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.

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.

Preclinical Verification of Modulated Electro-Hyperthermia —Part I. In Vitro Research

Modulated electro-hyperthermia (mEHT) targets tissue’s natural electric and thermal heterogeneities to heat the cancer cells selectively. The applied 13.56 MHz radiofrequency (RF) is a carrier of the low-frequency modulation. The high-frequency part was chosen to select the malignant lesion using the specialties of the tumor: the higher conductivity and dielectric constant of the tumor than its host. The electric field selects the tumor, and the low-frequency amplitude modulation polarizes and excites the transmembrane proteins of the malignant cells. The dominant absorption of the energy by the microscopic clusters of the membrane rafts acts like nanoparticle heating. Exciting the membrane produces various apoptotic signals. The processes were modeled using silico and phantom experiments, which proved the concept. The preclinical verification was made in vitro and in vivo, and in the end, clinical proofs validated the method. Our objective is to follow all the development steps from the laboratory to the clinics in a trilogy of articles. This present is the first part, which deals with in silico, phantom, and in vitro research.

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.

科研成果融入中药药剂学实验——不同粒径天麻粉粉体学性质及体外溶出度

为改变现阶段中药药学实验教学缺乏前沿性和创新性的现状,对中药药剂学实验课程的开设进行了初步探索;以“不同粒径天麻粉粉体学性质及体外溶出度”为依托,通过科研反哺教学,引导学生运用科研思维开展实验,提高学生学习主动性,激发学生的创新性和科研积极性;促进实验教学由“验证型”向“探索型、创新型”转变,提高教学效果和人才培养质量。

前沿科研成果融入药剂学实验教学——木犀草素可溶性微针的制备

为打破原有实验教学的壁垒,本文将科研前沿与药剂学实验相结合,开展实验创新改革,以提高学生的创新能力与动手能力。首先,采用模具法制备木犀草素可溶性微针,然后对其显微形态、皮肤穿刺性能、载药量及体外透皮性能进行考察。本实验可以锻炼学生制备可溶性微针的能力和创新能力,并掌握高效液相色谱仪等仪器设备的使用,认识微针的微观结构,拓宽学生的科研视野,提升科学素养。

保健食品清除自由基作用的体外测定方法和原理

综述了抗氧化保健食品清除O2·、·OH、H2O2和DPPH·自由基作用的体外实验方法和原理。检测保健食品清除自由基的体外实验方法有多种,一般采用化学方法在体系中产生自由基,通过测定自由基与体系中的化学物质或离体组织成分反应所产生的颜色变化、发光现象等间接测定自由基含量或根据氧张力的变化等直接测定自由基含量。当体系中加入有清除自由基作用的保健食品功效成分后,体系中的自由基含量会减少,由此确定保健食品清除自由基能力的高低。体外实验的特点是快速、灵敏,可对保健食品清除自由基作用进行初步分析评价,适用于筛选抗氧化保健食品功能材料。

兔成骨细胞体外培养体系的研究

为了建立兔成骨细胞体外培养模型,为骨替代材料的研究提供成骨细胞,选用了新生15 d兔颅盖骨,采用酶消化法和组织块法相结合的方法进行成骨细胞的体外分离和培养,通过倒置显微镜观察其形态,并对其碱性磷酸酶(ALP)活性及矿化能力进行鉴定,结果显示培养细胞具有体内成骨细胞的形态学特征和生物学行为。体外培养兔成骨细胞的实验方法切实可行,成功建立了兔成骨细胞体外培养模型,为骨替代材料的实验研究提供了一种客观而有效的手段。

醋蛋水解液抗氧化活性的研究

采用传统泡制工艺泡制醋蛋液,用胃蛋白酶-胰酶复合酶体系水解醋蛋液。分别采用连苯三酚(PA)自氧化法、邻二氮菲-Fe2+氧化法、二苯代苦味肼基自由基(DPPH·)法3种体外实验方法来测定醋蛋液酶解产物清除自由基的能力。研究结果表明,醋蛋液酶解产物有较强的抗氧化活性,其抗氧化活性与浓度呈良好的量效关系。醋蛋液酶解产物用去离子水为洗脱液经凝胶色谱分离,得到抗氧化活性较高的组分Ⅲ,其0.2mg/mL时DPPH·自由基清除率为50.56%,经高压液相色谱(HPLC)测定,组分Ⅲ的相对分子质量主要分布在600～131之间。

体外培养大鼠成骨细胞实验模型的建立

目的 :建立大鼠成骨细胞 (osteoblast)体外培养模型 ,并研究其生长规律。方法 :本实验用新生24h内大鼠颅盖骨 ,采用多次胶原酶消化法进行细胞体外培养。观察细胞形态 ,对其进行鉴定 ,测定细胞生长曲线 ,钙化结节染色。结果 :新生大鼠成骨细胞体外培养模型建立成功 ,证实所培养细胞具有体内成骨细胞的多种生物学特性。生长曲线研究发现 ,成骨细胞培养前7天处于缓慢增长期 ,第8天开始进入对数增长期 ,第15天进入稳定期 ,第21天进入抑制期。结论 :体外培养大鼠成骨细胞实验方法切实可行 。

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.

中药益气化瘀方对体外培养的大鼠颈椎间盘软骨细胞生物学功能的影响

为研究中药益气化瘀方对体外培养的大鼠颈椎间盘软骨细胞生物学功能的影响。取健康 10周龄SD大鼠 70只 ,体重 2 0 0± 5 0g ,雌雄各半 ,随机分为空白组 (E)、芬必得组 (D)、中药低剂量组 (C)、中药中剂量组 (B)、中药高剂量组 (A) ,每组 14只。定时、定量胃饲 ,4天后分别处死取血清 ,相同条件下制备益气化瘀方含药血清和对照血清 ,取体外培养的大鼠颈椎间盘软骨细胞进行实验研究 ,采用MTT法观察软骨细胞生长、增殖 ;流式细胞仪检测细胞凋亡率。结果显示不同中药浓度对培养的软骨细胞生长、增殖均有促进作用 ,以 10 %血清浓度高剂量含药血清组促进软骨细胞增殖的作用最为明显 ,与其他各组相比 ,有非常显著的差异 (P <0 .0 1)。细胞周期分析 ,中药组能促进细胞DNA合成 (S期 ) ,与对照组相比 ,有非常显著的差异 (P <0 .0 1)。表明益气化瘀方具有促进大鼠颈椎间盘软骨细胞生长、增殖及促进细胞DNA合成的作用。

新型可回收下腔静脉滤器的体外实验研究

目的通过建立体外模型,评估新型可回收下腔静脉滤器的滤过效果及稳定性。方法在体外建立下腔静脉模型,在不同管径、不同大小栓子及不同位置上分别评价滤器的稳定性及对栓子的捕获能力,并与Recovery可回收滤器进行对比。用牛血管重复实验,观察新型滤器滤过效果及稳定性。结果在体外下腔静脉模型中,新型可回收滤器对直径为2、3、5 mm的栓子滤过率分别为91.50%、96.50%和99.50%,管径为21、28 mm时滤过率为别为98.00%、93.67%,水平及垂直条件下栓子滤过率为94.33%、97.33%,总体滤过率为95.83%。Recovery可回收滤器在以上条件下的滤过率分别为81.00%,91.00%,95.00%;92.33%,85.67%及87.67%,90.33%,总体滤过率为89.00%。用牛血管取代硅胶管后,新型滤器在不同条件下的滤过率分别为88.50%,94.00%,98.00%;94.67%,92.33%及91.33%,95.67%,总体滤过率为93.50%。在实验过程中,两种滤器均未发现明显移位及偏斜现象。结论新型可回收下腔静脉滤器滤过效果及稳定性可靠,体外实验滤过效果优于Recovery滤器。

宁舒洗剂的体外抗菌作用

目的:研究宁舒洗剂的体外抗菌作用特点。方法:80株细菌全部为近期临床分离所得,其中G+菌34株,G-菌46株;采用试管内药液二倍稀释法,测定宁舒洗剂的MIC、MBC,对于产ESBLs的大肠杆菌(耐药菌株)与不产ESBLs的普通大肠杆菌的MIC和MBC进行比较,另外,观察了细菌接种量和药物pH值分别对MIC、MBC的影响。结果:①宁舒洗剂对所有试验菌株均有抑菌作用,对G+菌的作用较好,其MIC50为1∶32,而G-菌的MIC50为1∶8。②对试验菌均具有杀菌作用,仍对G+菌的作用较好,其MBC50为1∶32,而G-菌的MBC50为1∶4。每种细菌的MBC50与MIC50和MIC90等值均十分接近甚至相等。③对于产ESBLs大肠杆菌的抗菌作用与普通大肠杆菌相同,MIC50、MIC90、MBC50、MBC90均为1∶4。④细菌接种量为102cfu/ml的效价均高于104cfu/ml。⑤药物pH值为8.0时药物的效价高于pH6.0。结论:宁舒洗剂具有体外抑菌和杀菌作用,对G+菌的作用较好,对产ESBLs的大肠杆菌也具有抗菌作用。

Box-behnken效应面法优化水飞蓟素缓释滴丸的处方

目的采用固体分散技术将水飞蓟素制成缓释滴丸,以增加其溶出速度,提高生物利用度。以Box-behnken效应面优化法对制备工艺进行优化,筛选最佳处方。方法采用熔融法制备水飞蓟素缓释滴丸,以药物的质量分数(A)、水溶性基质与难溶性基质用量比(B)、硬脂酸与单硬脂酸甘油脂用量比(C)为考察因素,以2 h累积释放度,10 h累积释放度,R(R=250r-150+(f10-f2)×100/2)为评价指标,利用三因素三水平Box-behnken效应面设计法筛选水飞蓟素缓释滴丸的最佳处方。结果优化后最优处方为含药量20%,水溶性基质-难溶性基质(2.4∶1),硬脂酸-单硬脂酸甘油脂(1.3∶1)。最佳处方滴丸的溶出度的理论预测值与实测值偏差较小,模型具有良好的预测性。结论 水飞蓟素制成缓释滴丸后具有良好的缓释效果,为研发水飞蓟素新制剂提供参考。

诃子醇提物抗HBV的体外实施研究

目的:体外观察诃子抗HBV的作用。方法:以2.2.15细胞为靶细胞,实验共进行11天。采用酶联免疫吸附试验测定诃子对培养上清液HBsAg、HBeAg的抑制作用。结果:诃子醇提物浓度为6.25mg/ml地,对HBsAg、HBeAg抑制率分别为99.67％、71.40％。同一浓度下诃子对细胞的破坏率为30.65％。以HBsAg、HBeAg为指标,治疗指数分别为208.33、3.42。结论:在无毒浓度下,诃子醇提物在2.2.15细胞上有显著的体外抗HBV作用。

离体蛙心灌流实验方法的改进及实验教学的应用

离体蛙心灌流实验是功能学科经典实验之一。文章通过选择合适的实验动物,制作了一种新型简便实用的蛙心插管,提出的改进插管方法等措施提高了离体蛙心灌流实验的成功率。目前该方法运用到实验教学中,收到良好教学效果。