Reversal of tamoxifen resistance by artemisinin in ER+breast cancer:bioinformatics analysis and experimental validation

Breast cancer is the leading cause of cancer-related deaths in women worldwide,with Hormone Receptor(HR)+being the predominant subtype.Tamoxifen(TAM)serves as the primary treatment for HR+breast cancer.However,drug resistance often leads to recurrence,underscoring the need to develop new therapies to enhance patient quality of life and reduce recurrence rates.Artemisinin(ART)has demonstrated efficacy in inhibiting the growth of drug-resistant cells,positioning art as a viable option for counteracting endocrine resistance.This study explored the interaction between artemisinin and tamoxifen through a combined approach of bioinformatics analysis and experimental validation.Five characterized genes(ar,cdkn1a,erbb2,esr1,hsp90aa1)and seven drug-disease crossover genes(cyp2e1,rorc,mapk10,glp1r,egfr,pgr,mgll)were identified using WGCNA crossover analysis.Subsequent functional enrichment analyses were conducted.Our findings confirm a significant correlation between key cluster gene expression and immune cell infiltration in tamoxifen-resistant and-sensitized patients.scRNA-seq analysis revealed high expression of key cluster genes in epithelial cells,suggesting artemisinin’s specific impact on tumor cells in estrogen receptor(ER)-positive BC tissues.Molecular target docking and in vitro experiments with artemisinin on LCC9 cells demonstrated a reversal effect in reducing migratory and drug resistance of drug-resistant cells by modulating relevant drug resistance genes.These results indicate that artemisinin could potentially reverse tamoxifen resistance in ER-positive breast cancer.

Antimalarial qinghaosu/artemisinin: The therapy worthy of a Nobel Prize

Malaria is a major cause of human morbidity and mortality in the tropical endemic countries worldwide. This is largely due to the emergence and spread of resistance to most antimalarial drugs currently available. Based on the World Health Organization recommendation, artemisinin-based combination therapies are now used as first-line treatment for Plasmodium falciparum malaria. Artemisinin or qinghaosu(Chinese name) and its derivatives are highly potent, rapidly acting antimalarial drugs. Artemisinin was discovered in 1971 by a Chinese medical scientist Youyou Tu, who was awarded the Nobel Prize in 2015 on her discovering the antimalarial properties of qinghaosu from the traditional Chinese qinghao plant. Nevertheless, artemisinin resistance in falciparum malaria patients has first emerged on the Thai-Cambodian border in 2009, which is now prevalent across mainland Southeast Asia from Vietnam to Myanmar. Here, we reviewed malaria disease severity, history of artemisinin discovery, chemical structure, mechanism of drug action, artemisinin-based combination therapies, emergence and spread of drug resistance, including the recent findings on mechanism of resistance in the falciparum malaria parasite. This poses a serious threat to global malaria control and prompts renewed efforts for the urgent development of new antimalarial drugs.

Designing Artemisinins with Antimalarial Potential, Combining Molecular Electrostatic Potential, Ligand-Heme Interaction and Multivariate Models

Artemisinins tested against W-2 strains of malaria falciparum are investigated with molecular electrostatic potential (MEP), in an attempt to identify key features of the compounds that are necessary for their activities, as well as to investigate likely interactions with the receptor in a biological process and to use that information to propose new molecules. In order to discover the best geometry involving the ligand-receptor complexes (heme) studied and help in the proposition of the new derivatives, molecular simulations of interactions between the most negative charged region around the peroxide and heme locates (the ones around the Fe2+ ion) were carried out. In addition, PCA (principal components analysis), HCA (hierarchical cluster analysis), SDA (stepwise discriminant analysis), and KNN (K-nearest neighbor) multivariate models were employed to investigate which descriptors are responsible for the classification between the higher and lower antimalarial activity of the compounds, and also this information was used to propose new potentially active molecules. The information accumulated in studies of MEP, molecular docking, and multivariate analysis supported the proposal of new structures with potential antimalarial activities. The multivariate models constructed were applied to the new structures and indicated numbers 19 and 20 as the most prominent for syntheses and biological assays.

仪器分析实验课程思政案例设计与实践——以青蒿素的提取与结构鉴定的探究实验为例

中医药文化被喻为中华优秀传统文化的璀璨明珠,是中华传统文化的重要组成和典型代表。而作为抗疟明星的中医药青蒿素来源于天然产物,因此,天然产物中的青蒿素的提取和结构鉴定显得十分必要。课程团队结合课程特点,将青蒿素的提取和结构鉴定引入实验教学并进行课程思政教学案例设计。以此项目为载体,培养学生自主查阅文献、设计实验方案和运用仪器分析技术表征分子结构的科学素养。该教学案例的建立实施有助于教师充分挖掘课程中的思政元素,激发学生的学习兴趣,培养其自主创新意识,增强爱国情怀和民族文化自信,取得了良好的课程思政教学示范效果。

高效液相色谱-三重四级杆质谱测定黄花蒿中青蒿素含量方法的设计

利用高效液相色谱-三重四级杆质谱仪建立了黄花蒿中青蒿素含量的测定方法,并进行线性与范围、精密度、回收率及实验室比对方法学验证。结果表明:青蒿素在3~300 ng/mL线性良好(R^(2)=0.9994),检出限和定量限分别为0.25和0.75μg/g;该方法精密度良好,重复性RSD在2.0%~4.8%;平均加样回收率在93.8%~109.8%。分别在3个实验室对青蒿素含量比对检测,各实验室检测分析同一样品相对标准偏差在0.5%~3.3%。该实验项目难度适中,在保证可操作的同时具一定的创新性,可培养学生的科研探索精神,适用于研究生的中药成分分析开放性实验教学。

青蒿素改善咪喹莫特诱导银屑病样小鼠的作用研究

目的研究青蒿素通过调控微小RNA-129-5p(miRNA-129-5p)/钙/钙调蛋白依赖性蛋白激酶Ⅳ(CaMK4)轴介导信号转导及转录激活因子3(STAT3)信号通路对银屑病样小鼠的影响。方法将所有小鼠随机分为对照组(正常饲养)、模型组(0.9%NaCl)、阳性对照组(1 mg·kg^(-1)甲氨蝶呤)、实验组(100 g·mL^(-1)青蒿素)、miR-129-5p inhibitor组(在实验组的基础上尾静脉注射miR-129-5p inhibitor)、siCaMK4组(在实验组的基础上尾静脉注射siCaMK4)。除对照组外,其余各组小鼠均用咪喹莫特构建银屑病模型。用实时荧光定量聚合酶链反应法检测miR-129-5p、CaMK4 mRNA的相对表达水平,用双荧光素酶报告基因法验证miR-129-5p与CaMK4的靶向关系,用银屑病面积与严重性指数评分(PASI)评估皮损情况,用酶联免疫吸附试验法检测白细胞介素-17(IL-17)、IL-23、IL-22的水平,用蛋白质印迹法检测STAT3通路蛋白的相对表达水平。结果对照组、模型组和实验组的miR-129-5p相对表达水平分别为1.00±0.18、0.54±0.07和0.88±0.13,CaMK4 mRNA相对表达水平分别为1.00±0.17、1.68±0.31和1.20±0.22;对照组、模型组、阳性对照组、实验组、miR-129-5p inhibitor组和siCaMK4组的第7天PASI分别为0、(10.20±1.22)、(7.20±0.68)、(6.30±0.66)、(7.50±0.88)和(4.80±0.63)分,IL-17表达水平分别为(1.89±0.19)、(3.56±0.38)、(2.22±0.28)、(2.55±0.33)、(3.19±0.41)和(1.99±0.22)pg·mL^(-1),IL-23表达水平分别为(4.48±0.61)、(6.28±0.77)、(5.22±0.51)、(5.33±0.61)、(6.01±0.66)和(4.64±0.55)pg·mL^(-1),IL-22表达水平分别为(5.14±0.68)、(7.84±0.87)、(5.86±0.66)、(5.99±0.62)、(6.88±0.73)和(5.22±0.60)pg·mL^(-1),p-STAT3/STAT3比值分别为0.38±0.04、0.68±0.07、0.45±0.05、0.48±0.05、0.57±0.07和0.42±0.04。模型组的上述指标与对照组比较,阳性对照组、实验组的上述指标与模型组比较,miR-129-5p inhibitor组、siCaMK4组的上述指标与实验组比较,在统计学上差异均有统计学意义(P<0.05,P<0.01,P<0.001)。结论青蒿素可以改善银屑病小鼠皮损状态,抑制炎症反应,可能与调控miR-129-5p/CaMK4轴介导STAT3信号通路有关。

青蒿素抑制葡萄糖刺激下结直肠癌细胞恶性生物学行为的机制研究

目的探讨青蒿素(ART)对葡萄糖刺激下结直肠癌(CRC)细胞的恶性生物学行为的影响及其作用机制。方法以0、5、10、20、40、60μmol/L ART为浓度梯度处理人结直肠癌细胞株SW480,然后采用CCK-8法检测细胞活力。流式细胞术检测细胞凋亡情况;Transwell检测细胞迁移和侵袭情况;Western blot检测细胞凋亡、上皮-间质转化(EMT)及Janus激酶2(JAK2)/信号转导与转录激活因子3(STAT3)相关蛋白表达。结果与0μmol/L ART相比,5、10、20、40、60μmol/L ART处理下SW480细胞活力降低(P<0.05),IC_(50)为36.91μmol/L。故以10、20、40μmol/L ART处理的细胞为ART低、中、高剂量组,以0μmol/L ART处理的细胞为对照组,以ART 40μmol/L+Coumermycin A110μmol/L处理的细胞为Coumermycin A1组。与对照组相比,ART低剂量组、ART中剂量组、ART高剂量组细胞划痕愈合率、侵袭能力及Bcl-2、N-cadherin、Vimentin、p-JAK2、p-STAT3表达显著下降(P<0.05),细胞凋亡率及Bax、Caspase-3、E-cadherin表达上升(P<0.05);与ART高剂量组相比,Coumermycin A1组细胞划痕愈合率、侵袭能力及Bcl-2、N-cadherin、Vimentin、p-JAK2、p-STAT3表达水平显著上升(P<0.05),细胞凋亡率及Bax、Caspase-3、E-cadherin表达水平下降(P<0.05)。结论ART可能通过抑制JAK2/STAT3信号通路,抑制葡萄糖刺激下CRC细胞活力、迁移、侵袭和EMT,促进其凋亡。

青蒿素类化合物抗肿瘤作用机制研究进展

青蒿素及其衍生物不仅具有抗疟活性,还展现出了广泛的抗肿瘤活性。这类化合物通过多种途径实现抗肿瘤作用,包括诱导活性氧生成、阻滞肿瘤细胞增殖周期、引发肿瘤细胞凋亡、抑制肿瘤血管生成、抑制肿瘤细胞的侵袭和转移、克服肿瘤耐药性以及调节肿瘤免疫。本文对青蒿素类衍生物的抗肿瘤作用机制方面的最新研究进展进行了综述。

Effects of Fungal Elicitors on Cell Growth and Artemisinin Accumulation in Hairy Root Cultures of Artemisia annua

The artemisinin accumulation in the hairy root cultures of Artemisia annua L. was enhanced via a treatment of three fungal elicitors separately ( Verticillium dahliae Kleb., Rhizopus stolonifer (Ehrenb. ex Fr.) Vuill and Colletotrichum dematium (Pers.) Grove). Among these three elicitors, V. dahliae had the highest inducing efficiency, but none of them manifests any noticeable effects on the cell growth of the hairy root cultures. The artemisinin content of the hairy root cultures treated with V. dahliae elicitor was 1.12 mg/g DW, which was 45% higher than the control (0.77 mg/g DW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. In addition, the authors found that for V. dahliae , the optimum concentration was 0.4 mg carbohydrate per millilitre medium, the strongest response of A. annua hairy root cultures to the elicitation was at the late exponential growth stage, and the highest artemisinin content of the hairy root cultures was on the 4th day post treatment.

物理化学课程教学中的思政设计——以“理想稀溶液中任一组分的化学势”为例

立德树人是高校教育的根本任务,将思政元素无声融入课堂教学之中,有利于引导学生树立正确的世界观、价值观和人生观,从而成为合格的社会主义接班人。屠呦呦先生是中国第一位荣获诺贝尔自然科学奖的本土科学家。本文以其获得诺贝尔奖的工作“青蒿素高效提取并成功应用于疟疾防治”为例,结合物理化学多组分系统热力学章节中的“分配定律”,改进“理想稀溶液中任一组分的化学势”的教学过程,引导学生充分认识原理对实践的直接指导作用和中华传统文化在解决实际问题中的重要价值,理解科学发现的必然历程以及科学家在不断攀登科学高峰过程中所付出的艰辛和收获,将自己的聪明才智和所学所得应用于祖国的社会主义建设。

Elicitation on Artemisinin Biosynthesis in Artemisia annua Hairy Roots by the Oligosaccharide Extract from the Endophytic Colletotrichum sp. B501

The oligosaccharide elicitor from the mycelial wall of an endophytic Colletotrichum sp. B501 promoted the production of artemisinin in Artemisia annua L. hairy root culture. When hairy roots of 22-day-old cultures (later growth phase) were exposed to the elicitor (20 mg/L) for 4 d, the maximum content of artemisinin reached 1.15 mg/g, a 64.29% increment over the control. The electron X-ray microanalysis disclosed the rapid accumulation of Ca 2+ in the elicited cortical cells of hairy root. The electronic microscope observation revealed the high electron density area in vacuole of elicited cells. During the first day of elicitation the peroxidase activity of hairy roots was improved sharply. Some cellular morphological changes including cell shrinkage, condensation of cytoplasm and nuclear fragmentation, coincident with the appearance of DNA ladders, were observed after the third day of elicitation. It was suggested that the oligosaccharide elicitor triggered the programmed cell death, which may provide the substance or chemical signal for artemisinin biosynthesis.

青蒿素在肉鸡生产中的应用研究进展

青蒿素是从黄花蒿叶片中分离出来具有过氧基团的新型倍半萜内酯类的一种物质,也是青蒿发挥药理学功能的最主要活性成分。青蒿素作为饲料添加剂具有促进生长、抗菌消炎、抗球虫病、保护肠道健康和缓解热应激等作用。文章综述了青蒿素的理化性质、生物学功能、作用机理及其在肉鸡养殖中使用方法和使用量,为进一步将青蒿素作为新型饲料添加剂在肉鸡生产中的应用提供参考,从而推动肉鸡养殖业的绿色健康发展。

Selective Effect of Qinghaosu on Different Stages of Plasmodium falciparum in Vitro

Using highly synchronous cultures of Plasmodium falciparum in vitro,the susceptibi- lity of the different stages of the intraerythrocytic parasites to Qinghaosu (QHS) was assessed.The anti- parasitic effect of QHS was measured by comparing the changes of irradiation of^3 H-hypoxanthine in- corporated into the nucleic acids of parasites exposed to various concentrations of QHS at different stages of growth.It was found that the trophozoite stage of the parasite was the most sensitive to QHS, whereas the early ring stage was the least sensitive,and the sensitivities of the late ring and schizont stages fell between those of the early ring and trophozoite stages.The results revealed the correlation of stage-dependent effects of QHS with the blockade of the protein metabolism of the parasite.

HPLC法同时测定黄花蒿水提浸膏中青蒿素、青蒿乙素、猫眼草黄素和猫眼草酚D的含量

目的建立高效液相色谱(HPLC)法同时测定黄花蒿水提浸膏中青蒿素、青蒿乙素、猫眼草黄素、猫眼草酚D的含量。方法采用Agilent ZORBAX SB-C18(250 mm×4.6 mm,5μm)色谱柱;流动相:乙腈(A)-纯水(B),梯度洗脱;体积流量:0.8 mL·min^(-1);柱温:30℃;进样量:10μL;检测波长:210 nm。结果青蒿素、青蒿乙素、猫眼草黄素和猫眼草酚D进样量分别在1.6088~16.088μg(r=0.9999)、0.0141~0.1414μg(r=1)、0.1851~1.8509μg(r=0.9999)、0.1441~1.4414μg(r=0.9999)范围内与峰面积呈良好的线性关系,平均加样回收率(n=6)分别为102.44%、97.82%、95.07%、95.55%,RSD分别为1.12%、1.44%、1.29%、1.53%。结论该方法简便准确、可靠稳定、重复性好,可用于同时测定黄花蒿水提浸膏中青蒿素、青蒿乙素、猫眼草酚D、猫眼草黄素的含量。

青蒿素通过TIGIT/CD155信号轴调控4T1乳腺癌细胞转移与凋亡

乳腺癌是严重影响女性生命健康的恶性肿瘤之一。近年来,中药植物活性成分-青蒿素(artemisinin, ART)的抗肿瘤作用愈显突出。然而,目前仍不清楚ART在治疗乳腺癌转移方面的效果及其作用机制。本文以小鼠乳腺癌细胞4T1及小鼠乳腺上皮细胞HC11为研究对象,探索ART抑制4T1细胞转移并诱导凋亡的作用,并揭示其可能的分子机制。MTS(CellTiter 96?AQueous One Solution Cell Proliferation Assay, MTS)实验表明,ART能够明显抑制4T1细胞活力,且具有ART浓度及处理时长依赖性。通过细胞集落形成、Transwell及细胞划痕结果分析发现,ART能够显著抑制乳腺癌4T1细胞转移。流式细胞术结果显示,ART能够促进肿瘤细胞的ROS产生、线粒体膜电位丧失、细胞周期阻滞进而引起细胞凋亡。生物信息分析方法及研究证实,TIGIT极显著高表达(P<0.001)与乳腺癌转移密切相关,ART能够显著降低TIGIT/CD155信号轴的基因表达水平(P<0.001,P<0.01)。综上所述,本研究初步揭示了ART可能通过阻断TIGIT/CD155信号轴抑制乳腺癌转移的调控机制,为ART作为乳腺癌的潜在治疗药物提供依据,有助于探索利用中药有效成分治疗肿瘤的新途径。

基于网络药理学和分子对接研究青蒿素抗胃癌的作用机制及初步验证

目的:利用网络药理学和实验验证探究青蒿素抗胃癌的潜在作用机制。方法:利用中药系统药理学数据库和疾病靶点数据库筛选青蒿素的作用靶点。使用Cytoscape软件构建“药物-疾病-靶点”网络图,利用String数据库构建蛋白质相互作用(PPI)网络图。DAVIDA数据库对交集基因进行基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)富集分析,最终构建活性成分-靶点-关键通路网络。利用AutoDock Vina进行分子对接并计算结合亲和力。使用GEPIA和HPA等数据库分析核心靶点的mRNA和蛋白表达水平。结果:通过网络分析最终得到ABCB1、HSP90AA1、VEGFA三个核心靶点。功能富集分析显示,这些靶点基因通过PI3K-Akt信号通路对胃癌发挥良好的治疗效果。体外实验验证了青蒿素对胃癌细胞增殖和迁移的抑制作用,并且发现青蒿素能够下调核心靶基因ABCB1、HSP90AA1、VEGFA的表达,可能和PI3K-Akt信号通路有关。结论:本研究阐明了青蒿素在抗胃癌中的潜在靶点和分子机制,为传统中药治疗胃癌提供了新的思路和线索。

热毒宁吸入粉雾剂的制备与质量标准研究

目的:建立热毒宁吸入粉雾剂的质量标准。方法:通过实验对热毒宁吸入粉雾剂进行制备工艺优化,采用高效液相色谱法(HPLC)测定热毒宁吸入粉雾剂中绿原酸、栀子苷和青蒿素的含量。运用激光粒度仪测定热毒宁吸入粉雾剂的粉末粒径分布,结合药典和相关法规的要求,制定热毒宁吸入粉雾剂的质量标准。结果:15 g金银花和12 g青蒿在3倍体积的水中提取6 h,可以获得最佳的挥发油提取量,在6倍水体积,pH=2.5的条件下,提取240 min可以获得最优的绿原酸和浸膏得率。在乙醇浓度为70%,提取时间180 min, 6倍加液条件下,获得最佳的栀子醇提物得率。在温度80℃、风速600 L/h,进药量3 mL/min的条件下进行喷雾干燥,发现乙醇含量为60%时为最优条件,其粒径分布在1~5μm的体积百分数为67.6%,且得粉率为71.2%。喷雾微粉在25℃条件下储存1个月和3个月,药物分别降解0.25%和2.13%,水分分别增加0.02%和0.05%,且在避光和正常光照条件下无明显变化。热毒宁喷吸入雾剂中绿原酸含量不低于5.0%、青蒿素含量不低于3.2%、栀子苷含量不低于6.8%。热毒宁喷吸入雾剂中总菌落数低于1 000 cfu/g,真菌和酵母低于100 cfu/g,未检出大肠杆菌。结论:该优化后工艺可靠,含量测定的精密度、准确性、重复性均符合方法学要求,可用于热毒宁吸入粉雾剂的生产和质量控制。

熊果酸拼接抗肿瘤活性分子衍生物的合成及抗肺癌活性研究

研究熊果酸拼接抗肿瘤活性分子衍生物的抗肺癌活性及其机制。以熊果酸为原料,琥珀酰基作为linker,分别与鬼臼毒素、吲哚、奎宁、喜树碱、双氢青蒿素进行拼接,得到5个目标化合物。采用CCK-8法评价其对人非小细胞肺癌细胞株(A549)、人肺腺癌耐顺铂细胞株(A549/DDP)的抗增殖活性;不同浓度的化合物3d作用于A549/DDP后,AO/EB双荧光染色法检测,Annexin V-FITC/PI双染法检测细胞凋亡,细胞划痕实验检测细胞的迁移能力,PI染色法检测细胞周期。化合物3d对A549/DDP的体外抗增殖活性优于阳性对照药物(顺铂、依托泊苷),且表现较好的选择性,初步机制研究表明:化合物3d作用于A549/DDP之后,呈正常形态的活细胞不断减少,呈核染色质固缩状等形态的细胞不断增加,显著地阻滞A549/DDP细胞G_(2)/M期,干扰细胞分裂过程诱导其发生凋亡,同时抑制其细胞迁徙能力,且与化合物3d的浓度呈正相关。

青蒿药材中青蒿素快速检测的实验设计

文章介绍了课题组的科研成果“青蒿药材中青蒿素的快速检测”转化为教学实验的设计。以石油醚为溶剂,通过超声波从青蒿药材粉末中提取出青蒿素后,提取液挥干石油醚后,残渣用95%乙醇溶解,再用NaOH将溶液调至碱性,50℃水浴反应30 min后,0.22μm滤膜过滤,292 nm处测定吸光度,利用标准对照法测得青蒿药材中青蒿素的含量。课题组将最新研究成果设计成综合性实验,应用于学生课堂教学,旨在训练学生基本技能的同时,拓展学生的知识面,并激发学生的科研兴趣,进一步培养学生分析问题、解决问题的能力,同时也将科研成果落到实处。

青蒿素-丙酮层共组装纳米药制备、表征及体外抗肿瘤初步评价

为解决青蒿素及其丙酮层(以多甲氧基黄酮为主成分的青蒿素工业提取废料)的水溶性差导致其抗肿瘤应用受限的问题,采用乳化-溶液挥发法,构建质量均一、结构稳定的青蒿素-丙酮层超分子共组装纳米药。应用激光粒度仪、傅里叶变换红外光谱仪及扫描电子显微镜等对其进行表征,并以MTT、划痕试验测定纳米药对小鼠源CT26结肠癌细胞增殖、迁移能力的影响。结果表明:所制备的纳米药平均粒径106.93 nm,载药量7.78%,包封率72%;MTT试验检测共组装药物(青蒿素、丙酮层量之比1∶2)IC_(50)为青蒿素、丙酮层浓度比17.13 mg·L^(-1)∶34.27 mg·L^(-1),显著优于各药单用组;划痕试验表明共组装药物对肿瘤细胞迁移具有显著抑制作用。综上,制备的青蒿素-丙酮层超分子共组装纳米药具有较强抗肿瘤活性,为抗肿瘤药物开发和中药提取废弃物循环利用奠定了重要基础。