Dehydrozaluzanin C (DC) is a sesquiterpene lactone isolated from Asteraceae plant Ainsliaea macrocephala. To investigate the antitumor effects of DC and possible molecular mechanisms for treating cancer. The antitumor effect of DC was studied using HT-29 and HCT-116 human colon tumor cell lines and Balb/c nude mice models. The anti-proliferative, proapoptotic effects, and cycle arrest of DC were observed by cell viability, colony formation, apoptosis, and cycle assays. The changes of protein expression level were examined by Western blot analysis. The transcription activity of PPAR gamma was determined by Luciferase reporter assay. The role of PPAR gamma activation in the antitumor activity of DC was verified using PPAR gamma antagonist GW9662 and si-PPAR gamma HT-29 cells. DC treatment significantly decreased colon tumor cell viability, cell clone number, and increased apoptosis rate and arrested cell cycle at S phase. Furthermore, DC treatment significantly decreased Bcl-2, CDK2, and cyclin A2 protein levels while increasing the expression of cleaved caspase 3 and Bax in HT-29 and HCT-116 cells. Further investigations indicated that cell survival, induction of apoptosis, and cycle arrest by DC could be significantly reversed following treatment with the PPAR gamma antagonist GW9662 or in si-PPAR gamma cells. In vivo, DC treatment significantly decreased the weight and volume of xenograft tumor tissues in mice and apoptosis-related protein levels. The results suggest that DC effectively inhibits colon tumor cell proliferation, clone formation, apoptosis, and cell cycle arrest through PPAR gamma activation. These results support the potential of DC as an anti-tumor lead compound for further investigation.

Cytochrome P450 1A1 (CYP1A1), a heme-containing monooxygenase, is of particular importance for human health because of its vital roles in the metabolic activation of pro-carcinogenic compounds to the carcinogens. Deciphering the relevance of CYP1A1 to human diseases and screening of CYP1A1 modulators require reliable tool(s) for probing this key enzyme in complex biological matrices. Herein, a practical and ultrasensitive fluorescence-based assay for real-time sensing CYP1A1 activities in biological systems has been developed, via designing an isoform-specific fluorogenic sensor for CYP1A1 (CHPO). The newly developed fluorogenic substrate for CYP1A1 has been carefully investigated in terms of specificity, sensitivity, precision, quantitative linear range and the anti-interference ability. The excellent selectivity, strong anti-interference ability and fast response kinetics, making the practicability of CHPObased CYP1A1 activity assay is better than that of most reported CYP1A1 activity assays. Furthermore, CHPO has been successfully used for imaging CYP1A1 activities in living cells and human tissues, as well as for high-throughput screening of CYP1A1 inhibitors using tissue preparations as enzyme sources. Collectively, this study provided a practical fluorogenic sensor for real-time sensing CYP1A1 in complex biological systems, which would strongly facilitate the investigations on the relevance of CYP1A1 to human diseases and promote high-throughput screening of CYP1A1 modulators for biomedical applications. (C) 2020 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.

Dipeptidyl peptidase-IV (DPP-IV), an important prolyl-specific peptidase in the serine hydrolase family, hydrolyzes a large number of endogenous oligopeptides. In clinical studies, the detection of DPP-IV, especially the detection of its activity, is particularly important. We previously reported a specific fluorescent two-photon probe for DPP-IV. In this study, to further verify that our probe can be used for DPP-IV activity detection, we developed an accurate and sensitive method for the determination of DPP-IV activity in human tissue microsomes, cell homogenates or cell supernatants by liquid chromatography-fluorescence detection (LC-FD). This method is based on the fluorescence generated from BAN obtained from the hydrolysis of GP-BAN by detecting DPP-IV. The linearity, sensitivity, recovery, precision and stability of the method were fully validated. The method based on LC-FD has a lower limit of quantitation for BAN (product of DPP-IV), as low as 5 nM (only needs 2 μL), which is much lower than that of the others. This method also demonstrated good accuracy and precision; variances of both were less than 15% between the intra-and inter-assay. In addition, the method was successfully applied to the determination of DPP-IV activity in various cell homogenates and cell supernatants. The development of the LC-FD method will help to understand the expression and function of DPP-IV in different biological samples in humans for future studies. © The Royal Society of Chemistry 2020.

目的:药物肾毒性的传统评价方法多采用体内动物模型,通过观察短期或长期给药后动物血清、尿液中尿素氮（BUN）、肌酐（Scr）等生物标志物以及试验终点肾脏病理组织形态学的改变来评价药物肾毒性,并推断药物对人体的可能毒性,该方法耗时、耗资、耗力,且反应指标不敏感。而体外2D肾细胞系模型不能准确反应药物在体内生理环境中的毒性作用。现代药物毒理学研究开始由体内研究向体内和体外研究相结合发展,建立高通量肾毒性体外评价模型可以提高目标化合物的筛选效率,最新研究表明人诱导多能干细胞在适宜的体外条件下可分化再生形成三维的球状类器官（Organoid）结构,其生理功能与肾类似。本研究基于肾3D类器官构建一种新颖的评价药物肾毒性的方法,并将其应用于中药单体商陆皂苷甲的肾毒性评价。方法:首先向铺满基质胶的培养皿中接种人诱导多能干细胞,待细胞达到40～50%汇合度时,去除无饲养层培养基,加入含细胞因子的基础培养基,进行单细胞培养分化,收集细胞置于Transwell培养装置中,制作肾类器官,并对其进行形态观察及验证,确保其生长状态良好。其次,使用阳性化合物（顺铂、庆大霉素）对3D类器官模型进行验证,具体包括给药后3D类器官形态的变化。最后利用该模型对中药单体商陆皂苷甲进行肾毒性评价。结果:通过显微观察可以看到转入Transwell 3D培养后类器官的雏形从开始的细胞团,逐渐分化出管状结构,且随着时间推移,管状结构越清晰,未分化细胞即阴影部分越来越少。在高倍镜下观察类器官边缘部分,可见其明显出现多个高折光的管状结构轮廓,特异性表达蛋白验证肾类器官。当培养基中分别加入不同浓度的顺铂、庆大霉素时,随着药物浓度增加,肾类器官形态破坏的程度和数量明显增加。通过高内涵染色观察,在给与不同浓度的商陆皂苷甲后,肾3D类器官中凋亡细胞的数量及肾损伤因子（KIM-1）蛋白表达明显增加,活细胞数量明显减少。同时培养基中BUN含量与阴性对照相比明显升高,呈量效及时效关系。结论:本研究成功构建了基于3D类器官的肾毒性评价方法,并将其应用于中药单体商陆皂苷甲的肾毒性评价,其具体的损伤机制还有待进一步深入研究。基于3D类器官进行药物肾毒性评价不仅可减少受试物用量,利于早期对化合物潜在肾毒性进行深入评价,更能快速反应药物对肾脏毒性作用的具体部位,而且可在短期内完成对目标化合物肾毒性机制研究,缩短研究周期,减少实验动物的使用。

摘要： 目的 基于药代动力学研究山豆根对角叉菜胶致大鼠足跖肿胀模型的抗炎作用及机制。方法 雄性SD大鼠分为空白对照组、氢化可的松组(40 mg·kg^-1 )、山豆根水提浸膏粉高剂量组(生药0. 6 g·kg^-1 )和低剂量组(生药0. 3 g·kg^-1 )，测定致炎前和致炎后0. 5、1、2、和3 h足跖肿胀度、致炎组织中炎症因子TNF-α、IL-1β和IL-6含量及血中苦参碱、氧化苦参碱药代动力学参数。结果 氢化可的松组致炎后肿胀率明显降低（P <0. 01），山豆根水提浸膏粉组致炎后肿胀率亦降低（P <0. 05）且体内苦参碱含量呈剂量依赖性，高剂量组AUC值约为低剂量组两倍，达峰时间与抗炎作用高峰时间一致;山豆根水提浸膏粉可显著下调炎症因子含量（P <0. 01），抑制其表达的作用呈剂量依赖性。结论 山豆根对角叉菜胶致大鼠足跖肿胀模型具有抗炎作用，且呈剂量依赖性，与体内苦参碱系统暴露量成正相关，抗炎机制与抑制炎症因子表达有关。

Accumulating studies have indicated that reactive oxygen species (ROS) may be implicated into the destructive pathological events of rheumatoid arthritis (RA). As an effective antioxidant, artesunate (ARS) was reported to exert antiarthritic effects. However, whether ARS attenuates the bone erosion during RA progression by regulating ROS production remains to be defined. To address this problem, the inhibitive effects of ARS on osteoclastogenesis were observed in vitro. Mechanically, ARS significantly inhibited the NFATc1 signaling accompanied by markedly suppressing ROS production, which was abnormally enhanced during the pathological process of bone erosion. In addition, ARS may function as a potent ROS scavenger and significantly elevate the expression of HO-1 and NQO1 by activating Nrf2. Moreover, p62 accumulation induced by ARS was responsible for the activation of Nrf2, while the knockdown of p62 in osteoclast precursor cells diminished the suppressive effect of ARS on ROS production during osteoclastogenesis. Consistently, we also demonstrated that ARS effectively suppressed ROS production, leading to the inhibition of arthritic bone destruction by activating antioxidant enzyme and Nrf2/p62 signaling in the knee and ankle tissues of CIA rats. Collectively, our data offer the convincing evidence that ARS may inhibit osteoclastogenesis and ameliorate arthritic bone erosion through suppressing the generation of ROS via activating the p62/Nrf2 signaling.

具有良好导电性能的碳纳米材料,如碳纳米管及石墨烯,有望恢复心肌梗死后梗死区瘢痕组织所致的心肌组织电传导阻滞及心脏电活动紊乱,在心肌组织工程领域展现出重要的应用价值。文章综述了短碳纳米管特性、合成方法和导电纳米材料在心肌再生领域研究进展,阐述石墨烯的性能、作为生物支架材料在心肌组织工程中的应用、毒性等,并探讨了基于碳纳米材料的工程化心肌组织向临床转化的局限性及应用前景。碳纳米材料常通过心肌内注射或心脏贴片等方式植入体内,为了减少开胸手术对患者造成的伤害,需开辟微创介入等植入工程化组织的新途径。

In an earlier study, we had demonstrated ameliorative effects of Dendrobium nobile Lindl. on dyslipidemia and fatty liver in mouse fed a high fat diet. In the present study, we have examined the effect of D. nobile on the lipid metabolism in normal healthy rats after intragastric treatment for four times a day for five days. The treatment followed a metabolomics analysis of the plasma samples for metabolites by ultra-performance liquid chromatography-mass spectroscopy and the expression levels of genes for lipid metabolism. The metabolite profiles were substantially altered by D. nobile. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis, metabolic pathways were ranked first in terms of the number of referenced metabolites. There were 112 metabolites that were changed significantly in the pathways. The results revealed that D. nobile increased the content of epoxy-octadecenoic acids in plasma more than 19-folds. Moreover, the expression of genes Cyp1a2, Cyp2e1, Cyp2j2, Cyp3a1, Ppar gamma, Lpl, Acsl1, and Hmgcs2 were all induced in D. nobile treatment group (P < 0.05). Our results supported that D. nobile has a significant role in regulating lipid metabolism in healthy rats. Therefore, it is tempting to speculate that D. nobile would be beneficial in protecting human against the disorders of lipid metabolism.

随着组织工程、分子生物学、荧光探针、细胞成像及图像采集等技术的发展,近年来越来越多模拟人肝功能的细胞培养技术以及更灵敏的细胞功能检测技术被开发出来用于药物肝毒性评价,这些技术的应用有效的提高了药物肝毒性预测和评价的准确性。本文综述了用于肝毒性评价的细胞培养和检测技术的最新进展。在细胞培养技术方面,研究人员陆续开发出了肝细胞夹心培养（Sandwich-Cultured Hepatocyte,SCH）、三维培养（3D culture）、共培养（co-culture system）等技术。其中,肝细胞夹心培养（Sandwich-Cultured Hepatocyte,SCH）技术即是把肝细胞置于两层特殊培养基之间培养的常用细胞系统。SCH能较长时间的维持肝细胞生理功能,增加代谢酶活性,恢复细胞多边形态,增强蛋白分泌,其价值体现在培养过程中形成了与胆汁摄取和分泌相关的胆小管结构,这使SCH更适于研究胆管毒性和胆汁淤积性肝毒性。SCH己被用来评价了利福平、波生坦等大量西药对胆汁分泌和排泄的影响,近年来也有研究者将SCH用于小檗碱、巴马汀和药根碱等中药成分研究中,以评价这些成分在肝细胞中胆汁外排特征。三维培养（3D culture）技术是使用特殊的方法、材料及装置,使肝细胞呈立体方式生长,形成类似器官的微结构,从而表现出肝脏特有的功能。根据所用方法、材料和装置的不同,三维培养可分为生物反应器三维培养、聚球体培养、微流控芯片三维培养等,该类模型的共同特点是能长期维持肝细胞的功能并允许药物在培养体系内反复暴露,临床上多数药物需要反复暴露才能表现出肝毒性,此方法模拟药物在人体内长期暴露的状态,从而更有效预测药物对人体肝毒性。已有大量研究用三维培养肝细胞评价了曲伐沙星、对乙酰氨基酚、左氧氟沙星等西药长期暴露的肝毒性。李丹丹等人用3D HepG2细胞分别评价了中药何首乌及补骨脂的单次给药毒性和7天给药毒性,结果发现3D HepG2细胞对重复给予上述药物表现出更高的毒性敏感性。共培养（co-culture system）技术是将两种以上细胞混合共同培养的细胞模型,肝细胞常可与肝非实质细胞（Kupffer细胞、巨噬细胞等）或非肝细胞（成纤维细胞、脂肪细胞等）共培养,有研究者将人肝细胞、星状细胞、巨噬细胞和内皮细胞进行了混合培养,结果发现细胞的生物转化功能、对炎症刺激的反应、细胞间的联系和细胞桥粒的形成均得到改善。该模型能模拟肝脏微环境,有利于评价细胞与细胞之间的相互作用,以及发现药物肝毒性的作用机制及可能作用靶点。在分析技术方面,除了传统的细胞活力测定以外,也新发展出了高内涵分析（High-Content Analysis,HCA）技术,HCA是基于指示毒性参数的荧光探针和自动化细胞成像分析技术的细胞毒性评价方法,其特点是能够定量监测由药物引起的细胞形态、生化参数、亚细胞功能的改变,实现了对单个细胞的多参数、多靶点动态分析,并能按时间顺序呈现亚细胞器的损伤变化,有利于阐明细胞损伤机制。马喆等人将HCA用于何首乌肝毒性成分的分析及致毒机制的探讨,结果发现芦荟大黄素、大黄素、大黄酸和没食子酸可能是致肝毒性的主要成分其致肝毒性机制与线粒体介导的细胞凋亡有关。程源等人也用HCA评价了诃子的肝毒性,发现具有快速、高效、通量化检测细胞毒性的特点的HCA技术更适合于多成分、多作用靶点中药的毒理学研究。体外肝毒性预测和评价在中药毒性成分筛选和毒性机制阐明上有不可替代的作用,上述培养和检测技术均能显著提高药物肝毒性评价的可靠性,部分技术已应用于中药提取物及成分的肝毒性研究中,并用于阐明中药肝毒性成分和作用机制。但上述方法在使用上也均存在局限性,我们期待随着技术发展,能建立近似模拟体内环境的肝细胞培养模型,甚至将肝细胞培养系统与其他组织模型连接起来,形成"人体芯片"的微生理系统,为中药肝毒性的准确评价提供更完善的工具。

目的：探索ROS对类风湿关节炎（RA）骨破坏的影响研究。方法：建立Ⅱ型胶原诱导性类风湿关节炎（CIA）大鼠模型，观察大鼠关节红肿畸形等症状；苏木精-伊红染色（HE）和Masson三色染色观察关节滑膜、软骨、骨组织等病理变化；抗酒石酸酸性磷酸酶（TRAP）染色观察关节破骨细胞生成情况；二氢乙锭（DHE）荧光探针检测关节组织中ROS的表达。体外巨噬细胞集落刺激因子（M-CSF）和核因子κB受体活化因子配体（RANKL）诱导小鼠原代骨髓巨噬细胞（BMMs）48h、96h后，TRAP和鬼笔环肽染色观察破骨细胞形成和分化情况，RT-PCR和免疫荧光技术检测TRAP、基质金属蛋白酶9（MMP-9）、组织蛋白酶K（CTSK）mRNA和TRAP、MMP-9蛋白的表达；DCFH-DA探针检测破骨细胞中ROS变化水平。结果：CIA大鼠关节组织呈现炎性细胞浸润、滑膜异常增生、TRAP阳性细胞大量形成，骨破坏严重区域ROS表达明显增强。此外，成功构建体外破骨细胞分化模型，48h开始出现多核破骨细胞，第96h达到最多。溶骨功能相关因子mRNA和蛋白的表达呈诱导时间依赖性增加的同时，ROS的表达呈现趋势性累积；而且，ROS抑制剂NAC明显抑制了破骨细胞的分化。结论：ROS可能通过参与破骨细胞的分化影响RA骨破坏，抑制ROS产生可明显抑制破骨细胞分化。