Combinatorial Chemistry & High Throughput Screening - Current Issue

The incidence of metabolic-associated fatty liver disease (MAFLD) increases annually. Modified Zexie Decoction (MZXD) can treat this disease; however, their mechanisms of action are uncertain. This study evaluated the mechanisms of MZXD against MAFLD based on network pharmacology, molecular docking, and in vivo experiments.

The main active compounds, targets and signaling pathways of MZXD against MAFLD were obtained using network pharmacological analysis. Underlying mechanisms were validated by molecular docking and in vivo assays.

Forty-one active ingredients and 197 intersection targets were identified. The main active ingredients include quercetin, luteolin, isorhamnetin, 3-methylhexane, and 3β-acetoxyatractylone. The main targets were TP53, JUN, HSP90AA1, MAPK1, MAPK3, AKT1, NF-κB p65, TNF, ESR1, FOS, and IL-6. The pathway enrichment analysis indicated that MZXD was related to the IL-17, TNF, and PI3K-AKT signaling pathways. Molecular docking suggested that these active ingredients bound strongly to TNF, IL-6, and NF-κB p65, which are integral components of the TNF pathway. In the rat MAFLD model, MZXD attenuated high-fat diet(HFD)-induced liver injury and lipid accumulation, decreased the serum levels of the inflammatory mediators TNF-α, IL6, and IL-1β, and inhibited the protein expression of TNF-α, IL6, p-IKB-α and p-NF-κB p65. Furthermore, immunohistochemistry results showed that MZXD attenuated the F4/80 staining intensity of the liver compared with the model group.

Collectively, our results suggested that MZXD could improve MAFLD by downregulating TNF/NF-κB signaling mediated macrophage activation.

A major factor in type 1 diabetes mortality is Ischemic Heart Disease (IHD). In order to treat IHD, blood flow must be restored to the heart, which results in myocardial ischemia-reperfusion (MI/R) damage. While rosmanol inhibits MI/R damage, its role in streptozotocin-MI/R (STZ-MI/R) injury remains unclear. Both microRNA (miR) 126 and the PI3K/AKT signaling pathway have been linked to preventing MI/R damage.

The aim of this investigation was to determine whether rosmanol decreases STZ-MI/R-injury in diabetic rats and whether the protective effect is associated with the miR-126-phosphatidylinositol 3-kinase (PI3K)/protein kinase B axis (miR-126-PI3K/AKT).

For 30 days, diabetic rats received either distilled water or the drug rosmanol (40 mg/kg, orally) before being subjected to MI/R.

The findings from the current study demonstrated how rosmanol reduced MI/R damage in rats with diabetes caused by streptozotocin (STZ). Using spectrophotometry, it was possible to measure the decrease in myocardial enzyme levels, the rise in cardiac viability, the inhibition of myocardial oxidative stress, the increase in cardiac function, and the detection of these changes using a hemodynamic monitoring system. In addition, rosmanol augmented the miR-126-PI3K/AKT in the hearts of ischemic rats. After stimulating the myocardial miR-126-PI3K/AKT axis, our results showed that rosmanol protected the heart against MI/R in STZ-induced diabetic rats.

According to the most recent research, rosmanol may be a useful tool in the therapy of diabetic IHD since it is an effective agent against STZ-MI/R damage.

Melatonin (MT) has been demonstrated to have cardioprotective effects. Nevertheless, the precise mechanism through which MT provides protection against the etiology of LPS-induced myocardial injury remains uncertain. In this investigation, our objective was to explore the impact of MT on LPS-induced myocardial injury in an in vitro setting.

H9C2 cells were categorized into four groups: a control group (H9C2 group), an MT group, an LPS group, and an MT + LPS group. The H9C2 group received treatment with sterile saline solution, the LPS group was exposed to 5 μg/mL LPS for 24 hours, the MT + LPS group underwent pretreatment with 150 μmol/L MT for 2 hours, followed by exposure to 5 μg/mL LPS for 24 hours, and the MT group received only 150 μmol/L MT for 2 hours. Cell viability and lactate dehydrogenase (LDH) release were assessed using the CCK-8 assay and LDH activity assay, respectively. The levels of reactive oxygen species (ROS) were quantified in each group of cells, and the percentage of propidium iodide (PI)-stained apoptotic cells was determined by flow cytometry. The mRNA levels of caspase11, GSDMD, and IL-18 in each group of cells were quantified.

MT treatment significantly protected H9C2 cells from LPS-induced damage, as evidenced by decreased LDH release. LPS treatment markedly increased ROS levels in H9C2 cells, which were subsequently reduced by MT. LPS caused a substantial decrease in superoxide dismutase (SOD) activity and a significant increase in malondialdehyde (MDA) levels, while MT treatment significantly reversed these effects. Additionally, MT markedly enhanced the proportion of viable H9C2 cells compared to LPS-treated controls, as evidenced by the PI staining assay. LPS upregulated both mRNA levels and protein levels of IL-18 in H9C2 cells. However, MT treatment effectively mitigated this LPS-induced increase. Furthermore, MT significantly decreased LPS-induced protein levels of cleaved-caspase 11 and GSDMD-N in H9C2 cells.

Overall, our findings suggest that MT inhibits the Caspase11-GSDMD signaling pathway via pyroptosis-related proteins (caspase-11 and GSDMD-N) and reduces the expression of inflammation-related cytokines (IL-18), thereby exerting a protective effect on H9C2 cells after LPS injury.

The establishment and validation of methods for testing biological samples are crucial steps in pharmacokinetic studies. Currently, several methodological reports have been published on the detection of rapamycin plasma concentrations.

The objective of this study was to explore an effective method for detecting rapamycin in rat whole blood biological samples.

In this study, we designed a rapid, sensitive, and specific liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) methodology for detecting rapamycin in rat whole blood biological samples. We comprehensively validated the specificity, linear range, lower limit of quantification (LLOQ), precision, accuracy, recovery, and stability of this method.

The findings of this study confirmed the successful implementation of LC-MS/MS for the detection of rapamycin, demonstrating its sensitivity, specificity, and reliability in quantitative analysis. This method ensures the accuracy and reliability of subsequent study data through our validated LC-MS/MS approach.

The results demonstrated the successful implementation of an LC-MS/MS method for sensitive, specific, and reliable quantitative analysis of rapamycin in rat whole blood samples. This method ensures the accuracy and reliability of subsequent study data.

The importance of this study lies in the successful establishment of a rapid, sensitive, and specific LC-MS/MS method for detecting rapamycin concentration in rat whole blood, ensuring the accuracy and reliability of subsequent research data. This provides a crucial tool and foundation for further understanding the metabolism and pharmacological effects of rapamycin in vivo, aiding in the advancement of drug research and clinical applications in related fields.

Precancerous Lesions of Gastric Cancer (PLGC) are critical in the secondary prevention of gastric cancer. Despite the notable effects of natural products on PLGC, the specific mechanisms by which compounds, like 6-gingerol, influence these lesions are not fully understood.

This study aimed to confirm the effect and mechanism of 6-gingerol in the treatment of precancerous lesions of gastric cancer (PLGC).

The objective of this study was to elucidate the effects and mechanisms of 6-gingerol against PLGC using network pharmacology and in vitro experiments.

We employed network pharmacology to identify potential targets and pathways influenced by 6-gingerol, followed by validation through in vitro experiments using a PLGC cell model.

Network pharmacology analysis highlighted the PI3K/Akt signaling pathway as significantly influenced by 6-gingerol. In vitro experiments confirmed that 6-gingerol effectively inhibited proliferation, invasion, and metastasis of MC cells, promoted apoptosis, and induced cell cycle arrest, primarily through modulation of the PI3K/Akt pathway. Statistical analysis revealed significant inhibition (p < 0.05) across these cellular processes in a dose-dependent manner.

This study demonstrated that 6-gingerol acts as an effective agent against PLGC, with clear dose-dependent effects that pave the way for further experimental and clinical exploration.

Cuproptosis, a novel form of cell death mediated by protein lipoylation, is intricately linked to mitochondrial metabolism. However, the clinical association of cuproptosis-related genes (CRGs) in thyroid cancer remains unclear. In this study, we performed a systematic investigation on the differential expression and genetic alterations of CRGs in papillary thyroid carcinoma (PTC) and constructed a CRG signature to predict the prognosis of PTC patients.

We integrated the data of The Cancer Genome Atlas (TCGA) database and analyzed the expression of 10 CRGs in PTC. CRG signature was constructed using univariate Cox regression and the least absolute shrinkage and selection operator (LASSO) Cox regression. In addition, the signature-related molecular features were validated by a combination of functional enrichment, Cox regression, and immune infiltration analysis. Independent validation cohort and quantitative real-time polymerase chain reaction (qRT-PCR) were used to validate the expression of differentially expressed CRG (CDKN2A).

Thyroid cancer patients could be divided into two subtypes (high and low CRG score groups). We found that the overall survival (OS) of patients was lower in the high CRG score group (HCSG) than in the low CRG score group (LCSG) (P < 0.001). The area under the curve (AUC) values for 3 years, 5 years, and 8 years were 0.872, 0.941, and 0.976, respectively. Cox regression analysis indicated that the CRG score could serve as an independent prognostic indicator for PTC. Functional enrichment analysis indicated that the CRG prognostic signature was also associated with the tumor immune microenvironment. In HCSG, the immune suppression cell score was significantly higher than in LCSG. In addition, we identified the expression of CRG (CDKN2A) by qRT-PCR, and the results aligned with the TCGA database.

Our CRG signature demonstrates excellent predictive capabilities for the prognosis of PTC patients. CRGs may play an important role in tumorigenesis and could be used to predict the immunotherapy efficacy of PTC.

Glioblastoma (GBM) severely disrupts the quality of life of patients. Anoikis represents a significant mechanism in cancer invasion and metastasis. Our study focused on the prognostic relationship between the anoikis-associated gene and GBM and its effect on GBM cell progression.

We downloaded 656 and 979 GBM sample data from TCGA and CGGA cohort datasets, respectively. Fifteen anoikis-associated genes were obtained from the GeneCards database and were subsequently clustered to identify differential genes associated with them. After univariate analysis and survival random forest dimensionality reduction, the anoikis score was the sum of the coefficients * gene expression values of the five prognostic genes. Survival analysis and ROC curve analysis of anoikis scores were performed using the TCGA training and CGGA validation sets. The prognostic factors were analyzed using Cox regression analysis in GBM. Moreover, CCK-8, colony formation, wound healing, and transwell assay were used to evaluate GBM cell proliferation and migration.

Significant differences were observed in the 5-year survival of GBM patients between the two subgroups. Then, our analysis demonstrated that high OCIAD2, FTLP3, IGFBP2, and H19 levels were associated with lower 5-year GBM survival rates, whereas high SFRP2 levels were associated with higher survival rates. Univariate Cox analysis indicated that GBM risk was linked to both anoikis score and grade, while multivariate Cox analysis indicated that GBM risk was associated with both anoikis score and age. Additionally, OCIAD2 was highly expressed in U251MG and T98G cells. Moreover, OCIAD2 silencing inhibited GBM cell proliferation and migration.

This study demonstrated the potential of the anoikis-associated gene OCIAD2 as a prognostic biomarker for GBM. Furthermore, we validated in vitro that OCIAD2 promoted GBM cell progression. OCIAD2 silencing inhibited GBM cell proliferation and migration.

Sex hormones are important factors in maintaining brain function and acting as brain protectors. Recent research suggests that neuronal damage in brain aging may be linked to the methylation of the estrogen receptor α (ERα). However, the mechanism of Zuogui Pills (ZGW) in brain-aging ERα DNA methylation and neuronal repair remains unknown.

D-galactose-induced ovary removal mice were used as a model of aging. Changes in estrous cycle were detected in mice by vaginal cell smear. Animal behavior tests, including the Morris water maze (MWM) and new object recognition (NOR) test, were conducted. Hematoxylin-eosin (HE) and Nissl-staining were carried out to assess hippocampal neurogenesis. Enzyme-linked immunosorbent assay (ELISA) was performed for 5-methylcytosine methylation levels, and immunohistochemistry (IHC) and western blotting (WB) experiments were performed to assess ERα/DNA methyltransferase 1 (DNMT1) expression after ZGW treatment. Finally, bisulfite sequencing PCR (BSP) analysis was performed to identify methylated differentially expressed estrogen receptor 1 (ESR1) gene in D-gal-induced senescent neurons before and after ZGW treatment.

We found that ERα methylation was involved in the delayed brain ageing process of ZGW. Mechanistically, ZGW can improve the learning and memory ability of brain-aging mice, reduce the expression of 5-methylcytosine (5-mc) in serum, increase the amount of ERα, inhibit the expression of DNMT1, and significantly reduce methylated expression of the ESRI gene.

Our data suggested that ZGW slowed down D-gal-induced brain aging in mice, and these results showed that ZGW is beneficial for aging. It may be used for neuronal protection in aging.

The aim of this study is to explore the mechanism of HRAS and HSPB1 in ferroptosis. Primary liver cancer is the third leading cause of tumor death worldwide. Hepatocellular carcinoma (HCC) constitutes 75%-85% of cases of primary liver cancer. HRAS and HSPB1 co-express in multiple cells and participate in tumor progression regulation. However, their expression regulation and role in HCC have not been reported.

We investigated the effects of HRAS and HSPB1 on ferroptosis in in vitro experiments. Here, the role and mechanism of HRAS and HSPB1 on ferroptosis were investigated by transfecting the specific siRNA or overexpressing plasmids in HCC cells.

Bioinformatics analysis proved that HRAS and HSPB1 were highly expressed in HCC tissues and associated with poor prognosis of patients with HCC. In vitro, HRAS overexpression reduced the level of intracellular iron, ROS, and MDA production in HCC cells. Mechanistically, HRAS increased GPX4 expression and decreased the levels of ACSL4 and P53. HRAS also increased HSPB1 expression, and HRAS knockdown downregulated HSPB1 levels in HCC cells. Importantly, overexpression of HSPB1 reversed HRAS-increased concentration of iron, MDA, and ROS and eliminated HRAS-induced ferroptosis. Moreover, HRAS enhanced the proliferation and invasion by targeting HSPB1.

The regulation of HSPB1 by HRAS enhanced the resistance of HCC cells to ferroptosis. HRAS promoted proliferation and invasion by upregulating HSPB1. This research provides a new potential strategy for HCC treatment.

Systemic Lupus Erythematosus (SLE) is a multifactorial and complex immune disease; however, the relevance of COVID-19 infection in SLE patients remains uncertain.

This study aims to explore the key candidate genes and pathways in patients with SLE. It also seeks to employ bioinformatics analysis to unravel the molecular signatures inherent in both SLE and COVID-19 patients. The ultimate aim is to identify potential targets and markers specifically relevant to SLE patients who contract SARS-CoV-2.

Datasets (GSE12374, GSE20864, GSE61635, GSE81622, and GSE144390) from the Gene Expression Omnibus (GEO) database were analyzed using Robust Rank Aggregation (RRA) method to identify differential expression genes (DEGs) in SLE patients compared to healthy individuals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, tissue-specific gene analysis, and Protein-protein interaction (PPI) network were performed. Finally, the Venn diagram was employed to identify the intersections of COVID-19 genes, serving as potential targets for SLE patients with COVID-19 infection.

A total of 154 DEGs were discovered, with GO enrichment indicating a predominant involvement in the defense response against the virus (P<0.001). KEGG pathway analysis showed enrichment in the NOD-like receptor signaling pathway and coronavirus disease, specifically COVID-19 (P<0.001). Tissue-specific genes related to the hematological and immune systems were emphasized (74%). The PPI network highlighted 22 genes, and 5 key genes, namely, IFIT1, IFIT3, MX1, MX2, and OAS3, which were identified after intersecting with COVID-19 patients’ data.

IFIT1, IFIT3, MX1, MX2, and OAS3 exhibiting differential expression, as well as the pathways associated with COVID-19, could potentially function as biomarkers and therapeutic targets for individuals with SLE infected with COVID-19.

In the ongoing fight against bacterial resistance to antibiotics, this study focuses on synthesizing and evaluating 1,2,4-triazole derivatives to explore their potential as new antibacterial agents. 1,2,4-Triazole compounds are promising drug candidates with a wide range of therapeutic effects, including pain relief, antiseptic, antimicrobial, antioxidant, antiurease, anti-inflammatory, diuretic, anticancer, anticonvulsant, antidiabetic, and antimigraine properties.

The structures of all the synthesized compounds were identified using their physicochemical properties and spectral techniques, such as IR and NMR. These compounds were then evaluated in molecular docking studies against antimicrobial activity in vitro and further supported by molecular dynamics studies.

Compound 7, featuring a 6-chloro group on the phenyl ring, emerged as the most effective against Gram-positive S. aureus compared to the standard antibiotic ciprofloxacin. Docking studies revealed high and comparable affinities for all ten ligands, with compounds 4 and 6 showing the best-docked activity against Penicillin Acylase mutants. Further, compounds 6 and 10 displayed significant affinity against D-alanine-D-alanine ligase (DDL) from Yersinia pestis during 100 ns MD simulation.

Notably, compound 7 demonstrated the highest binding score to the 5C1P protein, suggesting its potential as a lead molecule for the development of potent and safer antimicrobial agents. This research contributes valuable insights into addressing the escalating challenge of bacterial resistance.

Normal keratinocyte differentiation is important for epidermal wound healing. ΔNp63 is a major gene regulating epidermal formation and differentiation. We identified miRNAs targeting ΔNp63 and studied the association between the miRNAs and DNA methylation in keratinocyte differentiation.

This study aimed to explore the mechanisms regulating ΔNp63 expression during keratinocyte differentiation.

Bioinformatics analysis was performed to screen the miRNAs targeting ΔNp63 and uncover potential pathway mechanisms. The differentiation model of keratinocytes was established by CaCl2 treatment. Furthermore, the effects of the miRNA transgenic technique on Δ Np63 and keratinocyte differentiation were studied. In addition, the RNA FISH experiment was conducted to detect the location of different miRNAs. A double luciferase reporter experiment was carried out to verify the potential bindings between the miRNAs and ΔNp63. A rescue experiment was also performed to assess the effects of different miRNAs targeting ΔNp63 on keratinocyte differentiation. We analyzed the methylation patterns of the promoter regions of miRNAs using keratinocytes treated with 5-Aza-2’-deoxycytidine. Finally, we designed a methylation rescue experiment to verify the effects of miRNA promoter methylation on keratinocyte differentiation.

Bioinformatics analysis showed that the miR-125b-5p and miR-199b-5p binding to the ΔNp63 3’UTR region decreased during skin development. Moreover, such binding may downregulate the PI3K/AKT/mTOR pathway. The expression levels of CK10, Inv, TG1, ΔNp63, and PI3K/AKT/mTOR were all significantly increased during keratinocyte differentiation. Both miR-125b-5p and miR-199b-5p were localized in the cytoplasm. Luciferase assay results showed that both miR-125b-5p and miR-199b-5p can bind to the 3’UTR region of ΔNp63. Overexpression of ΔNp63 can significantly counteract the inhibitory effect of miRNA mimics on keratinocyte differentiation. Moreover, the promoter regions of both miR-125b-5p and miR-199b-5p had methylation sites, and the methylation levels in those promoter regions were significantly increased during keratinocyte differentiation. 5-Aza-2’-Deoxycytidine treatment increased the expression of miR-125b-5p and miR-199b-5p and inhibited the differentiation of keratinocytes. Finally, miRNA inhibitors reversed the inhibitory effects of 5-Aza-2’-deoxycytidine on keratinocyte differentiation.

Promoter hypermethylation in miR-125b-5p and miR-199b-5p seem to promote keratinocyte differentiation via upregulation of ΔNp63 expression and the activation of the PI3K/AKT/mTOR pathway. The findings of this study are helpful for future research on skin development and clinical scar-free healing.

In this study, we used immune repertoire (IR) sequencing technology to profile the diversity of peripheral blood T cell receptors and used transcriptomics to profile the gene expression of peripheral blood neutrophil mRNA in patients with mild-moderate knee osteoarthritis (KOA) before and after electroacupuncture (EA) treatment.

An 8-week intervention with EA was performed on 3 subjects with KOA. IR sequencing of complementarity determining region 3 (CDR3) was performed using RNA extracted from peripheral blood T cells of KOA subjects prior to and at the end of the intervention, as well as healthy volunteers (controls) who matched the subjects in sex and age. Neutrophils were extracted from the plasma of healthy individuals, pretreatment patients, and posttreatment patients for further transcriptome sequencing.

The D50, diversity index (DI), and Shannon entropy values of circulatory T-cells were significantly lower in pretreatment KOA patients compared to healthy controls. Posttreatment KOA samples displayed significant decreases in serum proinflammatory factors, IL-8 and IL-18 (P < 0.01), as well as a substantial reduction in serum matrix MMP-3 and MMP-13 (P < 0.01, P < 0.05). Transcriptome analysis revealed that the expression of CXCL2, IRF8, and PEAR1 (P < 0.05) was significantly higher in patients before the treatment than in the healthy population and was significantly down-regulated after the treatment. In contrast, the expression of SMPD3 (P < 0.05) showed the opposite trend.

EA may alleviate KOA by rebalancing T-cell homeostasis and improving systemic inflammation. At the same time, EA treatment can significantly enhance TCR diversity, reduce levels of proinflammatory factors, and increase levels of anti-inflammatory factors, thereby achieving therapeutic effects.

NCT 03366363.