Combinatorial Chemistry & High Throughput Screening - Volume 25, Issue 8, 2022

Background: Traditional Chinese medicine has accumulated rich resources and experience through clinical research to explore the prevention and treatment of chronic cerebral circulatory insufficiency, but current medicine lacks in-depth research and confirmation on the established protocols and mechanism of prescribed TCMs at the macro and micro levels. Objective: To explore the prescription of Chinese medicines for the treatment of Chronic Cerebral Circulation Insufficiency (CCCI) and to explore the mechanism of core drugs. Methods: 229 Chinese prescriptions for CCCI were collected from CNKI, CBM, VIP and WANFANG databases for this study. The frequency and association rules of drugs were analyzed and the core drugs by TCMISSV2.5 software was extracted. The active ingredients and targets were obtained by TCMSP, and genes of CCCI were collected from the DisGeNET, OMIM, DrugBank disease databases. The intersection targets of herbal medicine and disease were imported into the STRING database for PPI network. The key targets were screened by the network topology algorithm. The Systems Dock website was used to verify the molecular docking. The GOEAST and DAVID tools were used to perform GO and KEGG pathway analysis with the key target genes. Results: 117 drugs involved in 229 prescriptions were identified, 2 core drugs were identified. We identified 8 active ingredients, which were mandenol, myricanone, perlolyrine, senkyunone, wallichilide, sitosterol, beta-sitosterol and stigmasterol. 371 herbal targets predicted and 335 disease targets. The enrichment analysis showed that the core herbal medicines could prevent CCCI by 15 key signaling pathways. Conclusion: There are direct or indirect connections in key signaling pathways, which not only participate in energy metabolism, hormone regulation, signal transduction, but also play a role in the comprehensive intervention of nervous system, immune system, circulatory system and other systems, which is consistent with the comprehensive pathogenesis of CCCI induced by multiple factors.

Background: Epithelial-mesenchymal transformation (EMT) promotes cancer metastasis, including hepatocellular carcinoma. Therefore, EMT-related gene signature was explored. Objective: The present study was designed to develop an EMT-related gene signature for predicting the prognosis of patients with hepatocellular carcinoma.. Methods: An integrated gene expression analysis based on tumor data of the patients with hepatocellular carcinoma from The Cancer Genome Atlas (TCGA), HCCDB18, and GSE14520 dataset was conducted. An EMT-related gene signature was constructed by the least absolute shrinkage and selection operator (LASSO) and COX regression analysis of univariate and multivariate survival. Results: A 3-EMT gene signature was developed and validated based on gene expression profiles of hepatocellular carcinoma from three microarray platforms. Patients with a high-risk score had significantly worse overall survival (OS) than those with low-risk scores. The EMT-related gene signature showed a high performance in accurately predicting prognosis and examining the clinical characteristics and immune score analysis. Univariate and multivariate Cox regression analyses confirmed that the EMT-related gene signature was an independent prognostic factor for predicting survival in hepatocellular carcinoma patients. Compared with the existing models, our EMTrelated gene signature reached a higher area under the curve (AUC). Conclusion: Our findings provide novel insight into understanding EMT and help identify hepatocellular carcinoma patients with poor prognosis.

Aims and Objective: Wedelolactone and demethylwedelolactone are the two major coumarin constituents of Herba Ecliptae. The objective of this work was to develop and validate a sensitive, rapid, and robust UPLC-MS/MS method for the simultaneous quantification of wedelolactone and demethylwedelolactone in rat plasma. Materials and Methods: Wedelolactone and demethylwedelolactone were extracted from rat plasma by protein precipitation with acetonitrile. Electrospray ionization in negative mode and selected reaction monitoring (SRM) were used for wedelolactone and demethylwedelolactone at the transitions m/z 312.8→298.0 and m/z 299.1→270.6, respectively. Chromatographic separation was conducted on a Venusil C18 column (50 mm × 2.1 mm, 5 μm) with isocratic elution of acetonitrile-0.1% formic acid in water (55:45, v/v) at a flow rate of 0.3 mL/min. A linear range was observed over the concentration range of 0.25-100 ng/mL for wedelolactone and demethylwedelolactone. Results: They reached their maximum plasma concentrations (Cmax, 74.9±13.4 ng/mL for wedelolactone and 41.3±9.57 ng/mL for demethylwedelolactone) at the peak time (Tmax) of 0.633 h and 0.800 h, respectively. The AUC0-t value of wedelolactone (260.8±141.8 ng h/mL) was higher than that of demethylwedelolactone (127.4±52.7 ng h/mL) by approximately 2-fold, whereas the terminal elimination half-life (t1/2) of wedelolactone (2.20±0.59 h) showed the approximately same as that of demethylwedelolactone (2.08±0.69 h). Conclusion: Based on full validation according to US FDA guidelines, this UPLC-MS/MS method was successfully applied to a pharmacokinetic study in rats.

Background: There is a possible relation between red blood cell distribution width (RDW) and various clinical conditions. These conditions can render RDW disadvantageous in its relation with cardiovascular disease. There may be a relation between the severity of acute coronary syndrome (ACS) and the percentage of hypochromia (hypo%), percentage of hyperchromia (hyper%), percentage of macrocytosis (MAC%), and percentage of microcytosis (MIC%) values measured using new-generation hematological devices. Objective: We aimed to examine the relation between the SYNTAX score and the hypo%, hyper%, MAC%, and MIC% values in patients admitted with ACS. Methods: A group of 55 patients who underwent coronary angiography with diagnosis of ACS (STEMI and NSTEMI) and a control group of 48 patients with normal coronary arteries were included in the study. Venous blood samples were collected in the morning after a fasting of at least 8 h and analyzed using standard laboratory methods. Hemogram parameters were studied using Alinity HQ (Abbott, USA) a completely automated hemogram autoanalyzer. Biochemical parameters were studied using Architect c16000 (Abbott, USA) a completely automated biochemistry autoanalyzer. Results: Significant difference was observed in erythrocyte morphology-related tests (mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, RDW, hypo%, hyper%, MIC%, and MAC%) between the groups. Correlation analysis showed a positive correlation between the SYNTAX score and MAC% (r = 0.315, p = 0.019). Multivariate logistic regression analysis was performed for MAC% to identify the independent predictors of the SYNTAX score (β = 0.315, p = 0.019). Conclusion: Changes in MAC% test can be measured in emergencies with new-generation hematological devices and used as independent predictors of the presence of severe coronary artery disease.

Background: Anal fistula is one of the most common colorectal and perirectal diseases in the world. Cuyuxunxi (CYXX) prescription is an efficient herbal fumigant used to promote the surgical wound healing of anal fistulas. Objectives: This study aimed to explore the underlying molecular mechanism of CYXX prescription on surgical wound healing of anal fistulas. Methods: Ten patients with anal fistula were randomized into a control group or treatment group. The wound surface of patients in the control group was rinsed by normal saline, while that in the treatment group was rinsed by CYXX prescription. The wound tissues of patients with anal fistulas seven days after the surgery were collected for hematoxylin-eosin (HE) staining and RNA sequencing. The expressions of differentially expressed genes (DEGs) were validated by real-time quantitative PCR (RT-qPCR). Results: HE staining showed that CYXX treatment reduced the infiltration of inflammatory cells. A total of 472 DEGs, including 141 up-regulated genes and 331 down-regulated genes, were identified. These genes were significantly related to skin development, xenobiotic stimulus, and inflammation. In addition, the consistency rate of RT-qPCR and sequencing results was 83.33%, which showed a high relative reliability of the sequencing results. Conclusion: CYXX prescription could improve epidermis repair and reduce inflammatory responses.

Aims and Objectives: Fructose, as a ubiquitous monosaccharide, can promote ATP consumption and elevate circulating Uric Acid (UA) levels. Our previous studies have confirmed that the macroporous resin extract of Dendrobium officinale leaves (DoMRE) could reduce the UA level of rats with hyperuricemia induced by a high-purine diet. This study aimed to investigate whether DoMRE had a UA-lowering effect on rats with hyperuricemia caused by fructose combined with potassium oxonate, so as to further clarify the UA-lowering effect of DoMRE, and to explore the UAlowering effect of DoMRE on both UA production and excretion. Materials and Methods: Rats with hyperuricemia induced by fructose and potassium oxonate were administered with DoMRE and vehicle control, respectively, to compare the effects of the drugs. At the end of the experiment, the Serum Uric Acid (SUA) and Creatinine (Cr) levels were measured using an automatic biochemical analyzer, the activities of xanthine oxidase (XOD) were measured using an assay kit, and the protein expressions of Urate Transporter 1 (URAT1), glucose transporter 9 (GLUT9), and ATP-Binding Cassette Superfamily G member 2 (ABCG2) were assessed using immune-histochemical and western blot analyses. Hematoxylin and eosin staining was used to assess the histological changes in the kidney, liver, and intestine. Results: Fructose and potassium induced hyperuricemia in rats. Meanwhile, the activities of XOD were markedly augmented, the expression of URAT1 and GLUT9 was promoted, and the expression of ABCG2 was reduced, which were conducive to the elevation of UA. However, exposure to DoMRE reversed these fructose- and potassium oxonate-induced negative alternations in rats. The activities of XOD were recovered to the normal level, reducing UA formation; the expressions of URAT1, ABCG2, and GLUT9 returned to the normal level, resulting in an increase in renal urate excretion. Conclusion: DoMRE reduces UA levels in rats with hyperuricemia induced by fructose combined with potassium oxonate by inhibiting XOD activity and regulating the expression of ABCG2, URAT1, and GLUT9. DoMRE is a potential therapeutic agent for treating hyperuricemia through inhibiting UA formation and promoting UA excretion.

Background: Polycystic ovary syndrome (PCOS) is a common endocrine disease in women that seriously interferes with patient's metabolic and reproductive functions. The current diagnostic criteria for PCOS are expert-based and still disputed. Previous studies have identified changes in DNA methylation in peripheral blood of women with PCOS, but their diagnostic potential for PCOS remains to be studied. Objective: The present study aimed to identify potential methylation biomarkers for the diagnosis of PCOS in blood. Methods: Methylation profiling of peripheral blood was downloaded from a public database, Gene Expression Omnibus (GEO), including 30 PCOS patients (diagnosed with the revised 2003 Rotterdam consensus criteria) and 30 age-matched healthy women recruited from Centre of Reproductive Medicine, Linyi People’s Hospital, Shandong, China. Weighted gene co-expression network analysis (WGCNA) was utilized to identify PCOS-related co-methylation CpG sites (co- MPs). Functional enrichment analysis was performed on the localized genes of PCOS-related co- MPs. The least absolute shrinkage and selection operator (LASSO) regression was used to screen out CpG methylation signatures for PCOS diagnosis, and receiver operating characteristic (ROC) analysis was conducted to evaluate their diagnostic accuracy. To assess the accuracy of the combination of the investigated indicators, multivariate ROC analysis was performed on the predicted probability values obtained using binary logistic regression on the methylation levels of selected CpGs. Results: Seven co-methylation modules were obtained, among which the turquoise module is the most relevant to PCOS, containing 194 co-MPs. The genes that these co-MPs located in were mainly associated with the immune-related pathway. According to LASSO regression, three Co- MPs (cg23464743, cg06834912, cg00103771) were identified as potential diagnostic biomarkers of PCOS. ROC analysis showed an AUC (area under curve) of 0.7556 (sensitivity 60.0%, specificity 83.3%) for cg23464743, 0.7822 (sensitivity 70.0%, specificity 80.0%) for cg06834912, and 0.7611 (sensitivity 63.3%, specificity 83.3%) for cg00103771. The diagnostic accuracy of the combination of these 3 indicators presented to be higher than any single one of them, with the AUC of 0.8378 (sensitivity 73.3%, specificity 93.3%). Conclusion: The combination of 3 CpG methylation signatures in blood was identified with a good diagnostic accuracy for PCOS, which may bring new insight into the development of PCOS diagnostic markers in the future.

Background: Chalcones with methoxy substituent are considered as a promising framework for the inhibition of monoamine oxidase (MAO) enzymes. Methods: A series of nine trimethoxy substituted chalcones (TMa-TMi) was synthesized and evaluated as a multifunctional class of MAO inhibitors. All the synthesized compounds were investigated for their in vitro MAO inhibition, kinetics, reversibility, blood-brain barrier (BBB) permeation, and cytotoxicity and antioxidant potentials. Results: In the present study, compound (2E)-3-(4-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)prop- 2-en-1-one (TMf) was provided with a MAO-A inhibition constant value equal to 3.47±0.09 μM with a selectivity of 0.008, thus comparable to that of moclobemide, a well known potent hMAOA inhibitor (SI=0.010). Compound (2E)-3-(4-bromophenyl)-1-(3,4,5-trimethoxyphenyl)prop-2- en-1-one (TMh) show good MAO-B inhibition with inhibition constant of 0.46±0.009 μM. The PAMPA assay demonstrated that all the synthesized derivatives can cross the BBB successfully. The cytotoxicity studies revealed that TMf and TMh have 88.22 and 80.18 % cell viability at 25 μM. Compound TMf appeared as the most promising antioxidant molecule with IC50 values, relative to DPPH and H2O2 radical activities equal to 6.02±0.17 and 7.25±0.07 μM. To shed light on the molecular interactions of TMf and TMh towards MAO-A and MAO-B, molecular docking simulations and MM/GBSA calculations have been carried out. Conclusion: The lead molecules TMf and TMh with multi-functional nature can be further employed for the treatment of various neurodegenerative disorders and depressive states.

Background: The most prevalent malignant tumor in women is breast cancer (BC). As autophagic therapies have been identified to contribute to BC cell death, the potential prognostic role of long non-coding RNA (lncRNA) related to autophagy in patients with BC was examined. Methods: The lncRNAs expression profiles were derived from The Cancer Genome Atlas (TCGA) database. Throughout univariate Cox regression and multivariate Cox regression test, lncRNA with BC prognosis have been differentially presented. We then defined the optimal cut-off point between high and low-risk groups. The receiver operating characteristic (ROC) curves were drawn to test this signature. In order to examine possible signaling mechanisms linked to these lncRNAs, the Gene Set Enrichment Analysis (GSEA) has been carried out. Results: Based on the lncRNA expression profiles for BC, a 9 lncRNA signature associated with autophagy was developed. The optimal cut-off value for high-risk and low-risk groups was used. The high-risk group had less survival time than the low-risk group. The result of this lncRNA signature was highly sensitive and precise. GSEA study found that the gene sets have been greatly enriched in many cancer pathways. Conclusion: Our signature of 9 lncRNAs related to autophagy has prognostic value for BC, and these lncRNAs related to autophagy may play an important role in BC biology.

Background: Various phenolics show inhibitory activity towards xanthine oxidase (XO), an enzyme that generates reactive oxygen species which cause oxidative damage. Objective: This study investigated the XO inhibitory activity of Euphorbia peplus phenolics. Methods: The dried powdered aerial parts of E. peplus were extracted, fractioned and phenolics were isolated and identified. The XO inhibitory activity of E. peplus extract (EPE) and the isolated phenolics was investigated in vitro and in vivo. Results: Three phenolics were isolated from the ethyl acetate fraction of E. peplus. All isolated compounds and the EPE showed inhibitory activity towards XO in vitro. In hyperuricemic rats, EPE and the isolated phenolics decreased uric acid and XO activity. Molecular docking showed the binding modes of isolated phenolics with XO, depicting significant interactions with the active site amino acid residues. Molecular dynamics simulation trajectories confirmed the interaction of isolated phenolics with XO by forming hydrogen bonds with the active site residues. Also, the root mean square (RMS) deviations of XO and phenolics-XO complexes achieved equilibrium and fluctuated during the 10 ns MD simulations. The radius of gyration and solvent accessible surface area investigations showed that different systems were stabilized at ≈ 2500 ps. The RMS fluctuations profile depicted that the drug binding site exhibited a rigidity behavior during the simulation. Conclusion: In vitro, in vivo and computational investigations showed the XO inhibitory activity of E. peplus phenolics. These phenolics might represent promising candidates for the development of XO inhibitors.

Background: Abnormal expression of miRNA is a common feature in many diseases. Some studies have also emphasized that miRNAs play an important role in asthma and Allergic Rhinitis (AR). This study attempts to reveal the differences between miRNAs expression and normal nasal mucosa in AR patients by microarray method so as to further understand the molecular mechanism of AR development. Methods: MiRNA microarrays were used for analyzing six samples of the nasal mucosa of AR and six samples of nonallergic patients. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of some differently expressed miRNAs was used to confirm the array results. Furthermore, pathway analysis was carried out. Results: The microarray identified that 64 miRNAs showed altered expression in the nasal mucosa of the AR group when compared with the control group. Moreover, the expression levels of ten miRNAs were significantly altered in the AR group. To verify the results of microarray, three differentially expressed miRNA were determined by RT-PCR, and the results also confirmed these changes. Ten differentially expressed miRNAs were present in the nasal mucosa of AR patients compared with the control group, and three differentially expressed miRNAs as miR-1244, miR- 4651, and miR-7641 were determined by RT-PCR. The results also confirmed the changes, indicating that they play important roles in the process of AR. Conclusion: MiR-1244, miR-4651, and miR-7641 may play important roles in the process of AR. Sequencing analysis indicated that three kinds of mutations existed in MAPK8 3’UTR, which may play a role in binding with miR-7641, and then influence the AR process. Single miRNA or, more probably, their sets hold the promise for their use as biomarkers of allergic rhinitis. They are also a promising target of future therapies.

Background: Metabolic Syndrome (MetS) is defined by a clustering of metabolic abnormalities associated with an increased risk of cardiovascular disease and type 2 diabetes mellitus. There has been an increasing interest in the associations of genetic variant involved in diabetes and obesity in the FABP1 pathway. The relationship between the rs2241883 polymorphism of FABP1 and risk of MetS remains unclear. Objective: We aimed to examine the association between this genetic polymorphism and the presence of MetS and its constituent factors. Methods: A total of 942 participants were recruited as part of the Mashhad Stroke and Heart Atherosclerosis Disorders (MASHAD study) Cohort. Patients with MetS were identified using the International Diabetes Federation (IDF) criteria (n=406) and those without MetS (n=536) were also recruited. DNA was extracted from peripheral blood samples that was used for genotyping for the FABP1 rs2241883T/C polymorphism using Tetra-Amplification Refractory Mutation System Polymerase Chain Reaction (Tetra-ARMS PCR). Genetic analysis was confirmed by gel electrophoresis and DNA sequencing. Results: Using both univariate and multivariate analyses after adjusting for age, sex and physical activity, carriers of C allele (CT/CC genotypes) in FABP1 variant was related to an increased risk of MetS, compared to non-carriers (OR: 1.38, 95%CI: 1.04,1.82, p=0.026). Conclusion: The present study shows that C allele in FABP1 variant can be associated with an increased risk of MetS. The evaluation of these factors in a larger population may help further confirm these findings.

Background: Gastric Cancer (GC) remains a major global health problem due to a poor understanding of its progression at the molecular level and a lack of early detection or diagnosis. Early detection is highly crucial for improving prognosis. The incidence of GC is very high in countries, like India, due to the limitations among the established biomarkers for GC owing to poor sensitivity and specificity. Objective: This study aimed to identify the novel biomarkers from serum samples obtained from GC patients compared to healthy subjects. Methods: Serum samples from GC patients were analyzed by two-Dimensional Gel Electrophoresis (2DGE) coupled with tandem Mass Spectrometry (MS), including both Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) and Liquid Chromatography-MS (LC-MS/MS) analysis. Identified proteins were further analyzed by gene ontology and protein interaction studies. Results: A total of 73 protein spots were detected in 2DGE image analysis. Among them, seven differentially-expressed proteins were identified using MS analyses, including serotransferrin/ transferrin, albumin, ceruloplasmin, C-reactive protein (CRP), fibrinogen γ-chain (FGG), and two unreported novel proteins, immunoglobulin kappa constant (IgΚC) region and Homo sapiens zinc finger protein 28 (ZNF28) homolog. Among these proteins, serotransferrin, albumin, ceruloplasmin, FGG, and ZNF28 were down-regulated in GC samples (p<0.05), while IgΚC region and CRP were up-regulated significantly. Conclusion: Most of the differentially expressed proteins were involved in angiogenesis, plasminogen-activating cascade, and blood coagulation pathways which are known to play a critical role in gastric tumorigenesis. Our current results provide a panel of candidate biomarkers for GC with novel biomarkers which have not been reported earlier.

Background: Esophageal carcinoma (ESCA) is a malignant tumor with high invasiveness and mortality. Autophagy has multiple roles in the development of cancer; however, there are limited data on autophagy genes associated with long non-coding RNAs (lncRNAs) in ESCA. The purpose of this study was to screen potential diagnostic and prognostic molecules and to identify gene co-expression networks associated with autophagy in ESCA. Methods: We downloaded transcriptome expression profiles from The Cancer Genome Atlas and autophagy-related gene data from the Human Autophagy Database, and analyzed the co-expression of mRNAs and lncRNAs. In addition, the diagnostic and prognostic value of autophagy-related lncRNAs was analyzed by multivariate Cox regression. Furthermore, Kyoto Encyclopedia of Genes and Genomes analysis was carried out for high-risk patients, and enriched pathways were analyzed by gene set enrichment analysis. Results: The results showed that genes of high-risk patients were enriched in protein export and spliceosome. Based on Cox stepwise regression and survival analysis, we identified seven autophagy-related lncRNAs with prognostic and diagnostic value, with the potential to be used as a combination to predict the prognosis of patients with ESCA. Finally, a co-expression network related to autophagy was constructed. Conclusion: These results suggest that autophagy-related lncRNAs and the spliceosome play important parts in the pathogenesis of ESCA. Our findings provide new insight into the molecular mechanism of ESCA and suggest a new method for improving its treatment.

Background: Wilms Tumor (WT) is the most common primary renal malignancy in children. Autophagy plays dual roles in the promotion and suppression of various cancers. Objective: The goal of our study was to develop a novel autophagy-related gene (ARG) prognostic nomogram for WT. Methods: The Cancer Genome Atlas (TCGA) database was used. We screened the expression profiles of ARGs in 136 WT patients. The differentially expressed prognostic ARGs were evaluated by multivariate Cox regression analysis and survival analysis. A novel prognostic nomogram based on the ARGs and clinical characteristics was established using multivariate Cox regression analysis. Results: First, 69 differentially expressed ARGs were identified in WT patients. Then, multivariate Cox regression analysis was used to determine 4 key prognostic ARGs (CC3CL1, ERBB2, HIF-α and CXCR4) in WT. According to their ARG expression levels, the patients were clustered into high- and low-risk groups. Next, survival analysis indicated that high-risk patients had significantly poorer overall survival than low-risk patients. The results of functional enrichment analysis suggested that autophagy may play a tumor-suppressive role in the initiation of WT. Finally, a prognostic nomogram with a Harrell's concordance index (C-index) of 0.841 was used to predict the survival probability of WT patients by integrating clinical characteristics and the 4-ARG signature. The calibration curve indicated its excellent predictive performance. Conclusion: In summary, the ARG signature could be a promising biomarker for monitoring the outcomes of WT. We established a novel nomogram based on the ARG signature, which accurately predicts the overall survival of WT patients.