Development of a New High-throughput Screening Model for Human High Density Lipoprotein Receptor (CLA-1) Agonists

To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA （cDNA） of CLA-1 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus （AcNPV） into donor vector pFastBacl and recombinant pFastBacl-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacrnid DNA. Recombinant bacrnid-CLA-1 was transfected into Spodopterafrugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA- 1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized. Results Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries. Conclusion Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.

Accurate construction of cell membrane biomimetic graphene nanodecoys via purposeful surface engineering to improve screening efficiency of active components of traditional Chinese medicine

Biomimetic nanoengineering presents great potential in biomedical research by integrating cell membrane(CM) with functional nanoparticles. However, preparation of CM biomimetic nanomaterials for custom applications that can avoid the aggregation of nanocarriers while maintaining the biological activity of CM remains a challenge. Herein, a high-performance CM biomimetic graphene nanodecoy was fabricated via purposeful surface engineering, where polyethylene glycol(PEG) was used to modifying magnetic graphene oxide(MGO) to improve its stability in physiological solution, so as to improve the screening efficiency to active components of traditional Chinese medicine(TCM). With this strategy, the constructed PEGylated MGO(PMGO) could keep stable at least 10 days, thus improving the CM coating efficiency. Meanwhile, by taking advantage of the inherent ability of He La cell membrane(HM) to interact with specific ligands, HM-camouflaged PMGO showed satisfied adsorption capacity(116.2 mg/g) and selectivity. Finally, three potential active components, byakangelicol, imperatorin,and isoimperatorin, were screened from Angelica dahurica, whose potential antiproliferative activity were further validated by pharmacological studies. These results demonstrated that the purposeful surfaceengineering is a promising strategy for the design of efficient CM biomimetic nanomaterials, which will promote the development of active components screening in TCM.

Compound Danshen tablets downregulate amyloid protein precursor mRNA expression in a transgenic cell model of Alzheimer's disease Effects and a comparison with donepezil

After gene mutation, the pcDNA3.1/APP595/596 plasmid was transfected into HEK293 cells to establish a cell model of Alzheimer＇s disease. The cell model was treated with donepezil or compound Danshen tablets after culture for 72 hours. Reverse transcription-PCR showed that the mRNA expression of amyloid protein precursor decreased in all groups following culture for 24 hours, and that there was no significant difference in the amount of decrease between donepezil and compound Danshen tablets. Our results suggest that compound Danshen tablets can reduce expression of the mRNA for amyloid protein precursor in a transgenic cell model of Alzheimer＇s disease, with similar effects to donepezil.

Traditional Chinese medicines for non-small cell lung cancer: Therapies and mechanisms

The most common subtype of lung cancer is non-small cell lung cancer(NSCLC), which has a poor prognosis and seriously threatens the health of human beings. The multidisciplinary comprehensive treatment model has gradually become the mainstream of NSCLC treatment. Traditional Chinese medicine(TCM) can be used effectively either as an adjunctive therapy or alone throughout the NSCLC therapy,which has a significant impact on survival, quality of life, and reduction of toxicity. Therefore, this paper reviewed the theoretical basis, the latest clinical application, and combined treatment mechanisms in order to explore the advantage stage of TCM treatment and the synergistic therapeutic mechanisms.

Comparison of intestinal absorption characteristics between rhubarb traditional Chinese medicine preparation and activity ingredients using in situ and in vitro model

Objective: The intestinal absorption characteristics of active ingredients are very important for oral administration of traditional Chinese medicine(TCM) to achieve the desired therapeutic effect.However, a deeper understanding about active ingredients absorption characteristics is still lack. The aim of this study was to investigate the absorption properties and mechanism of rhubarb active ingredients in TCM preparation and pure form.Methods: The intestinal absorption behavior of active ingredients in Shenkang extract(SKE) and rhubarb anthraquinone ingredients(RAI) were investigated by in situ single-pass intestinal perfusion model. And the bidirectional transport characteristics of these active ingredients were assessed by in vitro Caco-2 cell monolayer model.Results: In situ experiment on Sprague-Dawley rats, the effective permeability coefficient values of aloeemodin, emodin and chrysophanol in RAI were higher than those in SKE, and the value of rhein in RAI was lower than that in SKE. But the easily absorbed segments of intestine were consistent for all ingredients,whether in SKE or in RAI. In vitro experiment, the apparent permeability coefficient values of rhein, emodin and chrysophanol in RAI were higher than those in SKE, and this value of aloe-emodin in RAI was lower than that in SKE. But their efflux ratio(ER) values in SKE and RAI were all similar.Conclusion: Four rhubarb anthraquinone ingredients in SKE and RAI have similar absorption mechanism and different absorption behavior, and the microenvironment of the study models influenced their absorption behavior. The results may provide an aid for understanding of the absorption characteristics of the TCM active ingredients in complex environments and the complementarities of different research models.

Inhibition effect of Chinese herbal medicine on transcription of hepatitis C virus structural gene in vitro

AIM: To investigate the inhibitory effect of Chinese herbal medicine on the transcription of hepatitis C virus (HCV) structural gene in Hela D cells.METHODS: Hela cell line was transfected with recombinant pBK-CMV-HCV containing HCV structural gene by Lipofectamine. RT-nested-PCR and Western blot assay were used to testify the HCV gene expression in Hela cells. The Hela cells expressing HCV structural protein were named Hela D cells. Prescriptions of Xiao chaihu Decoction (XCHD),Fufang Huangqi (FFHQ) and Bingganling (BGL) wererespectively added to Hela D cells in various concentrations. Semi-quantitative RT-nested-PCR product analysis was performed according to the fluorescent density between HCV DNA band and GAPDH DNA band in gel electrophoresisafter screened. RESULTS: Recombinant pBK-CMV-HCV could correctly express the HCV structural gene in Hela D cells. After coculture of Hela D cells with three prescriptional different concentrations for 48 h respectively, the transcription of HCVgene decreased with increasing of the concentration of each prescription. The lightness ratio of HCV product bands to GAPDH product bands was 0.24, 0.10 and 0.12 in Hela D cells incubated with 0.1 g/mL of XCHD, FFHQand BGL respectively and the lightness ratio HCV product bands to GAPDH product bands was 0.75, 0.67 and 0.61respectively in the control cells. CONCLUSION: The prescriptions of XCHD, FFHQ and BGL partly inhibit the transcription of HCV structural gene inHela D cells.

Application of mesenchymal stem cells derived from human pluripotent stem cells in regenerative medicine

Mesenchymal stem cells(MSCs)represent the most clinically used stem cells in regenerative medicine.However,due to the disadvantages with primary MSCs,such as limited cell proliferative capacity and rarity in the tissues leading to limited MSCs,gradual loss of differentiation during in vitro expansion reducing the efficacy of MSC application,and variation among donors increasing the uncertainty of MSC efficacy,the clinical application of MSCs has been greatly hampered.MSCs derived from human pluripotent stem cells(hPSC-MSCs)can circumvent these problems associated with primary MSCs.Due to the infinite selfrenewal of hPSCs and their differentiation potential towards MSCs,hPSC-MSCs are emerging as an attractive alternative for regenerative medicine.This review summarizes the progress on derivation of MSCs from human pluripotent stem cells,disease modelling and drug screening using hPSC-MSCs,and various applications of hPSC-MSCs in regenerative medicine.In the end,the challenges and concerns with hPSC-MSC applications are also discussed.

Brain organoids are new tool for drug screening of neurological diseases

At the level of in vitro drug screening,the development of a phenotypic analysis system with highcontent screening at the core provides a strong platform to support high-throughput drug screening.There are few systematic reports on brain organoids,as a new three-dimensional in vitro model,in terms of model stability,key phenotypic fingerprint,and drug screening schemes,and particula rly rega rding the development of screening strategies for massive numbers of traditional Chinese medicine monomers.This paper reviews the development of brain organoids and the advantages of brain organoids over induced neurons or cells in simulated diseases.The paper also highlights the prospects from model stability,induction criteria of brain organoids,and the screening schemes of brain organoids based on the characteristics of brain organoids and the application and development of a high-content screening system.

Familial Alzheimer's disease modelling using induced pluripotent stem cell technology

Alzheimer’s disease(AD)is a progressive neurodegenerative disease in which patients exhibit gradual loss of memory that impairs their ability to learn or carry out daily tasks.Diagnosis of AD is difficult,particularly in early stages of the disease,and largely consists of cognitive assessments,with only one in four patients being correctly diagnosed.Development of novel therapeutics for the treatment of AD has proved to be a lengthy,costly and relatively unproductive process with attrition rates of】90%.As a result,there are no cures for AD and few treatment options available for patients.Therefore,there is a pressing need for drug discovery platforms that can accurately and reproducibly mimic the AD phenotype and be amenable to high content screening applications.Here,we discuss the use of induced pluripotent stem cells(iPSCs),which can be derived from adult cells,as a method of recapitulation of AD phenotype in vitro.We assess their potential use in high content screening assays and the barriers that exist to realising their full potential in predictive efficacy,toxicology and disease modelling.At present,a number of limitations need to be addressed before the use of iPSC technology can be fully realised in AD therapeutic applications.However,whilst the use of AD-derived iPSCs in drug discovery remains a fledgling field,it is one with immense potential that is likely to reach fruition within the next few years.

Photosensitizer Screeningfrom Chinese Medicinal Herbs and Photodynamic Anticancer Studies

Objective: To find new photosensitizers from Chinese medicinal herbs for cancer photodynamic therapy. Methods: The extracts of thirteen herbs were examined: (1) Their fluorescence excitation wave lengths and emission wave lengths; (2) Their fluorescence intensity in living cells and (3) Their distribution and localization in the living cells and the fixed cells both stained in each extract, and responses of cell fluorescence intensity to pH value change. Furthermore, the herb's anticancer photosensitive efficiencies were studied by using BGC823 human stomach cancer cells. Results: Cortex Phellodendri and Rhizoma Coptidis, were found with optimal fluorescence properties as photosensitizers in this test. The latter could remarkably reduce the cell metabolic viability, proliferative ability and increase the cell mortality when the cells exposed to both drugs and luminance but not to drug only. Conclusions: The potential of Chinese medicine as a new kind of photosensitizer and its possibility for using in anticancer photodynamic therapy are existed.

Zhichan decoction induces differentiation of dopaminergic neurons in Parkinson's disease rats after neural stem cell transplantation

The goal of this study was to increase the dopamine content and reduce dopaminergic metabolites in the brain of Parkinson’s disease rats. Using high-performance liquid chromatography, we found that dopamine and dopaminergic metabolite（dihydroxyphenylacetic acid and homovanillic acid） content in the midbrain of Parkinson’s disease rats was increased after neural stem cell transplantation ＋ Zhichan decoction, compared with neural stem cell transplantation alone. Our genetic algorithm results show that dihydroxyphenylacetic acid and homovanillic acid levels achieve global optimization. Neural stem cell transplantation ＋ Zhichan decoction increased dihydroxyphenylacetic acid levels up to 10-fold, while transplantation alone resulted in a 3-fold increment. Homovanillic acid levels showed no apparent change. Our experimental findings show that after neural stem cell transplantation in Parkinson’s disease rats, Zhichan decoction can promote differentiation of neural stem cells into dopaminergic neurons.

Systematic review of robust experimental models of rheumatoid arthritis for basic research

Rheumatoid arthritis(RA)is a common autoimmune disease characterized by progressive joint inflammation and destruction,deformity,loss of mobility,and permanent disability.Although the cellular and molecular mechanisms involved in RA are understood in detail,no drugs or therapies can completely cure RA.Many long-term efforts have been directed towards a better understanding of RA pathogenesis and the development of new classes of therapeutics.Thus,the ongoing elucidation of pathogenic events underlying RA mostly relies on studies of animal models.Herein,we comprehensively review and discuss the characteristics,challenges,and unresolved of issues of various experimental models of RA to provide a basis and reference for the rational selection of experimental RA models for basic investigations into traditional Chinese medicine(TCM).

Screening of natural compounds with neuronal differentiation promoting effects in a cell-based model

The purpose of this study was to establish a drug screening method for small molecules extracted from traditional Chinese medicines(TCM) that have neuronal differentiation promoting effects, using P19 embryonic carcinoma cell as a cell-based model. First, the constructed plasmid(p Tα1-Luc) was transfected into P19 cells to establish a screening model. Second, several TCMs were screened using the established model and all-trans-retinoic acid as a positive control. Finally, the underlying molecular mechanism was explored using immunofluorescence staining, q T-PCR, and Western blot analysis. Our results indicated that the drug screen model was established successfully and that both honokiol and hyperoside induced P19 differentiation into neurons, with the possible molecular mechanism being modulating the Wnt signaling pathway. In conclusion, the drug screening model developed in the present study provides a rapid, cell-based screening platform for identifying natural compounds with neuronal differentiation effects.

基于细胞膜色谱技术的中药复杂体系目标物筛选分析装备

中药是临床防治疾病的有效药物。由于中药物质基础的复杂性以及成药过程的多环节因素,实现中药全面质量控制的难度相对较大。如果能够在明确中药有效物质与有害物质的基础上,在成药过程中重点控制其含量和限量,则能在很大程度上“纲举目张”,有效保障中药质量的可控性和重复性。当前,我国药物研发源头创新技术不足,研发效率有待提升,缺乏高通量的精准分析装备。我国医药分析装备长期依赖进口,高端智能分析装备缺乏,亟需构建“医药智能分析系统”。本项目在国家自然科学基金重大仪器研制项目的资助下,利用细胞膜色谱技术的原创性与独创性,结合生物工程技术和人工智能技术,研制二维细胞膜色谱(2D/CMC)分析仪,对成型的“2D/CMC-中药药效物质分析仪”和“2D/CMC-中药注射液类过敏物分析仪”开展了示范应用,提升了中药药效物质和过敏组分的筛选、发现的效率,并同步进行定性、定量分析,为中药提质增效提供高效分析工具。

消脂化纤汤联合骨髓间充质干细胞移植对实验性非酒精性脂肪性肝炎肝硬化小鼠的干预作用及机制

目的观察消脂化纤汤联合骨髓间充质干细胞(BM-MSCs)移植对实验性非酒精性脂肪性肝炎(NASH)肝硬化小鼠的干预作用及其可能机制。方法将40只健康SPF级C57BL/6小鼠随机分出8只作为空白组,其余32只小鼠采用蛋氨酸-胆碱缺乏(MCD)饮食联合10%四氯化碳(CCl4)腹腔注射诱导NASH肝硬化模型。造模成功后随机分为模型组、中药组、干细胞组、联合组,每组8只。其中中药组予消脂化纤汤灌胃,干细胞组行尾静脉注射BM-MSCs移植1次,联合组予消脂化纤汤灌胃的同时,行尾静脉注射BM-MSCs移植1次,空白组和模型组则不予任何干预。干预4周后取材,检测肝功能指标[丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、白蛋白(ALB)],酶联免疫吸附法测定肝组织维A酸相关孤儿受体γt(RORγt)、白细胞介素23(IL-23)、IL-21、IL-17含量,酶联免疫吸附法测定血清及肝组织肝细胞生长因子(HGF)含量,逆转录聚合酶链式反应(RT-PCR)测定肝脏淋巴细胞RORγt、IL-23、IL-21、IL-17 mRNA表达水平。结果(1)与空白组比较,模型组血清ALT、AST含量均升高,ALB含量降低(P<0.01),与模型组比较,不同干预组各指标均有不同程度改善,以联合组变化最显著(P<0.01)。(2)肝组织IL-23、IL-21、RORγt、IL-17水平比较:与模型组比较,各干预组小鼠肝组织各指标均降低,联合组优于各单纯干预组(P<0.01)。(3)RT-PCR结果显示:与模型组比较,各干预组小鼠肝脏淋巴细胞RORγt、IL-23、IL-21、IL-17 mRNA表达降低,联合组优于各单纯干预组(P<0.05)。(4)各组小鼠血清和肝组织HGF表达水平比较:联合组优于中药组与干细胞组(P<0.01)。结论消脂化纤汤联合BM-MSCs移植可抑制肝脏过度炎症反应,从而促进肝脏细胞再生,其作用机制可能与调节辅助性T细胞17(Th17)免疫学功能相关。

细胞膜色谱技术在中药活性成分筛选中的应用新进展

细胞膜色谱(CMC)是一种发展迅速的新兴仿生色谱技术,该技术将活性表征与色谱分离相结合,克服了传统“分离-评价”模式的缺点,凭借其在线、高通量、高灵敏度、自动化等多种方法学优势,在中药活性成分筛选研究中备受关注。通过对CMC在中药抗肿瘤、抗类过敏、抗心血管疾病、抗骨质疏松、抗炎镇痛、抗病毒、抗前列腺增生等有效成分及中药注射剂致敏成分筛选中的最新研究进展(2018-2023年)进行综述,旨在为未来更多中药相关活性成分的高通量筛选提供方法学参考依据,同时为创新药物发现、药效作用机制研究、中药及其制剂的质量控制提供参考。

药物筛选模型和技术及其在中药活性成分研究中的应用

现代生物技术的发展和药物作用靶标的揭示 ,为中药活性成分的研究提供了新的技术和手段。本文就整体动物模型、受体模型和分子生物色谱、细胞模型和细胞膜色谱、基因芯片技术 4种药物筛选模型和技术及其在中药活性成分研究中的应用进行了分析、综述 ,为推动中药活性成分研究、加速中药现代化步伐提供参考。

以丙型肝炎病毒体内感染裸鼠模型筛选20种常用清热解毒类中药

目的:用HCV体内感染裸鼠模型筛选20种常用清热解毒类中药,以寻找有效的抗HCV药物。方法:模型鼠用药3个月,用透射电子显微镜观察裸鼠脾内移植的人胎肝细胞中是否仍有HCV样颗粒,用定量RT-PCR技术检测用药前后血清HCVRNA含量。结果:(1)各中药组,用药3个月在裸鼠脾脏内移植的人胎肝细胞中均可找到HCV样颗粒。(2)仅龙胆草、黄芩、山豆根、栀子、苦参5味中药组,用药3个月后血清HCVRNA含量明显下降,其他中药组,用药3个月前后血清HCVRNA含量无明显下降。结论:该20种中药均无直接清除HCV的作用,但龙胆草、黄芩、山豆根、栀子及苦参5味中药可明显抑制HCVRNA的复制。

巨噬细胞甘露糖受体结合物筛选模型的初步建立

目的建立巨噬细胞甘露糖受体(MMR)结合物筛选模型,用于筛选以甘露糖受体(MR)为靶标的潜在活性物质。方法将小鼠腹腔巨噬细胞分别与不同浓度的D-甘露糖和D-半乳糖共孵,用流式细胞仪和荧光显微镜检测D-甘露糖和D-半乳糖对Mφ结合异硫氰酸荧光素标记的甘露糖化牛血清白蛋白(M-FITC-BSA)的拮抗作用,优化实验条件,建立MMR筛选模型。结果两种检测方法均显示:随D-甘露糖浓度的上升,M-FITC-BSA标记的Mφ检出率逐步下降(P<0.01),而这种现象在D-半乳糖组中未观察到;当D-甘露糖达到0.080mmol.L-1浓度时,便能明显拮抗Mφ结合M-FITC-BSA(P<0.05或P<0.01)。中药多糖的筛选结果显示:在一定浓度下,黄芪、白术、防风、大枣等中药多糖组分能浓度依赖性地拮抗小鼠腹腔Mφ与M-FITC-BSA的结合(P<0.05或P<0.01)。结论MMR模型对MR结合成分的筛选作用稳定而敏感,对快速筛选中药免疫调节成分、研究中药免疫调节机制具有重要意义。

Caco-2细胞模型的建立及其在中药吸收研究中的应用探讨

目的建立Caco-2细胞模型并探讨其在中药吸收研究中的应用。方法通过显微镜观察细胞形态学特点、测定跨细胞膜电阻和荧光黄通透量等指标,对Caco-2细胞模型进行评价。结果各项指标的测定值符合要求,本实验建立的Caco-2细胞模型的完整性、紧密性和通透性良好。结论建立的Caco-2细胞模型可用于研究中药化学成分转运的吸收机制;高灵敏度分析仪器可以促进Caco-2细胞模型在中药化学成分吸收研究中最大限度的应用;通过两个或多个中药化学成分相互作用的研究,可以阐明中药配伍和中药复方应用的科学性。