淫羊藿苷对异丙肾上腺素所致乳鼠心肌细胞损伤的保护作用

目的在异丙肾上腺素(isoproterenol,ISO)诱导原代培养乳鼠心肌细胞损伤的模型上,探讨淫羊藿苷对心肌细胞的保护作用。方法采用ISO损伤心肌细胞,通过MTT法检测心肌细胞存活率,以荧光染色激光共聚焦显微镜检测线粒体膜电位的变化,流式细胞仪检测细胞凋亡的发生。结果ISO处理心肌细胞72h后降低心肌细胞的存活率,诱导心肌细胞发生凋亡,凋亡率达35%,线粒体的膜电位明显降低。预先给予0.1～10μmol.L-1淫羊藿苷,可提高心肌细胞的存活率,改善线粒体的膜电位,降低心肌细胞的凋亡率。结论淫羊藿苷对ISO诱导原代培养心肌细胞损伤具有明显的保护作用。     

淫羊藿苷对破骨细胞的分化及骨吸收功能的影响

目的研究淫羊藿苷对破骨细胞的分化及骨吸收功能的影响,评价淫羊藿苷对骨质疏松的预防及治疗作用。方法采用1,25-(OH)2D3诱导兔骨髓单核细胞形成破骨细胞样细胞以及原代分离的乳兔成熟破骨细胞与骨片共培养的方法,考察淫羊藿苷对破骨细胞的分化及骨吸收功能的影响。结果浓度为100、50μmol.L-1的淫羊藿苷明显抑制1,25-(OH)2D3诱导兔骨髓单核细胞形成破骨细胞样细胞的数目,当其浓度为100、50、10μmol.L-1时,明显抑制破骨细胞的骨吸收功能,具体表现在吸收陷窝数目及表面积均明显减少。结论淫羊藿苷不仅可以明显抑制破骨细胞的分化,同时具有抑制破骨细胞的骨吸收功能的作用,提示淫羊藿苷具有改善骨吸收功能亢进的潜力。     

淫羊藿苷对心肌缺血再灌注损伤模型大鼠心肌组织的保护作用

目的:研究淫羊藿苷对心肌缺血再灌注损伤(IR)模型大鼠心肌组织的保护作用。方法:取SD大鼠随机分为假手术(等容生理盐水)组、模型(等容生理盐水)组和淫羊藿苷低、中、高剂量(5、10、20 mg/kg)组,除假手术组6只外其余每组12只。各组大鼠ig给予相应药物,每日1次,连续14 d,末次给药1 h后行冠状动脉结扎术复制IR模型。采用2,3,5-氯化三苯基四氮唑(TTC)染色法考察各组大鼠心肌梗死比例;原位末端标记法检测各组大鼠心肌细胞凋亡率;western blot法检测各组大鼠心肌组织中p38和JNK磷酸化(p-p38、p-JNK)蛋白水平。结果:与假手术组比较,模型组大鼠心肌梗死比例、心肌细胞凋亡率、心肌组织中p-p38和p-JNK蛋白水平均增加,差异具有统计学意义(P<0.01或P<0.05)。与模型组比较,淫羊藿苷各剂量组大鼠上述指标均降低,差异具有统计学意义(P<0.01或P<0.05),其中高、中剂量组效果较低剂量组更明显(P<0.05)。结论:淫羊藿苷预处理对IR模型大鼠的心肌组织具有保护作用,并呈浓度依赖性。其作用机制可能与降低心肌细胞凋亡,下调p-p38和p-JNK蛋白水平有关。     

RP-HPLC测定淫羊藿不同品种和部位中柔藿苷和淫羊藿苷

目的:为了对中药淫羊藿中柔藿苷和淫羊藿苷含量进行系统研究,测定了淫羊藿不同品种和药用部位的柔藿苷和淫羊藿苷含量。方法:采用RP-HPLC法对3个品种多个样品进行测定,以BECKMAN COULTER_(TM)-C_(18)柱(带预柱)为色谱柱,流动相为乙腈-水(25∶75),流速1.0mL·min~(-1),检测波长为270nm。结果:柔藿苷在0.19642-1.9642μg范围内呈良好的线性关系,淫羊藿苷在0.19335-1.9335μg范围内呈良好的线性关系。巫山淫羊藿同一植株体的不同部位中柔藿苷和淫羊藿苷的分布均为叶>根>茎。巫山淫羊藿同一植株的任何部位都是柔藿苷含量高于淫羊藿苷,而淫羊藿和箭叶淫羊藿叶中都是淫羊藿苷含量高于柔藿苷。结论:巫山淫羊藿中以柔藿苷为主要成分,淫羊藿和箭叶淫羊藿中以淫羊藿苷为主要成分。     

淫羊藿苷对过氧化氢诱导的血管内皮细胞损伤的保护作用

目的研究淫羊藿苷对过氧化氢所致血管内皮细胞损伤的影响。方法体外培养血管内皮细胞,淫羊藿苷预处理24h后,采用过氧化氢诱导氧化损伤模型,MTT法测定细胞活性,测定细胞培养液中乳酸脱氢酶（LDH）的释放、细胞中丙二醛（MDA）含量、超氧化物歧化酶（SOD）的活力。结果淫羊藿苷能明显提高细胞存活率,增加细胞内SOD活性,降低培养液中LDH释放和细胞内MDA含量。结论淫羊藿苷对过氧化氢诱导的血管内皮细胞损伤有明显的保护作用,可能与其抗氧化有关。     

淫羊藿苷体外抑制SIV感染诱导的CEMx174细胞凋亡

目的探讨淫羊藿苷体外抑制猴免疫缺陷病毒(Simi-an immunodeficiency virus,SIV)复制作用和抑制SIV感染诱导的CEMx174细胞凋亡的信号转导机制。方法细胞病变和间接免疫荧光法测定了淫羊藿苷对SIV复制的抑制作用,Annexin V和Propidiumiodide(PI)双染法测定淫羊藿苷对SIV感染导致CEMx174细胞凋亡的作用,放免法测定了SIV感染CEMx174细胞内cAMP含量和依赖cAMP的蛋白激酶活性。Western blot法测定了PKA依赖的组蛋白磷酸化的水平。结果用不同浓度的淫羊藿苷处理细胞后结果表明,其半数毒性浓度为134mg·L-1,半数抑制浓度是26mg·L-1,治疗指数为5.15。淫羊藿苷对感染细胞凋亡的抑制要比正常细胞为高(P<0.05)。50mg·L-1淫羊藿苷作用病毒感染细胞15min后降低正常和感染细胞内的cAMP含量,但对感染细胞的cAMP含量影响更加明显。50mg·L-1淫羊藿苷可以显著下调感染和正常细胞的PKA活性(P<0.05)。病毒感染可以使磷酸化的组蛋白-H3升高,50 mg·L-1淫羊藿苷作用2h后,可以降低磷酸化组蛋白-H3的水平。结论淫羊藿苷体外对SIV复制有一定抑制作用,SIV-mac251感染导致的细胞凋亡是cAMP-PKA信号转导通路激活的结果,而短时间淫羊藿苷处理SIVmac251感染的CEMx174细胞可以暂时减缓SIVmac251诱导的细胞凋亡过程。     

淫羊藿苷抗抑郁及对皮质酮致PC12细胞损伤的保护作用研究

目的：研究淫羊藿苷的抗抑郁及对皮质酮致PC12细胞损伤的保护作用。方法：采用大／小鼠强迫游泳、小鼠悬尾三种实验模型,将动物随机分为对照组、淫羊藿苷低剂量组、淫羊藿苷高剂量组、阿米替林组,观察药物对大／小鼠强迫游泳不动时间、小鼠悬尾不动时间的影响;并在细胞水平建立皮质酮损伤PC12细胞模型,观察淫羊藿苷的细胞保护作用。结果：在大／小鼠强迫游泳和小鼠悬尾实验中,淫羊藿苷可显著缩短大／小鼠的强迫游泳不动时间和小鼠悬尾不动时间,与对照组相比差异有统计学意义（P〈0．05）;在皮质酮损伤PC12细胞的模型上,淫羊藿苷可显著提高PC12细胞的存活率,拮抗皮质酮诱导的细胞损伤作用。结论：淫羊藿苷具有明显的抗抑郁效果,其抗抑郁作用与神经细胞保护作用有关。     

基于cAMP-PKA信号通路的淫羊藿苷促进骨形成研究

目的研究淫羊藿苷是否通过c AMP-PKA信号通路来促进成骨细胞(osteoblast,OB)成熟矿化。方法以1×10?5 mol·L?1淫羊藿苷分别处理体外培养的大鼠颅骨OB细胞和人类乳腺癌细胞(MCF-7)不同时间后,免疫荧光染色法检测2种细胞内雌激素受体(ERα)的核转位情况。待P1代OB细胞铺满80%皿底后,采用1×10?5 mol·L?1的2’,3’-双脱氧腺苷(2’,3’-dideoxyadenosine,DDA)抑制胞内腺苷酸环化酶(adenylate cyclase,AC),同时以1×10?5 mol·L?1淫羊藿苷分别作用于正常组和信号阻断组不同时间后,ELISA检测细胞内c AMP的含量。以终浓度为1×10?6 mol·L?1蛋白激酶A(protein kinase A,PKA)抑制剂KT5720处理细胞,同时以1×10?5 mol·L?1淫羊藿苷分别作用于信号阻断组和正常组,3 d和6 d后检测胞内碱性磷酸酶(ALP)活性;药物处理48 h后,Real-time PCR检测I型胶原(collagen I,COL I)、Runx-2、ALP m RNA的表达量;Western blot检测COL I、Runx-2蛋白表达量。结果淫羊藿苷作用于细胞4 h后,MCF-7胞内ERα发生明显的核转位,而OB细胞在所有时间点都未检测到明显核转位现象,结合本课题组前期的淫羊藿苷雌激素比较试验说明,淫羊藿苷促进成骨细胞成骨性分化并不主要依赖于其雌激素活性。淫羊藿苷促OB细胞后,胞内c AMP迅速升高,处理1 h后与对照组相比具有显著性差异。加入DDA阻断AC后,淫羊藿苷促胞内c AMP升高的作用消失。1×10?5 mol·L?1淫羊藿苷能够显著地促进胞内ALP的活性,成骨性分化相关的因子COL I、Runx-2和ALP的基因表达也相应增高,同时COL I和Runx-2蛋白表达量也显著增加。当采用KT5720抑制PKA的活性之后,ALP活性下降,成骨性分化的指标也随之降低。结论淫羊藿苷促进OB细胞成骨性分化并不主要依赖于其雌激素活性,而是通过迅速提高成骨细胞内c AMP的含量,激活胞内c AMP-PKA信号通路,进而促进成骨性相关因子的表达,来促进OB细胞成熟矿化。     

高效液相色谱法测定更年灵胶囊中淫羊藿苷的含量

更年灵胶囊收载于中华人民共和国卫生部药品标准中药成方制剂[1]中,由淫羊藿、维生素B1、女贞子、谷维素、维生素B6等药味组成,温肾益阴,调补阴阳.用于妇女更年期综合征属阴阳两虚者.为了更好地监控其成品质量,对原质量标准[1]进行了完善提高,建立其君药淫羊藿的含量测定项,并以淫羊藿苷作为定量分析的指标成分.淫羊藿药材及其制剂中淫羊藿苷的含量测定方法较多[2,3],而高效液相色谱法具有灵敏、准确、分离度好等优点,故本文选用高效液相色谱法测定更年灵胶囊中的淫羊藿苷含量[4].经方法学考察,表明该方法操作简便,专属性强,重现性好,可作为更年灵胶囊中淫羊藿苷的含量测定方法.     

淫羊藿属主要资源种类的化学品质比较研究

本文对淫羊藿属主要资源种类药材化学品质进行综合比较研究。应用HPLC法建立不同品种淫羊藿药材中朝藿定A、朝藿定B、朝藿定C、淫羊藿苷及宝藿苷I等5种指标成分的含量测定方法,通过紫外-可见分光光度法测定淫羊藿总黄酮含量,并考察样品中水分、灰分、酸不溶性灰分和醇溶性浸出物等含量,比较不同品种淫羊藿药材化学品质。结果显示,不同品种淫羊藿药材中含有朝藿定A、朝藿定B、朝藿定C、淫羊藿苷及宝藿苷I等异戊烯基黄酮苷类成分的种类及含量各不相同,结合总黄酮含量及水分、灰分等常规理化实验项目测定结果,表明不同品种淫羊藿药材化学品质差异较大。本研究为不同品种淫羊藿药材综合质量评价体系的建立提供理论和实验依据。     

Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test

The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a reliable, scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts. In order to identify more objective endpoints of differentiation other than the microscopic evaluation of "beating areas" and to adapt the EST to applications in high-throughput screening systems we improved and expanded the EST protocol by establishing molecular endpoints of differentiation. The quantitative expression of sarcomeric myosin heavy chain (MHC) and alpha-actinin genes under the influence of test compounds was studied employing intracellular flow cytometry. Strong embryotoxicants exerted a dose-dependent effect on both the expression levels of MHC and alpha-actinin and the differentiation into beating cardiomyocytes. Furthermore, quantitative FACS (fluorescence-activating cell sorting) analysis showed the same sensitivity for the classification of substances as the conventional endpoint but allowed a significant reduction of the test period. Within 7 days, maximal expression of sarcomeric marker proteins was observed. Our findings indicate that structural proteins of the sarcomere apparatus, alpha-actinin and myosin heavy chain (MHC), seem to be promising candidates to predict developmental toxicity in vivo from in vitro data. Thus, the improved EST holds promise as a new predictive screen for risk assessment with respect to developmental toxicity using stem cell technology and technological advances in the field of gene expression analysis.     

Embryonic stem cell-derived cardiac, neuronal and pancreatic cells as model systems to study toxicological effects

Primary cultures or established cell lines of vertebrates are commonly used to analyse the cytotoxic potential of chemical factors, drugs and xenobiotics in vitro. An alternative approach will be provided by permanent lines of pluripotent embryonic stem (ES) cells, which are able to differentiate into specialised somatic cell types in vitro. Here, we demonstrate the capacity of ES cells to generate functional cardiac, neuronal and pancreatic cells. We show that during ES cell differentiation, tissue-specific genes, proteins as well as functional properties are expressed in a developmentally regulated manner recapitulating processes of early embryonic development. We present data that show the use of ES-derived cardiomyocytes and dopaminergic neurons in toxicological studies and the potential of ES-derived pancreatic beta-like cells in future in vitro assays. The application of these differentiation systems to human ES cells opens up new perspectives in basic and applied toxicology.     

Preclinical assessment of cardiac toxicity

The exact prediction of the clinical behavior of drugs represents one of the most difficult duties in preclinical drug development. The use of cell-based assay systems underpins the development of many drug candidates, but owing to the artificial character of many of these systems, cell response and physiological behavior seem to be mutually exclusive. Embryonic stem cell-derived cells represent a system that may address the disconnect between the behavior of cultured cells and cells in situ. While undifferentiated ES cells allow standardization, expansion and genetic manipulation, the differentiated cells provide a reflection of the normal physiological image of their primary counterpart. We compare common models to detect cardiac toxicity with an assay system comprising in vitro differentiated pure cardiomyocytes.     

Establishment of an in vitro reporter gene assay for developmental cardiac toxicity.

This study is based on the unique potential of pluripotent embryonic stem (ES) cells to differentiate in vitro into embryoid bodies containing cell lineages representative of most cell types found in the mammalian fetus. However, the use of wild type ES cells as an in vitro assay for embryotoxicological studies is complicated by the simultaneous development of various cellular phenotypes. This prevents a quantitative assessment of drug effects on one specific cell type. Here we report the effects of 15 chemicals on cardiac differentiation as determined by various specific toxicological endpoints such as morphological inspection (contractile activity), quantitative mRNA analysis and cardiac-specific expression of green fluorescent protein (GFP), used as a quantitative reporter. The data from the different endpoints have been subjected to a statistical analysis, and a preliminary prediction model is proposed. The results demonstrate that genetically-engineered ES cells could provide a valuable tool for estimating the developmental cardiotoxic potential of compounds in vitro and form the basis for automated analysis in a high-throughput system.