黄花蒿Dof基因家族鉴定及在GA-UV处理下对青蒿素生物合成的影响＊

目的 Dof（DNA binding with one finger）家族是高等植物中特有的一类转录因子家族，参与植物中光、激素、非生物胁迫等多种胁迫响应调控。本研究基于全基因组数据对黄花蒿Dof（AaDof）转录因子家族进行鉴定及表达模式分析，探究Dof家族基因在青蒿素合成调控中的作用。方法 经PFAM数据库鉴定获得AaDof序列，通过生物信息学软件分析其理化性质、亚细胞定位、基因结构、蛋白保守结构以及启动子序列结合元件等，并基于赤霉素（Gibberellic acid， GA）、紫外线B（UV-B）及二者协同胁迫下黄花蒿转录组数据对其表达模式进行分析。结果 本研究从全基因组水平共鉴定出51个AaDof基因，均含有保守的C₂-C₂单锌指结构，依据系统发育分析分为8个亚族，同一亚族内基因结构与蛋白保守结构域相对保守。亚细胞定位预测显示12个AaDof蛋白定位在细胞外，其余均定位在细胞核。启动子元件分析发现AaDof家族基因启动子区富含光、激素等多种响应元件。对AaDof在GA、UV-B和GA+UV-B处理下的表达模式分析发现，AaDof基因对GA胁迫处理响应较弱，仅有少量基因敏感，其表达主要受到UV-B胁迫影响。C1及C2.1亚族大部分基因在UV-B胁迫下上调表达，而A亚族大部分基因在UV-B胁迫下下调表达。qRT-PCR验证表明AaDof1、AaDof17和AaDof44在GA和UV-B处理下表达量显著上调，推测其可能通过参与GA和UV-B调控网络，正向调控青蒿素生物合成。结论 本研究系统鉴定了黄花蒿AaDof家族基因并筛选了3个可能正向调控青蒿素生物合成的候选AaDof基因，为黄花蒿Dof家族基因功能研究及其在青蒿素生物合成中的调控机制解析奠定基础。     

Updates on k13 mutant alleles for artemisinin resistance in Plasmodium falciparum

Background

In the fight against malaria caused by Plasmodium falciparum, the successes achieved by artemisinin were endangered by resistance of the parasites to the drug. Whole genome sequencing approach on artemisinin resistant parasite line discovered k13 gene associated with drug resistance. In vitro and in vivo studies indicated mutations in the k13 gene were linked to the artemisinin resistance.

Evaluation of Apoptotic and Antileishmanial Activities of Artemisinin on Promastigotes and BALB/C Mice Infected with Leishmania major

Background:

In leishmaniasis, some drugs prescribed for treatment have toxic effects and there are reports about drug resistance in some countries. Due to this fact, using herbal drugs such as artemisinin with good efficacy and low toxic effect might be suitable.

Methods:

We evaluated the apoptotic effect of artemisinin on Leishmania major in vitro and the antileishmanial activities of artemisinin on leishmaniasis in BALB/c mice and at the end INF-γ and IL-4 cytokines levels were detected by ELISA in spleen cell culture supernatants. During treatment the lesion size and survival rate were measured each four and ten days, respectively.

Results:

Percentage of early and late apoptosis in promastigotes of control group and promastigotes treated with 10, 25, 50 and 100 μg/ml of artemisinin after 48 h were 0.13, 16.04, 41.23, 49.03 and 81.83, respectively. The IFN-γ in ointment treated group were higher than those of other groups (P<0.05). The in vivo results showed that ointment compounds healed the lesions more effectively rather than intraperitoneal injection method (P<0.05). The survival rate of mice 150 days after challenge in treated group with ointment of artemisinin was 66% while all mice in control groups were died.

Conclusion:

All of in vitro results represented that this drug had antileishmanial effects and these results were confirmed by evaluation effects in vivo condition of leishmaniasis. Interestingly, according to these results it can be concluded that this drug has antileishmanial effects in vitro and in vivo conditions. Artemisinin induces cytotoxic effect on L. major via apoptosis-related mechanism.     

青蒿素衍生物对卡氏肺孢子虫基因的影响

目的：研究双氢青蒿素，蒿甲醚，青蒿琥酯对卡氏肺孢子虫5S rRNA基因，线粒体rRNA大亚基基因（mt LSU rRNA)，胸腺嘧啶核苷酸合成酶（TS）基因的影响，方法：以上述3种药物治疗实验大鼠肺孢子虫肺炎，治疗结束后收集鼠肺，以酚－氯仿抽提法抽提肺总DNA，以PCR方法扩增肺孢子虫的上述3基因，以双脱氧末端终止法测定PCR产物的核苷酸序列，并研究双氢青蒿素对基因序列的影响，结果：3种药物作用过的虫体均未见TS基因扩增产物，部分虫体无5S rRNA基因，mt LSU rRNA基因扩增产物，经双氢青蒿素作用过的与正常肺子虫的5S rRNA基因，mt LSU rRNA基因序列发生变异。     

青蒿素对ApoE-/-基因敲除小鼠动脉粥样硬化的影响

目的探讨青蒿素对ApoE-/-基因敲除小鼠动脉粥样硬化以及炎症因子的影响。方法将20只ApoE-/-基因敲除小鼠分为青蒿素组和PBS处理组,每组10只,经高脂喂养8周,与对照组小鼠(C57BL/6J标准饮食小鼠,10只)比较,通过血管大体油红O染色评价斑块面积;HE染色观察病变形态及血浆中炎症因子的变化。结果与对照组比较,PBS处理组及青蒿素组均可见动脉硬化斑块,炎症因子水平升高。青蒿素组的动脉硬化程度及炎症因子水平均明显低于PBS处理组。结论青蒿素可以改善ApoE-/-基因敲除小鼠动脉粥样硬化进展。     

青蒿素类抗疟药的作用机制及耐药机制研究进展

青蒿素作为重要的抗疟药物,因其抗疟作用效率高、速度快、毒性低并且与大部分其他类别的抗疟药无交叉抗性等优点,成为目前全球抗疟的主要药物,虽然在泰柬边境地区已出现了青蒿素耐药性,但就目前全球各．地使用青蒿素及其衍生物为基础的联合疗法（ACT）疗效来看仍能达到90％以上,因此必须对刚刚出现的青蒿素耐药性现象迅速采取遏制行动。本文主要通过描述青蒿素的抗疟机制,讨论其耐药性机制,以及对青蒿素的发展前景作一综述。     

青蒿素调控p53蛋白诱导肝癌细胞凋亡的机制研究

目的 通过探讨青蒿素调控p53表达进而诱导肝癌细胞凋亡的情况研究青蒿素抗肿瘤的机理.方法 20只C57小鼠随机分为空白组、PBS组、青蒿素组及青蒿素+Pifithrin-α组,每组5只.以PBS组、青蒿素组及青蒿素+Pifithrin-α组小鼠联合应用DEN/CCM/乙醇构建肝癌模型.空白组小鼠正常饲养3周,PBS组腹腔注射PBS 3周,青蒿素组腹腔注射青蒿素(100mg/kg)3周,青蒿素+Pifithrin-α组应用Pifithrin-α (50 mg/kg)隔日作用1周后,再与青蒿素作用3周.处死各组小鼠,分离小鼠肿瘤组织,Western blot检测肿瘤组织中Caspase3及p53的表达.结果 成功构建小鼠肝癌模型,青蒿素组荷瘤小鼠经作用后肿瘤组织Caspase3及p53蛋白表达显著增加,青蒿素+Pifithrin-α组荷瘤小鼠经Pifithrin-α预处理后,青蒿素诱导的细胞凋亡蛋白Caspase3显著降低.结论 青蒿素通过调控p53的表达诱导肝癌细胞凋亡,进而发挥抗肿瘤作用.     

青蒿素高含量地区黄花蒿种质资源的简单重复序列区间分析

目的:分析广西、贵州和重庆3个青蒿素高含量地区的野生黄花蒿资源的遗传多样性。方法:以40份黄花蒿为试材,通过简单重复序列区间(ISSR)分析,运用POPGENE version1.31软件和TREECONW软件计算相关参数,UPGMA方法聚类,构建亲缘关系树状图。结果:18条ISSR引物扩增出84条带,多态比率为79.76%;物种水平上,ISSR标记揭示的Nei's基因多样性指数为0.2019,Shannon信息指数为0.3244;3个地区之间ISSR标记揭示的Nei's基因多样性指数范围为0.1867～0.1943,Shannon信息指数范围为0.2936～0.3073;ISSR检测不同材料间遗传距离范围为0.1467～0.4493,平均为0.3132。结论:ISSR标记能有效地揭示青蒿素高含量地区野生黄花蒿极为丰富的遗传多样性,可为进一步的选择育种提供更多的种质资源。     

3种青蒿素衍生物对日本血吸虫吡喹酮抗性株童虫的体内作用效果观察

目的观察3种青蒿素衍生物双氢青蒿素、青蒿琥酯和蒿甲醚对日本血吸虫吡喹酮抗性株童虫的体内作用效果。方法以经11轮亚治疗剂量吡喹酮筛选的日本血吸虫为吡喹酮抗性株,以未暴露于吡喹酮的日本血吸虫作为吡喹酮敏感株,收集2虫株尾蚴感染小鼠,以300mg/kg双氢青蒿素、青蒿琥酯和蒿甲醚对感染后7~8 d童虫分别进行2次灌服用药(总剂量600 mg/kg),所有小鼠于感染后45 d解剖,收集小鼠体内成虫并计数,计算减虫率和减雌率。结果 300 mg/kg双氢青蒿素、蒿甲醚和青蒿琥酯2日疗法(总剂量600 mg/kg)对日本血吸虫吡喹酮敏感株7~8 d童虫的减虫率为69.8%~71.0%,减雌率为75.4%~79.8%;对日本血吸虫吡喹酮抗性株7~8 d童虫的减虫率为64.6%~66.1%,减雌率为69.3%~71.1%,差异均无统计学意义(均p0.05)。结论 日本血吸虫吡喹酮抗性株对青蒿素类衍生物双氢青蒿素、青蒿琥酯和蒿甲醚依然敏感,青蒿素衍生物与吡喹酮在日本血吸虫中不存在交叉抗药性。     

Artemisinin inhibits inflammatory response via regulating NF-κB and MAPK signaling pathways

Artemisinin, isolated from the Chinese plant Artemisia annua, has been used for many years to treat different forms of malarial parasites. In this study, we explored the anti-inflammatory activity of artemisinin and the underlying mechanism of this action. We demonstrated that the anti-inflammatory effects of artemisinin in TPA-induced skin inflammation in mice. Then the artemisinin significantly inhibited the expression of NF-κB reporter gene induced by TNF-α in a dose-dependent manner. Artemisinin also inhibited TNF-α induced phosphorylation and degradation of IκBα, p65 nuclear translocation. Artemisinin also has an impact on upstream signaling of IKK through the inhibition of expression of adaptor proteins, TNF receptor-associated factor 2 (TRAF2) and receptor interacting protein 1 (RIP1). Furthermore, pretreatment of cells with artemisinin prevented the TNF-α-induced expression of NF-κB target genes, such as anti-apoptosis (c-IAP1, Bcl-2, and FLIP), proliferation (COX-2, cyclinD1), invasion (MMP-9), angiogenesis (VEGF), and major inflammatory cytokines (TNF-α, iNOS, and MCP1). We also proved that artemisinin potentiated TNF-α-induced apoptosis. Moreover, artemisinin significantly impaired the ROS production and phosphorylation of p38 and ERK, but did not affect the phosphorylation of JNK. Taken together, artemisinin may be a potentially useful therapeutic agent for inflammatory-related diseases.