ABSTRACT
ObjectiveTo study the mechanism of matrine in the treatment of inflammatory bowel disease （IBD） based on the zebrafish model and network pharmacology. MethodThe IBD model of zebrafish was established using 2，4，6-trinitro-benzenesulfonicacid （TNBS）， and the intestinal phagocytic function， goblet cell secretion， and neutrophil aggregation were evaluated using neutral red staining， alcian blue staining， and neutrophil number changes. Changes in tumor necrosis factor （TNF）-α and cholecystokinin （CCK） content in zebrafish were determined by using relevant reagent kits. Network pharmacology and molecular docking techniques were used to predict the potential mechanism of matrine in the treatment of IBD. Gene expression of relevant targets was verified through Real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the model group， the matrine administration group can increase the neutral red staining area in a dose-dependent manner and improve intestinal phagocytic function（P<0.05，P<0.01）. It can reduce the staining area of alcian blue and affect the secretion of intestinal goblet cells（P<0.01）. It can reduce the number of neutrophil granulocytes， relieve its aggregation， significantly reduce TNF-α content（P<0.01）， and increase the CCK content. Network pharmacology analysis identifies 28 potential targets for matrine in the treatment of IBD. The top five targets by protein-protein interaction （PPI） network analysis are CHRNA7， DRD1， CHRNA4， SLC6A3， and GRM5. The Kyoto encyclopedia of genes and genomes （KEGG） results show that the treatment of IBD with matrine may be related to neuroactive ligand-receptor interaction， cholinergic synapse， and neutrophil extracellular trap formation. Real-time PCR results show that matrine can affect the expression level of related target genes. Conclusionmatrine has a certain therapeutic effect on IBD and can affect the inflammatory response of IBD. Its therapeutic effect may be related to neuroactive ligand-receptor interaction and other pathways.
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Polyphyllin Ⅰ(PPⅠ)and polyphyllin Ⅱ(PⅡ)are the main active substances in the Paris polyphylla.However,liver toxicity of these compounds has impeded their clinical application and the potential hepatotoxicity mechanisms remain to be elucidated.In this work,we found that PPⅠ and PⅡ exposure could induce significant hepatotoxicity in human liver cell line L-02 and zebrafish in a dose-dependent manner.The results of the proteomic analysis in L-02 cells and transcriptome in zebrafish indicated that the hepa-totoxicity of PPⅡ and PⅡwas associated with the cholesterol biosynthetic pathway disorders,which were alleviated by the cholesterol biosynthesis inhibitor lovastatin.Additionally,3-hydroxy-3-methy-lglutaryl CoA reductase(HMGCR)and squalene epoxidase(SQLE),the two rate-limiting enzymes in the choles-terol synthesis,selected as the potential targets,were confirmed by the molecular docking,the over-expression,and knockdown of HMGCR or SQLE with siRNA.Finally,the pull-down and surface plasmon resonance technology revealed that PPⅠ could directly bind with SQLE but not with HMGCR.Collectively,these data demonstrated that PPⅠ-induced hepatotoxicity resulted from the direct binding with SQLE protein and impaired the sterol-regulatory element binding protein 2/HMGCR/SQLE/lanosterol synthase pathways,thus disturbing the cholesterol biosynthesis pathway.The findings of this research can contribute to a better understanding of the key role of SQLE as a potential target in drug-induced hepatotoxicity and provide a therapeutic strategy for the prevention of drug toxic effects with similar structures in the future.
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Objective To investigate the applicability of zebra fish thrombosis model in antithrombotic activity screening of Chinese materia medica.Methods The living zebra fish thrombosis model was induced by adrenaline hydrochloride. Zebra fish were randomly divided into blank control group, model group, positive medicine group and medication group. Each group was given the corresponding medicine or embryo culture water. O-anisidine staining solution was used to stain and calculate the staining intensity of erythrocytes in zebra fish heart, and quantitative analysis was carried out. The platelet aggregation of transgenic zebra fish was observed and under qualitative analysis. Results Compared with the model group, 100μg/mL salvianolic acid B, 300, 900μg/mL aqueous extract of Salvia miltiorrhiza, 45μg/mL 95% ethanol extract and 400, 1200μg/mL hypothalamus could significantly inhibited the formation of zebra fish thrombosis (P<0.01).ConclusionZebra fish thrombosis model has good applicability in antithrombotic activity screening of Chinese materia medica.
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This study was aimed to develop HPLC for determination of vitexin and isovitexin inM. dodecandrum. The HPLC column was SunFireTM C18 (4.6 mm× 150 mm, 5μm). The detection wavelength was 365 nm. The mobile phase was methanol-0.2% formic acid aqueous solution. The column temperature was 40℃. The flow rate was 1.0 mL·min-1. The results showed that the regression equations of vitexin and isovitexin wereY = 1× 106X– 14 396, Y = 1× 106X– 13 900, respectively. The linear ranges were 0.210μg - 1.050μg (r = 0.999) and 0.186μg - 0.930μg (r = 1.000), respectively. The recovery rates were 97.48% and 104.64%, respectively. The RSD were 2.32%and 1.51%, respectively. The sample contents of vitexin and isovitexin were 1.25 and 1.86 mg·g1, respectively. It was concluded that the method was simple, feasible and reproducible for the content determination of vitexin and isovitexin inM. dodecandrum.