【作者】 梁晓雯；

【导师】 许立；

【作者基本信息】 南京中医药大学 ， 药理学， 2011， 硕士

【摘要】 环维黄杨星D (Cyclovirobuxine D,简称CVB-D)是从黄杨科植物小叶黄杨(Buxus microphylla Sieb.et Zucc.Var.Sinica Rehd.et Wils)及其同属植物中提取得到的活性单体—生物碱,属孕甾烷衍生物,是黄杨宁片的主要成分。在临床上广泛用于心律失常、心绞痛、心肌缺血等治疗。近年来,对CVB-D的基础研究主要集中在其药理作用及其机制方面,毒理学研究报道较少。本实验室前期研究发现长期应用CVB-D能引起大鼠肾毒性,具有一定的剂量和时间依赖性关系,且病变在停药后不能彻底恢复。尽管如此,关于CVB-D肾脏毒性的机制目前尚无报道。基于此,我们将对CVB-D|肾脏损伤机制进行较为系统的研究,为临床应用提供合理依据。本研究主要包括以下内容：1.大鼠连续腹腔注射CVB-D两周后,检测各组大鼠尿液和血清中相关指标；检测肾皮质中生物膜和氧化相关指标；并做病理检查；此外基因芯片技术检测大鼠连续腹腔注射CVB-D 20 mg/kg 14天后与对照组大鼠肾脏基因表达的差异。结果显示,CVB-D各剂量组大鼠尿液中葡萄糖(Glu)和尿蛋白(TP)含量升高,血清中尿素氮(BUN)、肌酐(Cr)、Cl-含量升高并且降低大鼠肾皮质中Na+-K+-ATPase和Ca2+-Mg2+-ATPase活性,但是对脂质过氧化方面的指标没有显著影响。病理检测结果显示CVB-D可致肾小球毛细血管球细胞增生,肾小管上皮细胞有变性、坏死,肾间质有少量炎细胞浸润等现象；基因芯片检测结果显示,CVB-D对大鼠肾脏中凋亡、细胞周期、死亡受体等方面的相关通路和基因有所影响。以上结果显示CVB-D在较短时间内腹腔注射给药也会引起肾脏毒性,毒性机制与其引起肾小球、’肾小管损伤和抑制生物膜活性相关。2.我们以HK-2细胞株为研究对象,在体外观察了CVB-D对肾小管上皮细胞的直接毒性。用比色法检测不同浓度CVB-D作用不同时间后HK-2细胞的活力变化、CVB-D对细胞LDH释放量的影响；倒置显微镜下观察CVB-D对细胞整体形态的影响；以及CVB-D对细胞周期和细胞内活性氧(ROS)水平的影响。结果发现,CVB-D可显著抑制HK-2细胞的增殖,促进细胞LDH的释放,且呈现时间和剂量依赖性,同时观察到细胞形态的变化如变圆、收缩,脱壁等变化。说明在实验设定的剂量和时间范围内,CVB-D对HK-2细胞具有明显的毒性作用。经CVB-D处理48h后,HK-2细胞出现S期阻滞,同时亚二倍体峰随着剂量的增加而增加；此外,CVB-D并未刺激HK-2细胞产生ROS。以上结果提示CVB-D对HK-2细胞的毒性可能与凋亡有关。3.CVB-D处理HK-2细胞后,用透射电镜观察细胞形态学改变,并用Hoechst染色法观察细胞核形态变化；用Annexin V/PI双染法在流式细胞仪上检测CVB-D对HK-2细胞的凋亡诱导；用分光光度法检测CVB-D对HK-2细胞caspase-3活化程度的影响；基因芯片技术检测60μg/ml CVB-D作用HK-2细胞48h对其相关通路及基因表达变化的影响。结果显示,HK-2细胞出现了明显的染色质凝聚、边集等凋亡早期现象,细胞核染色同时显示出凋亡特征；AnnexinV/PI染色确证CVB-D处理后HK-2细胞出现早期凋亡,并且几乎没有晚期凋亡/坏死现象；CVB-D可活化HK-2细胞caspase-3;基因芯片结果显示,CVB-D可通过Caspase cascade, Fas和P53等凋亡通路影响HK-2细胞的凋亡,并且上调DIABLO, FADD和Gadd45a等促凋亡基因。上述现象说明,CVB-D对HK-2细胞的毒性作用主要是通过细胞凋亡的诱导而产生的,并且和caspase-3有关。此外,CVB-D可通过Caspase cascade, Fas和P53等凋亡通路影响HK-2细胞的凋亡。4.观察CVB-D对Na+-K+-ATPase活性的影响,并用Western Blot方法检测CVB-D对Na+-K+-ATPaseα1和β1亚基蛋白表达的影响；此外,测定不同浓度CVB-D作用48h后对HK-2细胞线粒体膜电位和胞内Ca2+浓度的影响。结果显示,CVB-D对HK-2细胞Na+-K+-ATPase活性有显著抑制,具有浓度和时间的依赖性,并且显著下调了Na+-K+-ATPaseα1和Na+-K+-ATPaseβ1蛋白表达；CVB-D降低了HK-2细胞线粒体膜电位并且显著升高了胞内Ca2+浓度。以上实验结果显示,CVB-D通过下调HK-2细胞中Na+-K+-ATPaseα1和β1蛋白的表达从而抑制Na+-K+-ATPase的活性,导致细胞内Ca2+浓度升高,介导HK-2细胞凋亡更多还原

【Abstract】 Cyclovirobuxine D (CVB-D), an active compound extracted from the Chinese medicinal herb Buxus microphylla Sieb. et Zucc. var. sinica Rehd. et Wils, is the active component of huangyangning tablets, which are widely used in China for the treatment of arrhythmias, angina pectoris, and myocardial ischemia. Numerous studies have examined the pharmacodynamic effects and mechanism of CVB-D, but few studies have examined its toxicity. We have found that long-term using CVB-D may cause nephrotoxicology, with the relationship between dose-dependent and time-dependent manners:and the nephrotoxicology cannot be recovered completely after injection. However, few studies have examined with its mechanism of kidney toxicity. In this study, we investigated the mechanism of CVB-D on nephrotoxicity to provide the reasonable reference for clinical practice.1. After CVB-D was ip. given for two weeks, and we tested the relative indicators of urine and serum of rats. We also detected the biomembrane and oxidation indicators of renal cortex and observed pathological changes in renal tissue. Besides, we tested the differential gene expression between the CVB-D20 mg/kg group and control group using gene chip technology. Results showed compared with control group, Glu and TP in urine and BUN, Cr and Cr in serum of CVB-D group was increased; while the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase was significant decreased in CVB-D group. CVB-D had few effects on oxidation indicators of renal cortex. Histopathology showed CVB-D caused capillary ball cell hyperplasia of glomerulus, denaturation and necrosis of renal tubular epithelial cells, and little inflammatory cell in renal interstitial. Gene chip showed CVB-D influenced the pathway about apoptosis, cell cycle, death receptor and so on. Those evidences above suggested that CVB-D caused the nephrotoxicity with ip.for shorter time, and the mechanism was associated with damage of glomerulus and tubles and inhibition of biomembrane activity.2. We observed direct cytotoxic of CVB-D on HK-2cell in vitro by cell viability inhibition, LDH leakage, morphological changes, cell cycle changes and ROS generation. Results showed that CVB-D inhibited HK-2 cells viability and induced LDH leakage at a dose-and time-dependent manner. Treatment of CVB-D increased S phase and the SubGl phase appeared to be simultaneously. In addition, CVB-D had few effects on ROS generation. Those evidences above, especially the appearance of SubGl phase and turned round cells, have suggested that the cytotoxicities of CVB-D may related to apoptosis.3. We observed cell morphology by electron microscope and cell nucleus morphology by Hoechst 33258 staining. Not only did we detect HK-2 cell apoptosis by annexinⅤ/PI staining combined with FCM analysis. We also detected the effects of CVB-D on caspase-3 activity by spectrophotometry and tested the differential gene expression between the CVB-D60μg/ml group and control group using gene chip technology. Results showed CVB-D group HK-2 cell exhibited chromatin margination, which was due to early apoptotic events. AnnexinV/PI staining confirmed the apoptosis was induced by CVB-D in HK-2 cell and without significant necrosis. In the meantime, CVB-D can activated caspase-3 and gene chip results showed it influenced HK-2 cell apoptosis according to caspase cascade, Fas and P53 pathway. All of above showed the CVB-D is able to induce apoptosis on HK-2 cell and caspase-3 is involved.4. We observed the effects of CVB-D on Na+-K+-ATPase activity and tested expression of Na+-K+-ATPaseα1andβ1by western blotting. Besides, we tested the effects of CVB-D on mitochondrial membrane potential and intracellular Ca2+of HK-2 cell. Results showed that CVB-D can significant inhibited Na+-K+-ATPase activity and down regulated expression of Na+-K+-ATPaseα1 andβ1 at a dose-and time-dependent manner. In the meanwhile, CVB-D decreased mitochondrial membrane potential of HK-2 cell and increased intracellular Ca2+. All of above showed a significant decrease in Na+-K+-ATPase activity in cells treated with CVB-D, probably caused by the decrease in the expression of theα1 andβ1 subunit of Na+-K+-ATPase in HK-2 cells that may be associated with the CVB-D-induced apoptosis of HK-2 cells.更多还原