【作者】 郝旭亮；

【导师】 李青山；

【作者基本信息】 山西医科大学 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 目的建立罗布麻总黄酮（TFF）标准色谱指纹图谱,对TFF进行抗血栓药理作用及体内代谢过程研究,探讨其抗血栓作用的物质基础,通过体外人脐静脉血管内皮细胞（HUVECs）的培养研究TFF及其主要成分金丝桃苷（hyperin）抑制过氧化氢诱导的HUVECs凋亡的作用及其机制。方法（1）采用HPLC-DAD法建立TFF标准色谱指纹图谱,确保为后续药理试验和作用机理探讨提供质量均一、稳定可控的供试样品;（2）通过大鼠静脉血栓模型实验,胶原蛋白-肾上腺素诱导小鼠血栓模型实验,小鼠凝血时间测定及凝血酶原时间测定,放免法对大鼠血浆中TXB₂、6-keto-PGF1α含量的测定来研究TFF的抗血栓作用;（3）通过HPLC, LC/MS/MS及UPLC/MS/MS技术,研究TFF在大鼠胃肠内的吸收与转化、研究口服和静注给药TFF后大鼠血浆和血清相关成分及初步药代动力学;（4）建立过氧化氢诱导的HUVECs凋亡模型,采用MTT观察不同浓度TFF和Hyperin对HUVECs增殖的影响,流式细胞仪检测对HUVECs细胞周期变化、凋亡率及ROS释放的影响,AO/EB染色法观察对HUVECs形态学改变的影响,比色法检测对细胞培养液中NO和SOD的活性、MDA和LDH的含量的改变,免疫组化法测定对NF-κB和VEGF的表达的影响,采用双抗夹心ELISA法测定对细胞培养上清液中TF和TFPI的浓度的影响;（5）应用激光共聚焦显微镜,采用新型荧光染料Flou3-AM检测细胞内Ca²⁺浓度的变化,以罗丹明123作为染料检测线粒体跨膜电位变化,采用RT-PCR检测Caspase-3、Bcl-2、Bax基因表达,Western blot分析Cyt C、Caspase-9和Caspase-3等蛋白的表达,探讨TFF和Hyperin抑制H₂O₂诱导的HUVECs细胞凋亡的机制。结果（1）建立了TFF标准指纹图谱,以金丝桃苷为参比峰共确定了17个共有峰,与在相同的色谱条件下测定的罗布麻叶药材图谱有很好的相关性。建立的方法精密度、稳定性及重现性较好,专属性强,可全面控制TFF的质量,确保为后续药理试验和作用机理探讨提供质量均一、稳定可控的供试样品;（2）对静脉血栓形成的影响实验中,TFF具有明显的抑制静脉血栓的作用,其中高、中剂量组血栓干重与湿重与正常对照组相比均有显著性差异（p<0.001）,对血栓湿重的抑制率分别为73.0%,83.4%;不同剂量的TFF对胶原-肾上腺素诱导的小鼠血栓性偏瘫或死亡数与正常对照组相比明显减少,均有明显的保护作用,但各给药组比较无显著性差异;对凝血功能的影响实验中,TFF中、高剂量组能显著延长正常小鼠的凝血时间,影响小鼠的凝血功能（p<0.05）,能显著延长家兔凝血酶原时间和部分凝血酶原时间（p<0.05）,提示TFF可能通过抑制内源性、外源性凝血途径产生抗凝血作用;对大鼠血浆中TXB₂、6-keto-PGF1α含量的影响实验表明,与正常对照组比较TFF中、高剂量组使大鼠血浆中TXB₂含量明显下降,6-keto-PGF1α的含量明显升高（p<0.05或p<0.01）;（3）以金丝桃苷、异槲皮苷为考察指标,采用大鼠在体灌流模型,通过HPLC测定灌流TFF前后两成分的含量,计算其在胃肠中的吸收率,结果在大鼠胃中2h的平均吸收率:金丝桃苷为36.92%,异槲皮苷为36.49%,在大鼠肠中2h的平均吸收率:金丝桃苷为47.7%,异槲皮苷为48.25%,表明金丝桃苷和异槲皮苷在大鼠胃肠部均有吸收,但两成分在肠部的吸收比胃部较好;通过检测静脉及口服给药TFF后大鼠血浆中有关物质,结果大鼠静脉给药5分钟和15分钟后大量测到异槲皮苷和金丝桃苷,同时也检测到少量的槲皮素二项结合物（槲皮素的葡萄糖醛酸结合和硫酸酯化结合代谢产物）。口服给药TFF后于15分钟和4小时在血中没有发现异槲皮苷和金丝桃苷,但却发现大量槲皮素的二相代谢产物;通过检测静脉及口服给药TFF后大鼠血清中有关物质,结果两种给药途径血清成分有很大差别,口服给药30min血清中总黄酮的主要成分金丝桃苷和异槲皮苷均未以原型成分出现,提示发生了转化,而静脉给药30min后血清中可见金丝桃苷和异槲皮苷原型成分,以及口服后出现的一些成分,表明静注给药部分发生了转化。并根据相应的质谱图m/z结合文献初步推测可能含有的成份有:槲皮素、槲皮素+葡萄糖醛酸、酸化的三叶豆苷（或紫云英苷）、柽柳素、柽柳素-7-O-葡萄糖醛酸等,转化途径可能有甲基化、葡萄糖醛酸化和硫酸酯化等;初步药代动力学结果显示未经水解的血样中测到的槲皮素浓度很低,而水解后的同一批血样中测到了大量的槲皮素,推测水解后测到的大量的槲皮素是由吸收入体的异槲皮苷和金丝桃苷（槲皮素糖苷成分）或槲皮素代谢物所贡献的。比较水解后口服和静脉给药槲皮素的曲线下面积,得到体内黄酮类化合物总的口服生物利用度为59.8%。（4）终浓度为200μmol/lH₂O₂可明显诱导HUVECs形成包括细胞固缩,染色质凝聚等特征的凋亡模型,为进一步探讨TFF和Hyperin对血管内皮细胞氧化损伤保护作用及其机制提供手段;MTT、FCM等方法测定结果显示,与正常细胞组比,H₂O₂模型组细胞增殖活性下降,细胞凋亡率明显增加,G0/G1期细胞比率增加,S期细胞和G2/M期细胞比率明显降低,可诱导产生大量的ROS,并出现明显的细胞凋亡特征,培养上清液中NO和SOD活性明显下降,MDA和LDH的释放量明显增加,NF-κB的表达明显增加VEGF表达明显下降（P<0.01）,不同浓度的TFF和Hyperin作用后,均能明显抑制和改变H₂O₂引起的上述改变（P<0.05或P<0.01）。表明TFF和Hyperin对H₂O₂引起HUVECs损伤具有确切的保护作用,能明显抑制H₂O₂诱导的HUVECs凋亡,其作用机制可能与其确切的抗氧化作用及调节NF-κB和VEGF的表达有关;测定细胞培养上清液中TF和TFPI的浓度实验结果显示,与模型组比较,不同浓度TFF和hyperin能剂量依赖性地抑制H₂O₂刺激引起的TF含量升高,而TFPI的变化与TF相反（P<0.05或P<0.01）,这种变化也可能是TFF和Hyperin抗血管内皮细胞损伤和抗血栓形成的机制之一;（5）与正常组比较,H₂O₂作用后的HUVECs胞内Ca²⁺严重超载,线粒体跨膜电位显著降低（P<0.01）;Bax基因表达明显增加,Bcl-2基因表达明显减小,Caspase-3,Caspase-9和Cyt C蛋白表达明显增加（P<0.05或P<0.01）,证实H₂O₂通过激活经典的线粒体途径诱导细胞凋亡,与文献报道一致。给予不同浓度的TFF和Hyperin作用后,与H₂O₂模型组比较,可明显抑制H₂O₂所引起的HUVECs细胞内Ca²⁺浓度升高,能明显促进Bcl-2基因表达,减少Bax基因的表达,从而抑制线粒体的通透性,恢复氧化损伤降低的膜电位（P<0.05或P<0.01）,同时能明显降低Cyt C,Caspase-9和Caspase-3蛋白表达（P<0.05或P<0.01）,表明TFF和Hyperin可能是通过抑制Ca²⁺超载,促进Bcl-2表达,减少Bax的表达,抑制Bax由胞浆向线粒体转位,使得线粒体通透性降低,膜电位得到恢复,阻止线粒体Cyt C的释放,阻止了Caspase-9和Caspase-3的激活,从而通过阻断线粒体凋亡通路来抑制H₂O₂诱导的HUVECs细胞凋亡。结论（1）建立了TFF标准指纹图谱,为后续药理试验和作用机理探讨提供质量均一、稳定可控的供试样品提供保障;（2）通过抗血栓主要药理学研究,证明TFF具有确切的抗血栓作用;（3）采用HPLC、LC/MS/MS及UPLC/MS/MS技术基本阐明了TFF给药后体内的吸收转化及代谢过程等,初步探讨了TFF发挥药效作用的物质基础;（4）确证了TFF和Hyperin对H₂O₂引起HUVECs损伤具有明显的保护作用,能明显抑制H₂O₂诱导的HUVECs凋亡,其作用机制可能与其确切的抗氧化作用及调节NF-κB和VEGF的表达有关,也可能与细胞培养上清液中TF和TFPI的浓度变化有关;（5）研究证实H₂O₂主要通过激活经典的线粒体途径诱导细胞凋亡,故本文重点探讨了线粒体途径Caspases凋亡信号转导通路,研究表明TFF和Hyperin可能是通过抑制Ca²⁺超载,促进Bcl-2表达,减少Bax的表达,抑制Bax由胞浆向线粒体转位,使得线粒体通透性降低,膜电位得到恢复,阻止线粒体Cyt C的释放,阻止了Caspase-9和Caspase-3的激活,从而通过阻断线粒体凋亡通路来抑制H₂O₂诱导的HUVECs细胞凋亡。更多还原

【Abstract】 Objective:To establish a standard HPLC fingerprinting of total flavonoids of Folium Apocyni veneti（TFF）. To study anti-thrombi pharmacological action and intracorporal process（substance bases）of TFF, and investigate the protective effects and mechanism of TFF and hyperin on H₂O₂-induced human umbilical vein endothelial cells（HUVECs）insults through cultured in vitro.Methods:（1）The standard fingerprint of TFF was established by HPLC-DAD,which can supply samples of controllable quality for further experiments with animals and research mechanism of action. （2） Rat model of venous thrombosis was made to measure the thrombus weight. Mouse model of thrombosis by injecting collagen-adrenaline to the tail vein was applied to observe numbers of dead mice in 5 minutes and recovery mice from hemiplegia in 15 minutes. The clotting time（CT） of mouse, and the prothrombin time （PT） and partial thromboplastin time（PTT） of rabbits were tested by Clotting Method.. The contents of TXB₂、6-keto-PGF1αin rat plasma were detected by radioactive immunization. （3）The gastrointestinal absorption and biotransformation, correlation ingredient of rat plasma and serums and the initiative pharmacokinetics of TFF were investigated by HPLC,LC/MS/MS and UPLC-MS/MS techniques. （4） The cultured HUVECs were used as the experimental model. H₂O₂ was used as the inducer of oxidative stress. Cell viability was measured by MTT assay. Cell apoptosis, cell circles and the level of ROS were detected by flow cytometry（FCM）. The cell morphology change was assessed by AO/EB fluorescent staining. The contents of MDA and LDH, the activities of SOD and NO were determined by spectrophotometry. The immunohistochemistry was used to observe the expression of NF-κB and VEGF. The concentration of TF and TFPI were determined by ELISA.（5）Intracellular Ca²⁺ and the mitochondria transmembrane potential variance （ΔΨm） were measured with Flou3-AM and Rh123 fluorescent staining respectively by LSCM. The gene expression of Bcl-2, Bax and caspase 3 were analysized by RT-PCR. The protein expression of cytochrome C, caspase-9 and caspase-3 were analysized by western blotting to further investigatation of the mechanism of TFF and hyperin in inhibiting HUVECs apoptosis induced by H₂O₂.Results:（1）The chromatographic fingerprinting of TFF was established which showed 17 charavcteristic peaks from 7 patches of total flavonoids products. The similarity from different lots was 0.95¹.00 by the software of“Computer-aided Similarity Evaluation”and showed high similitude in peak numbers and the retention time. Moreover, comparison of the HPLC profiles of total flavonoids and the Folium Apocyni veneti indicated that they were closely related to each other. The chromatographic fingerprinting with high specificity can be used to control its quality and assure to supply samples of controllable quality for further experiments with animals and research mechanism of action. （2）The study indicated that TFF had obvious restriction to venous thrombosis in the test of phlebothrombosis. Compared with the control group , the high and middle dose group of TFF had notable difference in dry weight and wet weight of thrombosis （p<0.001） and the restrain rate of thrombus wet weigh were 73.0% and 83.4% respectively. The hemiplegy and death rate was decreased in mouse by injecting collagen-adrenaline to the tail vein with different dosage TFF compared with control group, but has no notable difference. The high and middle dose group can obviously prolong the clotting time（CT） of mouse （p<0.05）, can prolong the prothrombin time （PT） and partial thromboplastin time（PTT） of rabbits, which show TFF has anticoagulant action through the restrain to intrinsic and extrinsic coagulation pathway. The high and middle dose group can decline the content of TXB2 and increase the 6-keto-PGF1α（p<0.05 or p<0.01 vs control group） in the detection of TXB2、6-keto-PGF1αin rat plasma by radioactive immunization. （3）The absorptivity of hyperin and isoquercitrin in gastrointestinal was counted through detecting the content of hyperin and isoquercitrin in former and behind of perfusion TFF by HPLC. The results showed that the average absorptivity in stomach of hyperin and isoquercitrin were 36.92% and 36.49%, while in intetines were 47.7% and 48.25% respectively. All these indicated that hyperin and isoquercitrin have good bsorptivity in intetines. The detective results of correlation ingredient of rat plasma indicated that hyperin, isoquercitrin, glucuronic acid complex compound of quercetin and sulphating complex compoundof quercetin （the phaseⅡmetabolites of the quercetin） can be detected in vivo after ig and iv TFF. Hyperin, isoquercitrin and measly phaseⅡmetabolites of the quercetin can be detected after iv TFF 5 and 15 min. However, only substantive phaseⅡmetabolites of the quercetin can be detected after ig TFF 4 h and 15 min. The detective results of correlation ingredient of rat serums indicated that the prototype ingredient（hyperin and isoquercitrin） can not be detected after ig TFF 30 min, while can be detected after iv TFF 30 min. The results indicated biotransformation have happened after ig TFF. The potential inclusive substance included quercetin, quercetin+glucuronic acid , astragalin, tamarisktwig, tamarisktwig-7-O-glucuronic acid and so on through initiative speculate based mass spectrum by UPLC-MS analysis. The biotransformation pathways in vivo perhaps comprised of methylation reaction, sulphating reaction and metabolic product association reaction with glucuronic acid etc. Preliminary pharmacokinetics study showed that quercetin concentration were low in no hydrolyzing blood sample,while were high in hydrolyzing blood sample, which confered that quercetin was obtained by absorption of hyperin and isoquercitrin after hydrolysis.The oral bioavailability of flavonoid was 59.8% through area under concentration （AUC）. （4） The apoptosis model of human umbilicus vascular endothelial cell （HUVECs） with cell volume reduction, cytoplasm shrinkage, condensation and fragmentation of chromatin can be induced by 200μmol/l H₂O₂. It offered a good instrument for further study the protective effect and mechanism of TFF and hyperin on H₂O₂-induced HUVECs injury. Compared with the normal group, H₂O₂ can obviously increase the release of ROS, decrease cell viability, induce cell apoptosis, increase contents of LDH and MDA, decrease the activities of SOD and NO of the cultured medium, reduce the percentage of cells of S phase and G2/M phase, down-regulate the expression VEGF, up-regulated the expression of NF-κB, （P<0.01）. Compared with the H₂O₂ model group, different concentrations of TFF and hyperin were shown to alike distinctly attenuate these responses elicited by H₂O₂（P<0.05或P<0.01）. The mechanism may be related to its exact antioxidant effects and regulate the expression of NF-κB and VEGF. At the same time, the concentration determination of TF and TFPI indicated that different concentrations of TFF and hyperin can remarkably inhibit the increase of TF content, while the change of TFPI content was reversed with TF. The concentration change of TF and TFPI was may be a mechanism of TFF and hyperin inhibit the H₂O₂-induced apoptosis of HUVECs.（5） Compared with the normal group, the intracellular Ca²⁺ influx content was severely overloaded , while mitochondria membrane potential△Ψm was obviously decreased （P<0.01）, Bcl-2 down regulation and Bax up-regulation, the protein expression of caspase-3, caspase-9 and Cyt C remarkably increased （P<0.05 or P<0.01）. The study indicated that HUVECs apoptosis by H₂O₂ induced was carried out mainly by activating the mitochondrial pathway, according to the literature reporting. Compared with the H₂O₂ model group, different concentrations of TFF and hyperin were shown to distinctly inhibit the increase of intracellular Ca²⁺content , Bcl-2 up-regulation and Bax down-regulation, depress the mitochondrion permeability, increase the mitochondria membrane potential△Ψm （P<0.05 or P<0.01）. At the same time, the protein expression of caspase-3, caspase-9 and Cyt C remarkably decreased （P<0.05 or P<0.01）. The investigation indicated that the mechanism of TFF and hyperin in inhibiting the HUVECs apoptosis by H₂O₂ induced may be related to inhibit the increase of intracellular Ca²⁺content, Bcl-2 up-regulation and Bax down-regulation, depress the mitochondrion permeability, increase the mitochondria membrane potential△Ψm, prevent mitochondria to release cytochrome C, prevent the activation of Caspase-9 and Caspase-3 and inhibite the HUVECs apoptosis by H₂O₂ induced by preventing the mitochondria caspases signal transduction pathway.Conclusions:（1） The standard fingerprint of TFF was established which can ensure to supply samples of controllable and uniform quality for further experiments with animals and research mechanism of action of TFF. （2） In results, TFF exerted remarkable effects against thrombosis by the major pharmacology research of resist thrombosis. （3）The absorption, biotransformation and its chief metabolites were initiatively illuminated by HPLC /MS/MS and UPLC/MS/MS analysis. The substance bases of pharmacological action were discussed.（4） The study first indicated that TFF and hyperin had a potent protective effect on damage in cultured HUVECs, and obviously inhibit the H₂O₂-induced apoptosis of HUVECs.The mechanism may be related to its exact antioxidant effects and the regulative expression of NF-κB and VEGF, or may be related to the concentration changes of TF and TFPI.（5） The study indicated that HUVECs apoptosis by H₂O₂ induced was carried out mainly by activating the mitochondrial pathway. So, the research focal point was the mitochondria caspases signal transduction pathway. The dissociative Ca²⁺ was one of prerequisite to activate caspase-3, while homeostatic destroying of Ca²⁺ could directly cause mitochondria to release cytochrome C and some apoptosis factor. Cyt-C released from mitochondria can combine with apoptosis enzyme promoter and then activate Apaf-1 by deoxidation triphosadenine. Eventually, the activated Apaf-1 binding to procaspase-9 would start a series of caspases cascade reaction, thus activates typical mitochondria apoptosis pathway. The investigation indicated that different doses of TFF and hyperin were shown to distinctly inhibit the increase of intracellular Ca²⁺content , Bcl-2 up-regulation and Bax down-regulation, depress the mitochondrion permeability, increase the mitochondria membrane potential△Ψm, prevent mitochondria to release cytochrome C, prevent the activation of Caspase-9 and Caspase-3 and inhibit the H₂O₂ induced HUVECs apoptosis by preventing the mitochondria caspases signal transduction pathway.更多还原