Study on the effect and mechanism of Swertia mussotii Franch. in the treatment of primary biliary cholangitis based on bioinformatics and in vitro experiments

Background:Primary biliary cholangitis(PBC)is a chronic biliary autoimmune liver disease characterized by intrahepatic cholestasis.Swertia mussotii Franch.(SMF)is a Tibetan medicine with hepatoprotective and anti-inflammatory activities.In this study,the therapeutic effect and potential mechanisms of SMF on PBC were investigated by bioinformatics analysis and in vitro experimental validation,with the aim of promoting the progress of SMF and PBC research.Methods:We first explored the therapeutic effects and key targets of SMF on PBC using a network pharmacology approach,further screened the core targets using the GSE79850 dataset,and finally validated the results using molecular docking techniques and in vitro experiments.Results:By bioinformatics analysis,we identified core targets of SMF for PBC treatment(STAT3,JAK2,TNF-α,and IL-1β)and important signaling pathways:JAK-STAT,TNF,and PI3K-AKT.The molecular docking results showed that the significant components of SMF had good binding properties to the core targets.In vitro experiments showed that SMF extracts improved the extent of epithelial-mesenchymal transition in human intrahepatic biliary epithelial cells and had a significant reversal effect on epithelial-mesenchymal transition process markers and potential targets in PBC.Conclusion:SMF may exert its therapeutic effects on PBC by acting on important targets such as STAT3,JAK2,TNF-α,IL-1β,Vimentin,and E-cadherin and the pathways in which they are involved.

Mechanisms of imperatorin on regulating Pglycoprotein in blood-brain barrier based on network pharmacology and in vitro experiment

OBJECTIVE To explore mecha⁃nisms of imperatorin on regulating P-glycoprotein(P-gp)in blood-brain barrier(BBB)based on net⁃work pharmacology combined with in vitro experi⁃ment.METHODS Drug targets were predicted using the Pharmapper and Swiss targets data⁃bases;disease targets were obtained through the Genecards database;intersections between drugs and disease targets were screened by Cytoscape software;the obtained core targets were used to construct protein-protein interaction(PPI)network,gene ontology(GO)functions,and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis.The effects of imperatorin(20,50,100μmol·L^(-1))on P-gp activity were monitored in hCMEC/D3 in vitro BBB model,and the effects of imperatorin on the expression of target proteins were verified using Western blot method.RESULTS 55 drug targets and 3102 disease targets were obtained from the network pharmacology screening,and 37 core targets were obtained after the combination.Enrichment analysis showed that core targets were closely related to chemical synaptic trans⁃mission regulation,neurotransmitter receptor activity,protein kinase regulation activity,G proteincoupled receptor signaling pathway,neural active ligand receptor interaction pathway,PI3K-Akt sig⁃naling pathway,VEGF signaling pathway,etc..In vitro experimental validation suggested that all tested concentration groups of imperatorin signifi⁃cantly reduced the activity and expression of P-gp,which were achieved by significantly downregu⁃lating the phosphorylation levels of PI3K and Akt,and repressing the expression of VEGFR2 pro⁃tein.CONCLUSION Network pharmacology was used to predict the core targets and signaling pathways of imperatorin on regulating P-gp in BBB and relevant validation was conducted through in vitro experiments,providing a refer⁃ence basis for further exploration of the mecha⁃nisms of imperatorin on regulating P-gp in BBB.

INFLUENCE OF ALPHA-FETOPROTEIN ON THE GROWTH OF TUMOR CELLS IN VITRO

It has been recognized that alpha-fetoprotein (AFP),as an oncofetal antigen, re-expresses in large amounts inadult tumor cells and serves clinically useful purposes asa tumor marker assay. However, its biological activiticsare still far from clear. In thc present study, the ability ofAFP to stimulate tumor cell growth was observed by invitro test system. The new finding indicates that AFPcontributes to the generation and development of tumorand is an important target action site of tumor therapy.

Study on the in vitro activity of Hehuan Yin aqueous extract against hepatitis C 2a virus

Objective:To study the anti-HCV activity and mechanism of Hehuan Yin aqueous extract.Methods:Huh7.5.1 cells were used to establish the HCV2a virus infection model.Cell survival rate(%)and Renilla Luciferase Assay Kit(%)were calculated by Celltiter-GLO Assay for evaluating CC50,EC50 and SI values.To observe the drug resistance of the virus to different concentrations of Hehuan Yin within 72 hours by detecting luciferase activity,western-blot was used to detect the protein expression levels of NS5A,NS3 and NS5B.Results:the CC50,EC50 and SI of Hehuan Yin against HCV2a were 132.50g/ml,1.90g/ml and 67.90 respectively.The EC50 after 24h,48h and 72h administration were 18g/ml,5.8g/ml and 2.3g/ml respectively.Within the range of drug concentration,the aqueous extract Hehuan Yin had inhibitory effect on the expression of NS5A and NS5B proteins in a dose-effect relationship,but had no obvious effect on the expression of NS3 protein.Conclusion:The aqueous extract of Hehuan Yin may inhibit the replication of HCV2a virus by changing the protein expression levels of NS5A and NS5B,and the virus has no tolerance to the aqueous extract of Hehuan Yin.

Methodological factors affecting gas and methane production during in vitro rumen fermentation evaluated by meta-analysis approach

Effects of some methodological factors on in vitro measures of gas production（GP, mL/g DM）, CH4production（mL/g DM） and proportion（% CH4 on total GP） were investigated by meta-analysis. These factors were considered：pressure in the GP equipment（0 = constant; 1 = increasing）, incubation time（0 = 24; 1 = ≥ 48 h）, time of rumen fluid collection（0 = before feeding; 1 = after feeding of donor animals）, donor species of rumen fluid（0 = sheep; 1 =bovine）, presence of N in the buffer solution（0 = presence; 1 = absence）, and ratio between amount of buffered rumen fluid and feed sample（BRF/FS; 0 = ≤ 130 mL/g DM; 1 = 130–140 mL/g DM; 2 = ≥ 140 mL/g DM）. The NDF content of feed sample incubated（NDF） was considered as a continuous variable. From an initial database of 105 papers, 58 were discarded because one of the above-mentioned factors was not stated. After discarding 17 papers,the final dataset comprised 30 papers（339 observations）. A preliminary mixed model analysis was carried out on experimental data considering the study as random factor. Variables adjusted for study effect were analyzed using a backward stepwise analysis including the above-mentioned variables. The analysis showed that the extension of incubation time and reduction of NDF increased GP and CH4 values. Values of GP and CH4 also increased when rumen fluid was collected after feeding compared to before feeding（＋26.4 and ＋9.0 mL/g DM, for GP and CH4）,from bovine compared to sheep（＋32.8 and ＋5.2 mL/g DM, for GP and CH4）, and when the buffer solution did not contain N（＋24.7 and ＋6.7 mL/g DM for GP and CH4）. The increase of BRF/FS ratio enhanced GP and CH4production（＋7.7 and ＋3.3 mL/g DM per each class of increase, respectively）. In vitro techniques for measuring GP and CH4 production are mostly used as screening methods, thus a full standardization of such techniques is not feasible. However, a greater harmonization of analytical procedures（i.e., a reduction in the number of available protocols） would be useful to facilitate comparison between results of different experiments.

Puerarin exhibits greater distribution and longer retention time in neurons than astrocytes in a co-cultured system

The phytoestrogen puerarin has been shown to protect neurons and astrocytes in the brain, and is therefore an attractive drug in the treatment of Alzheimer’s disease, Parkinson’s disease and cerebral ischemia. Whether puerarin exhibits the same biological processes in neurons and astro-cytesin vitro has rarely been reported. In this study, cortical neurons and astrocytes of newborn Sprague-Dawley rats were separated, identiifed and co-cultured in a system based on Transwell membranes. The retention time and distribution of puerarin in each cell type was detected by lfuorescence spectrophotometry and lfuorescence microscope. The concentration of puerarin in both co-cultured and separately cultured neurons was greater than that of astrocytes. Puerarin concentration reached a maximum 20 minutes after it was added. At 60 minutes after its addi-tion, a scant amount of drug was detected in astrocytes; however in both separately cultured and co-cultured neurons, the concentration of puerarin achieved a stable level of about 12.8 ng/mL. The results indicate that puerarin had a higher concentration and longer retention time in neu-rons than that observed in astrocytes.

Artificial gut and the applications in poultry:A review

Artificial gut models including both the gastric and intestinal phases have been used in poultry research for decades to predict the digestibility of nutrients,the efficacy of feed enzymes and additives,and caecal fermentation.However,the models used in the past are static and cannot be used to predict interactions between the feed,gut environment and microbiome.It is imperative that a standard artificial gut model for poultry is established,to enable these interactions to be examined without continual reliance on animals.To ensure the validity of an artificial model,it should be validated with in vivo studies.This review describes current practices in the use of artificial guts in research,their importance in poultry nutrition studies and highlights an opportunity to develop a dynamic gut model for poultry to reduce the number of in vivo experiments.