Through a network pharmacology method, we screened the main active compounds of Citri Reticulatae Pericarpium (CRP), constructed a drug-ingredient-disease-target network, explored the molecular mechanism of its treatment of myocardial hypertrophy, and validated it by using molecular biology approach.  Traditional Chinese Medicine Systems Pharmacology (TCMSP) and GeneCards were utilised to collect the effective component in CRP and the targets of CRP and myocardial hypertrophy. The STRING database constructed the protein interaction network. The drug-ingredient-disease-target network was outlined by the Cytoscape 3.9.0 software. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the Metascape database. Real-time PCR (RT-PCR) and Western blotting were utilised to determine the mRNA and protein level of the critical targets of CRP therapy for myocardial hypertrophy.  We found that five practical components of CRP exerted therapeutic effects on myocardial hypertrophy by modulating 41 targets. Further analysis revealed that naringenin was the essential active compound in CRP that regulated myocardial hypertrophy. In addition, we showed that the active compounds of CRP might exert antihypertrophy effects via regulating essential target proteins such as AKT1-, MAPK3-, PPARA-, PPARG-, and ESR1-mediated signaling pathways such as cell proliferation, nuclear receptor activation, and oxidative stress. The molecular biology experiments demonstrated that naringenin inhibited the mRNA level of NPPA and NPPB induced by Ang II and regulated related targets such as AKT1, MAPK3, PPARA, PPARG, and ESR1.  CRP could inhibit myocardial hypertrophy through multitarget and multiapproach. Studies show that CVD is a primary cause of death in China, accounting for more than 40% of worldwide deaths, affecting hundreds of millions of people each year –3. Myocaity rate –12. Howeity rate .Citri reticulatae pericarpium (CRP), commonly called Chenpi in Chinese, is the dried and ripe peel of Citrus reticulata Blanco and its cultivated varieties, aromatic, spicy, and bitter . As one This present study constructs the interaction network between the active components of CRP, drug targets, and myocardial hypertrophy-related target genes, initially investigating the molecular mechanism of action of CRP in the treatment of myocardial hypertrophy. Moreover, we also investigate the effect of naringenin on myocardial hypertrophy and related targets predicted before. The workflow is shown in Naringenin (purity >95%) was purchased from Aladdin Chemistry Co., Ltd. . Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco . Real-time PCR kit was purchased from Takara Co., Ltd. . Rabbit anti-ERK1/2 (#4695), anti-p-ERK1/2 (#8544), anti-AKT (#4691), and anti-p-AKT (#4060) antibodies were purchased from CST Company . The chemiluminescent substrate was purchased from Pierce .The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was applied to retrieve the active ingredients of CRP with the keywords of Citri reticulatae pericarpium. Oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were set as the screening criteria to screen out the active components of CRP. The active ingredients of CRP-related target genes were also obtained from the TCMSP database.https://www.genecards.org/), an online tool, is a comprehensive resource of human genes, providing all known and predicted human-related genes in proteome, genome, genetics, transcription, and function. In this study, the GeneCards database was used to search for myocardial hypertrophy-related genes using the keyword of myocardial hypertrophy. The gene target names were corrected using Perl's computer programming language (https://www.perl.org/).GeneCards , and an optical network topology diagram of CRP-active ingredient-myocardial hypertrophy was constructed based on the Cytoscape software. The network described the relationship between the active components of CRP and myocardial hypertrophy. The active ingredients, drugs, and disease genes were nodes of the web, and the line between each node represented the relationship between the three.The information of active ingredients of CRP and myocardial hypertrophy targets was imported into the network visualization software Cytoscape 3.9.0 (https://string-db.org/), the species selected was human. Then, the protein interaction network relationships were mapped, and the data were analyzed by Cytoscape 3.9.0 to filter out the core components of the protein interaction network. Then, the intersection target genes were placed into the Metascape database, and species were selected as “Homo sapiens,” and P < 0.01 were set for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.With the intersection gene of CRP-myocardial hypertrophy entered on the STRING database  when they reached 80%-90% fusion and then passaged. Before treatment, the cells were treated with DMEM medium containing 0.1% FBS for 12 h to treatment to synchronize the cells, and then, subsequent experiments were performed.H9C2 cardiomyocytes were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 95% air-5% COAfter H9C2 cardiomyocytes were treated with naringenin and Ang II, the culture medium was discarded, and the total RNA of cardiomyocytes was extracted with TRIzol. The mRNA concentration was detected by Bio-Rad quantitative PCR kit. PCR primers were designed using the sequences shown in The extracted total cell protein was added to 2× SDS buffer, followed by SDS-PAGE gel electrophoresis, membrane transfer, blocking, the addition of primary antibody , incubation at 4°C overnight, incubation at room temperature for one h on the next day with secondary antibody, detection of target protein expression by chemiluminescence, and grayscale analysis of bands by the ImageJ software.P < 0.05.We use the SPSS 13.0 software for statistical analysis. The data of each group were presented as mean ± SD. One-way ANOVA was used for comparison between multiple groups. In all cases, differences were considered statistically significant with Based on the TCMSP search results, 63 active ingredients of CRP were collected. Each component's chemical information was standardized by molecular ID, molecular name, molecular weight, OB value, and DL value to establish the chemical composition information database of the drug. Subsequently, with OB ≥ 30% and DL ≥ 0.18 as criteria, five compounds with high activity were obtained by further screening, as shown in OB: oral bioavailability; DL: drug-likeness.With the intersection targets of CRP and myocardial hypertrophy being imported into the STRING database, the free nodes outside the network being hidden, and the self-defined confidence score value > 0.4, the protein-protein interaction network of CRP-myocardial hypertrophy was carried out . The whoThe intersections of active ingredient targets of CRP and myocardial hypertrophy disease targets were placed into Cytoscape 3.9.0, and the network of CRP-active ingredient-disease-intersection targets was mapped to elucidate the connection between the four targets . A totalP < 0.05), 15 molecular functions (MF), and 10 cell composition (CC) corresponding to the target of the practical components of CRP for treating myocardial hypertrophy , and the top 9 pathways with higher enrichment were screened (Based on the annotated database of biological information, Metascape, GO bioprocess enrichment analysis, and KEGG pathway analysis of CRP-myocardial hypertrophy disease were established. The results of GO analysis obtained 20 biological processes ( Figures . Hormonescreened . The resTo clarify the effect of naringenin on cardiomyocyte hypertrophy, H9C2 cells were pretreated with naringenin for 1 h and then treated with Ang II for 24 h. The mRNA expressions of hypertrophy-related genes NPPA and NPPB were detected. Ang II was able to induce increased mRNA level of NPPA and NPPB, while naringenin pretreatment inhibited the mRNA expression of NPPA and NPPB Figures , which sTo clarify the effect of naringenin on the screened cardiac hypertrophic targets, we gave H9C2 cardiomyocytes naringenin pretreatment for 1 h, followed by Ang II treatment for 24 h. Real-time PCR detected the mRNA expression of PPARA, PPARG, and ESR1. The phosphorylation levels of AKT and ERK1/2 were detected by Western blotting. The results showed that naringenin pretreatment could inhibit the Ang II-induced decrease in mRNA expression of PPARA, PPARG, and ESR1 Figures . At the Cyberpharmacology provides new ideas for Chinese medicine research by searching databases such as proteomics, genomics, and bioinformatics to perform a systematic analysis of Chinese medicine at the molecular and holistic levels to obtain the core chemical components and protein targets of TCM and to clarify the mechanism of action of TCM , 20. As β and mTOR to initiate the expression of myocardial hypertrophy marker genes such as NPPA and NPPB [Numerous extracellular and intracellular signals synergistically regulate the onset and progression of myocardial hypertrophy . Myocardand NPPB , 27. MAPand NPPB . MAPK-deand NPPB . The MAPand NPPB . In cardand NPPB . ASK1 acand NPPB . ERK1 (Mand NPPB . Previouand NPPB . In thisAs one of the high energy-consuming tissues in the body, cardiac mitochondrial energy metabolism dysfunction is closely associated with many CVD . The PPAIn summary, we found that naringenin may be the critical active component in CRP that regulates myocardial hypertrophy. Moreover, we also showed that naringenin could exert inhibitory effects on myocardial hypertrophy through AKT1, MAPK3, PPARA, PPARG, and other essential target proteins mediating cell proliferation, receptor activation, oxidative stress, and different signaling pathways. Further molecular biology experiments also verified this prediction. The present study provides a scientific basis for further research on the mechanism of action of CRP against myocardial hypertrophy.


CYP27B1, CYP24A1, GC, and VDR genes were analyzed through real-time PCR. A total of 63 patients were enrolled. Calprotectin, hemoglobin, and C-reactive protein levels were influenced by SNPs in VDR, CYP27B1, and GC genes. After 3 months of therapy, clinical remission was predicted by smoke, systemic steroids, and VDR BsmI, whereas at 12 months by GC 1296AA/AC and VD supplementation. This study reports the association between VD pathway-related genetics and ADA treatment. Further studies are needed to confirm these promising data.Adalimumab (ADA) is a human anti-tumor necrosis factor (TNF-α) monoclonal antibody used in inflammatory bowel diseases, such as Crohn’s disease (CD). Vitamin-D (VD) is important for biological functions, such as the modulation of expression of genes encoding enzymes and transporters involved in drug metabolism and transport. ADA trough levels were associated with VD concentrations in patients with IBD, but no data are present in the literature concerning VD pathway-related gene single-nucleotide polymorphisms (SNPs) in affecting clinical outcomes. For this reason, the aim of this study was to evaluate the ability of VD-related genetics to predict clinical remission at 3 and 12 months in patients affected by CD treated with ADA. Patients affected by CD were included in this study. SNPs in  Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are multifactorial, lifetime, inflammatory diseases of the gastrointestinal tract, with an increasing prevalence worldwide . There iWorldwide, vitamin D (VD) deficiency represents a public health problem in all age groups, although studies are still lacking in most countries, particularly at -risk groups. The National Health and Nutrition Examination Survey (NHANES) shows that 79% of the elderly adult population have VD deficiency or insufficiency ,13. ThisMany studies displayed a correlation between VD reduced plasma levels and the onset of bone, neurological, cardiovascular diseases, diabetes, metabolic syndrome, infections, cognitive decline, and neurodegenerative diseases and neoplasms and autoimmune disorders ,18,19,20Ultraviolet light is responsible for VD synthesis in the skin; cholecalciferol is hydroxylated to calcifediol  in the liver through cytochrome P-450 (CYP) 27A1 and CYP2R1 and, in the kidney, calcitriol  is synthesized through CYP27B1. 1,25-VD is then transported in the bloodstream through the vitamin D binding protein (VDBP). Cholecalciferol is inactivated to calcitroic acid by the CYP24A1 enzyme, whose expression is partially affected by calcium and parathyroid blood concentrations and partially induced by 1,25-VD, which can also directly down-regulate the CYP27B1 gene. CYP3A4 and, to a lesser extent, CYP2B6 and CYP2C9 genes expression in human hepatocytes. In fact, they revealed that VDR recognizes and transactivates CYP3A4, CYP2B6, and CYP2C9 promoters binding to xenobiotic-responsive elements; in addition, it is targeted by the constitutive androstane receptor (CAR) and/or the pregnane X receptor (PXR). Moreover, the authors suggested that VDR, PXR, and CAR modulate several CYP genes’ expression by competitively interacting with the same battery of responsive elements [Through its receptor VDR, VD modulates the expression of genes encoding transporters and enzymes responsible for drug transport and metabolism, such as cytochromes (CYPs) . Drocourelements .Several drugs are metabolized by CYP3A4; this gene shows VD responsive elements (VDRE) and its expression is upregulated in presence of 1,25-VD. Lindh et al. suggested tacrolimus and sirolimus seasonal variability according to changes in VD levels, with lower drug concentrations in July to September than in January to March. Furthermore, VD influence on CYP3A4 expression results in altered metabolized drugs concentration as shown for immunosuppressants ,25,26.VD may interact with several drugs, potentially altering drug toxicity or efficacy, but drugs may also affect VD metabolism and status : The 25-VD plays an important role in IBD during anti-tumor necrosis factor (TNF) treatment: Higher rates of clinical response and clinical remission correlate with increased VD levels . Santos-An association between vitamin D pathway-related gene single-nucleotide polymorphisms (SNPs) and ADA clinical outcome, from the perspective of a putative use as a biomarker of the therapeutic response, has never been assessed. For this reason, the aim of this study was to evaluate the ability of VD-related genetics to predict clinical remission in patients affected by CD treated with ADA, for the first time in the literature.A total of 63 patients were enrolled in Turin and Pisa; their characteristics are reported in Subsequently, patients’ clinical characteristics were analyzed according to SNPs of genes related to VD metabolism, transport, and activity.CYP27B1-1260 TT was able to affect all three time points (CYP27B1 + 2838 CT/TT (p = 0.010), VDR ApaI CA/AA (p = 0.021), VDR Cdx2 AG/GG (p = 0.036), and GC 1296 CC (p = 0.034) had an influence; finally, a role was suggested for CYP27B1 + 2838 CT/TT (p = 0.023), VDR ApaI CA/AA (p = 0.021), and VDR TaqI TC/CC (p = 0.046) at 3 months of therapy. The levels of calprotectin were different according to the genetic variants ; particue points . At BL, CYP27B1-1260 TT was able to affect the three analyzed time points . Hemoglobin (Hb) levels were influenced by genetic polymorphisms ; also, ie points . CYP24A1VDR TaqI CC (p = 0.036) at BL, CYP27B1-1260 TT (p = 0.017) at 3 months, and, finally, VDR ApaI AA (p = 0.037) at 12 months.The C-reactive protein (CRP) was affected by VDR BsmI AA remained in the multivariate regression, whereas GC 1296 AA/AC and VD supplementation were retained in the final regression model at 12 months of therapy 27A1 and CYP2R1 hydroxylate cholecalciferol to calcifediol ; and in the kidney, CYP27B1 synthesizes calcitriol . 1,25-VD is then transported in the bloodstream through the vitamin D binding protein (VDBP) encoded by the  1,25-VD , which c7B1 gene ,35. VD l7B1 gene ,37.VD deficiency is highly prevalent in IBD, involving up to 80% of apparently well-nourished patients . HoweverIn this study, 63 patients affected by CD were enrolled and evaluated; particularly, predictive factors of biochemical biomarkers, such as calprotectin, Hb, and CRP, and clinical remission at three and twelve months of therapy were investigated.We focused on VD-related genetics, since no data are available in literature in this context. Polymorphisms in genes encoding its activation enzyme (CYP27B1), its inactivation enzyme (CYP24A1), its receptor (VDR), and its transporter (VDBP) were analyzed and related to clinical features.CYP27B1 -1260 TT was found to be associated with calprotectin and Hb at BL, three, and twelve months of therapy. In particular, this genotype patients showed higher calprotectin levels and lower Hb concentrations. These two findings could be related, since increased calprotectin exposure could be associated with a high grade of inflammation and blood loss, with consequently reduced Hb levels [CYP27B1 -1260 TT was associated with increased colorectal cancer risk; the CYP27B1 gene encodes a member of the CYP monooxygenases family (the 1α-hydroxylase enzyme), localized to the inner mitochondrial membrane of renal cells [CYP27B1 -1260 rs10877012 promoter polymorphism are inconsistent: Lange and colleagues suggested its influence on 1,25-VD serum concentrations, in contrast of what was observed by Kitanaka et al. [Interestingly, b levels ,40. Furtal cells . Availaba et al. ,43,44,45VDR BsmI AA genotype, in addition to smoke and use of systemic steroids, was a predictive factor at three months of therapy, whereas GC 1296 AA/AC was at twelve months. In particular, all the factors, with the exception of GC 1296 AC/CC, are negative predictors.For the first time, clinical remissions at three and twelve months after starting therapy were analyzed according to VD-related genetic variants. We suggested that the GC 1296 is unknown: It encodes the VDBP, which is a multifunctional glycoprotein produced by hepatic parenchymal cells and secreted into the circulation [The role of culation . Its funculation . Our recculation .49. It was supposed that VDR mRNA expression and protein levels could be lower in IBD affected subjects [VDR-mRNA expression, affecting its stability, confirming previous studies [VDR is the VD receptor that regulates the transcription of several genes; furthermore, it modulates several physiological systems, such as immune, calcium, and phosphorous homeostasis or apoptosis. Several VDR gene SNPs were able to affect certain features in different diseases, for example non-small-cell lung cancer or autoimmune hepatitissubjects . HoweverConcerning clinical remission at 12 months of therapy, VD supplementation remained in the final model as a negative predictive factor: It could be possible that these patients have lower VD levels, which could not be normalized, despite the supplementation. This finding is in line with the current literature, which displayed how the vast majority of patients with IBD have low levels of VD, regardless of their clinical or nutrition conditions ,50.The present study has several important limitations. Firstly, the number of patients was small; however, it is worth noting that this factor did not affect the significance of the analyses, thus suggesting that the putative associations of VD genetics and clinical outcome of ADA treatment in CD are conceivable. Secondly, data concerning VD exposures would have significantly improved our results, but unfortunately, these data were unknown. Finally, an endoscopic assessment at twelve months would improve the significance of the results, although fecal calprotectin is currently well recognized as a marker of endoscopic remission ,51.We performed a prospective, multicenter study at the Gastroenterology Unit of “Città della Salute e della Scienza di Torino”, Italy and the IBD Unit, Pisa University Hospital, Pisa, Italy. From January 2018 to January 2020, we recruited consecutive patients: (1) Affected by CD with indications to treatment with ADA; (2) naive to anti-TNF drugs or other biological drugs; (3) older than or equal to 18 years; (4) who agreed to sign the informed consent to participate in the study. We treated patients with moderate-to-severe disease activity or steroid-dependent disease with previous failure or intolerance to thiopurines with ADA. Exclusion criteria were: (1) Refusal to participate to the study; 2) pregnancy; 3) other concomitant auto-immune diseases . Clinical history, data on physical examination, recent biochemical examinations, and signed informed consent for the purpose of enrolment in the study were collected. All patients were treated with a total dose of 160 mg at day 1, 80 mg at day 15, and 40 mg of ADA by subcutaneous administration every two weeks. Before starting ADA therapy, 9 mL of venous blood was collected. The blood samples were associated with an anonymous numerical identification code and stored frozen at −80 °C. In a subgroup of patients, randomly identified, a second blood sample was collected after 3 months of treatment (T1).®, Trieste, Italy).Clinical characteristics were evaluated at BL , 3, and 12 months of therapy. At the same time-points, fecal calprotectin was assessed by ELISA . Clinical response to ADA therapy was defined as a decrease in the Harvey–Bradshaw index (HBI) score used to foster a systematic collection of clinical data related to Crohn’s disease: It considers five parameters, exclusively clinical . For each parameter, a specific score is assigned: Greater than or equal to 3 in the absence of corticosteroid therapy and with ongoing ADA therapy, clinical remission, and HBI <= 4, in agreement with the literature . The stuWhole blood was collected in EDTA tubes and genomic DNA was obtained from blood samples ; allelic discrimination was assessed through the real-time polymerase chain reaction system . The investigated gene SNPs were VDR (encoding VD receptor) rs7975232 (ApaI) C>A, rs731236 (TaqI) T>C, rs10735810 (FokI) T>C, rs11568820 (Cdx2) A>G, and rs1544410 (BsmI) G>A, CYP27B1  rs4646536 (+2838) C>T and rs10877012 (-1260) G>T, CYP24A1  production) rs2248359 (3999) T>C, rs927650 (22776) C>T, and rs2585428 (8620) A>G, and finally GC  rs7041 (1296) A>C.p-value < 0.05. Stepwise multivariate logistic regression analysis was then performed including variables with a p-value below 0.05 in univariate analysis to evaluate factors able to predict clinical remission at three and twelve months of therapy .All variables were tested for normality through the Shapiro–Wilk test. Normal variables were presented as the average and standard deviation, whereas non-normal variables were median values and the interquartile range (IQR) and categorical variables were numbers and percentages. Allele frequencies were analyzed for Hardy–Weinberg equilibrium. Kruskal–Wallis and Mann–Whitney tests were adopted for differences in continuous variables between genetic groups, considering a statistical significance with a two-sided Since no previous studies have analyzed the ability of VD genetics to predict clinical response to ADA in patients with CD, a prior calculation of the power of the study was not possible.In conclusion, this is the first study investigating the role of VD-associated genes in evaluating the clinical remission at three and twelve months of therapy with ADA in patients with CD. The novelty of our results is the most important strength of the present study. However, further studies are required in order to confirm these preliminary data and to evaluate their potential role in clinical management.


Ankylosing spondylitis (AS) is a rheumatism that mainly affects the axial bones and joints. Xinfeng capsule (XFC) is a preparation with a remarkable clinical effect that is used in our hospital. And it has definite curative effect and less side effects in the treatment of AS.  Data mining and network pharmacology were used to analyze the efficacy of Chinese medicine Xinfeng capsule on treating the hypercoagulable state of ankylosing spondylitis and the underlying mechanism behind it.  Clinical data were collected and compiled from the Department of Rheumatology and Immunology of the First Affiliated Hospital of Anhui University of Chinese Medicine. Cluster analysis was used to investigate herbs that frequently used to treat AS, Apriori module was used to analyze the association rules between herbs and laboratory indexes, and the random walk model was used to reveal the therapeutic efficacy of XFC against AS. The TCMSP database was used to acquire the active components and targets of XFC, and the GeneCards and OMIM database were used to obtain the targets of AS. Afterward, an active ingredient-target network was established and core targets were screened for; overlapping targets were screened for the protein-protein interaction (PPI) network analysis, the Gene Ontology (GO) enrichment analysis, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Molecular docking was adopted to investigate the interactions between main active components and core targets.  Frequently used herbs could be divided into three groups, and according to the analysis of Apriori module, there is a strong correlation between XFC and the improvement of ESR and hs-CRP, and the results of the random walk model demonstrated that the effect of XFC on improving PLT, ESR, and hs-CRP was superior to the use of traditional Chinese medicine alone. In total, 103 active compounds of XFC and 59 overlapping targets were obtained. The PPI relationships were obtained through the STRING database, and 13 core targets were identified. 1786 GO enrichment results and 205 KEGG enrichment results were obtained, including NF-kappa B signaling pathway, TNF signaling pathway, and IL17 signaling pathway. The outcomes of molecular docking revealed a close relationship between the active compounds of XFC and core targets.  This study demonstrated that XFC can effectively improve the hypercoagulable state and the inflammatory indices of AS patients through data mining, and it has a strong correlation with the clinical improvement of inflammation. The active compounds of formononetin, triptolide, quercetin, and kaempferol may be the key active components of XFC in regulating AS, possibly through inhibiting the activation of NF-kappa B signaling pathway to improve hypercoagulable state. Ankylosing spondylitis (AS) is an autoimmune disease that has a high incidence, mainly manifested as spinal inflammation and chronic connective tissue lesions . It is cXinfeng capsule (XFC) is a preparation with a remarkable clinical effect that is used in our hospital. It is mainly composed of four herbs: Astragalus membranaceus (Huang Qi:HQ), Coix seed (Yi Yi Ren:YYR), Tripterygium wilfordii (Lei Gong Teng:LGT), and centipede (Wu Gong:WG). XFC has been used in clinic for more than 10 years, and it has definite curative effect and less side effects in the treatment of AS .As a new scientific method, network pharmacology is based on the analysis of network data and system biology. The combination of traditional Chinese medicine and network pharmacology has bridged the gap between modern medicine and traditional medicine .The clinical efficacy of traditional Chinese medicine on treating AS is valid, as it improves the immune inflammations indices of AS patients , 8, but Clinical data from hospitalized AS patients in the Department of Rheumatology and Immunology of the First Affiliated Hospital of Anhui University of Chinese Medicine between July 2009 and June 2021 were collected and compiled. The dataset includes usage records of XFC and Chinese herbal medicines, and the value of laboratory indicators includes PLT, hs-CRP, and ESR. 1040 datasets were acquired, Among them, 710 received Chinese medicine alone as control groups, and 330 were treated with XFC combined with Chinese medicine as experimental groups.Any Chinese herbal medicine that was prescribed to individual patients was assigned 1, and those not used were assigned 0. The compatibility of Chinese herbal medicine was studied by systematic clustering in the SPSS v. 23.0 software. In cluster analysis, each herb was considered as a cluster and then combined based on their similarity to form a new class. The formula is as follows:Any Chinese herbal medicine that was prescribed to individual patients was assigned 1, and those not used were assigned 0; any improvement in the laboratory indices was also assigned 1, otherwise 0. The Apriori module in SPSS Clementine client v. 11.1 was used to determine the correlation between Chinese herbal medicine and laboratory indicators. The minimum support rate was 30%, the confidence level was 50%, and the lift was >1. Apriori was used to establish relationships between items. Individual drug and index were used as variables. The formula is as follows:X⟶Y represents an association rule, X (left-hand side (LHS)) represents the set of laboratory indices, and Y (right-hand side (RHS)) represents herbs, and σ(X) is the probability of the itemset X occurring; X ∪ Y is the union of itemsets X and Y, and σ(X ∪ Y) is the probability when X and Y occur together. Support(X⟶Y) represents the probability of itemset  occurring within the total itemset, and confidence(X⟶Y) is the probability of Y occurring in the presence of X. Lift is the ratio of the probability that itemset Y appears in the presence of X to the probability of Y appearing. Support and confidence are usually used to eliminate insignificant associations, and lift indicates the effectiveness of the association rules.where The random walk model evaluation in the index laboratory was realized using the Oracle 10 g tool; Peng's random walk model was used to refer [https://tcmspw.com), which is a Chinese herbal medicine platform [The compounds in XFC consisting of four Chinese herbs were acquired from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  and OMIM databases (http://www.omim.org/); AS related targets were recognized and filtered, with duplicate targets deleted to establish the targets of AS. Targets of AS and XFC were collected and visualized through R 4.1.1.The keyword “ankylosing spondylitis” was searched for in the GeneCards , in which the species was set as “Homo sapiens” for retrieving a PPI network with strong confidence. The network was then analyzed in the Cytoscape 3.8.2 software, in which the CytoNCA plug-in [Overlapping targets relating to both AS and XFC were screened to clarify the interaction between XFC and AS by R 4.1.1. Potential targets were then uploaded to the STRING database ( plug-in  was usedBoth Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were carried out using the R 4.1.1 clusterProfile V3.12.0. The Go enrichment analyzed biological processes, cellular composition, and molecular functions, whereas the KEGG pathway analyzed important biological pathways.https://www.rcsb.org/), the molecular docking of the target and active components was carried out by the AutoDock 4.2.6 software, and the visualization was achieved by PyMOL 2.5.2. The best affinity and minimum binding energies are conformations as final docking results.The core targets and main components were selected for molecular docking. The mol2 format file of active compound structure was downloaded on the TCMSP platform, along with the 3D structure of the corresponding target from PBD , Coix seed (Yi Yi Ren:YYR), Tripterygium wilfordii (Lei Gong Teng:LGT), and centipede (Wu Gong:WG) were acquired from the TCMSP database and searched from literature. 103 active compounds and 212 potential targets of XFC were obtained. 1961 AS targets were obtained by screening the GeneCards database and OMIM database. Through inspecting the intersection of drug and disease targets, 59 overlapping targets were identified .The active component target network, which can intuitively reflect the interactions between active ingredients and targets, was designed using the Cytoscape v3.8.2 software . Red nodβ, VEGFA, IL10, CCL2, PTGS2, CXCL8, EGF, STAT3, NFκBIA, and IFNG.59 overlapping targets were uploaded to the STRING database to export the TSV text. The PPI network built by Cytoscape 3.8.2 is shown in The Go analysis contained the following aspects: biological process (BP), cellular components (CC), and molecular function (MF). In the end, 1786 GO enrichment results were obtained. The upper 10 of BP, CC, and MF are shown in q value < 0.05), and some played essential roles, such as the NF-kappa B signaling pathway, TNF signaling pathway, and IL17 signaling pathways. The potential pathways were mainly involved with inflammatory responses, antiviral functions, and immune regulation.205 KEGG enrichment results were obtained, and The main active components, such as formononetin, triptolide, quercetin, and kaempferol, were docked with IL6, CCL2, TNF, and IL4 of the core targets. The results showed that all targets and molecules were successfully docked, as shown in κB signaling pathway was related to overactivation [Previous studies of our team have discovered that the formation of AS might be related to cytokine disorder, and the NF-tivation , the clitivation ; the usetivation , 16.The present study made use of data mining to explore the usage rules of Chinese herbal medicine for AS treatment in our hospital and verified its efficacy. Three groups of commonly used Chinese herbal combinations for AS treatment were obtained through cluster analysis, and treatment modalities mostly adopted the methods of tonifying Qi and strengthening of the spleen, dispelling wind, and dehumidification, promoting blood circulation and removing blood stasis. The results of association rules showed that XFC has a strong correlation with the improvement of ESR and hs-CRP. The improvement of PLT, ESR, and hs-CRP in the experimental group was superior to those in the control group as shown in the random walk model, and the experimental group had a lower number of walking steps compared to the control group, hence supporting the effectiveness of XFC in treating AS. Data mining results showed that XFC can effectively improve the hypercoagulable state and inflammatory indexes of as patients, regulate the hypercoagulable state and inflammatory response, and has a strong correlation with the improvement of hypercoagulable state and inflammation.Network pharmacology , 18, whiβ, VEGFA, IL10, CCL2, PTGS2, CXCL8, EGF, STAT3, NFκBIA, and IFNG. These targets are related to host immunity, oxidative stress, and other pathogenic microorganisms. IL6 is a pleiotropic cytokine with numerous biological functions, which is involved in the pathogenesis of a variety of rheumatic diseases [α, IL1β, and IL6 in the serum, and aggravate the inflammatory response [The topological parameters of identifying key nodes were calculated through the PPI network, and 13 targets were found to be the core targets for XFC in the treatment of AS, including IL4, IL6, TNF, IL1diseases . IL10 isdiseases . Throughdiseases . The higresponse .Go and KEGG enrichment analyses showed that XFC could control immune pathway processes. XFC was involved in biological process such as responses to lipopolysaccharide, bacterially derived molecules, cellular responses to biotic stimulus, cytokine activity, receptor ligand activity, and signaling receptor activator activity. Results of the KEGG pathway enrichment analysis illustrated that most targets were enriched in NF-kappa B signaling pathway, TNF signaling pathway, and the IL17 signaling pathway.κB signaling pathway [Previous studies from our group found that XFC could significantly improve the status of AS disease activity and immune-inflammatory indicators, and the mechanism could be contributed to inhibiting the activation of the NF- pathway –30. NF-kMolecular docking was also performed to forecast complex interactions between four active components and protein targets. The binding energy further confirmed the reliability of the docking results and showed great bonding characteristics. The results demonstrated that formononetin, triptolide, quercetin, and kaempferol could have crucial roles to play in the treatment of AS.The study has demonstrated that XFC can improve the hypercoagulable state and the inflammatory indices of AS patients, and it has a strong correlation with the clinical improvement of inflammation. The mechanism of XFC used to treat hypercoagulable state of AS was analyzed through network pharmacology, and its possible molecular mechanism involves multiple components. The active compounds of formononetin, triptolide, quercetin, and kaempferol may be the key components of XFC in regulating hypercoagulable state of AS and may do so through the NF-kappa B signaling pathways. However, there are some limitations in this present study, and clinical experimental validation of superior quality is needed to support the results concluded.


This study aimed to predict the key targets and endocrine mechanisms of Guizhi Fuling Wan (GZFLW) in treating adenomyosis (AM) through network pharmacology, molecular docking, and animal experiment verification.  The related ingredients and targets of GZFLW in treating AM were screened out using TCMSP, BATMAN-TCM, SwissTargetPrediction, and PubChem Database. Then, the protein-protein interaction (PPI) analysis and the network of compound-hub targets were constructed. At the same time, the key targets were uploaded to the Metascape Database for KEGG pathway enrichment analysis. After that, the molecular docking technology of the main active components and hub targets was performed. Furthermore, animal experiments were used to verify the results of network pharmacology analysis. α and EGFR. More importantly, the animal experiments confirmed that the GZFLW could downregulate the abnormal infiltration of the endometrial epithelium into the myometrium and had no interference with the normal sexual cycle. This effect may be directly related to intervening the local estrogen signaling pathway of the endometrial myometrial interface (EMI). It may also be associated with the myometrium cells' estrogen resistance via GPER/EGFR signaling pathway.  A total of 55 active ingredients of GZFLW and 44 overlapping targets of GZFLW in treating AM were obtained. After screening, 25 hub targets were collected, including ESR1, EGF, and EGFR. Then, the KEGG pathway enrichment analysis results indicated that the endocrine therapeutic mechanism of GZFLW against AM is mainly associated with the estrogen signaling pathway, endocrine resistance, and an EGFR tyrosine kinase signaling pathway. Then, molecular docking showed that the significant compounds of GZFLW had a strong binding ability with ER The endocrine mechanism of GZFLW in treating AM was explored based on network pharmacology, molecular docking, and animal experiments, which provided a theoretical basis for the clinical application of GZFLW. In 2020, 1.03% of the women were suffering from adenomyosis (AM), and approximately 90% of them were accompanied by menorrhagia, progressive dysmenorrhea, pelvic pain, dysmenorrhea, and infertility , 5. TherPoria cocos (PC), Cortex Moutan (CM), Radix Paeoniae Rubra (RPR), and Peach Kernel (PK). The complex composition of GZFLW makes it not easy to conduct mechanistic studies on it. Thus, the “multicomponent and multitarget” therapeutic mechanism of GZFLW in treating AM is still obscure, hindering its clinical use. Hence, for a comprehensive understanding of the mechanism of action of GZFLW, a novel analysis method is needed [Traditional Chinese medicine (TCM), which has been widely used in clinical practice in China for thousands of years, has made considerable contributions to the relief of AM . Guizhi s needed .In this study, a network pharmacology strategy was adopted for investigating the mechanism of GZFLW against AM. We screened representative compounds in five herbs of GZFLW. Their potential targets in treating AM were identified. Furthermore, molecular docking technology was used to verify the binding ability of active ingredients to several potential targets. In addition, animal experiments have further confirmed some of the hypotheses. The results revealed that GZFLW might modulate the estrogen signaling pathway, endocrine resistance, and EGFR tyrosine kinase signaling pathway to exert an effect. These results may provide novel sights into the anti-AM effect of GZFLW.http://lsp.nwu.edu.cn/tcmsp.php) and the Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine , which are both bioinformatics analysis tools for the main components of TCM.The active ingredients of the five herbs contained in GZFLW were obtained using the Traditional Chinese Medicine Systems Pharmacology Database  and drug-likeness (DL) were used as screening indexes. OB is the percentage of oral drugs absorbed into the systemic circulation, which is a frequently used pharmacokinetic parameter. DL refers to the property of measuring the absorption, distribution, metabolism, and excretion (ADME) of drug molecules, which could help optimize pharmacokinetic and pharmaceutical properties. The OB and DL indices of all the related ingredients are presented in the TCMSP. We set the indexes of OB ≥ 30% and DL ≥ 0.18 as the screening criteria of active ingredients. Briefly, components that satisfied the screening threshold of OB and DL were regarded as the candidate compounds.http://www.swisstargetprediction.ch/index.php), and PubChem Database (https://pubchem.ncbi.nlm.nih.gov/), with the species limited as ‘‘Homo sapiens.”The targets of active ingredients were searched in TCMSP, BATMAN-TCM, SwissTargetPrediction Database (https://www.genecards.org/) and Online Mendelian Inheritance in Man Database . We searched these databases with the keyword “adenomyosis” to obtain related targets. After deletion of duplications, candidate targets against AM were identified by matching the targets of the active ingredients of GZFLW and the genes related to AM. These targets were then uploaded to STRING Database (https://string-db.rog/) to obtain the protein-protein interaction (PPI) information between two proteins or multiple proteins with the confidence ≥0.7.Targets associated with AM were collected by GeneCards Database . Nodes greater than or equal to the median are screened as hub nodes for GZFLW against AM.http://metascape.org/) to obtain the potential pathways of GZFLW in treating AM. P < 0.05 was regarded as the cutoff value.We used the Metascape Database (http://autodock.scripps.edu/) was a molecular docking software for network pharmacology based prediction and analysis, which could be used to dock the hub targets and active ingredients. In this process, structures files in the formats of 3D of hub targets we chose were obtained from RCSB Protein Data Bank : EGFR (PDB ID: 2GS2) and ERα (PDB ID: 6CHW); structures files in the formats of 3D of active compounds were provided from ZINC Database (http://zinc.docking.org/) and PubChem Database (https://pubchem.ncbi.nlm.nih.gov/). During the docking process, the PDBQT files of these structures were used in AutoDock software, as these files contained the information of potential ligands and receptors required for the process. After that, the Lamarckian genetic algorithm was used to obtain docking results and the binding energy of ligands and receptors. At last, the results were output in .dlg files and uploaded to PyMOL software for establishing the 3D results binding models. Binding energy was used as a docking score to evaluate the protein-ligand binding potential of active ingredients to hub targets. Among them, those results with value ≤ −5 were selected and considered to have moderate binding potential and tight combination.Molecular docking is a computational tool to predict the binding ability and connection type of proteins and ligands. It can calculate and predict the conformation and directions of ligands at active protein sites. AutoDock 1.5.6 (μm). The mobile phase was ammonium acetate (A) and water (B). The flow rate was kept constant at 1 mL/min. The system operated at 30°C and the injection volume was 10 μL  and 5 μL (CM). The detection wavelength was, respectively, kept at 285 nm (CT), 230 nm (RPR), 274 nm (CM), and 210 nm (PK). The reference substances  were prepared by Chengdu Ruifensi Biotechnology Co., Ltd. .The HPLC finger printing of CT, RPR, PK, and CM in GZFLW was constructed using Series LC-2010A Liquid Chromatograph . Octadecyl silane bonded silica was used as the stationary phase  was used with a spectral range of 4000∼400 cmVirgin female ICR mice  were provided by the Experimental Animals Center of China Three Gorges University. All the animals were housed at a temperature of 22 ± 2°C, humidity of 55–65%, and light controlled (12 h light/12 h dark cycle) vivarium, with food and water ad libitum. Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the China Three Gorges University Medical Animal Ethics Commission .n = 14) and GZFLW group (n = 11). After intraperitoneally anesthetizing with emulsion , pituitary obtained from age-matched male mice was grafted into the right uterine lumen in female mice. Then, the gentamycin solution  was dropped intraperitoneally to prevent infection. Mice receiving no pituitary grafts served as controls (n = 8). One day after the pituitary grafting, 11 mice were intragastriced with decocting-free granules of GZFLW  and dissolved in aqueous solution every day for 8 weeks (GZFLW group). The remaining 22 mice  were intragastriced with the same dosage of saline as a control vehicle. All mice were killed at 15 weeks of age by decapitation, after the estrous cycle had been checked by vaginal smear.Twenty-five mice were implanted with a single pituitary gland each under the capsule of the right uterus at 7 weeks of age and then were divided randomly into 2 groups: model group  .Haematoxylin and eosin (HE) staining was used to observe the depth of invasion. Immunofluorescence was adopted to identify the expressions of ERP < 0.05 was regarded as a statistically significant difference.Data were analyzed using SPSS 20 software and presented as mean ± standard deviation (SD). Statistical differences between groups were evaluated by one-way analysis of variance (ANOVA). The Chi-square test was applied to analyze the categorical variables. Pearson analysis was used for correlation. By retrieving from the databases, there were 498 related ingredients of GZFLW in total: 222 ingredients in CT, 33 ingredients in PC, 56 ingredients in CM, 121 ingredients in RPR, and 66 ingredients in PK. After eliminating the redundancy and screening with OB and DL, 55 active components and 364 corresponding targets of GZFLW were obtained. It is worth noting that cinnamaldehyde , cinnamic acid , benzoic acid , paeonol , gallic acid , and amygdalin  have a relatively low OB or DL value, but they were included in this study because they were the vital pharmacological compounds identified, which were confirmed to the previous study  Table S.Via the keyword of “adenomyosis,” the therapeutic targets of AM were obtained from GeneCards and OMIM Database with a total of 211 . A totalFrom the STRING Database of “multiple proteins” interaction of targets, the data of the protein-protein interaction (PPI) network of those 48 targets were subsequently obtained . With coP-value of each enriched pathway in ascending order, and the top 20 pathways were selected, which mainly related to endocrine resistance, EGFR tyrosine kinase inhibitor resistance, and estrogen signaling pathway  had binding energies lower than −5, which verified that those active components may be well binding with ERα and EGFR , estrus (mainly nonnucleated keratinocytes), metestrus , and diestrus (mainly a large number of leukocytes). We divided the estrous cycles into two phases, combining metestrus with diestrus because of the leucocytic predominance . There iα and GPER were expressed in glandular epithelial cells, stromal cells, and muscularis cells. EGFR was expressed in stromal cells, muscularis cells, and some glandular epithelial cells  analogs, have been used to control menstrual pain and menorrhagia by inhibiting uterine ERα expression. Still, they interfere with the menstrual cycle of AM women [α expression decreased and the pathological infiltration depth reduced in GZFLW group, while the diestrus/metestrus stages time was not prolonged. The result suggested that the treatment of TCM did not interfere with the estrous cycle in mice, which may be attributed to the selective action of the GZFLW on the local lesion.As a hormone-dependent disease, the disorder of estrogen and its receptor is known to promote and exacerbate AM , 17. It α , 27–29. ry cells . SeveralAM women . Those tAM women , which wAM women . Our datOf note, the endocrine resistance pathway and EGFR tyrosine kinase signaling pathway may also contribute to the therapeutic mechanism of GZFLW against AM predicted by the network pharmacology analysis. Indeed, endocrine resistance is a complex pathophysiological process, known to lead to unresponsiveness of the regular endocrine therapy in a particular proportion of AM patients. It was reported that the EGFR gene polymorphism is involved in the endometriosis's susceptibility . EGF/EGFα estrogen signaling pathway and suppress estrogen resistance of myocytes. This effect has no apparent interference in the normal endocrine.In conclusion, based on the results of network pharmacology and animal experiments, we think that GZFLW can synergistically inhibit the abnormal proliferation and differentiation of AM via downregulating the ERα, GPER1, and EGFR, and this is not enough and comprehensive for data analysis. Finally, the number of animals is relatively small and may lead to study biases. Although network pharmacology strategy could help researchers simplify the complex system of TCM formulas and offer a novel research strategy for studying the mechanism of other TCM formulas, the mechanism remains confirmed by further experiments.There are several limitations in our study. Firstly, the databases we used can help predict the potential mechanism of GZFLW in treating AM. Nevertheless, most of them are based on the current research results, limiting the discovery of new therapeutic targets. Moreover, the primary detection indexes of the animal experiment only include ER


Accurately defining hierarchical relationships between human stem cells and their progeny, and using this knowledge for new cellular therapies, will undoubtedly lead to further successful treatments for life threatening and chronic diseases, which represent substantial burdens on patient quality of life and to healthcare systems globally. Clinical translation relies in part on appropriate biomarker, in vitro manipulation and transplantation strategies. CD164 has recently been cited as an important biomarker for enriching both human haematopoietic and skeletal stem cells, yet a thorough description of extant human CD164 monoclonal antibody (Mab) characteristics, which are critical for identifying and purifying these stem cells, was not discussed in these articles. Here, we highlight earlier but crucial research describing these relevant characteristics, including the differing human CD164 Mab avidities and their binding sites on the human CD164 sialomucin, which importantly may affect subsequent stem cell function and fate. Importantly, a 1998 special focus report in ‘Blood’4 highlighted the findings that hCD164 was a sialomucin involved in human haematopoietic progenitor-stromal interactions, and that engagement of specific glycosylated hCD164 epitopes on quiescent human CD34+ haematopoietic stem/progenitor cells (HSPCs) could prevent their recruitment into the cell cycle5. Two decades on, by analysing human bone marrow Lineage (Lin)neg, CD34+, CD34low, and CD34neg HSPCs with single cell RNAseq and improved surrogate transplantation models, Pellin and colleagues, in their article ‘A comprehensive single cell transcriptional landscape of human hematopoietic progenitors’ identified hCD164 as an important, reliable biomarker for defining the earliest branch points of hHSPC specification, which incorporates the basophil lineage and shows close similarities to murine lineage specification6. Interestingly, just prior to this observation, Chan and colleagues, in their paper entitled ‘Identification of the human skeletal stem cell’ meticulously purified, from hypertrophic zones of the growth plate in long bones, a subset of human PDPN+CD73+CD164+ skeletal stem cells (hSSC), which were negative for CD45, CD146, CD235, Tie2 and CD31, and at the apex of a hierarchy of stem cells that could transition into an early bone-cartilage-stromal progenitor8. These cells were demonstrated to meet the rigorous definition of hSSCs10, as being locally restricted to the bone, with the ability to clonally self-renew, differentiate into multiple lineages and reconstitute a haematopoietic microenvironment both in vitro and in vivo in surrogate serial transplantation models11. Importantly, and reminiscent of initial studies4, regulatory cross-talk was shown to exist between CD164+ hHSCs and these CD164+ hSSCs11.Just over two decades ago, human (h) CD164 was identified as a functional biomarker on human haematopoietic precursors and their associated bone marrow stromal cells5, namely the choice of hCD164 antibody used for these purposes. As described below, at least three epitopes defined by three distinct Classes of hCD164 antibodies (Mabs) exist on hCD164 and representative of each class do not stain cells equivalently.While it is gratifying to see hCD164 identified as a potentially improved biomarker for hHSPC and hSSC isolation, we wish to highlight a critical issue not addressed in the three recent manuscripts cited abovehCD164 was cloned from a bone marrow stromal cDNA expression library using two Mabs, 103B2/9E10 and 105A55, and was demonstrated to be partially homologous to the MGC-24 cDNA12. The hCD164 gene spans at least 22 kb of genomic DNA, is located on chromosome 6q21, comprises six exons (E1–6) interspersed with five introns, and encodes a sialomucin15. Its extracellular domain comprises two highly O-glycosylated mucin domains interrupted by a cysteine-rich non-mucin domain17. Four splice variants exist, three involving differential splicing of complete exons and one involving splicing in the 3′ UTR17. The former comprises hCD164(E1–6) containing all six exon-encoded domains, hCD164(EΔ5) lacking the exon 5-encoded domain and hCD164(EΔ4) lacking the exon-4-encoded domain, and comprising the respective 197, 178, and 184 amino acid polypeptides  is located in its non-mucin domain , association with other components  or the existence of an hCD164 tetrameric form may increase the molecular weight to >220 kD17.The full length hCD164(E1–6) isoform is predicted to contain 9 N-linked and 32 O-linked glycans, and a glycosaminoglycan (GAG) attachment site at the end of E5 and beginning of E6ain Fig. . Figure 23. While our initial studies defined the epitope reactivity of four Mabs, 103B2/9E10, 105A5, N6B6, and 67D2 with the hCD164 sialomucin, our further studies identified an additional five hCD164 Mabs, 96.1H5, 96.2D2, 96.3F5, 96.10H10, and 96.12H11, which had been produced against hCD34+ cells17. Cross‐competition experiments between these different hCD164 Mabs indicated that the 105A5 or 103B2/9E10 Mabs were not in hibited in their binding to the hCD164+ hHSPC line KG1A by each other nor by any of the other hCD164 Mabs. Conversely, the N6B6, 67D2, 96.1H5, 96.2D2, 96.3F5, 96.10H10, and 96.12H11 Mabs all cross competed with each other suggesting that they recognise very similar or identical epitopes17.Nine Mabs  of chimaeric recombinant domain-truncation constructs17. These latter studies revealed that hCD164 contains sialic acid moieties in both α2-3 and α2-6 linkages on O-glycans and N-glycans, but with O-glycans having a much higher degree of sialylation. Sialyl LewisX (sLeX) was not detected17. The N-glycans are more complex than the O-glycans, with high mannose, hybrid and mono-antennary, di-antennary, tri-antennary, and tetra-antennary complex forms (with or without bisecting GlcNAC). Most O-glycans are small core 1 or 2. Predominantly large neutral tri-antennary and tetra-antennary structures occur on N-glycans in mucin domain II, while smaller, bi-antennary structures are present in mucin domain I17.Given this, we examined the glycosidase sensitivities of the cell surface hCD164 sialomucin and subsequently of recombinant soluble chimaeric domain-truncation hCD164-Fc/hCD164-His-tagged constructs17. The N6B6, 67D2 and 96 series Mabs reacted with the hCD164E1‐3‐Fc, hCD164E1‐4‐Fc, hCD164E1‐5‐Fc, hCD164E1‐6′‐Fc and hCD164EΔ5‐Fc proteins, but not the hCD164E1‐Fc, hCD164E1‐2‐Fc, hCD164E1,2,4‐Fc, and hCD164E1,3,4‐Fc constructs, demonstrating that they require at least exons 2 and 3 for epitope recognition17. Notably however, none of these Mabs distinguish between the four known hCD164 splice variants17.Using a set of nine soluble hCD164 domain truncation mutants comprising different exons (E) but lacking the transmembrane region, namely hCD164E1‐Fc, hCD164E1‐2‐Fc, hCD164E1‐3‐Fc, hCD164E1‐4‐Fc, hCD164E1‐5‐Fc, hCD164EΔ5‐Fc, hCD164E1‐6′‐Fc, hCD164E1,2,4‐Fc, and hCD164E1,3,4‐Fc, we demonstrated that the 103B2/9E10 and 105A5 Mabs recognised all nine soluble proteins, indicating that they react with the region encoded by exon 1N-glycanase, O-glycosidase, sialidase, α-fucosidase, and O-sialoglycoprotease treatments, either separately or together , like their Class I and II counterparts, identify hCD164 monomers or homodimers, while the Class IIIB antibodies  also bind to a > 220 kD hCD164 protein species17. As indicated earlier, the latter may be an hCD164 tetramer, a hCD164 monomer or homodimer in association with other, possibly cytoskeletal or growth factor, components, or a GAG containing hCD164 species, an issue not yet resolved -Fc soluble protein and hCD164 purified from KG1A cells using her Fig. 13–17. Th31 revealed their expression on CD45+CD34+ hHSPCs throughout ontogeny. Notably, they are present on CD34+ clusters located on the ventral floor of the dorsal aorta in the week 5–6 developing human embryo, and on a proportion of CD34+ cells from foetal liver, cord blood, bone marrow, and mobilised peripheral blood23. Furthermore, LinnegCD34lo/neg or CD34int+extnegCD38lo/neg, CD133hiCD34hiCD38lo/neg, and CD133hiCD34lo/neg repopulating HSPCs express abundant hCD164, of which the CD34+CD164hi HSPCs expand more in ex vivo cultures than bulk CD34+ HSPCs31. Recent studies by Pellin’s group used the Class IIIB 67D2 Mab to demonstrate that CD34+CD164hi, but not CD34+CD164lo, cells sustained early and late in vivo human haematopoietic reconstitution in NBSGW immunodeficient mice6. Importantly, hCD164 is also a biomarker for hMSCs/hSSCs, including CD271hiCD45lo/neg and CD56+ or CD56neg primary bone marrow hMSC26. Notably, the Class IIIB Mab (67D2) has been used to identify and isolate PDPN+CD146neg CD73+CD164+ self-renewing, serially transplantable hSSCs with multipotent  potential from the human bone growth plate and diaphyseal zones7. Significantly, these hSSCs exist at the apex of the human skeletogenic differentiation hierarchy, are present in human foetal and adult bones, can be generated from BMP-2 stimulated human adipose MSCs and iPSCs, expand locally following acute skeletal injury and maintain human haematopoiesis11.Assessment of the cellular distribution of Class I and II hCD164 epitopes+ and hCD133+ hHSPC subsets and on hBM MSCs, this is in direct contrast to their differential expression in other postnatal haematopoietic and non-haematopoietic tissues23. We observed that the epitopes detected by the Class I and II Mabs can be differentially expressed and distributed on reciprocal cell subsets in some tissues, while the Class III Mabs stained cells that bound both or either of the Class I and/or II hCD164 Mabs23. For example, the epitope recognised by the 103B2/9E10 Class II Mab, but not that by the 105A5 Class I Mab, is present on most vascular endothelium, on some high endothelial venules in lymphoid tissues, on the venous sinuses in spleen, on the thymic subcapsular epithelia which is associated with pre-T lymphoid cell trafficking and on the basal-layer epithelia of the tonsil and skin. In contrast, the epitope recognised by the 105A5 Class I Mab is present on lymphoid cells in tissues that are involved in lymphoid/blood cell recirculation, such as in the tonsil, spleen, and gut23. However, the CD1+ and cortical and medullary CD43+ thymic lymphoid cells stain weakly with the Class I, but not at all with the Class II, hCD164 Mabs, while thymic macrophages express both the hCD164 epitopes defined by the Class I and II Mabs23.While all the hCD164 epitopes described to date are present on hCD34+ or CD133+hHSPC proliferation, differentiation, adhesion to, and retention or migration in different microenvironmental niches. As a first exemplar, the hCD164 103B2/9E10 Class II Mab-defined epitope partially blocks adhesion of CD34+ and CD133+ hHSPCs to bone marrow MSCs31. Ligating this epitope with the 103B2/9E10 Class II Mab also inhibits cytokine-driven  recruitment of single CD34+CD38lo/neg hHSPCs into the cell cycle in vitro14. Such effects are indicative of cognate ligands for hCD164 acting in cis or trans, albeit not yet fully defined. Our findings show that hCD164 lacks sLeX moieties required for P-selectin or E-selectin binding, while its sialylation in the absence of fucosylation suggests that hCD164 is a potential Siglec ligand, with preliminary studies indicating hSiglec-5  binding17. The second example demonstrates that culturing hBM CD34+ cells with IL-1β, IL-3, IL-6, G-CSF, GM-CSF, and SCF for 21 days in the presence of the Class I 105A5 or Class II 103B2/9E10 hCD164 Mabs reduces the absolute number of both total nucleated cells generated, and, when erythropoietin is added as well, decreases the development of clonogenic BFU-E and CFU-GM15. Furthermore, hCD164 is a key component of the CXCR4-VLA-4-VLA-5 complex, regulating CD133+ hHSPC CXCL12-mediated migration or retention in the hHSC niche and promoting CXCR4-mediated signalling, a process inhibited by the 103B2/9E10 Class II hCD164 Mab31. Although functional effects of engaging the hCD164 epitopes with Class III Mabs have not been fully assessed, the full length hCD164 contains a potential cytokine binding pocket located in its non-mucin domain, as well as a potential GAG attachment site at the E5-E6 junction of hCD16417, that could act in concert to modulate cytokine-mediated, chemokine-mediated, or adhesion-mediated effects on the same or opposing cells. While this is purely hypothelical, it is important to keep in mind that these and other Mabs chosen to isolate hHSCs might inadvertently affect the function or fate of the hHSCs when assessed in vitro or in vivo.The epitopes defined by the different Classes of hCD164 Mabs are implicated in possessing functional roles in haematopoiesis as illustrated by in vitro studies or based on hCD164 structural studies. Engaging the hCD164 molecule with specific hCD164 Mabs can modulate CD3436. These, when coupled with the increasingly sophisticated technological developments44 of cell sorting, surrogate in vivo models, gene editing, single cell barcoding, single cell deep sequencing, lineage tracing and fate mapping in vitro and in vivo, and human transplantation or regenerative medicine, have fuelled a series of debates on the hierarchical relationships of these human stem cells with their immediate progeny, and on selecting the best biomarkers and cell subsets for therapeutic use. To this end, biomarkers, lineage trees and lineage relationships have been constantly changing with progressive technological developments and research. Examples of these are indicated in the attached references62.Biomarkers that identify definitive, but rare, repopulating human bone marrow stem cells have been the “holy grail” of stem cell research since the seminal discovery of monoclonal antibodies by Koehler and Milstein over 5 decades ago62, and must not be hindered by the use of reagents that modulate hHSPC function in vivo. Hypotheses are hotly debated; the classical model describes a structured hierarchy of lineage commitment, with early branching of lymphoid and erythro-myeloid lineages. Other hypotheses include lineage-biased HSCs, early megakaryocyte lineage branching followed by erythroid, myeloid, and lymphoid lineage transitions, early branching of erythroid-megakaryocyte precursors from lymphoid–myeloid precursors, and a continuum of low-primed hHSPCs from which unilineage cells differentiate (CLOUD-HSPCs)62. Pellin et al.6 have stated that hCD164 can ‘preserve the resolution of the single-cell events of the more-primitive compartments, whereas at the same time maintaining a full representation of the late cell fate branching’, leading them to propose a modification of the classical hierarchical model, in which ‘human hematopoiesis develops along early cell fate bifurcations occurring in a continuum of states forming a hierarchical-like structure’ with early priming to either lymphoid/dendritic cell/monocyte/granulocyte or erythroid/megakaryocyte /basophil commitment.Deciphering lineage commitment processes with biomarkers is critical to our understanding of human haematopoiesis6 and Chan et al.7 used the 67D2 Class IIIB hCD164 antibody to identify hHSCs and hSSCs, different classes of hCD164 antibodies possess different avidities for this molecule and, by interfering with ligand binding sites, these may alter human hHSPC and hSSC function and hence fate. The information garnered from previous research should be carefully reviewed when considering the choice of hCD164 antibodies in future clinical studies. This said, Class II, and to a lesser extent, Class I hCD164 antibodies, with isotypes distinct from the Class III antibodies, may prove beneficial for confirming the purity of and hence establishing release criteria for the hCD164 Class III-enriched hHSC and hSSC subsets for in vivo therapy.When defining lineage hierarchies and developing new cellular products with hCD164 as a biomarker, it is important to remember that, while Pellin et al.64.In conclusion, using this knowledge in association with new cellular therapies will undoubtedly contribute to successful treatments for life threatening and chronic diseases, which are substantial burdens on both patient quality of life and healthcare systems globally


Clear cell renal cell carcinoma (ccRCC) is the most common malignant type of kidney cancer. The present study aims to explore the underlying mechanism and potential targets of the traditional Chinese medicine Bu-Shen-Jian-Pi-Fang (BSJPF) in the treatment of ccRCC based on network pharmacology. After obtaining the complete composition information for BSJPF from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, we analyzed its chemical composition and molecular targets and then established a pharmacological interaction network. Twenty-four significantly differentially expressed genes and nine pathways mainly related to tumor proliferation were identified and screened. Functional enrichment analysis indicated that the potential targets might be significantly involved in glycolysis and the HIF-1 signaling pathway. To further confirm the effect of BSJPF on ccRCC cell proliferation, a BALB/c xenograft mouse model was constructed. Potential targets involved in regulating glycolysis and the tumor immune microenvironment were evaluated using RT-qPCR. VEGF-A expression levels were markedly decreased, and heparin binding-EGF expression was increased in the BSJPF group. BSJPF also inhibited tumor proliferation by enhancing GLUT1- and LDHA-related glycolysis and the expression of the immune checkpoint molecules PD-L1 and CTLA-4, thereby altering the immune-rejection status of the tumor microenvironment. In summary, the present study demonstrated that the mechanism of BSJPF involves multiple targets and signaling pathways related to tumorigenesis and glycolysis metabolism in ccRCC. Our research provides a novel theoretical basis for the treatment of tumors with traditional Chinese medicine and new strategies for immunotherapy in ccRCC patients. With the growth of the economy and improvements in living standards, the prevalence and mortality rates of kidney cancer are continuously increasing. Currently, kidney cancer accounts for ∼4% of all cancers worldwide. In recent years, there have been approximately 400,000 new kidney cancer cases and 180,000 related deaths per year ,3. ClearCoptis chinensis, Ligustrum, Curcuma and Coix Seed. Previous studies have shown that some components in BSJPF play a role in inhibiting tumor growth [Recently, traditional Chinese medicine prescriptions have been widely used to treat intractable diseases, such as tumors, diabetes, arthritis and others . Bu-Shenr growth . Interesr growth .To identify novel ccRCC treatment strategies, we investigated whether traditional Chinese medicine could inhibit the invasion and growth of ccRCC. To this end, we analyzed the key molecular targets and signaling pathways of BSJPF to clarify the mechanism underlying its specific biological effects and guide the clinical treatment of ccRCC. The present study aimed to explore the multi-component, multi-target and multi-pathway interactions and regulatory networks by which BSJPF inhibits ccRCC cell growth, induces cell cycle arrest and increases apoptosis based on network pharmacological analysis techniques. We hypothesized that an in-depth investigation of the underlying mechanism and potential targets of BSJPF could provide valuable information for the development of new effective drugs and strategies for the clinical treatment of ccRCC.http://tcmspw.com/tcmsp.php) [https://www.ncbi.nlm.nih.gov/geo). Gene enrichment analysis data were obtained from the Bioinformatics Annotation Database .The composition and molecular target data of BSJPF were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (msp.php) , and theWe compared the expression of genes in ccRCC cells and datasets with normal kidney tissue expression. In addition, we only selected datasets containing more than 20 samples. We screened the titles and abstracts of these datasets. Finally, only 44 datasets in GSE105261 were selected for further study. The GSE105261 dataset based on the GPL10558 platform was downloaded from the National Biotechnology Information Center and included 9 normal kidney tissues and 35 tumor samples.Cytoscape software (version 3.7.2)  was usedUsing the Cytoscape plug-in CytoNCA , we perfWe defined the tightly connected regions of molecular complexes in the obtained PPI network as topology modules with network characteristics ,21. The P value less than 0.05 was considered statistically significant. The ggplot2 plug-in package of R software (version 3.6.2) was used to visualize the above data.We performed three biological analyses of the screened genes by GO analysis , includihttp://kmplot.com/analysis/) was utilized to perform survival analysis to identify the prognostic values of hub genes for BSJPF in ccRCC patients. Each survival analysis was implemented, all possible cutoff values between the lower and upper quartiles are computed, and the best performing threshold is used as a best cutoff in this study.A total of 530 ccRCC patients from the TCGA database with available mRNA sequence and survival outcome data were included. Kaplan–Meier Plotter  premixed with Matrigel at a ratio of 1:2 was subcutaneously injected into the right rear back region. When tumors reached 120 mm3, mice were randomized into two groups (n=5 each), treated with a decoction of BSJPF or normal feeding and monitored for tumor size and volume weekly. Then, BALB/c mice were anesthetized with 2,2,2-Tribromoethanol  through intraperitoneal injection into the lower-right quadrant of the abdomen. The onset of anesthesia  was defined as the time between the disappearance of the conditioned reflex and the recovery. After 6 weeks, the mice were killed using spondylolisthesis. All animal experiment procedures were carried out in the Medical Institution of Fudan University Shanghai Cancer Center  and approved by the Animal Care and Use Committee (No. 2008222-xp49).For Total RNA was isolated after 3 days of culture using TRIzol reagent  in accordance with the manufacturer’s instructions. Potential targets involved in regulating glycolysis and the tumor immune microenvironment were detected. Significant hub genes and RT-qPCR reactions were carried out as previously described . The mRNt-test or one-way ANOVA was used to measure differences between groups. All tests were two-sided, and P values less than 0.05 were considered significant.In the present study, R (Version 3.3.2), RStudio (Version 1.2) and GraphPad Prism (Version 8.0) were used to perform most data analyses. The Kaplan–Meier method with the 95%CI and log-rank test were used in separate survival curves. A two-tailed Student’s Our research was divided into three sequential stages, as illustrated in NDUFA4L2, FABP7 and CA9 showed the highest up-regulation. AQP2, UMOD and FXYD4 were the most down-regulated genes. We generated a heat map  and 24 gene nodes  and treated with a decoction of BSJPF or normal feeding. Tumor size and volume were significantly decreased in the BSJPF group compared with the normal control group (To further confirm the effect of BSJPF on ccRCC cell proliferation, a xenograft mouse model was constructed by subcutaneously injecting nude mice with RENCA cells. When tumors reached 120 mmol group A–C. To dol group D. Importol group E. Intereol group F. TherefIn the present study, we first screened the target genes related to ccRCC and identified the genes that affect the occurrence, development and prognosis of ccRCC. Then, the active ingredients and potential targets of BSJPF that may affect prognosis were explored using online pharmacology databases. Our research lays the foundation for further research on the treatment of ccRCC with Chinese medicine.In the past, scientists have explored the potential targets of various drugs and verified these candidates in basic science experiments or clinical trials . HoweverP value of each enrichment pathway and its relationship with ccRCC, we identified nine relatively important related signaling pathways, which are closely associated with tumor proliferation and invasion.Studies conducted in Asia have shown that some components of BSJPF play an important role in the treatment of renal disease. For example, BSJPF could provide nutrients and improve the circulatory system of the kidney . ResearcTumors maintain the balance of energy supply and demand through a metabolic reprogramming mode that is different from normal tissues, thereby protecting their own survival. The occurrence and development of ccRCC often involve changes in a wide range of metabolic pathways, such as glycolysis, oxidative phosphorylation and hypoxia. The proliferation, metastasis and other activities of tumor cells require glucose decomposition for energy. In the present study, potential targets of BSJPF involved in glycolysis and the tumor immune microenvironment were evaluated. The expression levels of VEGF-A were markedly decreased, and heparin binding-EGF expression was increased in the BSJPF group. Importantly, this suggested that BSJPF could inhibit tumor proliferation by enhancing GLUT1- and LDHA-related glycolysis. The HIF-1 signaling pathway is regulated by Von Hippel–Lindau tumor suppressor protein and induces glucose metabolism, cell proliferation and angiogenesis, thereby playing a significant role in ccRCC progression . This rein silico and based on complex bioinformatics methods. Second, experimental sorting was not as generally expected. The present study failed to perform fundamental experiments with BSJPF and did not investigate the interactions among all potential targets. Third, the present study did not provide sufficient clinical evidence to prove the effects of BSJPF on the recurrence and long-term survival of ccRCC patients, and there is no real-world verification of the identified biomarkers.There are some limitations to this research. First, most experiments were performed In summary, the present study demonstrated that the mechanism of BSJPF involves multiple targets and signaling pathways related to tumorigenesis and glycolysis metabolism in ccRCC. Our research provides a novel theoretical basis for the treatment of tumors with traditional Chinese medicine and new strategies for immunotherapy in ccRCC patients.Click here for additional data file.


Fibroblasts are the essential cell type of skin, highly involved in the wound regeneration process. In this study, we sought to screen out the novel genes which act important roles in diabetic fibroblasts through bioinformatic methods. A total of 811 and 490 differentially expressed genes (DEGs) between diabetic and normal fibroblasts were screened out in GSE49566 and GSE78891, respectively. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways involved in type 2 diabetes were retrieved from miRWalk. Consequently, the integrated bioinformatic analyses revealed the shared KEGG pathways between DEG-identified and diabetes-related pathways were functionally enriched in the MAPK signaling pathway, and the MAPKAPK3, HSPA2, TGFBR1, and p53 signaling pathways were involved. Finally, ETV4 and NPE2 were identified as the targeted transcript factors of MAPKAPK3, HSPA2, and TGFBR1. Our findings may throw novel sight in elucidating the molecular mechanisms of fibroblast pathologies in patients with diabetic wounds and targeting new factors to advance diabetic wound treatment in clinic. As the global population ages, the incidence of diabetes is rapidly increasing during recent decades . DiabetiFibroblasts are the essential cell type of skin, highly involved in the wound regeneration process, and acted in wound healing by interacting with other cells including keratinocytes and endothelial cells . ExosomaIn this study, we sought to identify the DEG modulation in diabetic fibroblasts by using bioinformatic methods. These findings may provide useful insights into understanding the molecular mechanisms of fibroblast pathologies in patients with DFUs.http://www.ncbi.nlm.nih.gov/geo). DEGs were performed by Limma in R, and p values < 0.05 were considered as statistically significant. R package pheatmap was used to visualize Log2 mRNA gene expression. Using Circlize and ComplexHeatmap in R, common DEGs from different datasets were identified and visualized. The circular visualization of chromosomal information of common DEGs was achieved with circular visualization in R.Microarray data of datasets comparing diabetes and the healthy controls were screened out from the Gene Expression Omnibus database  to construct the PPI network. Then, the TSV file of PPI network was imported into Cytoscape 3.7.2. The interactions between enriched KEGG pathways were calculated and visualized by Cytoscape 3.7.2.p ≤ 0.05) and type 2 diabetes-related KEGG pathways was obtained with Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/). The top shared KEGG pathway with the smallest p value was selected. The enriched DEG-related part of the KEGG pathway was established with the PPT drawing tool.miRWalk is an online bioinformatics atlas tool. In this study, the KEGG pathways involved in type 2 diabetes were retrieved from miRWalk. Then, the intersection of enriched KEGG pathways  and the results suggested that ETV4 and NPE2 were the potential transcript factors for these genes. Thus, it was assumed that ETV4 and NPE2 may exert a critical role in the regulation of diabetic wound healing.Phosphorylation of transcription is one of the modifications of MAPK-dependent regulation in cellular responses . Three s pathway . Similar pathway . In the Some limitations also existed in this bioinformatic research. First, the current results were based on a public database and only two datasets were included in our study; the sample size should be enlarged to minimize the possible confounding factors. Furthermore, this is a pure bioinformatic research; more experimental validation is needed to confirm the candidate pathways and their potential transcript factors. Moreover, clinical specimens of different degrees of DFUs should be collected to validate our current findings.Our findings suggested a functionally enriched MAPK signaling pathway, with a focus on the potential role of ETV4 and NPE2 in the regulation of diabetic wound regeneration. The current study may provide novel therapeutic targets in diabetic wound treatment.