Network pharmacology study and in vitro experimental validation of Xiaojianzhong decoction against gastric cancer

BACKGROUND Cancer is one of the most serious threats to human health worldwide.Conventional treatments such as surgery and chemotherapy are associated with some drawbacks.In recent years,traditional Chinese medicine treatment has been increasingly advocated by patients and attracted attention from clinicians,and has become an indispensable part of the comprehensive treatment for gastric cancer.AIM To investigate the mechanism of Xiaojianzhong decoction(XJZ)in the treatment of gastric cancer(GC)by utilizing network pharmacology and experimental validation,so as to provide a theoretical basis for later experimental research.METHODS We analyzed the mechanism and targets of XJZ in the treatment of GC through network pharmacology and bioinformatics.Subsequently,we verified the impact of XJZ treatment on the proliferative ability of GC cells through CCK-8,apoptosis,cell cycle,and clone formation assays.Additionally,we performed Western blot analysis and real-time quantitative PCR to assess the protein and mRNA expression of the core proteins.RESULTS XJZ mainly regulates IL6,PTGS2,CCL2,MMP9,MMP2,HMOX1,and other target genes and pathways in cancer to treat GC.The inhibition of cell viability,the increase of apoptosis,the blockage of the cell cycle at the G0/G1 phase,and the inhibition of the ability of cell clone formation were observed in AGS and HGC-27 cells after XJZ treatment.In addition,XJZ induced a decrease in the mRNA expression of IL6,PTGS2,MMP9,MMP2,and CCL2,and an increase in the mRNA expression of HOMX1.XJZ significantly inhibited the expression of IL6,PTGS2,MMP9,MMP2,and CCL2 proteins and promoted the expression of the heme oxygenase-1 protein.CONCLUSION XJZ exerts therapeutic effects against GC through multiple components,multiple targets,and multiple pathways.Our findings provide a new idea and scientific basis for further research on the molecular mechanisms underlying the therapeutic effects of XJZ in the treatment of GC.

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.

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.

Biomechanical comparison of locking plate and crossing metallic and absorbable screws fixations for intra-articular calcaneal fractures

The locking plate and percutaneous crossing metallic screws and crossing absorbable screws have been used clinically to treat intra-articular calcaneal fractures, but little is known about the biomechanical differences between them. This study compared the biomechanical stability of calcaneal fractures fixed using a locking plate and crossing screws. Three-dimensional finite-element models of intact and fractured calcanei were developed based on the CT images of a cadaveric sample. Surgeries were simulated on models of Sanders type III calcaneal fractures to produce accurate postoperative models fixed by the three implants. A vertical force was applied to the superior surface of the subtalar joint to simulate the stance phase of a walking gait. This model was validated by an in vitro experiment using the same calcaneal sample. The intact calcaneus showed greater stiffness than the fixation models. Of the three fixations, the locking plate produced the greatest stiffness and the highest von Mises stress peak. The micromotion of the fracture fixated with the locking plate was similar to that of the fracture fixated with the metallic screws but smaller than that fixated with the absorbable screws. Fixation with both plate and crossing screws can be used to treat intra-articular calcaneal fractures. In general, fixation with crossing metallic screws is preferable because it provides sufficient stability with less stress shielding.

Identification,expression analysis,and functional characterization of ghrelin and its receptors in spotted sea bass(Lateolabrax maculatus)

Ghrelin(Ghrl),an appetite-inducing peptide hormone secreted by the stomach,is the endogenous ligand for the growth hormone secretagogue receptor(Ghs-r).In this study,we identified the preproghrelin gene and its receptors in spotted sea bass(Lateolabrax maculatus).The ghrl gene consisted of an open reading frame(ORF)of 324 nucleotides encoding 107 amino acids,and the premature protein contained a 20-amino-acid mature peptide.Through a syntenic analysis,we also validated the annotation of growth hormone secretagogue receptor la(ghs-rla)and growth hormone secretagogue receptor la-1ike(ghs-rla-like),which contained seven-transmembrane structures,in spotted sea bass.The ORF of ghs-rla consisted of 1152 bp that encoded a 383-amino-acid protein,and(ghs-rla-like) contained an ORF of 2631 bp and produced a protein with 876 amino acids.A phylogenetic analysis showed that spotted sea bass ghrl and its receptors clustered with those of other fishes and were more distantly related to those of other vertebrates.In situ hybridization revealed that ghrl was highly expressed in the stomach and localized in the mucosa and submucosa.The expression of these genes varied during short-term starvation in a time-dependent manner.In vitro studies showed that after incubation with Ghrl for 3 h enhanced the expression of motilin(mln),gastrin(gas)and cholecystokinin(cck)、but this effect vanished after 6 h of incubation.In summary,Ghrl and its receptors might play important roles in the regulation of food intake in spotted sea bass.

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.