makino  (banxia) 3–6 g/d, and Glycyrrhiza uralensis Fisch.  (gancao) 3–10 g/d. Five traditional Chinese medicines with the highest RR values and A total of 244 symptoms were extracted from EMRs. The fast unfolding algorithm was used to divide the symptoms into 14 network communities, as shown in As shown in The biometric information on the quantized compound was taken as the input information. The ROC curve of HN and its symptom community  shows thp < 0.05). This combination of botanical drugs was used as an effective prescription, Gao Shen Formula (GSF), for HN.According to the statistics on the optimal number of compounds in each module of 14 core traditional Chinese medicines, a distance matrix was established, and HCT was used to classify the combinations of botanical drugs . Salvia SVM = 0.845, AUCGBDT = 0.868, and AUCBN = 0.530). This shows that the training setting of the KNN model was reasonable and had a stable prediction performance.Based on the ROC curves obtained for each model , the KNNGSF contains 57 core targets. To show its biological characteristics, a 7 × 57 matrix was established for the heatmap . Among tGSF was enriched in 46 KEGG pathways and 24 GO terms. Combined with the enrichment results for each symptom community, 13 × 46 and 13 × 24 matrices were established based on the number of enriched genes. The enrichment pathways are presented as heatmaps . The KEGAmong the enriched KEGG pathways, Salvia miltiorrhiza Bunge, Codonopsis pilosula (Franch.) Nannf., Pinellia ternata (Thunb.) Makino, and Glycyrrhiza uralensis Fisch. were significantly enriched in inflammation-related pathways such as the NF-κB signaling pathway, the HIF-1 signaling pathway, the TNF signaling pathway, and the NOD-like receptor signaling pathway and can regulate vascular endothelial proliferation pathways such as the MAPK signaling pathway, the PI3K-Akt signaling pathway, and the VEGF signaling pathway. In addition, Salvia miltiorrhiza Bunge can regulate the cGMP-PKG signaling pathway, act on vascular smooth muscle, and regulate blood pressure. At the same time, Salvia miltiorrhiza Bunge can regulate the renin secretion pathway and the PI3K-Akt signaling pathway, regulate downstream inflammatory factors after renin activation, inhibit renal interstitial fibrosis, and antagonize the renin-angiotensin-aldosterone system. Glycyrrhiza uralensis Fisch. not only inhibits inflammation but also regulates the endocrine system, regulating protein, fatty acid, and glycogen metabolism by acting on the AMPK signaling pathway. Codonopsis pilosula (Franch.) Nannf. can also regulate fat and carbohydrate metabolism by affecting the PPAR signaling pathway. Angelica sinensis (Oliv.) Diels can regulate the cAMP pathway and affect the cytoskeleton. Euryale ferox Salisb. and Atractylodes macrocephala Koidz. can also act on cholinergic synapses and on the calcium signaling pathway, affect smooth muscle, and regulate blood pressure.Based on the obtained GO terms, GSF mainly regulates cytokines, chemokines, and growth factors. Salvia miltiorrhiza Bunge, Glycyrrhiza uralensis Fisch. and Pinellia ternata (Thunb.) Makino showed the most extensive regulation of cellular processes, including the regulation of DNA transcription factors and the production of a variety of amino acids.The treatment plans of a total of 286 patients with HN in the EMRs included GSF, and 279 individuals did not receive TCM treatment. Based on our calculation of sample size, 91 patients were needed in each group. To ensure that the baseline levels of the patients in the groups were consistent, the patients in each group were excluded according to IPTW,  and 91 pp > 0.05). After 6 months of treatment, the creatinine clearance rate in both groups increased (p < 0.05), the serum creatinine level in the GSF group was lower than that in the control group (p < 0.05), and more patients in the GSF group than in the control group experienced a decrease in the CKD stage . Based on this retrospective cohort validation, the addition of GSF to conventional Western medical treatment improves the renal function of patients and reduces their serum creatinine levels, and there are no evident adverse reactions.There was no significant difference in age, sex, length of stay, liver function, glucose metabolism, lipid metabolism, or renal function among the enrolled patients , Angelica sinensis (Oliv.) Diels  (9 g), Astragalus mongholicus Bunge  (9 g), Codonopsis pilosula (Franch.) Nannf.  (15 g), Pinellia ternata (Thunb.) Makino  (9 g), Curcuma phaeocaulis Valeton  (9 g), and Glycyrrhiza uralensis Fisch.  (6 g). The botanical drugs were boiled in water for 1 h, filtered, concentrated, dried, and pulverized to make granules. Granules of botanical drugs were purchased from Tiangjiang Pharmaceutical Co., Ltd, China.p < 0.05) and were lower than those in the model group (p < 0.05). There was no difference in blood pressure between the GSF group and the irbesartan group after treatment (p > 0.05). It is suggested that both GSF and irbesartan have hypotensive effects on SHR.After 8 weeks of drug treatment of SHR rats, systolic blood pressure and diastolic blood pressure decreased in the GSF group and in the irbesartan group (p < 0.05). The left ventricular ejection fraction of the rats in all four groups was in the normal range, and there was no significant difference among the groups (p > 0.05). It is suggested that GSF can antagonize ventricular hypertrophy caused by hypertension and that its effect is similar to that of irbesartan. As shown in p < 0.05), and there were no significant differences in the serum TNF-α levels of the high kidney formula group, the irbesartan group and the control group (p > 0.05). As shown in The interventricular septum at the end of diastole, the left ventricular posterior wall, and the left ventricular posterior wall at the end of systole were thinner in the GSF group and the irbesartan group than in the model group  increases, causing upregulation of the production of inflammatory cytokines such as TNF-α, IL-6, and IL-8 expression in kidney tissue. The mRNA and protein levels of type I and IV collagen are increased , and excAccording to the pathway enrichment results, the inflammatory pathway related to hypertensive renal damage that is regulated by GSF is mainly the NF-κB signaling pathway. GSF may regulate TNF-α, IL-1β, IL-6, and other inflammatory factors indirectly or directly, thereby inhibiting the NF-κB signaling pathway and further inhibiting the inflammatory response caused by inflammatory cytokines, the upregulation of the MAPK signaling pathway caused by MAPK8 and TNF, and the interstitial fibrosis caused by the increase in serine . GSF treIn the early stage of hypertensive renal damage, glomerular metabolism increases, especially GTP metabolism, lipid breakdown, and the production of various amino acids. Moreover, due to the increase in lipid oxidation, an inflammatory reaction can be induced , and thiThe drugs contained in GSF can regulate insulin resistance and the PPAR signaling pathway; improve the utilization of lipids and sugars by the body through specific effects on INS, PPARG, ADIPOQ, and other targets ; and improve the efficiency of utilization of sugars and lipids . GSF canTo verify the anti-inflammatory mechanism of GSF, this study used SHR rats to establish a disease model. After eight weeks of intervention with GSF, it was determined that GSF reduced renal NF-κB p65 and phosphorylated p65 levels, indicating that NF-κB dimer decreased, signal conduction was weakened, and NF-κB activation was inhibited, thereby reducing the production of inflammatory factors similar to serum TNF-α. Fewer inflammatory cells were present in the pathological sections obtained from the kidneys of the animals in the GSF group, and structural changes in the nephrons were not notable, indicating that GSF alleviates renal inflammation. The animals in the GSF group also had decreased blood pressure after intervention. The Color Doppler ultrasound showed that the left ventricular myocardium was thinner in the animals in the GSF group than in those in the model group, proving that it delays myocardial remodeling. No adverse reactions or death were observed during the experiment, which could preliminarily indicate its safety.This research innovatively combines network pharmacology with real-world data mining and uses the KNN model to process information on diseases and botanical drugs, closely linking systems biology with clinical data. To determine the efficacy of the model, it was compared with other classical machine learning models, and its efficacy was verified in a retrospective cohort. Finally, animal experiments were used to verify the core mechanism of action of GSF. The results show that compared with traditional models, the KNN model can better handle the heterogeneity and complexity of the relationship between genome data and TCM clinical information. GSF obtained using this method was shown to have a practical effect after verification, and the findings have a certain reference significance for the analysis of target organ damage after TCM treatment of hypertension.At the same time, this study also has limitations. First, although the symptoms of the patients are included in the evaluation system, this study still uses the patients as a whole dataset and fails to make full use of the complex clinical individualized differences reflected by the real-world data. Second, the sample size used for model training in this study was limited. Although we used a variety of models for comparative verification, further research is needed to improve the model’s performance. Third, the animal experiments conducted in this study only verified the most fundamental anti-inflammatory mechanism, and no experimental analysis of the changes in downstream pathways of the inflammatory response was performed; thus, further research on the pathways and mechanisms involved is needed. In terms of clinical effects, this study features a small sample size, multiple risks of bias, heterogeneity of GSF among patients, and limited measurement of results. In addition, randomized trials should be conducted to determine the efficacy and safety of GSF in the treatment of HN , 2.This study was based on EMR data for patients with HN collected at the Affiliated Hospital of Shandong University of Traditional Chinese Medicine from 2014 to 2017. It identified core botanical drugs for the treatment of HN. After establishment of the PPIN, machine learning model training was used to obtain an effective compound screening model based on information on the biological characteristics of compounds, and a hierarchical clustering screening of botanical drugs was conducted based on the model results, retaining the important botanical drugs as GSF for treating HN. From the perspective of targets and pathways, it was found that GSF plays a multilevel biological regulatory role in which it controls inflammatory reactions that lead to hypertensive kidney damage and reduces the damage caused by excessive activation of the RAAS system.


AR active components were obtained through TCMSP platform and literature search. The related targets of AR and PCOS were obtained through the disease and Swiss Target Prediction databases. An “active ingredient-target” network map was constructed using Cytoscape software, and gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis was conducted through Hiplot. Finally, Auto Dock Tools software was used to conduct molecular docking between active ingredients and core targets. The main bioactive ingredients of AR in the treatment of PCOS are acteoside, baicalin, caffeic acid, cistanoside F, geniposide, etc. These ingredients involve 10 core targets, such as SRC, HSP90AA1, STAT3, MAPK1, and JUN. The effect of AR on anti-PCOS mainly involves the AGE-RAGE signaling pathway, Relaxin signaling pathway, TNF signaling pathway, and ErbB signaling pathway. Molecular docking results showed that the main active components and key targets of AR could be stably combined. AR can improve hyperandrogen status, regulate glucose homeostasis, and correct lipid metabolism and other physiological processes through multi-component, multi-target, and multi-pathway. Thus, it could play a significant role in PCOS treatment. The results of our study provide a scientific foundation for basic research and clinical applications of AR for the treatment of PCOS.This study aimed to investigate the  PCOS is one of the major causes of infertility in women of reproductive age, accounting for 70% of ovulation-disordered infertility, and its prevalence in women of reproductive age is 6% to 10%. The etiology of polycystic ovary syndrome is complex, mainlycaused by genetic and environmental factors. In recent years, the incidence of PCOS has increased significantly, and it is often accompanied by complications such as diabetes, abnormal uterine bleeding, infertility, and high androgen expression, which has brought a heavy economic burden to patients and society. Therefore, PCOS treatment is a hot topic in current medical research. Currently, the treatment drugs for PCOS mainly include ovulation-stimulating drugs, insulin resistance-improving drugs, and androgen-lowering drugs. Still, these drugs are limited to symptomatic treatment, easy to relapse after drug withdrawal, require repeated treatment, and are prone to ovarian hyperstimulation syndrome, premature ovarian failure, lactic acid poisoning, increased cardiovascular burden, and other side effects. New treatments are urgently needed. Traditional Chinese medicines (TCM) have the advantages of having fewer side effects and acting on multiple targets and pathways. Several clinical studies have demonstrated that TCM has achieved significant clinical effectiveness in restoring the menstrual cycle, regulating sex hormone levels and glucose-lipid metabolism, as well as increasing ovulation rates and conception rates., so the study of TCM used in treating polycystic ovary syndrome has a broad application prospect.Polycystic ovary syndrome (PCOS) is the most common gynecological endocrine disorder among women of reproductive age, Compared with a single drug, herbal pairs can act synergistically to promote the treatment of various diseases. Modern research shows that Angelica sinensis has antitumour, liver protection, anti-inflammatory, hypoglycemic, immune regulation, cardiovascular and cerebral vascular system protection, etc, and Radix Rehmanniae has the effect of regulating blood lipids and blood glucose, antiaging, antitumour, antibacterial, and protecting gastric mucosa, etc. The pair “angelica sinensis - radix rehmanniae (AR)” was first recorded in “Taiping Huimin Hejiaobu Fang”, which is a commonly used pair in TCM. Angelica sinensis and Radix Rehmanniae are 2 Chinese medicines with the highest frequency of use and the most frequent combinations of medicines in treating PCOS. However, their mechanism of action in PCOS treatment is still poorly understood.Herbal pairs, the basic constituent units of herbal formulas, have special clinical significance in Chinese-medicine and have gradually become a hotspot of modern research.The progress of network pharmacology relies on a more profound grasp of how molecules and proteins interact with each other. There are great opportunities to gain a better understanding of diseases, how they develop TCM syndromes, and their treatment mechanisms. It has been applied to the field of TCM, including drug target discovery, efficacy evaluation, and mechanism research. A variety of applications have been made to TCM, including the discovery of drug targets, evaluation of efficacy, and investigation of mechanisms. In order to better understand the mechanism of action of TCM, network pharmacology is an effective method. In this paper, we explored the active ingredients of AR using network pharmacology methods and predicted their targets and signaling pathways related to PCOS, built a disease-drug-ingredient-target network, and analyzed the targets and signaling pathways that could be involved in the treatment of PCOS by AR.Angelica sinensis and Radix Rehmanniae were searched through the TCMSP database (https://tcmsp-e.com/tcmsp.php). Based on pharmacokinetic principles and platform recommendations, the chemical components were obtained as the active ingredients of the 2 Chinese medicines. In the search results, Angelica sinensis and Radix Rehmanniae had oral bioavailability of ≥ 30% and drug-like properties of ≥ 0.18. Also, based on a literature search, ingredients with pharmacodynamic effects were included as active ingredients. A database containing active ingredient information has been integrated with PubChem (https://pubchem.ncbi.nlm.nih.gov/). Download the Smiles information for each component and enter it into the Swiss Target Prediction database (http://www.Swisstargetprediction.ch/). The target proteins corresponding to each component were obtained by setting the attribute as “human” and filtering out targets with “Probability” > 0 as potential targets.In this study, the compound compositions of https://www.genecards.org/) and OMIM database (https://www.ncbi.nlm.nih.gov/omim/) were searched for disease targets related to PCOS using the keywords “polycystic ovary syndrome”. Obtaining PCOS-related targets was achieved by combining the results of the 2 databases and de-weighting them.The Genecard database (www.cytoscape.org/) was utilized. Compounds with the top 10 Degree values were then selected to screen them as the key compounds, considering them to have an essential role in the treatment of PCOS by AR.The drug target and disease target were taken to intersect to obtain the common target, which is the target of AR drug pair for PCOS treatment. In order to construct the drug-active ingredient therapeutic target network, Cytoscape v3.8.0 (https://string-db.org/), was used to construct protein interactions with therapeutic targets, with a threshold value > 0.900 for filtring nonspecific interactions. Cytoscape 3.8.0 software was employed for visualizing the protein interaction network. The 10 highest-degree targets are considered core targets for subsequent molecular docking.A String database (https://hiplot-academic.com/), was utilized for GO and KEGG pathway enrichment analysis. GO enrichment analysis provided results for Biological Processes, Molecular Functions, and Cellular components. We create a Bubble plot for the top 20 entries based on KEGG enrichment analysis with a P value < .05.The Hiplot platform (http://www.rcsb.org). We used a PubChem database to download SDF files of the 3D structures of the active ingredients. The target protein was dehydrogenated using Auto Dock software. The Box size and position were adjusted to make the ligand and receptor into the optimal conformation, and the results were output in “pdbqt” format. We visualized A molecular docking result with PyMol 2.4.0 software. Our flow chart of the study is shown in Figure By employing restricted species “homosapiens,” with an acceptable resolution of < 2.5, and small molecule compounds as reference criteria, we conducted screening of suitable target protein structures in the RCSB database (Angelica sinensis and 76 chemical components of Radix Rehmanniae were retrieved, and 14 active components of Angelica sinensis and 20 active components of Radix Rehmanniae were obtained after screening, among which 2 active components were common to both Angelica sinensis and Radix Rehmanniae (Table Angelica sinensis, and 215 for Radix Rehmanniae.A total of 125 chemical components of The Genecard and OMIM databases were searched for disease targets related to PCOS using the keyword “polycystic ovary syndrome”. After de-emphasis, 6675 disease targets were obtained; the intersection of component targets and disease targets was taken to get 222 potential targets of AR for PCOS treatment (Fig. P value < .05 as the screening condition, a total of 4525 entries were obtained for GO enrichment, and the top-ranked results were visualized in Figure With P value < .05, highlighting higher-ranking pathways such as 20 AGE-RAGE signaling pathways, Relaxin signaling pathways, TNF signaling pathways, ErbB signaling pathways, and others’’. The angelica-ripened dihuang pair may play a role in these pathways for treating PCOS, and the results are shown in Figure A total of 278 KEGG enrichment analysis results were screened with Molecular docking was performed to analyze how the core compounds interact with the core targets. The docking results will then be combined and displayed through heatmaps. PyMol was used to visualize the top-ranked results. A docking score lower than −4.25 kcal/mol indicates some binding activity, while a score lower than −7.0 kcal/mol indicates strong binding activity. The present results showed that the average docking score was −9.14 kJ/mol, among which the results with docking score ≤ −7.0 kJ/mol accounted for 91.17%, which reflected that the compounds in the AR pairs had good binding ability to the core target according to the scoring results. Heatmaps demonstrated all the scoring results, as in Figure  Clinical treatments for PCOS are mainly based on adjusting the menstrual cycle, lowering the level of androgens, and promoting ovulation or surgery, which has more adverse effects and is prone to recurrence.–28 It is found that most PCOS patients show increased androgen content, high blood sugar, and lipid metabolism disorders. TCM plays a role in treating PCOS through the advantages of multi-targets and multi-pathways. Clinical studies have shown that the pair of AR and the formula containing AR can significantly improve hyperandrogenism, regulate glucose homeostasis, and correct lipid metabolism abnormality.,31 It is therefore necessary to clarify the molecular basis and biological basis of AR against PCOS. This study could provide new directions for further research on the molecular mechanism of PCOS using network pharmacology, a new method for elucidating complex pharmacological problems.PCOS is a polygenic, multifactorial, systemic, inflammatory autoimmune disease. Its pathogenesis is not fully clear. HSP90AA1, a member of the heat shock family, encodes inducible molecular chaperones that aid in the folding of specific target proteins. HSP90 is mainly found in ovarian granulosa cells, with significantly higher levels in dominant follicles. It plays a role in promoting follicle maturation regulation. STAT3 is an intracellular signaling transcription factor that plays a crucial role in the immune response, cell cycle regulation, and cell survival. Up-regulation of STAT3 expression in porcine ovarian granulosa cells inhibits apoptosis and promotes cell proliferation. It is also involved in inflammation, with increased phosphorylation levels during inflammation. These findings suggest that up-regulating STAT3 expression or inhibiting its phosphorylation could improve ovarian function. MAPK is a crucial signaling pathway linked to androgen production and insulin resistance in PCOS. Its role in the development and maturation of oocytes, as well as the initiation of ovulation, is of utmost importance. Inhibiting the MAPK pathway pharmacologically leads to a decrease in the expression of mitotic cyclin D2 and a reduction in granulocyte proliferation; lower levels of granulocytes indirectly interfere with follicle development and maturation.,36 JUN kinase is a significant branch of the mitogen-activated protein kinase signaling pathway. In vivo, high expression of p-c-Jun promotes trophoblast migration and invasion and enhances ovarian function. HRAS belongs to the RAS gene family and acts as an upstream regulator of the RAS/RAF/MEK/ERK/MAPK pathway. In patients with polycystic ovary syndrome, the expression of serum HRAS correlates with metabolic profiles such as body mass index, fasting glucose levels, and fasting insulin levels. EGFR, a tyrosine kinase-type receptor, plays a role in crucial reproductive processes like embryo implantation and metaphase. It influences the proliferation and apoptosis of ovarian granulosa cells, thereby inducing oocyte division and maturation. The EGFR gene is predominantly expressed in granulosa cells, and patients with polycystic ovary syndrome exhibit higher levels of EGFR expression in their ovarian tissues, which can impact follicular development.–40According to the PPI study based on the STRING database and network topology parameters, we further screened 10 core targets: SRC, HSP90AA1, STAT3, MAPK1, JUN, HRAS, EP300, CREBBP, PIK3CA, and EGFR. C-src is a proto-oncogene that regulates cell proliferation, differentiation, and apoptosis. It promotes the activation of primordial follicles through the PI3K, PKC, and MAPK signaling pathways.Based on the core targets, we obtained 17 key elements that are associated with them. These components can serve as the fundamental active ingredients in the treatment of PCOS using the AR pair. Angelica sinensis contains coniferyl ferulate, ferulic acid, levistolide A, ligustilide, sedanolide, Senkyunolide A, and Senkyunolide B as its active ingredients. Radix Rehmanniae, on the other hand, contains Acteoside, Baicalin, Caffeic Acid, Cistanoside F, Geniposide, Isoacteoside, Kaempferol-3-O-Glucuronide, Rehmannioside A, Rehmapicroside, and Reineckiagenin. Through molecular docking analysis, it was found that Reineckiagenin had the highest docking score with each core target. The study also revealed that Reineckiagenin exhibits estrogen-like activity by binding to estrogen receptors ERα and GPR30. It regulates the hypothalamic-pituitary-ovarian axis, inhibits the secretion of luteinising hormone and follicle-stimulating hormone, and suppresses the production of inflammatory mediators such as iNOS, COX-2, and IL-6. Consequently, it generates anti-inflammatory effects by reducing the concentration of C-reactive protein in the blood plasma.Go enrichment analysis showed that the treatment of PCOS using AR primarily involves the regulation of nutrients, vascular lesions, and oxidative substance metabolism. Various organelles, hormone levels, and steroid metabolism influence this process. It encompasses multiple pathways associated with inflammation and metabolic processes. This indicates that the therapeutic effects of traditional Chinese medicines on this disease are achieved through multiple components, targets, pathways, and biological mechanisms.,43Based on the KEGG enrichment analysis, the AGE-RAGE signaling pathway was identified as the most significant therapeutic pathway. This pathway plays a crucial role in mediating insulin resistance and triggering inflammatory responses in polycystic ovaries. Factors like high glucose and obesity lead to non-enzymatic glycosylation of proteins, lipids, and DNA in the body. This, in turn, increases the production of advanced glycation end products, which bind to the RAGE receptor on cell membranes. The binding of advanced glycation end products to RAGE activates the AGE-RAGE signaling pathway, resulting in insulin resistance, inflammation, oxidative stress, and the development of PCOS.This study, even though it potentially identifies the targets and signaling pathways of AR in the treatment of PCOS, has certain limitations due to the fact that TCM has great potential in treating PCOS. Network pharmacology uses these newly discovered chemical components of AR as a research topic. TCM is not just a straight collection of chemical-compounds, but both its structure and chemical constituents are transparent. A variety of ingredients in AR can produce different effects depending on their concentrations, contents, and interactions. Moreover, AR pharmacological effects in the treatment of PCOS cannot be fully revealed by the databases used to compile data on targets, components, etc. A scientific hypothesis cannot be divorced from experimental verification - and network pharmacology research is no exception. To verify the accuracy and reliability of the prediction results of the network pharmacology of AR in the treatment of PCOS, it is necessary to combine computational prediction with experimental confirmation and to conduct biological experiments on the prediction results of the network pharmacology of AR. This provides strong evidence to support the development of new Chinese medical treatments for PCOS and other diseases.Angelica sinensis and Radix Rehmanniae primarily exert their therapeutic effects by targeting multiple molecules such as SRC, HSP90AA1, STAT3, MAPK1, JUN, and others. Through their 17 active ingredients, these herbs regulate lipid metabolism, insulin resistance, and androgen levels to treat PCOS. The main mechanism of action involves the modulation of the AGE-RAGE signaling pathway, which is crucial in PCOS treatment. This study provides insights into the potential pharmacological basis and mechanism of action of AR for PCOS treatment. The network pharmacology approach used here offers a comprehensive analysis of the multi-component, multi-target, functional, and signaling pathway aspects of these herbs in the context of PCOS treatment. This lays the groundwork for future in-depth research and clinical evaluations.In conclusion, the integration of network pharmacology and molecular docking technology revealed that Conceptualization: Keyuan Xiao.Data curation: Ihsan Ullah.Funding acquisition: Keyuan Xiao, Yuqiang Liu.Writing – original draft: Xinghua Li.Writing – review & editing: Chunxia Hou, Yuqiang Liu.


P < 0.05). Compared with the model group, the low-dose group of Qingyi II granules only significantly reduced IL-2 levels and had no effect on other indicators. The other groups could significantly reduce the levels of AMS, BAX, and CASP3 (P < 0.05). Compared with the model group, the octreotide group and Qingyi II granules high-dose group significantly increased the Bcl-2 level (P < 0.05), and there was no statistical difference in other drug-administered groups. Compared with the normal group, the expression of AKT1 and PIK3CA in the pancreas of the model group was significantly higher. Compared to the model group, the expression of PIK3CA was low in all drug-administered groups. In addition to the low-dose group, the other drug-administered groups significantly reduced the expression of AKT1. Qingyi can reduce the levels of AMS, BAX, IL-2, and CASP3 and increase the levels of Bcl-2. This mechanism may be related to the PI3K- AKT signaling pathway.Network pharmacology, reverse molecular docking, and rat acute pancreatitis (AP) models were used to analyze the mechanism of protection by Qingyi II granules. The chemical components of 7 Chinese herbal medicines in Qingyi II granules were searched through the TCMSP  database. The active ingredients were screened out in the OB  and DL (drug likeness) filters as a condition for inclusion. Then, the prediction analysis of potential targets was performed through databases. A GO (gene ontology) enrichment analysis of target proteins related to AP and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotation was performed using the DAVID  database. Finally, the “Herbal-Compound-Target” network was constructed using Cytoscape software. The active component structure and target name were uploaded to the Systems Dock database for reverse molecular docking. With octreotide as a positive control, Qingyi II decoction and Qingyi II granules were administered to AP rats at low, medium, and high doses. The pathological changes in the pancreas were observed using HE staining. The levels of Bcl-2, AMS, BAX, IL-2, and CASP3 in plasma were determined by an ELISA kit. Real-time PCR detected the expression of AKT1 and PIK3CA mRNA in the pancreas. The database predicted 94 active components of Qingyi II granules, 76 potential targets, and 64 signaling pathways. Twenty pathways were directly or indirectly associated with acute pancreatitis, including the TNF signaling pathway and the PI3K-AKT signaling pathway. In the reverse molecular docking experiment, the matching scores of the active components and the target were mainly between 6.0 and 7.0, with strong binding activity. Compared to the normal group, the plasma concentrations of BAX, IL-2, Bcl-2, AMS, and CASP3 in the model group were significantly increased ( Acute pancreatitis (AP) is a common clinical condition with rapid onset, rapid progression, and a high mortality rate that can quickly progress to severe acute pancreatitis (SAP). SAP is often accompanied by local or systemic inflammatory response syndrome and systemic multiple organ dysfunction , 2. The Rhei Radix Et Rhizoma (Da Huang), Paeoniae Radix Rubra (Chi Shao), Gardeniae Fructus (Zhi Zi), Aucklandiae Radix (Mu Xiang), Magnoliae Officinalis Cortex (Hou Po), Coptidis Rhizoma (Huang Lian), Corydalis Rhizoma (yanhusuo), Moutan Cortex (Mu Dan Pi), and Mirabilite (Mang xiao) [Qingyi II granules are derived from the clinical experience of the Affiliated Hospital of Zunyi Medical University. It is composed of ng xiao) –7. Da Hung xiao) . Many exng xiao) –13. Netwng xiao) , 15. Theng xiao) . Reverseng xiao) –19. The ng xiao) , 21. TheMale Sprague–Dawley rats, weighing 210–250 g, were purchased from Changsha TianQin Biotechnology Co., Ltd., license number: SCXK (Xiang) 2014–0011. This protocol was approved by the Animal Care and Use Committee of the Zunyi Medical University (2019-2-252), and all experimental procedures were in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.Qingyi II granules and Qingyi II broth are homemade buds (lot number: 180228-C-0301). Hefei Semenno Biotechnology Co., Ltd, lipopolysaccharide  Sigma, USA, octreotide acetate injection liquid (batch number: 180608) Taishan Pharmaceutical Co., Ltd, Animal Total RNA isolation Kit, RT EasyTM II, and Real-Time PCR EasyTM were purchased from FOREGENE. ELISA kits for Bcl-2, AMS, BAX, IL-2, and CASP3 were purchased from Shanghai Shenggong Bioengineering Co., Ltd., and anhydrous ethanol, sodium chloride, potassium dichromate, and concentrated concentration sulfuric acid were purchased from the Chengdu Branch. Long Chemical Reagent Factory, formaldehyde was purchased from Shanghai Titan Technology Co., Ltd, and xylene was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd. Hematoxylin dyeing solution (Cat. No. BA-4097) and Yihong dyeing liquid (Cat. No. BA-4099) were purchased from Zhuhai Besso Biotechnology Co., Ltd.Multiskan spectrum full-wavelength microplate reader , tissue microtome , BMJ-III type embedding machine, TSJ-II type fully automatic closed tissue dehydrator (Changzhou Zhongwei electronic instrument) Co., Ltd), BA400Digital microscope , PIKORed 96 real-time fluorescence meter , and TCA0096 thermal cycler  were used as primary instruments.https://sm.nwsuaf.edu.cn/lsp/tcmsp.php/), China Traditional Chinese Medicine Integration Database TCMID (https://www.megabionet.org/tcmid/), Taiwan Traditional Chinese Medicine Database TCM Database@Taiwan (https://tcm.cmu.edu.tw/), and other Chinese herbal medicines in the chemical composition database, we are collecting Qingyi II granules in the Rhubarb, Radix Paeoniae Rubra, Gardenia, Common Vladimiria Root, Magnolia Officinalis Rehd. Et Wils, Rhizoma Corydalis, and Cortex Moutan. All the chemical components were combined with the existing literature data for data mining and finishing, based on the chemical components contained in the TCMSP database. The chemical name and number shall be unified, and the duplicate numerator shall be deleted. By limiting the bioavailability of chemical components of traditional Chinese medicine (OB ≥ 30%) and the drug similarity (DL ≥ 0.18), [Through the databases TCMSP (≥ 0.18), , 23 combThe active components were imported into the TCMSP, STITCH, Target-Prediction, and Swiss Target-Prediction databases to obtain their effect targets. After the deduplication was summarized, the target name was input into Uniprot (the limited species was human and existed in rats simultaneously) and converted to a gene name. In the OMIM database, GAD database, TTD database, and PharmGKB database, input keywords “acute pancreatitis” and “Severe acute pancreatitis” to retrieve genes related to acute pancreatitis, remove duplicate and false-positive genes, and match the targets of active components. The potential target of Qingyi II granule active ingredient in the treatment of AP was obtained.Qingyi II granules compound medicinal materials, active components, and target data sets were constructed and introduced into the Cytoscape software. The medicinal materials, components, and targets were constructed into a ternary comprehensive network model that can reflect the medicinal materials, components, and targets using the combined function (Merge) in the software. The potential targets related to AP were introduced into Cytoscape, a construction component-potential target network.P < 0.05) related to AP was screened and plotted as a bar graph, and OMIC shares drew a high-level bubble map to screen the key pathways to construct a target-pathway network.The potential target was imported into the biomolecular function annotation system (DAVID), the “official gene symbol” was selected, the research object was defined as human, and the KEGG pathway annotation and GO enrichment annotation analysis were performed on the AP target. According to the principle of AP correlation reported in the literature, a significant enrichment pathway (https://systemsdock.unit.oist.jp/) database was used to perform molecular docking of key active components and key targets for the treatment of AP to further validate the target's reliability. The dock score of the active ingredient and the target protein was obtained by inputting the PDB ID of the target protein and introducing the structure of the active ingredient. The degree of match between the active ingredient and the target protein was judged based on the dock Score.The System Dock  15 mg/kg. After successful modeling, octreotide group (100 μg/kg), Qingyi II decoction group (13 g/kg), and Qingyi II granules in low-dose groups (6.5 g/kg), medium-dose groups (13 g/kg), and high (26 g/kg) dose groups, the three groups were dosed once a day for five consecutive days. Octreotide was injected subcutaneously, and the other groups were intragastrically administered. The control and model groups were given the same amount of normal saline.A total of 56 male Sprague–Dawley rats, weighing 200–250 g, were fed for one week. They were randomly divided into seven groups, eight in each group, including the control group, model group, octreotide group, Qingyi II decoction group, and Qingyi II granules in the low, medium, and high-dose groups. They were fasting for 12 h, drinking only water. AP rats were induced with intraperitoneal injections of caerulein (CAE) 60 Blood was collected from the abdominal aorta after the last administration, and the plasma was taken by centrifugation  and stored in a refrigerator at −20°C. After the rats died, the pancreas was quickly removed, placed in a Petri dish containing physiological saline, rinsed, and blotted dry using filter paper. The same portion of the pancreas was cut into a 10% formaldehyde solution for fixation. The gradient alcohol was dehydrated, made transparent with xylene, serially sliced with paraffin, and subjected to HE staining to observe pancreatic tissue lesions. The ELISA kit detected the levels of Bcl-2, AMS, BAX, IL-2, and CASP3 in plasma. Real-time PCR detected the expression of AKT1 and PIK3CA mRNA in the pancreas.P < 0.05 indicated that the difference was statistically significant.Statistical analysis was performed using SPSS 17.0 software. The differences were expressed as the mean ± standard deviation. A one-way analysis of variance was used between groups. The LSD test was used to compare groups with variance, and Dunnett's T3 test was used for groups with irregular variance. Dunnett's T3 test was performed for those with irregular variance. Rhubarb, 75 kinds of Radix Paeoniae Rubra, 77 kinds of Rhizoma Corydalis, 55 kinds of Cortex Moutan, 107 kinds of Common Vladimiria Root, 139 kinds of Magnolia Officinalis Rehd. Et Wils, and Gardenia 19 kind. After collecting and removing repetition and screening by OB ≥ 30% and DL ≥ 0.18, 104 active components were obtained. According to screening rules, 12 components such as Emodin, Physcion, Geniposide, Paeonol, Magnolol, and Honokiol were initially removed, and the Chinese pharmacopeia quantitatively identified these components. The literature has reported that these ingredients have a good effect on acute pancreatitis. Therefore, these components were manually searched for further analysis, and 116 chemical components were finally obtained. A total of 116 components were entered into databases such as TCMSP and STITCH, and the target points of Rhubarb, Radix Paeoniae Rubra, Gardenia, Common Vladimiria Root, Magnolia Officinalis Rehd. Et Wils, Rhizoma Corydalis, and Cortex Moutan were given 107, 80, 148, 82, 136, 118, and 223, respectively. A total of 336 targets were removed from the components, including repetitive targets that did not have corresponding targets. The selected Qingyi II granules, active ingredients, and target were sequentially introduced into Cytoscape software to construct a Qingyi II granules-activeingredient-target network , as shown in P value. The greater the rich factor, the greater the degree of enrichment.The potential targets were imported into the DAVID database for KEGG analysis, and 20 signaling pathways with a high correlation with AP were selected (P < 0.05). The Cytoscape software was used to construct a target-path network, as shown in κB signaling pathway. This indicates that the Qingyi II granules may achieve their purpose of treating AP by regulating these signaling path-related genes.Six key pathways for the treatment of AP-potential targets were screened out with AP-related pathways . Compared to the model group, the low-dose group of Qingyi II granules only significantly reduced IL-2 levels and did not affect other indicators. Compared with the model group, except for the low-dose group of Qingyi II granules, all the other administration groups could significantly decrease the levels of AMS, CASP3, and BAX (P  <  0.05). Compared with the model group, the octreotide group and the high-dose group of Qingyi II granules significantly increased the level of Bcl-2 (P  <  0.05), but there was no significant difference in other administration groups.As shown in P  <  0.05). There was no statistically significant difference between the low-dose group and the model group, but the expression of AKT1 mRNA in the other administration groups was significantly lower than that in the model group (P  <  0.05). Compared to the normal group (P  <  0.05). Compared to the model group, PIK3CA mRNA was not statistically significant in all the administration groups.Compared with the normal group , the expal group , the expNetwork pharmacology utilizes the analysis of pathway targets related to drugs and diseases to predict the therapeutic effect of drugs, which has the potential to improve clinical trials of new drugs. The success rate and the advantage of saving drug research and development costs . By refeIn clinical practice, Qingyi II decoction is used as medicine. Although decoction is suitable for the principle and flexibility of TCM syndrome differentiation and treatment, plus or minus symptoms, it is bulky, easy to deteriorate, and inconvenient to take, carry, and store. The previous research of our research group shows that after Qingyi II decoction is changed into Qingyi II granules, it can not only ensure the curative effect but also be convenient for patients to take, carry, and store and is superior to the decoction in terms of quality control and preparation stability. The etiology and pathogenesis of acute pancreatitis are complex multifactor pathological processes. Qingyi II granules are effective in the treatment of spleen-stomachdampness-heat acute pancreatitis. However, its mechanism is not clear. The integrity and comprehensiveness of the network pharmacology research strategy are consistent with the principle of multicomponent, multichannel, and multitarget synergy of traditional Chinese medicine and its compounds . It is bκB and the p53 signaling pathways in the pathogenesis of AP.This study discovered that Aloe-Emodin, Rhein, Emodin, and Physcion in Qingyi II granule compounds could directly act on PIK3CA, MAPK14, or IL-2, IL-1B, IL-6, and TNF via component and target prediction. The targets of PAEONOL are BCL-2, IL-2, and TNF. Geniposide can act on BCL-2, Costunolide, BAX, and CASP3. These targets are important for predicting six signaling pathways. These six signaling pathways have been reported in the literature. A PI3K-AKT signaling pathway is downstream of the toll-like receptor pathway and the TNF signaling pathway, but upstream of the NF-κB and mTOR to participate in the growth and survival of the body [The PI3K/AKT pathway is an important information pathway in the pathogenesis of AP. Current studies have confirmed that PI3K/AKT can treat acute pancreatitis through multitarget, multipathway, and multifunction. There are three types of PI3K: I, II, and III. Among them, the most widely studied class I PI3Ks are composed of the regulatory subunit P85 and the catalytic subunit P110. PI3K is the intermediate bridge of a variety of cellular responses and plays a key role in cell apoptosis, pyroptosis, autophagy, oxidative stress, and other pathways under the influence of a variety of upstream or downstream factors. AKT, also known as protein kinase B (PKB), is a serine/threonine protein kinase composed of AKT1, AKT2, and AKT3 subtypes. Activated PI3K phosphorylates AKT at phosphorylation sites Ser473 and Thr308, and AKT activates or inhibits its downstream targets such as NF-the body . The PI3the body , 35. It the body . Therefothe body . The relThe modeling method used in this experiment is simple, and its success rate is high. There are many methods to establish the acute pancreatitis model. CAE can only induce mild acute pancreatitis. In this experiment, CAE combined with LPS was selected to establish the SAP model. After modeling, it was observed that the surface of the pancreas was dark red, with hyperemia and edema. Under the microscope, the pancreatic tissue had cell degeneration and necrosis, edema, and interstitial inflammatory cell infiltration. The serum amylase was significantly increased by abdominal aortic blood collection. The model was successfully established.κB and downstream genes. Several studies have confirmed its vital role in the occurrence and development of acute pancreatitis. It is speculated that the mechanism of Qingyi II granules in the treatment of acute pancreatitis may be through inhibiting AKT1 phosphorylation, decreasing Bax, increasing Bcl-2 expression inhibiting the activation, and release of CASP3, and inhibiting apoptosis.The results of the pharmacokinetics test showed that Qingyi II granules could up-regulate the expression of AKT1 mRNA, decrease the concentration of AMS, IL-2, BAX, and CASP3, and increase the concentration of Bcl-2. PI3K-AKT is an important mediator for regulating the expression of NF-In conclusion, it is correct to study the mechanism of the Qingyi II granule in the prevention and treatment of acute pancreatitis using the combination of network pharmacology and molecular docking technology. This reveals the characteristics of multicomponent, multitarget, and multichannel treatment of Qingyi II granules. It also embodies the principles of integrity and systematicity in the treatment of diseases with traditional Chinese medicine. It is of great significance to explore the targeted regulation of the PI3K/AKT signaling pathway in the prevention and treatment of AP from the perspective of related targets and cell functions of the PI3K/AKT signaling pathway and traditional Chinese medicine treatment of AP, which is expected to provide new ideas for basic research and clinical treatment of AP. The synergistic effects of the multiple compounds in Qingyi II granules and the relationship between the effects of these compounds and their mechanisms in AP will be investigated in the future.


Cordyceps sinensis powder, has been applied to treat PCOS. However, the specific active ingredients of BL and its mechanisms of action are yet to be elucidated.Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder that is common in women of reproductive age. The clinical features of PCOS include hyperandrogenemia and polycystic ovarian changes. Bailing capsule (BL), a proprietary Chinese medicine that contains fermented Initially, the effectiveness of BL on PCOS model mice was evaluated. Subsequently, the active ingredients of BL were searched in the TCMSP and TCM Systems Pharmacology databases, and their targets were predicted using Swiss Target Prediction and SEA databases. Furthermore, the GEO gene database was used to screen for differentially expressed genes (DEGs) related to PCOS. Data from Gene Card, OMIM, DDT, and Drugbank databases were then combined to establish a PCOS disease gene library. Cross targets were imported into the STRING database to construct a protein–protein interaction network. In addition, GO and KEGG pathway enrichment analyses were performed using Metascape and DAVID databases and visualized using Cytoscape software and R 4.2.3. The core targets were docked with SYBYL-X software, and their expressions in PCOS mice were further verified using qPCR.JAK2, PPARG, PI3K, and AKT1 were upregulated, whereas those of ESR1 and IRS1 were downregulated in PCOS model mice. After the administration of BL, the expressions of associated genes were regulated. This study demonstrated that BL exerted anti-PCOS effects via PIK3CA, ESR1, AKT, PPARG, and IRS1 targets affecting PI3K-Akt signaling pathways.The core active ingredients of BL were identified to be linoleyl acetate, cholesteryl palmitate, arachidonic acid, among others. Microarray data sets from four groups containing disease and normal samples were obtained from the GEO database. A total of 491 DEGs and 106 drug–disease cross genes were selected. Estrous cycle and ovarian lesions were found to be improved in PCOS model mice following BL treatment. While the levels of testosterone, progesterone, and prolactin decreased, that of estradiol increased. qPCR findings indicated that the expressions of This research clarified the multicomponent, multitarget, and multichannel action of BL and provided a theoretical reference for further investigations on its pharmacological basis and molecular mechanisms against PCOS. Polycystic ovary syndrome (PCOS) is a heterogeneous endocrinological and metabolic disorder that occurs commonly in women of reproductive age. The main clinical symptoms include abnormal ovulation, ovarian changes, and androgen increase , 2. The The pathogenesis and causes of the disease have not been completely examined and may be linked to genetic and environmental factors. The drugs used to treat PCOS are mostly symptomatic interventions, such as oral contraceptives, antiandrogens, and insulin sensitizers . This trC. sinensis (Ophiocordyceps sinensis (Berk.) G.H. Sungetal). BL contains various active ingredients, including cordycepin and its derivatives, alkaloids, sterols, trace elements, fatty acids, mannitol, and amino acids. These chemical components exhibit diversified physiological effects, such as antioxidant, antifibrotic, antitumor, antiviral, and anti-inflammatory effects [C. sinensis has been used widely as a herbal medicine and functional food for centuries. Several studies have established that C. sinensis upregulates steroidogenic enzymes and ovarian 17β-estradiol in human granulosa lutein cells and affects the quality of mature oocytes [C. sinensis) can reduce the abnormalities in lipid metabolism by regulating the PPARα pathway, enhancing lipolysis, and lowering the accumulation of renal triglycerides in diabetic rats [In recent years, the efficacy of several classical Chinese medicine prescriptions in treating PCOS has been increasingly recognized –14. Acco effects , 17. C.  oocytes . Clinicatic rats . Diane-3tic rats . Moreovetic rats , 22. In tic rats . The comtic rats , 25; howOcimum sanctum–ginseng medication to prevent and treat diarrheal irritable bowel syndrome using gene expression integrated microarray data combined with network pharmacology. Wu et al. [Modern bioinformatics aids in exploring the mechanism of action of TCM. To identify the differential genes in clinical data samples, the GEO database was mined and analyzed using the R language. Subsequently, network pharmacology and molecular docking were used in a combined manner to link the active components of medications and target proteins and disease pathways to create a “drug–target–disease” relationship network. Sun et al.  investigu et al.  employedIn this study, PCOS mice models were constructed by feeding a high-fat and high-sugar diet combined with the subcutaneous injection of dehydrogenated epiandrosterone (DHEA) into the back of the neck . The estBL were purchased from Zhongmei Huadong Pharmaceutical Co., Ltd. ; High-sugar and high-fat model feed  was purchased from Nantong Trophy Feed Technology Co., Ltd. ; dehydrogenated epiandrosterone (DHEA) was purchased from Jiasheng Chemical Reagent Business Center ; Hematoxylin and Eosin (H&E) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. , mouse serum testosterone T ELISA kit, and mouse serum estradiol E2 ELISA kit from Jianglai Biotechnology Co., Ltd. ; SPARKeasy Improved Tissue/Cell RNA Kit, SPARKscript II RT plus Kit, and 2×SYBR Green qPCR Mix purchased from SparkJade .Twelve SPF grade female ICR mice weighing 20 ± 5 g were obtained from Shanghai Slake Experimental Animal Co., Ltd. . The animals were maintained in controlled feeding conditions with a temperature range of 22–26 ℃, humidity between 45–50%, and a 12-h cycle of light and darkness. Water and feed were changed daily, and hygiene was maintained by regular cleaning. All animal procedures followed the “Regulations on the Management of Experimental Animals” and were approved by the Zhejiang University of Technology’s ethics committee. The experimental flow is shown in Fig. After 3 days of adapted feeding, ICR mice were divided into weight-based groups at random and given diets high in fat and sugar along with subcutaneous injections of exogenous androgens into the back of the neck for a total of 28 consecutive days, with the exception of the normal control group. Subsequently, vaginal smears were collected for four consecutive days, stained with H&E, and observed under the microscope. The mice in the model group showed obvious inhibition and disturbance of the motility cycle, suggesting that the modeling of PCOS mice was successful. At the end of modeling, the mice were divided into three groups according to their body weight: (1) normal group (NC); (2) model group (PCOS); (3) BL group. The dose was converted to the dose according to the body surface area coefficient and given by gavage. Normal and model control mice were given purified water by gavage at a dosing volume of 1 mL/100 g. BL was configured to a concentration of 240 mg/mL and administered by gavage once daily for 4 weeks. At the end of the experiment, mice were fasted overnight and euthanized by anesthesia with 0.3% pentobarbital sodium solution . Serum was obtained via orbital blood collection, ovarian tissue was collected, washed with cold saline, and stored at -80 ℃.Vaginal smears were obtained from mice beginning on day 28 of modeling and stained with H&E for 4 days in a row. The morphology and coloration of cells were observed under a biological microscope using the following criteria: Proestrus , Estrus , Metestrus , and Diestrus . The presence of an inhibited or disturbed estrous cycle indicates successful modeling. After 4 weeks of administration, vaginal smears were observed for 8 consecutive days to analyze changes in the mice’s estrous cycle.The ovaries were embedded in paraffin after being treated in 4% formalin. All specimens were then cut into sections with a 4μm thickness and stained with hematoxylin and eosin (H&E). Finally, tissue images were captured using a biomicroscope.An orbital blood sampling technique was used to gather blood samples. The samples were then incubated for 30 min at 37 °C, followed by 10 min of centrifugation at 3000 rpm at 4 °C. After removing the supernatant, the procedure above was carried out one more. For upcoming investigations, the serum was collected in 1.5 mL Eppendorf tubes and kept at -80 °C.The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was utilized to collect the chemical constituents of BL. This was supplemented by the results of CNKI and Pubmed literature searches. TCMSP is a systematic pharmacology platform for TCM that provides information on TCM components, compounds, and pharmacokinetic effects of natural compounds . It incl≧1 and an adjusted P-value < 0.05 for differential gene identification [The keyword “polycystic ovary syndrome” was used to search for samples in NCBI’s GEO database. Studies with a sample size greater than 10 were then screened, and four datasets  containing disease samples and normal samples were selected as the study population. Subsequently, the samples were subjected to principal component analysis and clustering hierarchical analysis to evaluate the acceptable quality of each set of samples. Systematic analysis was performed by R 4.2.3, with joint analysis and batch correction using the SVA and Limma packages, with a threshold of |logFC|fication . Volcanofication , 39. Thefication , DrugBanfication , OMIM dafication  and TTD fication . FinallyThe objectives and active components gathered in the preceding phase were adjusted. The software Cytoscape 3.8.2 was used to map the drug–component–target modulation network after the network files and annotation files had been loaded.Targets connected to PCOS were used to map the prospective targets of BL. Venn diagrams were generated using TBtool software to identify cross-targets as regulatory targets of BL against PCOS . The STRP < 0.05 as the critical values for GO Molecular Functions, Biological Processes, and Cellular Components [P < 0.01, while the default values for other parameters were used. The visualization was plotted by https://www.bioinformatics.com.cn (last accessed on 10 Nov 2023), an online platform for data analysis and visualization [The core genes were analyzed for Gene Ontology (GO) enrichment using the Metascape database with the species set as H. sapiens, applying FDR < 0.05 and mponents . The Kyomponents –52 was pmponents . The crilization .Based on the results of network pharmacology and literature research, potential pathways of action were identified. Molecular docking was then performed for validation. The 2D structure of the matching active component was retrieved from the NCBI database, and the 3D structure of the protein target was obtained from the PDB database. SYBYL-X software was used for molecular docking with the Surflex-Dock Geom docking mode. The default number of dockings was set to 20. The total score was shown on a heat map, and the docking score was used to assess the binding capacity and activity of the target and active substance. The optimal result for ligand-receptor binding was determined by the binding mode with a Total score > 7 .Total RNA from mouse ovary tissues was extracted with SPARKeasy Improved Tissue/Cell RNA Kit  and reverse-transcribed using 2 × SPARKscript II RT Plus Master Mix  according to the manufacturer’s instructions. The primers used were synthesized by SparkJade  Table . And reap < 0.05.The bar charts in the study were produced using GraphPad Prism 6.0 and analyzed using SPSS 20.0 for one-way ANOVA. Results were expressed as mean ± standard deviation  and decreased E2 levels (p < 0.01) compared with normal mice. However, administration of BL resulted in significantly lower serum T levels and higher E2 concentrations relative to the PCOS group (p < 0.05).Apart from the observed alterations in ovarian tissue, treatment with BL also led to significant changes in serum T and E2 levels in PCOS-like mice. As illustrated in Fig. By applying the TCMSP database and literature search, a total of 38 chemical components of BL were found. These 38 chemical components were then further screened under the circumstances of OB ≥ 30% and DL ≥ 0.18 to find a total of 7 active components of BL Table . The afoWe imported data on the 7 active ingredients and their targets for BL into CytoScape 3.8.2 software to develop a visualization diagram of the drug targets of these active ingredients, as shown in Fig. Cluster analysis and PCA analysis of the four sample sets showed that the quality of the GSE1615 microarray samples was suitable for subsequent analysis. 491 DEGs were identified, comprising 130 up-regulated genes and 361 down-regulated genes. As shown in Fig. p-value < 1.0e-16 . We identified core PPI networks based on topological analysis using median ≥ 2× as a screening criterion. As shown in Fig. To investigate the mechanism of therapeutic action of BL on PCOS, we imported 106 shared targets into the STRING database to construct a PPI network. The number of edges of this network was 1265, and the average degree value of targets was 17.6 with PPI enrichment Several predicted targets identified in this study are related to various biological processes, including glycolipid metabolism , inflammatory response , immunomodulation , and hormone regulation . These findings suggest that the therapeutic effects of BL may involve multiple mechanisms, including modulation of inflammatory responses, regulation of glycolipid metabolism, immunomodulation, and hormone regulation.To explore the various mechanisms of BL involvement in the treatment of PCOS, we obtained 642 BF entries, 37 CC entries, and 46 MF entries through the Metascape database. The results revealed that BP was mainly involved in positive regulation of phosphorylation, protein phosphorylation, and response to peptide. MF was mainly involved in protein kinase activity, protein kinase binding, and protein domain-specific binding. Additionally, CC was mainly associated with caveola, nuclear envelope, and transcription regulator complex. To visualize the GO enrichment network, ClueGo was utilized . We interlinked the compounds with the 6 core targets  one by one and determined their matching degree based on Total Score, Crash, and Polar Table A. TypicaJAK2, PPARG, PI3K, and AKT1 gene expressions were significantly upregulated (p < 0.01), while ESR1 and IRS1 gene expressions were significantly downregulated (p < 0.01) in the PCOS model group compared with the normal group. These findings were consistent with the differential gene changes observed in microarray GSE1615 dataset. Additionally, compared to the model group, the BL group exhibited significant changes in gene expression, which were statistically significant   Fig. B.Fig. 10PCOS is characterized by androgen overproduction and ovarian dysfunction. This condition is categorized into PCOS in adolescence and PCOS in reproductive age, with the latter being more complex, more heterogeneous, and more prevalent among younger women. PCOS is the most common cause of infertility in women of reproductive age and accounts for 30–60% of all patients with infertility who exhibit ovulatory disorders . MoreoveThe pathogenesis and etiology of PCOS are yet to be elucidated, but emerging evidence hints that it may be a complex polygenic disease influenced by epigenetic and environmental factors, such as diet and lifestyle. However, IR has been reported to be a significant pathological basis for the development of PCOS . A studyHyperandrogenemia is the central factor in treating PCOS as it can lead to ovarian stromal hyperplasia, thickening of the envelope, acceleration of follicular atresia, and ultimately, female ovulatory infertility. Excessive insulin secretion triggered by IR can promote androgen secretion via three pathways, which ultimately results in hyperandrogenemia: direct activation of 17-β hydroxylase in follicular membrane cells, which promotes androgen production; stimulation of luteinizing hormone production via insulin receptors in the pituitary gland, which further enhances androgen production in the ovaries; inhibition of the synthesis of hepatic sex hormone-binding globulins, which leads to an increase in free androgen levels in the bloodstream , 63. TheC. sinensis mushroom powder, which has been confirmed to exert anti-PCOS effects in clinical studies [BL is a new Chinese medicine prepared from  studies –25 HowevIn this study, PCOS models were established via the subcutaneous injection of DHEA into mice combined with a high-sugar and high-fat diet. The research findings indicated that the model mice exhibited disrupted estrous cycles and evident polycystic ovary lesions, which suggested that PCOS models were successfully created. The mice were subsequently treated with BL, after which their estrous cycles and ovarian lesions tended to normalize. Additionally, when their serum testosterone and estradiol levels were tested, significant improvements were noted in the BL group. These experimental data confirmed the effectiveness of BL in DHEA-induced PCOS model mice. Nonetheless, the mechanisms underlying the ability of BL to alleviate PCOS remained unclear.Network pharmacology, which is based on the “drug–target–gene–disease” interaction network, systematically examines the intervention and influence of drugs on the disease network. This study can reveal the material basis and effect mechanism of the synergistic effect of Chinese medicine on the organism holistically and systematically. Molecular docking is a multidisciplinary technique that combines computer technology, molecular biology, and molecular pharmacology to aid in the molecular design of drugs. Furthermore, this technique is useful in elucidating the mechanism of action of the effector components and targets of traditional Chinese medicine at the molecular level. Therefore, this research made use of network pharmacology and molecular docking to explore the potential mechanism and material basis of BL in treating PCOS.Seven effective components were retrieved from the TCMSP database . Of these, beta-sitosterol inhibits cholesterol absorption in the small intestine and lowers its plasma level , whereasThe GEO database is a valuable repository of high-throughput gene expression data submitted by research institutions globally. Via computer language mining and analysis of clinical data samples, this database enables rapid and comprehensive investigation of disease-related targets. Relevant differential genes can be obtained by inputting the disease name and applying conditional filtering. In this study, PCOS was searched in the GEO database and four microarray datasets  were screened. These datasets were analyzed using the R language, which resulted in the identification of 491 differentially expressed genes (DEGs), including 130 upregulated genes and 361 downregulated genes. Additionally, disease target databases, such as GeneCards, DrugBank, OMIM, and TTD, were integrated to predict targets and establish a comprehensive database of differential genes associated with PCOS.Network pharmacology analysis revealed that the seven active ingredients of BL corresponded to 437 predicted targets, 143 pharmacophoric cross targets, and 30 key pathways. By combining protein–protein interaction network topology analysis with related literature reports, this study predicted that ESR1, MAPK1, PPARG, CD4, PIK3CA, JAK2, MAPK8, IL2, and TLR4 were the key targets of BL in treating PCOS. PPARG is a nuclear receptor that binds to peroxisome proliferators, such as fatty acids and lipid-lowering drugs. PPARG binds to certain PPAR response elements on the DNA and, once activated by ligands, controls the transcription of its target genes, including acyl-coenzyme A oxidase. This modulation of the peroxisomal β-oxidation pathway of fatty acids is a key regulator of adipocyte development and glucose homeostasis. PPARG further inhibits NF-κB-mediated proinflammatory responses, which makes it a key regulator of intestinal homeostasis –69. ESR1PCOS is a chronic low-grade inflammatory disease often associated with disturbed sex hormone levels and IR. Ovarian tissues from patients have been found to contain elevated levels of IL-6 and TNF-α, which may be a major factor in the ability of PCOS to sustain low-grade inflammation. TNF-α is a pertinent inflammatory factor released by adipocytes and macrophages. Studies have shown that the upregulation of TNF-α expression induces apoptosis of follicular granulosa cells, activates mitosis of follicular membrane mesenchymal cells, decreases follicular granulosa cells, and increases follicular membrane mesenchymal cells. These alterations ultimately impair estrogen synthesis and increase androgen synthesis. TNF-α is linked to IR, ovarian follicular membrane cell growth, glucolipid metabolism control, ovarian follicular membrane cell proliferation, and steroidogenesis , 77. WhePathway enrichment analysis signified that the effects of BL in treating PCOS may be related to PI3K-Akt signaling, IR, and toll-like receptor (TLR) signaling pathways. The PI3K-Akt signaling pathway is involved in cell survival, proliferation, growth, and metabolism, among other cellular processes. This pathway is activated by the binding of ligands, such as insulin and growth factors, to their corresponding receptors on the cell membrane. This binding activates PI3K, which mediates downstream AKT, thereby affecting NF-κB and further leading to the release of inflammatory factors. TLR1-TLR10 , which aThe results of molecular docking indicated that the active ingredients of BL have a high binding affinity for their targets, particularly JAK2-cholesteryl palmitate, PIK3CA–cholesteryl palmitate, PPARG–arachidonic acid, and PPARG–linoleyl acetate, which exhibited total scores of 10.719, 10.453, 10.321, and 9.613, respectively. These results imply that PI3K-Akt, TLR signaling, and IR pathways may be important for the efficacy of BL. These pathways may also play a crucial role in the effectiveness of BL. However, further investigations are required to confirm these results and determine the potential therapeutic applications of BL for managing diseases related to these pathways.p-value < 0.05 screening to obtain DEGs. The analysis revealed the upregulation of core targets JAK2, PPARG, PI3K, and AKT1 as well as downregulation of ESR1 and IRS1.Via analysis of the GEO database, samples were selected from an obese PCOS model that was compatible with the mouse model created using a high-sugar and high-fat diet and subcutaneous injection of DHEA. ANOVA was performed using R 4.2.3 with standard selection criteria of |logFC|≥ 1 and adjusted Overactivation of PI3K-Akt signaling has been reported in certain studies in patients with PCOS , 90, butThis study made use of bioinformatics, network pharmacology, molecular docking, and genetic validation to identify the active components, probable target pathways, and molecular processes of BL in preventing and treating PCOS. Linoleyl acetate, cholesteryl palmitate, and arachidonic acid were identified as the main components of BL in treating PCOS. These compounds affected the JAK-STAT and PI3K-Akt signaling pathways by binding to JAK2, PIK3CA, PPARG, ESR1, IRS1, and AKT. They improved IR and inflammatory response and exhibited multitarget and multi-pathway therapeutic effects in anti-PCOS. These findings could serve as a solid foundation for additional investigations on the effectiveness and molecular processes of BL in treating PCOS.