Uncovering the effects and molecular mechanism of Astragalus membranaceus (Fisch.) Bunge and its bioactive ingredients formononetin and calycosin against colon cancer: An integrated approach based on network pharmacology analysis coupled with experimental validation and molecular docking

Colon cancer is a highly malignant cancer with poor prognosis. Astragalus membranaceus (Fisch.) Bunge (Huang Qi in Chinese, HQ), a well-known Chinese herbal medicine and a popular food additive, possesses various biological functions and has been frequently used for clinical treatment of colon cancer. However, the underlying mechanism is not fully understood. Isoflavonoids, including formononetin (FMNT) and calycosin (CS), are the main bioactive ingredients isolated from HQ. Thus, this study aimed to explore the inhibitory effects and mechanism of HQ, FMNT and CS against colon cancer by using network pharmacology coupled with experimental validation and molecular docking. The network pharmacology analysis revealed that FMNT and CS exerted their anticarcinogenic actions against colon cancer by regulating multiple signaling molecules and pathways, including MAPK and PI3K-Akt signaling pathways. The experimental validation data showed that HQ, FMNT and CS significantly suppressed the viability and proliferation, and promoted the apoptosis in colon cancer Caco2 and HT-29 cells. HQ, FMNT and CS also markedly inhibited the migration of Caco2 and HT-29 cells, accompanied by a marked increase in E-cadherin expression, and a notable decrease in N-cadherin and Vimentin expression. In addition, HQ, FMNT and CS strikingly decreased the expression of ERK1/2 phosphorylation (p-ERK1/2) without marked change in total ERK1/2 expression. They also slightly downregulated the p-Akt expression without significant alteration in total Akt expression. Pearson correlation analysis showed a significant positive correlation between the inactivation of ERK1/2 signaling pathway and the HQ, FMNT and CS-induced suppression of colon cancer. The molecular docking results indicated that FMNT and CS had a strong binding affinity for the key molecules of ERK1/2 signaling pathway. Conclusively, HQ, FMNT and CS exerted good therapeutic effects against colon cancer by mainly inhibiting the ERK1/2 signaling pathway, suggesting that HQ, FMNT and CS could be useful supplements that may enhance chemotherapeutic outcomes and benefit colon cancer patients

Dynamic changes in the levels of metabolites and endogenous hormones during the germination of Zanthoxylum nitidum (Roxb.) DC. Seeds

Accumulating experimental data have shown that endogenous hormones play important roles in regulating seed dormancy and germination. Zanthoxylum nitidum is a medicinal plant that propagates via seeds, which require a long dormancy period for normal germination, and complex changes in metabolites occur during the germination process. However, the regulatory network of endogenous hormones and metabolites during the germination of Z. nitidum seeds remains unclear. This study investigated the dynamic changes in the levels of metabolites and endogenous hormones during the germination of Z. nitidum seeds. The results revealed an increase in the levels of gibberellin 3 (GA3), 12-oxophytodienoic acid (OPDA), 1-aminocyclopropane-1-carboxylic acid (ACC) and trans-zeatin (TZ) and decrease in the levels of abscisic acid (ABA), jasmonic acid (JA), N-[(-)-jasmonoyl]-(S)-isoleucine (JA-Ile) and trans-zeatin riboside (TZR). Overall, 112 differential metabolites (DAMs) were screened from 3 seed samples (Sa, Sb and Sc), most of which are related to primary metabolism. A total of 16 DAMs (including 3 monosaccharides, 3 phosphate lipids, 3 carboxylic acids, 1 amino acid, 2 pyrimidines, and 4 nucleotides) were identified in the three sample comparison pairs (Sa vs Sb, Sa vs Sc, and Sb vs Sc); these DAMs were significantly enriched in purine metabolism; glycerophospholipid metabolism, citrate cycle (TCA cycle), alanine, aspartate and glutamate metabolism and pyruvate metabolism. OPDA, ACC and GAs were significantly positively correlated with upregulated metabolites, whereas ABA and JA were significantly positively correlated with downregulated metabolites. Finally, a hypothetical metabolic network of endogenous hormones that regulate seed germination was constructed. This study deepens our understanding of the importance of endogenous hormonal profiles that mediate seed germination

Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum).

The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions

Associations of Exposure to Air Pollution with Insulin Resistance: A Systematic Review and Meta-Analysis

In this article, we review the available evidence and explore the association between air pollution and insulin resistance (IR) using meta-analytic techniques. Cohort studies published before January 2018 were selected through English-language literature searches in nine databases. Six cohort studies were included in our sample, which assessed air pollutants including PM2.5 (particulate matter with an aerodynamic diameter less than or equal to 2.5 μm), NO2(nitrogen dioxide), and PM10 (particulate matter with an aerodynamic diameter less than 10 μm). Percentage change in insulin or insulin resistance associated with air pollutants with corresponding 95% confidence interval (CI) was used to evaluate the risk. A pooled effect (percentage change) was observed, with a 1 μg/m3 increase in NO2 associated with a significant 1.25% change (95% CI: 0.67, 1.84; I2 = 0.00%, p = 0.07) in the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) and a 0.60% change (95% CI: 0.17, 1.03; I2 = 30.94%, p = 0.27) in insulin. Similar to the analysis of NO2, a 1 μg/m3 increase in PM10 was associated with a significant 2.77% change (95% CI: 0.67, 4.87; I2 = 94.98%, p < 0.0001) in HOMA-IR and a 2.75% change in insulin (95% CI: 0.45, 5.04; I2 = 58.66%, p = 0.057). No significant associations were found between PM2.5 and insulin resistance biomarkers. We conclude that increased exposure to air pollution can lead to insulin resistance, further leading to diabetes and cardiometabolic diseases. Clinicians should consider the environmental exposure of patients when making screening and treatment decisions for them

Development of a direct competitive enzyme‐linked immunosorbent assay for quantitation of sodium saccharin residue in food

Sodium saccharin is a common artificial sweetener. However, due to its possible carcinogenic effects and causing metabolic disorders, many countries have strictly regulated its use in food. In the study, we prepared a specific monoclonal antibody (mAb 2H11) using the new hapten (6-carboxylsaccharin) and developed a direct competitive enzyme-linked immunosorbent assay (dcELISA) for the screening of sodium saccharin residue in food. The half-maximum inhibition concentration (IC50 ) and working range (IC20 -IC80 , the concentrations causing 20% and 80% inhibition by sodium saccharin) were 32.5 and 6.47 to 164 ng/mL, which was 6.5 times more sensitive than the previously reported immunoassay. The average recoveries of sodium saccharin in spiked food samples detected by dcELISA ranged from 82.1% to 117%. Among 70 food samples bought in the physical stores and online, sodium saccharin residues were only detected in four samples purchased online (one canned pineapple, two winter jujube, and one kimchi). The content measured by dcELISA agreed well with those determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The developed dcELISA was proved to be a sensitive and accurate method for determining sodium saccharin in food. PRACTICAL APPLICATION: Quantitation of sodium saccharin residue in food is very necessary and important for consumers and regulatory agencies

Development of a specific monoclonal antibody-based ELISA to measure the artemether content of antimalarial drugs.

Artemether is one of the artemisinin derivatives that are active ingredients in antimalarial drugs. Counterfeit and substandard antimalarial drugs have become a serious problem, which demands reliable analytical tools and implementation of strict regulation of drug quality. Structural similarity among artemisinin analogs is a challenge to develop immunoassays that are specific to artemisinin derivatives. To produce specific antibodies to artemether, we used microbial fermentation of artemether to obtain 9-hydroxyartemether, which was subsequently used to prepare a 9-O-succinylartemether hapten for conjugation with ovalbumin as the immunogen. A monoclonal antibody (mAb), designated as 2G12E1, was produced with high specificity to artemether. 2G12E1 showed low cross reactivities to dihydroartemisinin, artemisinin, artesunate and other major antimalarial drugs. An indirect competitive enzyme linked immunosorbent assay (icELISA) developed showed a concentration causing 50% of inhibition for artemether as 3.7 ng mL⁻¹ and a working range of 0.7-19 ng mL⁻¹. The icELISA was applied for determination of artemether content in different commercial drugs and the results were comparable to those determined by high-performance liquid chromatography analysis. In comparison with reported broad cross activity of anti-artemisinin mAbs, the most notable advantage of the 2G12E1-based ELISA is its high specificity to artemether only