大黄酚脂质体对小鼠脑缺血再灌注损伤的神经保护作用及机制研究
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摘要
脑血管病是临床多发病、常见病之一，具有高发病率、高复发率、高致残率、高致死率的特点，并有年轻化趋势，影响患者的生活和工作，给社会和家庭带来沉重的医疗负担。在缺血性脑血管病的康复治疗中，恢复缺血区的血流供应是避免脑组织缺血损伤的首要条件，但与此同时带来的再灌注损伤也是目前医学研究热点。在脑缺血再灌注损伤发生发展过程中，有众多病理机制参与其中，如兴奋性氨基酸的过度释放、体内离子水平失衡、能量耗竭、炎症反应、氧化应激和细胞凋亡，这些因素相互作用最终导致脑组织的不可逆损害。因此，近年来化学药物如钙离子拮抗剂，自由基清除剂，以及神经保护剂等化学药物用于治疗脑缺血再灌注损伤。但是，用药后出现的一系列不良反应如抗药性、胃肠道反应，脑出血等超出了临床长期治疗所带来的治疗效果。近年来，临床应用和实验报道中药成分在治疗脑缺血再灌注损伤方面具有很大的优势。
大黄Radixet rhizoma Rhei为蓼科植物药用大黄Rheum officinale Baill、掌叶大黄Rheum palmatum L、或唐古特大黄Rheum tanguticum Maxim的干燥根及根茎，主产于四川、青海、甘肃等地。大黄始载于《神农本草经》，苦寒，入脾、胃、大肠、心包、肝经，主攻积滞，可清湿热、泻火、凉血、祛瘀、解毒之功能。现代药理研究表明，大黄除具有泻热通肠、凉血解毒、逐瘀通经的作用外，尚具有清除自由基、降血脂、抗动脉硬化、抗癌、延缓衰老等药理作用。大黄的主要药效成分是蒽醌类化合物，大黄酚(Chrysophanol，Chry)属羟基蒽醌类，具有抗病毒、抗癌、抗炎、抗菌、降压和解痉等作用。
本课题组前期实验证明大黄酚在体外可清除超氧自由基、DPPH自由基、羟基自由基，对离体大鼠肝、脑丙二醛(Malondiadehyde，MDA)，超氧化物歧化酶(Superoxide dismutase，SOD)生成有明显的抑制作用；在小鼠脑缺血再灌注损伤后，大黄酚改善学习记忆功能并提高耐缺氧的能力，改善脑组织病理形态学损伤，结果表明大黄酚具有脑保护作用，但大黄酚对脑的保护作用机制尚不明确。
大黄酚属于脂溶性化合物，在水中溶解度极小，见光易分解，性质极其不稳定，且对胃肠有刺激作用，且生物利用度不高，影响了药物的临床应用。因此，本研究首先从中药大黄中提取大黄酚粗品，再采用制备高效液相色谱法(PHPLC)进行单体分离，再完成大黄酚脂质体(Chrysophanolliposomes，Chr-lip)的制备，建立昆明种小鼠脑缺血再灌注模型，动态观察小鼠脑缺血再灌注后神经功能损伤评分，神经元超微结构，病理组织学改变以及SOD，谷胱甘肽过氧化物酶(Glutathione peroxidase，GSH-PX)，一氧化氮合酶(Nitric oxide synthase，NOS)的活性，MDA，一氧化碳(Nitricoxide，NO)含量，以及Bax，Bcl-2，Cytc，caspase3等表达变化。确定缺血后与氧化损伤及凋亡的关系，并选用大黄酚脂质体进行干预，评价干预后神经功能缺损评分，以及大黄酚脂质体对氧化应激，自由基损伤，细胞凋亡信号通路的作用，初步探讨其在缺血再灌注损伤后的脑保护作用机制。本研究分三部分，现将各部分内容概述如下。
第一部分大黄酚的提取纯化及大黄酚脂质体制备
目的：建立从大黄中分离纯化大黄酚的PHPLC方法，考察大黄酚脂质体的处方和制备工艺进行研究，并评价其质量。
方法：建立HPLC测定大黄酚的分析方法，采用Hypersil BDS C8(4.6×150mm，5μm)，流动相为0.1%磷酸-甲醇溶液(15∶85)，流速为1.0mL·min-1，柱温35℃，检测波长254nm；其次，建立从大黄提取液中分离纯化大黄酚的PHPLC法，采用ZORBAX SB-C18：(21.2mm×250mm，7μm)，流动相：0.1%磷酸∶甲醇(15∶85)，柱温：35℃，流速：20mL·min-1，检测波长：254nm，进样量：7mL，馏分收集：基于峰，阈值Min：2.2。制备所得大黄酚单体采用核磁共振(Nuclear Magnetic Resonance，NMR)进行结构鉴定，HPLC法检测其纯度；再次，对大黄酚脂质体的处方和制备工艺进行研究，采用薄膜-超声法制备大黄酚脂质体，并考察大黄酚脂质体的包封率、形态学、粒径分布、稳定性。
结果：
所得大黄酚单体经NMR结构鉴定为大黄酚，经HPLC法检测，其含量为98.9%。大黄酚脂质体包封率为88.5%。粒径较为均匀，相互之间无聚集现象，粒径均小于2μm，稳定性好。
小结：
建立PHPLC分离纯化大黄中大黄酚方法，该方法灵敏度高，操作简便，所得大黄酚单体含量为98.9%。大黄酚脂质体包封率为88.5%，可用于药理学研究。
第二部分大黄酚脂质体对小鼠脑缺血再灌注损伤所致氧化应激的影响
目的：动态观察小鼠脑缺血再灌注后神经功能损伤评分，神经元超微结构，病理组织学改变以及SOD，GSH-PX，NOS的活性，MDA，NO含量。研究大黄酚脂质体对脑缺血再灌注损伤所致氧化应激的作用，探讨大黄酚脂质体抗氧化作用的相关机制。
方法：应用线栓法建立小鼠脑缺血再灌注模型，采用健康雄性昆明种小鼠，体质量24~26g。实验1：小鼠随机分为正常对照组、假手术组、脑缺血再灌注组(脑缺血1h后再灌注，按再灌注不同时间点分为3h、6h、12h、24h、48h、72h6个亚组。实验2：小鼠随机分为正常对照组、假手术组、脑缺血再灌注24h组、大黄酚脂质体(10.0，5.0，0.5mg·kg-1)三个剂量组。大黄酚脂质体组于脑缺血再灌注前3天连续腹腔给予大黄酚脂质体，每天一次。于规定时间检测小鼠神经功能损伤评分；采用HE法，透射电镜法观察小鼠脑组织病理组织学，神经元超微结构改变；采用试剂盒法检测SOD，GSH-PX，NOS的活性和MDA，NO含量。
结果：
1大黄酚脂质体减轻神经功能损伤正常对照组和假手术组，未见明显神经功能损害。脑缺血再灌注组神经功能评分显著高于假手术组(P<0.01)；以24h神经功能评分最高。结果表明，脑缺血再灌注后可损伤小鼠神经功能。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组神经功能缺陷评分均显著降低(P<0.05~P<0.01)，以大黄酚脂质体(10.0mg·kg-1)组效果最为显著。结果表明，大黄酚脂质体减轻神经功能损伤。
2大黄酚脂质体改善神经元超微结构正常对照组和假手术组神经元结构正常，可见丰富的细胞器。脑缺血再灌注3h可见神经元染色质密度增高，核膜皱缩、边集，线粒体轻微肿胀，可见空泡。脑缺血再灌注6h神经元染色质密度增高，核膜严重皱缩、边集，线粒体部分溶解，内质网部分溶解，细胞器数目减少，细胞质高度水肿，可见空泡。脑缺血再灌注12h可见核固缩，细胞核水肿，染色质边集，胞浆内有大量的空泡形成，有线粒体完全溶解；脑缺血再灌注24h可见细胞器明显减少，线粒体结构空泡状，外膜隐约可见，可见凋亡小体，粗面内质网脱颗粒；脑缺血再灌注48h神经元核固缩，核膜内陷，核染色质成团块状边集于核膜下，细胞质高度水肿，细胞器数目少，线粒体不同程度脱空，可见溶酶体。脑缺血再灌注72h组可见神经元核固缩，核膜溶解，核染色质成团块状边集于核膜下，线粒体不同程度断裂，粗面内质网脱颗粒，细胞器少。结果表明，脑缺血再灌注损伤小鼠神经元超微结构。大黄酚脂质体(10.0，5.0mg·kg-1)组神经元染色体比较均匀，核膜清楚，线粒体数目有所减少，粗面内质网有轻度肿胀，核糖体数目有一定的减少；大黄酚脂质体(0.5mg·kg-1)组对神经元超微结构也有改善作用。结果表明，大黄酚脂质体改善神经元超微结构。
3大黄酚脂质体改善病理组织学损害正常对照组和假手术组未见明显病理损害改变。脑缺血再灌注组3h脑缺血半球组织细胞轻度水肿，大小不等的空泡在细胞间隙出现，神经元及胶质细胞固缩，无明显细胞坏死；脑缺血再灌注6h-12h之间时，胞质明显水肿等上述症状逐渐加重；脑缺血再灌注24h-48h时神经元核固缩、浓染，染色质浓缩集聚于核周围，呈凋亡前或凋亡改变，神经元结构明显破裂；脑缺血再灌注72h时神经元水肿逐渐减轻，部分神经元周围出现水肿。结果表明，脑缺血再灌注可致小鼠脑病理组织学改变。大黄酚脂质体治疗组明显改善脑缺血再灌注损伤后的病理组织学损伤。
4大黄酚脂质体可增强抗氧化能力脑缺血再灌注3h MDA含量升高，12h后MDA含量达到最大，并持续到24h，48h-72h后有所下降，与假手术组比，脑缺血再灌注各时间点MDA含量增加有显著性差异(P<0.01)。脑缺血再灌注3h SOD活性明显降低，并于12h后SOD活性达到最低，24h-72h后有所增加，与假手术组比，脑缺血再灌注各时间点SOD活性降低有显著性差异(P<0.01)。脑缺血再灌注3h GSH-PX活性明显降低，并于24h后GSH-PX活性达到最低，48h-72h后有所增加；与假手术组比，脑缺血再灌注各时间点GSH-PX活性降低有显著性差异(P<0.01)。脑缺血再灌注3h NO含量升高，并于6h后NO含量达到最大，并持续到24h，48h-72h后有所下降；与假手术组比，脑缺血再灌注各时间点NO含量增加有显著性差异(P<0.01)。脑缺血再灌注3h NOS活性明显增加，并于12h后NOS活性达到最高，并持续到24h，48h-72h后有所减少；与假手术组比，脑缺血再灌注组NOS活性增加有显著性差异(P<0.01)。结果表明，脑缺血再灌注后，小鼠体内抗氧化能力减弱。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组显著降低MDA含量(P<0.05~P<0.01)，NO含量(P<0.05~P<0.01)，NOS活性(P<0.01，P<0.05，P＞0.05)，显著增强SOD活性(P<0.01)，GSH-PX活性(P<0.05~P<0.01)。以大黄酚脂质体(10.0mg·kg-1)组效果最为显著。结果显示，大黄酚脂质体可增强抗氧化能力。
小结：
脑缺血再灌注后，神经功能评分，病理组织学损伤，神经元超微结构损伤，GSH-PX，SOD，NOS的活性，MDA，NO含量呈动态改变，并且脑缺血再灌注12h~24h是脑缺血再灌注损伤过程的重要时间转折点。大黄酚脂质体能改善小鼠脑缺血再灌注损伤后神经功能评分，病理组织学损伤，神经元超微结构损伤，提高脑组织中SOD，GSH-PX活性，抑制NOS的活性，降低脑组织中MDA，NO的含量。因此，我们推测大黄酚脂质体可能通过抗氧化应激实现其对小鼠脑缺血再灌注损伤的神经保护作用。
第三部分大黄酚脂质体对小鼠脑缺血再灌注损伤所致细胞凋亡的影响
目的：动态观察小鼠脑缺血再灌注后神经元凋亡及Bax，Bcl-2，Cytc，caspase3等动态表达变化。研究大黄酚脂质体对脑缺血再灌注损伤小鼠的抗凋亡作用，探讨大黄酚脂质体抗凋亡作用的相关机制。
方法：应用线栓法建立小鼠脑缺血再灌注模型，采用健康雄性昆明种小鼠，体质量24~26g。实验1：小鼠随机分为正常对照组、假手术组、脑缺血再灌注组(脑缺血1h后再灌注，按再灌注不同时间点分为3h、6h、12h、24h、48h、72h6个亚组。实验2：小鼠随机分为正常对照组、假手术组、脑缺血再灌注24h组、大黄酚脂质体(10.0，5.0，0.5mg·kg-1)三个剂量组。大黄酚脂质体组于脑缺血再灌注前3天连续腹腔给予大黄酚脂质体，每天一次。于规定时间采用Hoechst33258染色检测神经元凋亡；免疫组织化学法、western blot法、实时荧光定量PCR法分别检测Bax，Bcl-2，Cytc，caspase3阳性细胞数、蛋白、mRNA水平表达。
结果：
1大黄酚脂质体减少神经元凋亡数目结果显示，正常对照组和假手术组小鼠脑组织偶见神经元凋亡，脑缺血再灌注3h可有少量的染色体深染、核浓缩的凋亡细胞；随着再灌注时间的延长，神经元细胞核缩小，染色质断裂凝集显著增多，荧光强度逐渐增加。而在48h后核深染、核浓缩，凋亡神经元数目有所减少。与假手术组比，脑缺血再灌注各组神经元凋亡数目有显著性差异(P<0.05)。结果表明，脑缺血再灌注可致小鼠脑神经元凋亡。大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组小鼠脑组织部分神经元染色体深染、核浓缩，神经元凋亡数目较脑缺血再灌注24h组均显著减少(P<0.05~P<0.01)，以大黄酚脂质体(10.0mg·kg-1)组效果最为显著。结果显示，大黄酚脂质体可显著减少神经元凋亡数目。
2大黄酚脂质体对细胞凋亡相关阳性细胞数变化的影响脑缺血再灌注组时Bax，Cytc，caspase3阳性细胞呈逐渐增多的趋势，于24h时达最高峰，再灌注48h、72h时有所下降。与假手术组比，脑缺血再灌注组Bax，Cytc，caspase3阳性细胞数目明显增多，具有显著性差异(P<0.01)。脑缺血再灌注组Bcl-2阳性细胞呈逐渐减少的趋势，24h时达低谷，再灌注48h-72h时有所上升。与假手术组比，脑缺血再灌注各组Bcl-2阳性细胞数目减少具有显著性差异(P<0.01)。结果表明，脑缺血再灌注后Bax，Cytc，caspase3阳性细胞数增加，Bcl-2阳性细胞数降低。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组中Bax、Cytc、caspase3阳性细胞数均减少(P<0.05~P<0.01)，Bcl-2阳性细胞数提高(P<0.05~P<0.01)；以大黄酚脂质体(10.0mg·kg-1)组效果最为显著。
3大黄酚脂质体细胞凋亡相关蛋白表达变化的影响脑缺血再灌注组时Bax，Cytc，caspase3蛋白表达呈逐渐增多的趋势，于24h时达最高峰，再灌注48h、72h时有所下降。与假手术组比，脑缺血再灌注组Bax，Cytc，caspase3蛋白明显上调(P<0.01)；脑缺血再灌注组Bcl-2蛋白表达呈逐渐减少的趋势，24h时达低谷，再灌注48h-72h时有所上升，与假手术组比，Bcl-2蛋白表达下调(P<0.05~P<0.01)。结果表明，脑缺血再灌注增加Bax，Cytc，caspase3蛋白表达，降低Bcl-2蛋白表达。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0mg·kg-1)组Bax，Cytc，caspase3蛋白表达均减少(P<0.01)，Bcl-2蛋白表达均提高(P<0.01)；大黄酚脂质体(0.5mg·kg-1)组Bax，Bcl-2，Cytc，caspase3蛋白表达水平也有显著性差异(P<0.05~P<0.01)
4大黄酚脂质体对细胞凋亡相关mRNA表达变化结果显示，与假手术组比，脑缺血再灌注各组Bax，Cytc，caspase3mRNA水平明显上调(P<0.05~P<0.01)，于脑缺血再灌注24h时达到高峰；Bcl-2mRNA水平明显下调(P<0.01)，于脑缺血再灌注24h时达到低谷。结果表明，脑缺血再灌注后可上调Bax，Cytc，caspase3mRNA表达，下调Bcl-2mRNA表达。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组下调Bax，Cytc，caspase3mRNA水平(P<0.05~P<0.01)，上调Bcl-2mRNA水平(P<0.01)。
小结：
脑缺血再灌注后神经元凋亡数目，Bax，Bcl-2，Cytc，caspase3表达呈动态改变，且脑缺血再灌注24h是脑缺血再灌注损伤过程的重要时间转折点。大黄酚脂质体降低神经元凋亡数目，下调Bax，Cytc，caspase3，上调Bcl-2表达。因此，我们推测大黄酚脂质体可能通过抗凋亡实现其对小鼠脑缺血再灌注损伤的神经保护作用。
Cerebrovascular disease is becoming a prominent public health concern.Due to high rates of morbidity, disability and mortality, this type of diseasenot only has a strong impact on the quality of life, but also accompanied byheavy economic burdens for the patients’ families and society in general.Currently, treatment of this disease is accomplished by surgically removingblocked blood vessels in order to improve the blood supply in and around thelesion. Once the blood supply improves, this area is extremely prone to getsevere cerebral reperfusion injury. There are several pathological mechanismsin the process of cerebral ischemic such as excessive release of excitatoryamino acids, loss of ionic homeostasis, energy failure, inflammatory response,increased oxidative stress and apoptosis. These mechanisms eventually causeirreversible damage of the brain tissue. So recent years, chemical drugs suchas calcium ion antagonist and radical scavengers as well as neuroprotectiveagents have been used for the treatment of CIR injury. However, side effectssuch as resistance to drugs, cerebral hemorrhage and gastrointestinalirritationmay exceed the clinical benefits for long-term therapy. Fortunately,clinical applications and experimental reports of traditional Chinese medicinesagainst CIR injury have been ascendant.
Radixet rhizoma Rhei, dried roots and rhizome of Rheum officinale Baill,Rheum palmatum L, and Rheum tanguticum Maxim has been used as animportant traditional Chinese medicine in Chinese folk for a long history,which widely grows in Sichuan, Qinghai, and Gansu and so on. Sheng Nong'sherbal classic\said that it was “bitter in taste, cold-natured, into the spleen,stomach, large intestine, and pericardium, liver” and had the effects ofanti-indigestion, anti-fever, discharging fire, cooling blood, removing stasisand detoxicate. Modern pharmacological studies show that it could be used for purging heat flux of intestinal, removing pattogenic heat from the blood andtoxic material from the body, decreasing stagnated. Besides, it could have theeffects of anti-radical, anti-hyperlipidemia, anti-arteriosclerosis, and anti-cancer, anti-aging. The main medicinal composition of Rhubarb is anthraqui-none compound, which has the utility monomer Chry that belonging toanthraquinone compound. Chry have ability of anti-cancer, anti-bacterial,anti-inflammatory, depressurization, and spasmolysis.
Chry can scavenge O2-, DPPH free radical in vitro, and inhibit lipidperoxidation in liver and brain tissue of rat. Chry has also been shown toimprove learning and memory function and to improve tolerance ability ofhypoxia CIR injury in mice. However, because of the lack of researchevidence in level of molecule, whether Chry protects against CIR by blockingapoptosis pathway remains unknown.
However, Chry is indissolvable in water, irritates the stomach, has lowbioavailability, and its physical and chemical properties are unstable. Thesedisadvantages restrict applications of Chry in the clinical setting. In this study,we extracted crude product of Chry from Rhubarb, then separated monomerby PHPLC, finally completed the preparation of Chr-lip; kunming mice wereinduced into CIR by transient middle cerebral artery occlusion, neurologicaldeficits, neuronal ultrastructure, histopathological changes, the activity ofSOD, GSH-PX, NOS, the content of MDA, NO, expressions of Bax, Bcl-2,Cytc, caspase3were examined dynamic observed, then the relation wereconfirmed between oxidative damage, apoptosis with cerebral ischemia.Furthermore, Male mice were intraperitoneally injected with Chr-lip for threesuccessive days, and then subjected to brain ischemia induced by MCAO.After reperfusion24h, neurological deficits, neuronal ultrastructure,histopathological changes, oxidative stress-related biochemical parameters,neuronal apoptosis, and apoptosis-related proteins were assessed. The presentstudy was designed to evaluate the neuroprotective effects of Chr-lip, as wellas the underlying mechanisms by focusing on oxidative stress and neuronalapoptosis. The study was divided into three part list as below.
PartⅠ Extraction of Chrysophanol and preparation of Chrysophanolliposomes
Objective: PHPLC method was established for purifying the substancesof Chry from Rhubarb, the prescription and preparation technology of Chr-lipwas investigated; the quality of Chr-lip was evaluated.
Methods: HPLC method was developed for determineation of Chry. Thecolumn was a Hypersil BDS C8(4.6×150mm,5μm) with a mixture of0.1%phosphoric acid solution-methanol-acetonitrile (15:85) as the mobile phase, ata flow rate of1.0mL·min-1. The column temperature was at35℃. Thedetection wavelength was254nm. Second, PHPLC method was establishedfor purifying the substances of Chry. The column was ZORBAX SB-C18:(21.2mm×250mm,7μm) with a mixture of0.1%phosphoric acid solution-methanol-acetonitrile (15:85) as the mobile phase, at a flow rate of20mL·min-1. The column temperature was at35℃. The detection wavelengthwas254nm. Fraction was collected: based on the peak, the threshold beingMin:2.2. Both NMR and HPLC are used for the structure and quantitativeanalysis of Chry. The prescription and preparation technology of Chr-lip wasinvestigated; film-ultrasonic dispersion method was used to prepare Chr-lip;the encapsulation efficiency, morphology, size distribution and stability qualityof Chr-lip was evaluated.
Results: NMR analysis showed that the structure was Chry. HPLCanalysis showed that the purity of Chry was98.9%, the entrapment efficiencyof Chr-lip was88.5%. The grain diameter was relatively homogeneous, therewas no gathered phenomenon, and the grain diameter was less than2microns.
Conclusions: PHPLC method was high sensitivity, easy operated forpurifying the substances of Chry. The purity of Chry was98.9%; theentrapment efficiency of Chr-lip was88.5%. It can be used for pharmacolo-gical study.
PartⅡ Effects of Chrysophanol liposome on cereral ischemia-reperfu-sion injury induced oxidative stress in mice
Objective: Neurological deficits, neuronal ultrastructure, histopatholo- gical changes, the activity of SOD, GSH-PX, NOS, the content of MDA, NO,were dynamic observed, then the relation were confirmed between oxidativedamage with CIR injury. We estimated the anti-oxidative of Chr-lip on CIRinjury in mice and explore its possible mechanisms.
Methods: Kunming mice,24~26g weight, were subjected to middlecerebral artery occlusion. Experiment1: Kunming mice were randomlydivided into the control group, the sham group, the model group. The modelgroup were applied and reperfused after60min, and then the model groupswere divided into6subgroups according to different reperfusion time points,3h,6h,12h,24h,48h. Experiment2: Kunming male mice were randomlydivided into the control group, sham group, model group (reperfusion24h),and Chr-lip treatment (10.0,5.0,0.5mg·kg-1) groups. Chr-lip treatment groupsintraperitoneally injected with Chi-lip (10.0,5.0,0.5mg·kg-1) for threesuccessive days, then subjected to brain ischemia induced by MCAO.According to the different time points, neurological deficits of each mousewere determined; changes of neuronal ultrastructure, histopathological weremeasured by transmission electron microscopy, HE stainning; then the activityof SOD, GSH-PX, NOS, the content of MDA, NO were determined.
Results:
1Chr-lip alleviated neurological deficit: in the control and sham group,there were no obvious neurological functions in mice. In model groups theneurological scores remarkably increased comparing with sham group(P<0.01), the highest neurological scores of group was at reperfusion24h.The results showed that neurological deficit was induced by CIR in mice.After CIR24h, neurological deficit score of the model group wassignificantly higher than that of the sham group (P<0.01); whereas those ofChr-lip-treated groups (10.0,5.0,0.5mg·kg-1) were significantly lower thanthe value in the model group (P<0.05~P<0.01).
2Chr-lip improved neuronal ultrastructure injury: in the control and shamgroup, neuron structure was regular, abundant organelles could be seen. Afterischemia reperfusion3h, shrunken neucleus and aggregated chromatin toward the nuclea membrane were observed. Obviously, slightly swollen mitocho-ndrion and vacuolus were observed. After ischemia reperfusion6h, neuronnuclear chromatin density increased, the nuclear membrane have seriousshrinkage, side set, partly dissolved mitochondria, endoplasmic reticulumpartly dissolved, cytoplasm height edema, vacuoles were observed, thenumbers of organelles reduced. After ischemia reperfusion12h, karyopy-knosis, swollen nucleus, edge chromatin, a large number of vacuolation incytoplasm were observed, mitochondria had completely dissolved. Afterischemia reperfusion24h, organelles were significantly decreased, vacuolatedmitochondria structure, disappeared outer, apoptosis body, and roughendoplasmic reticulum degranulation were observed. After ischemia reperfu-sion48h, karyopyknosis, nuclear membrane invagination, chromatin clumpsblock edge set under nuclear membrane, height edema cytoplasm, less numberof organelles, different degree of empty mitochondria, and lysosome wereseen. After ischemia reperfusion72h, karyopyknosis, nuclear membranedissolved, chromatin clumps block edge set under nuclear membrane, heightedema cytoplasm, different level of mitochondrial breakage, rough endoplas-mic reticulum degranulation, less number of organelles were seen. The resultsshowed neuronal ultrastructure was changed by CIR in mice. In the treatmentgroups, the chromosome distribution was relatively uniform, and the nuclearmembrane was clear. The mitochondria were reduced to some extent, butswelling of the rough endoplasmic reticulum was mild.
3Chr-lip alleviated histopathological changes: the histopathologicalexamination revealed that there was no obvious damage in the control andsham group. After ischemia reperfusion3h, mild edema in infarcts ofsurrounding brain tissue, mott cell in intercellular space with a small amountof inflammatory cell infiltration, pyknosis degeneration of neurons and glialcells were seen; there was no clear cell necrosis; with the reperfusion6h-12h,the above change gradually aggravated; with the reperfusion24h-48h,karyopyknosis, thick dyeing, condensed chromatin gathered around thenucleus with the change before the apoptosis or apoptosis, significant damage of neuron structure were seen; after ischemia reperfusion72h, cell edemagradually alleviated, edema surrounding part of the nerve cells, inflammatorycell infiltration gradually reduced, gliocyte proliferation were observed. Theresults showed histopathological were changed by CIR in mice. Chr-lipmarkedly alleviated histopathological changes induced by CIR.
4Chr-lip enhanceds antioxidant ability: the content of MDA reached apeak by12h, and was maintaining from24to48hours. Compared with shamgroup, the content of MDA significantly increased at different time points ofreperfusion (P<0.01). The activity of SOD reached a nadir by12h, andincreased from24to72hours. Compared with sham group, the activity ofSOD significantly decreased at different time points of reperfusion (P<0.01).The activity of GSH-PX reached a nadir by24h, and increased from48to72hours. Compared with sham group, the activity of GSH-PX significantlydecreased at different time points of reperfusion (P<0.01). The content of NOreached a peak by6h, and maintained from12to24hours. Compared withsham group, the content of NO significantly increased at different time pointsof reperfusion (P<0.01). The activity of NOS reached a peak by12h, andincreased from48to72hours. Compared with sham group, the activity ofNOS significantly decreased at different time points of reperfusion (P<0.01).The results showed that the antioxidant ability was decreased induced by CIR.Compared with model group, treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)significantly reduced the content of MDA (P<0.05~P<0.01), NO (P<0.05~P<0.01), the activity of NOS (P<0.01, P<0.05, P＞0.05), enhanced theactivity of SOD (P<0.01), GSH-PX (P<0.05~P<0.01).
Conclusion: Neurological deficits, neuronal ultrastructure injury,pathologic histology injury, the activity of SOD, GSH-PX, NOS, the contentof MDA, NO, were dynamic changed, as well as cerebral ischemia reperfusion12to24hours was an important time turning point in the process of cerebralischemic injury. We demonstrated that Chr-lip protected against CIR injury byimproving neurological, neuronal ultrastructure and histological deficits, andthese beneficial effects were associated with inhibition of oxidative stress, such as elevation of SOD and GSH-PX activities, reduction of the activity ofNOS, the content of MDA, NO. So the protective mechanisms of Chr-lipagainst CIR injury might be involved to its anti-oxidant activities.
PartⅢ Effects of Chrysophanol liposome on cereral ischemia-reperfus-ion injury induced apoptosis in mice
Objective: neuronal apoptosis, the expression of Bax, Bcl-2, Cytc,caspase3, were dynamic observed, and then the relation was confirmedbetween oxidative damage with CIR injury. We estimated the apoptosis ofChr-lip on CIR injury in mice and explore its possible mechanisms.
Methods: Kunming mice,24~26g weight, were subjected to middlecerebral artery occlusion. Experiment1: Kunming mice were randomlydivided into the control group, the sham group, the model group. The modelgroup were applied and reperfused after60min, and then the model groupswere divided into6subgroups according to different reperfusion time points,3h,6h,12h,24h,48h. Experiment2: Kunming male mice were randomlydivided into the control group, sham group, model group (reperfusion24h),and Chr-lip treatment (10.0,5.0,0.5mg·kg-1) groups. Chr-lip treatment groupsintraperitoneally injected with Chr-lip (10.0,5.0,0.5mg·kg-1) for threesuccessive days, then subjected to brain ischemia induced by MCAO.According to the different time points, neuronal apoptosis were detected byHoechst33258stainning; Immunohistochemistry was used for measuring thepositive cells of Bax, Bcl-2, Cytc, caspase3. The protein and mRNAexpression of Bax, Bcl-2, Cytc and caspase3were detected by western blotand real-time quantitative PCR.
Results:
1Chr-lip attenuated the neuronal apoptosis: neuronal injuries in theischemic hemispheres were analyzed by Hoechst33258staining. Apoptoticcells were sparsely detected in the control and sham group; after ischemiareperfusion3h, there was a small amount apoptotic cell with chromosomehyperchromatic and pyknosis; with the reperfusion extend, the number ofapoptotic cell with shrink and chromatin agglutination increased; however, after ischemia reperfusion48h, the number of apoptotic cell with nuclearhyperchromatism, pyknosis decreased. Compared with sham group, there wasa significant difference in the number of neurons apoptosis between modelgroups (P<0.05). Neuronal injuries in the ischemic hemispheres were analyzedby Hoechst33258staining. The treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)effectively attenuated the neuronal apoptosis caused by CIR injury, asindicated by significant reduction of apoptotic rate (P<0.05~P<0.01).
2Chr-lip effected on the positive cell of apoptosis-related: the number ofBax, Cytc, caspase3positive cell increased gradually, reached a peak atreperfusion24h, decreased from reperfusion48to72hours; compared withsham group, the number of Bax, Cytc, caspase3positive cell obviouslyincreased at different time points of reperfusion (P<0.01). The number ofBcl-2positive cell decreased gradually from reperfusion3to24hours, thenumber of Bcl-2positive cell reached a nadir at reperfusion24h, increasedfrom reperfusion48to72hours; compared with sham group, the number ofBcl-2positive cell obviously decreased at different time points of reperfusion(P<0.01). The results showed that the number of Bax, Cytc, and caspase3positive cell increased, Bcl-2positive cell obviously decreased after CIR.Compared with model group, treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)significantly reduced the number of Bax positive cell, Cytc positive cell,caspase3positive cell (P<0.05~P<0.01), increased the number of Bcl-2positive cell (P<0.05~P<0.01).
3Chr-lip effected on the proteins of apoptosis-related: the proteins ofBax, Cytc, caspase3increased gradually, reached a peak at reperfusion24h,decreased from reperfusion48to72hours; compared with sham group, theprotein of Bax, Cytc, caspase3levels was obviously up-regulated (P<0.01);the protein of Bcl-2decreased gradually, reached a nadir at reperfusion24h,increased from reperfusion48to72hours; the protein of Bcl-2levels wasobviously down-regulated (P<0.05~P<0.01). The results showed that theproteins of Bax, Cytc, and caspase3increased, Bcl-2obviously decreased afterCIR. Compared with model group, treatment with Chr-lip (10.0,5.0mg·kg-1) significantly reduced the protein of Bax, Cytc, and caspase3(P<0.01),increased the protein of Bcl-2(P<0.01). Treatment with Chr-lip (0.5mg·kg-1)significantly reduced the protein of Bax, Bcl-2, Cytc, caspase3showedsignificant differences (P<0.05~P<0.01).
4Chr-lip effected on the mRNA expression of apoptosis-related:compared with sham group, the mRNA levels of Bax, Cytc, caspase3wasobviously up-regulated (P<0.05~P<0.01), reached a peak at reperfusion24h;the mRNA levels of Bcl-2was obviously down-regulated (P<0.05~P<0.01),reached a nadir at reperfusion24h. The results showed that the mRNA levelsof Bax, Cytc, and caspase3increased, Bcl-2obviously decreased after CIR.Compared with model group, treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)significantly reduced the mRNA expression of Bax, Cytc, and caspase3(P<0.05~P<0.01), increased the mRNA expression of Bcl-2(P<0.01).
Conclusion: Neuronal apoptosis, the expression of Bax, Bcl-2, Cytc andcaspase3, were dynamic changed, as well as cerebral ischemia reperfusion24h was an important time turning point in the process of cerebral ischemicinjury. We demonstrated that Chr-lip protected against CIR injury by reducingneuronal apoptosis, promoting of Bcl-2expression, inhibiting of Baxexpression and Cytc release, and suppressing of caspase3activation. So theprotective mechanisms of Chr-lip against CIR injury might be involved to itsanti-apoptotic.
大黄Radixet rhizoma Rhei为蓼科植物药用大黄Rheum officinale Baill、掌叶大黄Rheum palmatum L、或唐古特大黄Rheum tanguticum Maxim的干燥根及根茎，主产于四川、青海、甘肃等地。大黄始载于《神农本草经》，苦寒，入脾、胃、大肠、心包、肝经，主攻积滞，可清湿热、泻火、凉血、祛瘀、解毒之功能。现代药理研究表明，大黄除具有泻热通肠、凉血解毒、逐瘀通经的作用外，尚具有清除自由基、降血脂、抗动脉硬化、抗癌、延缓衰老等药理作用。大黄的主要药效成分是蒽醌类化合物，大黄酚(Chrysophanol，Chry)属羟基蒽醌类，具有抗病毒、抗癌、抗炎、抗菌、降压和解痉等作用。
本课题组前期实验证明大黄酚在体外可清除超氧自由基、DPPH自由基、羟基自由基，对离体大鼠肝、脑丙二醛(Malondiadehyde，MDA)，超氧化物歧化酶(Superoxide dismutase，SOD)生成有明显的抑制作用；在小鼠脑缺血再灌注损伤后，大黄酚改善学习记忆功能并提高耐缺氧的能力，改善脑组织病理形态学损伤，结果表明大黄酚具有脑保护作用，但大黄酚对脑的保护作用机制尚不明确。
大黄酚属于脂溶性化合物，在水中溶解度极小，见光易分解，性质极其不稳定，且对胃肠有刺激作用，且生物利用度不高，影响了药物的临床应用。因此，本研究首先从中药大黄中提取大黄酚粗品，再采用制备高效液相色谱法(PHPLC)进行单体分离，再完成大黄酚脂质体(Chrysophanolliposomes，Chr-lip)的制备，建立昆明种小鼠脑缺血再灌注模型，动态观察小鼠脑缺血再灌注后神经功能损伤评分，神经元超微结构，病理组织学改变以及SOD，谷胱甘肽过氧化物酶(Glutathione peroxidase，GSH-PX)，一氧化氮合酶(Nitric oxide synthase，NOS)的活性，MDA，一氧化碳(Nitricoxide，NO)含量，以及Bax，Bcl-2，Cytc，caspase3等表达变化。确定缺血后与氧化损伤及凋亡的关系，并选用大黄酚脂质体进行干预，评价干预后神经功能缺损评分，以及大黄酚脂质体对氧化应激，自由基损伤，细胞凋亡信号通路的作用，初步探讨其在缺血再灌注损伤后的脑保护作用机制。本研究分三部分，现将各部分内容概述如下。
第一部分大黄酚的提取纯化及大黄酚脂质体制备
目的：建立从大黄中分离纯化大黄酚的PHPLC方法，考察大黄酚脂质体的处方和制备工艺进行研究，并评价其质量。
方法：建立HPLC测定大黄酚的分析方法，采用Hypersil BDS C8(4.6×150mm，5μm)，流动相为0.1%磷酸-甲醇溶液(15∶85)，流速为1.0mL·min-1，柱温35℃，检测波长254nm；其次，建立从大黄提取液中分离纯化大黄酚的PHPLC法，采用ZORBAX SB-C18：(21.2mm×250mm，7μm)，流动相：0.1%磷酸∶甲醇(15∶85)，柱温：35℃，流速：20mL·min-1，检测波长：254nm，进样量：7mL，馏分收集：基于峰，阈值Min：2.2。制备所得大黄酚单体采用核磁共振(Nuclear Magnetic Resonance，NMR)进行结构鉴定，HPLC法检测其纯度；再次，对大黄酚脂质体的处方和制备工艺进行研究，采用薄膜-超声法制备大黄酚脂质体，并考察大黄酚脂质体的包封率、形态学、粒径分布、稳定性。
结果：
所得大黄酚单体经NMR结构鉴定为大黄酚，经HPLC法检测，其含量为98.9%。大黄酚脂质体包封率为88.5%。粒径较为均匀，相互之间无聚集现象，粒径均小于2μm，稳定性好。
小结：
建立PHPLC分离纯化大黄中大黄酚方法，该方法灵敏度高，操作简便，所得大黄酚单体含量为98.9%。大黄酚脂质体包封率为88.5%，可用于药理学研究。
第二部分大黄酚脂质体对小鼠脑缺血再灌注损伤所致氧化应激的影响
目的：动态观察小鼠脑缺血再灌注后神经功能损伤评分，神经元超微结构，病理组织学改变以及SOD，GSH-PX，NOS的活性，MDA，NO含量。研究大黄酚脂质体对脑缺血再灌注损伤所致氧化应激的作用，探讨大黄酚脂质体抗氧化作用的相关机制。
方法：应用线栓法建立小鼠脑缺血再灌注模型，采用健康雄性昆明种小鼠，体质量24~26g。实验1：小鼠随机分为正常对照组、假手术组、脑缺血再灌注组(脑缺血1h后再灌注，按再灌注不同时间点分为3h、6h、12h、24h、48h、72h6个亚组。实验2：小鼠随机分为正常对照组、假手术组、脑缺血再灌注24h组、大黄酚脂质体(10.0，5.0，0.5mg·kg-1)三个剂量组。大黄酚脂质体组于脑缺血再灌注前3天连续腹腔给予大黄酚脂质体，每天一次。于规定时间检测小鼠神经功能损伤评分；采用HE法，透射电镜法观察小鼠脑组织病理组织学，神经元超微结构改变；采用试剂盒法检测SOD，GSH-PX，NOS的活性和MDA，NO含量。
结果：
1大黄酚脂质体减轻神经功能损伤正常对照组和假手术组，未见明显神经功能损害。脑缺血再灌注组神经功能评分显著高于假手术组(P<0.01)；以24h神经功能评分最高。结果表明，脑缺血再灌注后可损伤小鼠神经功能。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组神经功能缺陷评分均显著降低(P<0.05~P<0.01)，以大黄酚脂质体(10.0mg·kg-1)组效果最为显著。结果表明，大黄酚脂质体减轻神经功能损伤。
2大黄酚脂质体改善神经元超微结构正常对照组和假手术组神经元结构正常，可见丰富的细胞器。脑缺血再灌注3h可见神经元染色质密度增高，核膜皱缩、边集，线粒体轻微肿胀，可见空泡。脑缺血再灌注6h神经元染色质密度增高，核膜严重皱缩、边集，线粒体部分溶解，内质网部分溶解，细胞器数目减少，细胞质高度水肿，可见空泡。脑缺血再灌注12h可见核固缩，细胞核水肿，染色质边集，胞浆内有大量的空泡形成，有线粒体完全溶解；脑缺血再灌注24h可见细胞器明显减少，线粒体结构空泡状，外膜隐约可见，可见凋亡小体，粗面内质网脱颗粒；脑缺血再灌注48h神经元核固缩，核膜内陷，核染色质成团块状边集于核膜下，细胞质高度水肿，细胞器数目少，线粒体不同程度脱空，可见溶酶体。脑缺血再灌注72h组可见神经元核固缩，核膜溶解，核染色质成团块状边集于核膜下，线粒体不同程度断裂，粗面内质网脱颗粒，细胞器少。结果表明，脑缺血再灌注损伤小鼠神经元超微结构。大黄酚脂质体(10.0，5.0mg·kg-1)组神经元染色体比较均匀，核膜清楚，线粒体数目有所减少，粗面内质网有轻度肿胀，核糖体数目有一定的减少；大黄酚脂质体(0.5mg·kg-1)组对神经元超微结构也有改善作用。结果表明，大黄酚脂质体改善神经元超微结构。
3大黄酚脂质体改善病理组织学损害正常对照组和假手术组未见明显病理损害改变。脑缺血再灌注组3h脑缺血半球组织细胞轻度水肿，大小不等的空泡在细胞间隙出现，神经元及胶质细胞固缩，无明显细胞坏死；脑缺血再灌注6h-12h之间时，胞质明显水肿等上述症状逐渐加重；脑缺血再灌注24h-48h时神经元核固缩、浓染，染色质浓缩集聚于核周围，呈凋亡前或凋亡改变，神经元结构明显破裂；脑缺血再灌注72h时神经元水肿逐渐减轻，部分神经元周围出现水肿。结果表明，脑缺血再灌注可致小鼠脑病理组织学改变。大黄酚脂质体治疗组明显改善脑缺血再灌注损伤后的病理组织学损伤。
4大黄酚脂质体可增强抗氧化能力脑缺血再灌注3h MDA含量升高，12h后MDA含量达到最大，并持续到24h，48h-72h后有所下降，与假手术组比，脑缺血再灌注各时间点MDA含量增加有显著性差异(P<0.01)。脑缺血再灌注3h SOD活性明显降低，并于12h后SOD活性达到最低，24h-72h后有所增加，与假手术组比，脑缺血再灌注各时间点SOD活性降低有显著性差异(P<0.01)。脑缺血再灌注3h GSH-PX活性明显降低，并于24h后GSH-PX活性达到最低，48h-72h后有所增加；与假手术组比，脑缺血再灌注各时间点GSH-PX活性降低有显著性差异(P<0.01)。脑缺血再灌注3h NO含量升高，并于6h后NO含量达到最大，并持续到24h，48h-72h后有所下降；与假手术组比，脑缺血再灌注各时间点NO含量增加有显著性差异(P<0.01)。脑缺血再灌注3h NOS活性明显增加，并于12h后NOS活性达到最高，并持续到24h，48h-72h后有所减少；与假手术组比，脑缺血再灌注组NOS活性增加有显著性差异(P<0.01)。结果表明，脑缺血再灌注后，小鼠体内抗氧化能力减弱。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组显著降低MDA含量(P<0.05~P<0.01)，NO含量(P<0.05~P<0.01)，NOS活性(P<0.01，P<0.05，P＞0.05)，显著增强SOD活性(P<0.01)，GSH-PX活性(P<0.05~P<0.01)。以大黄酚脂质体(10.0mg·kg-1)组效果最为显著。结果显示，大黄酚脂质体可增强抗氧化能力。
小结：
脑缺血再灌注后，神经功能评分，病理组织学损伤，神经元超微结构损伤，GSH-PX，SOD，NOS的活性，MDA，NO含量呈动态改变，并且脑缺血再灌注12h~24h是脑缺血再灌注损伤过程的重要时间转折点。大黄酚脂质体能改善小鼠脑缺血再灌注损伤后神经功能评分，病理组织学损伤，神经元超微结构损伤，提高脑组织中SOD，GSH-PX活性，抑制NOS的活性，降低脑组织中MDA，NO的含量。因此，我们推测大黄酚脂质体可能通过抗氧化应激实现其对小鼠脑缺血再灌注损伤的神经保护作用。
第三部分大黄酚脂质体对小鼠脑缺血再灌注损伤所致细胞凋亡的影响
目的：动态观察小鼠脑缺血再灌注后神经元凋亡及Bax，Bcl-2，Cytc，caspase3等动态表达变化。研究大黄酚脂质体对脑缺血再灌注损伤小鼠的抗凋亡作用，探讨大黄酚脂质体抗凋亡作用的相关机制。
方法：应用线栓法建立小鼠脑缺血再灌注模型，采用健康雄性昆明种小鼠，体质量24~26g。实验1：小鼠随机分为正常对照组、假手术组、脑缺血再灌注组(脑缺血1h后再灌注，按再灌注不同时间点分为3h、6h、12h、24h、48h、72h6个亚组。实验2：小鼠随机分为正常对照组、假手术组、脑缺血再灌注24h组、大黄酚脂质体(10.0，5.0，0.5mg·kg-1)三个剂量组。大黄酚脂质体组于脑缺血再灌注前3天连续腹腔给予大黄酚脂质体，每天一次。于规定时间采用Hoechst33258染色检测神经元凋亡；免疫组织化学法、western blot法、实时荧光定量PCR法分别检测Bax，Bcl-2，Cytc，caspase3阳性细胞数、蛋白、mRNA水平表达。
结果：
1大黄酚脂质体减少神经元凋亡数目结果显示，正常对照组和假手术组小鼠脑组织偶见神经元凋亡，脑缺血再灌注3h可有少量的染色体深染、核浓缩的凋亡细胞；随着再灌注时间的延长，神经元细胞核缩小，染色质断裂凝集显著增多，荧光强度逐渐增加。而在48h后核深染、核浓缩，凋亡神经元数目有所减少。与假手术组比，脑缺血再灌注各组神经元凋亡数目有显著性差异(P<0.05)。结果表明，脑缺血再灌注可致小鼠脑神经元凋亡。大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组小鼠脑组织部分神经元染色体深染、核浓缩，神经元凋亡数目较脑缺血再灌注24h组均显著减少(P<0.05~P<0.01)，以大黄酚脂质体(10.0mg·kg-1)组效果最为显著。结果显示，大黄酚脂质体可显著减少神经元凋亡数目。
2大黄酚脂质体对细胞凋亡相关阳性细胞数变化的影响脑缺血再灌注组时Bax，Cytc，caspase3阳性细胞呈逐渐增多的趋势，于24h时达最高峰，再灌注48h、72h时有所下降。与假手术组比，脑缺血再灌注组Bax，Cytc，caspase3阳性细胞数目明显增多，具有显著性差异(P<0.01)。脑缺血再灌注组Bcl-2阳性细胞呈逐渐减少的趋势，24h时达低谷，再灌注48h-72h时有所上升。与假手术组比，脑缺血再灌注各组Bcl-2阳性细胞数目减少具有显著性差异(P<0.01)。结果表明，脑缺血再灌注后Bax，Cytc，caspase3阳性细胞数增加，Bcl-2阳性细胞数降低。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组中Bax、Cytc、caspase3阳性细胞数均减少(P<0.05~P<0.01)，Bcl-2阳性细胞数提高(P<0.05~P<0.01)；以大黄酚脂质体(10.0mg·kg-1)组效果最为显著。
3大黄酚脂质体细胞凋亡相关蛋白表达变化的影响脑缺血再灌注组时Bax，Cytc，caspase3蛋白表达呈逐渐增多的趋势，于24h时达最高峰，再灌注48h、72h时有所下降。与假手术组比，脑缺血再灌注组Bax，Cytc，caspase3蛋白明显上调(P<0.01)；脑缺血再灌注组Bcl-2蛋白表达呈逐渐减少的趋势，24h时达低谷，再灌注48h-72h时有所上升，与假手术组比，Bcl-2蛋白表达下调(P<0.05~P<0.01)。结果表明，脑缺血再灌注增加Bax，Cytc，caspase3蛋白表达，降低Bcl-2蛋白表达。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0mg·kg-1)组Bax，Cytc，caspase3蛋白表达均减少(P<0.01)，Bcl-2蛋白表达均提高(P<0.01)；大黄酚脂质体(0.5mg·kg-1)组Bax，Bcl-2，Cytc，caspase3蛋白表达水平也有显著性差异(P<0.05~P<0.01)
4大黄酚脂质体对细胞凋亡相关mRNA表达变化结果显示，与假手术组比，脑缺血再灌注各组Bax，Cytc，caspase3mRNA水平明显上调(P<0.05~P<0.01)，于脑缺血再灌注24h时达到高峰；Bcl-2mRNA水平明显下调(P<0.01)，于脑缺血再灌注24h时达到低谷。结果表明，脑缺血再灌注后可上调Bax，Cytc，caspase3mRNA表达，下调Bcl-2mRNA表达。与脑缺血再灌注24h组比，大黄酚脂质体(10.0，5.0，0.5mg·kg-1)组下调Bax，Cytc，caspase3mRNA水平(P<0.05~P<0.01)，上调Bcl-2mRNA水平(P<0.01)。
小结：
脑缺血再灌注后神经元凋亡数目，Bax，Bcl-2，Cytc，caspase3表达呈动态改变，且脑缺血再灌注24h是脑缺血再灌注损伤过程的重要时间转折点。大黄酚脂质体降低神经元凋亡数目，下调Bax，Cytc，caspase3，上调Bcl-2表达。因此，我们推测大黄酚脂质体可能通过抗凋亡实现其对小鼠脑缺血再灌注损伤的神经保护作用。
Cerebrovascular disease is becoming a prominent public health concern.Due to high rates of morbidity, disability and mortality, this type of diseasenot only has a strong impact on the quality of life, but also accompanied byheavy economic burdens for the patients’ families and society in general.Currently, treatment of this disease is accomplished by surgically removingblocked blood vessels in order to improve the blood supply in and around thelesion. Once the blood supply improves, this area is extremely prone to getsevere cerebral reperfusion injury. There are several pathological mechanismsin the process of cerebral ischemic such as excessive release of excitatoryamino acids, loss of ionic homeostasis, energy failure, inflammatory response,increased oxidative stress and apoptosis. These mechanisms eventually causeirreversible damage of the brain tissue. So recent years, chemical drugs suchas calcium ion antagonist and radical scavengers as well as neuroprotectiveagents have been used for the treatment of CIR injury. However, side effectssuch as resistance to drugs, cerebral hemorrhage and gastrointestinalirritationmay exceed the clinical benefits for long-term therapy. Fortunately,clinical applications and experimental reports of traditional Chinese medicinesagainst CIR injury have been ascendant.
Radixet rhizoma Rhei, dried roots and rhizome of Rheum officinale Baill,Rheum palmatum L, and Rheum tanguticum Maxim has been used as animportant traditional Chinese medicine in Chinese folk for a long history,which widely grows in Sichuan, Qinghai, and Gansu and so on. Sheng Nong'sherbal classic\said that it was “bitter in taste, cold-natured, into the spleen,stomach, large intestine, and pericardium, liver” and had the effects ofanti-indigestion, anti-fever, discharging fire, cooling blood, removing stasisand detoxicate. Modern pharmacological studies show that it could be used for purging heat flux of intestinal, removing pattogenic heat from the blood andtoxic material from the body, decreasing stagnated. Besides, it could have theeffects of anti-radical, anti-hyperlipidemia, anti-arteriosclerosis, and anti-cancer, anti-aging. The main medicinal composition of Rhubarb is anthraqui-none compound, which has the utility monomer Chry that belonging toanthraquinone compound. Chry have ability of anti-cancer, anti-bacterial,anti-inflammatory, depressurization, and spasmolysis.
Chry can scavenge O2-, DPPH free radical in vitro, and inhibit lipidperoxidation in liver and brain tissue of rat. Chry has also been shown toimprove learning and memory function and to improve tolerance ability ofhypoxia CIR injury in mice. However, because of the lack of researchevidence in level of molecule, whether Chry protects against CIR by blockingapoptosis pathway remains unknown.
However, Chry is indissolvable in water, irritates the stomach, has lowbioavailability, and its physical and chemical properties are unstable. Thesedisadvantages restrict applications of Chry in the clinical setting. In this study,we extracted crude product of Chry from Rhubarb, then separated monomerby PHPLC, finally completed the preparation of Chr-lip; kunming mice wereinduced into CIR by transient middle cerebral artery occlusion, neurologicaldeficits, neuronal ultrastructure, histopathological changes, the activity ofSOD, GSH-PX, NOS, the content of MDA, NO, expressions of Bax, Bcl-2,Cytc, caspase3were examined dynamic observed, then the relation wereconfirmed between oxidative damage, apoptosis with cerebral ischemia.Furthermore, Male mice were intraperitoneally injected with Chr-lip for threesuccessive days, and then subjected to brain ischemia induced by MCAO.After reperfusion24h, neurological deficits, neuronal ultrastructure,histopathological changes, oxidative stress-related biochemical parameters,neuronal apoptosis, and apoptosis-related proteins were assessed. The presentstudy was designed to evaluate the neuroprotective effects of Chr-lip, as wellas the underlying mechanisms by focusing on oxidative stress and neuronalapoptosis. The study was divided into three part list as below.
PartⅠ Extraction of Chrysophanol and preparation of Chrysophanolliposomes
Objective: PHPLC method was established for purifying the substancesof Chry from Rhubarb, the prescription and preparation technology of Chr-lipwas investigated; the quality of Chr-lip was evaluated.
Methods: HPLC method was developed for determineation of Chry. Thecolumn was a Hypersil BDS C8(4.6×150mm,5μm) with a mixture of0.1%phosphoric acid solution-methanol-acetonitrile (15:85) as the mobile phase, ata flow rate of1.0mL·min-1. The column temperature was at35℃. Thedetection wavelength was254nm. Second, PHPLC method was establishedfor purifying the substances of Chry. The column was ZORBAX SB-C18:(21.2mm×250mm,7μm) with a mixture of0.1%phosphoric acid solution-methanol-acetonitrile (15:85) as the mobile phase, at a flow rate of20mL·min-1. The column temperature was at35℃. The detection wavelengthwas254nm. Fraction was collected: based on the peak, the threshold beingMin:2.2. Both NMR and HPLC are used for the structure and quantitativeanalysis of Chry. The prescription and preparation technology of Chr-lip wasinvestigated; film-ultrasonic dispersion method was used to prepare Chr-lip;the encapsulation efficiency, morphology, size distribution and stability qualityof Chr-lip was evaluated.
Results: NMR analysis showed that the structure was Chry. HPLCanalysis showed that the purity of Chry was98.9%, the entrapment efficiencyof Chr-lip was88.5%. The grain diameter was relatively homogeneous, therewas no gathered phenomenon, and the grain diameter was less than2microns.
Conclusions: PHPLC method was high sensitivity, easy operated forpurifying the substances of Chry. The purity of Chry was98.9%; theentrapment efficiency of Chr-lip was88.5%. It can be used for pharmacolo-gical study.
PartⅡ Effects of Chrysophanol liposome on cereral ischemia-reperfu-sion injury induced oxidative stress in mice
Objective: Neurological deficits, neuronal ultrastructure, histopatholo- gical changes, the activity of SOD, GSH-PX, NOS, the content of MDA, NO,were dynamic observed, then the relation were confirmed between oxidativedamage with CIR injury. We estimated the anti-oxidative of Chr-lip on CIRinjury in mice and explore its possible mechanisms.
Methods: Kunming mice,24~26g weight, were subjected to middlecerebral artery occlusion. Experiment1: Kunming mice were randomlydivided into the control group, the sham group, the model group. The modelgroup were applied and reperfused after60min, and then the model groupswere divided into6subgroups according to different reperfusion time points,3h,6h,12h,24h,48h. Experiment2: Kunming male mice were randomlydivided into the control group, sham group, model group (reperfusion24h),and Chr-lip treatment (10.0,5.0,0.5mg·kg-1) groups. Chr-lip treatment groupsintraperitoneally injected with Chi-lip (10.0,5.0,0.5mg·kg-1) for threesuccessive days, then subjected to brain ischemia induced by MCAO.According to the different time points, neurological deficits of each mousewere determined; changes of neuronal ultrastructure, histopathological weremeasured by transmission electron microscopy, HE stainning; then the activityof SOD, GSH-PX, NOS, the content of MDA, NO were determined.
Results:
1Chr-lip alleviated neurological deficit: in the control and sham group,there were no obvious neurological functions in mice. In model groups theneurological scores remarkably increased comparing with sham group(P<0.01), the highest neurological scores of group was at reperfusion24h.The results showed that neurological deficit was induced by CIR in mice.After CIR24h, neurological deficit score of the model group wassignificantly higher than that of the sham group (P<0.01); whereas those ofChr-lip-treated groups (10.0,5.0,0.5mg·kg-1) were significantly lower thanthe value in the model group (P<0.05~P<0.01).
2Chr-lip improved neuronal ultrastructure injury: in the control and shamgroup, neuron structure was regular, abundant organelles could be seen. Afterischemia reperfusion3h, shrunken neucleus and aggregated chromatin toward the nuclea membrane were observed. Obviously, slightly swollen mitocho-ndrion and vacuolus were observed. After ischemia reperfusion6h, neuronnuclear chromatin density increased, the nuclear membrane have seriousshrinkage, side set, partly dissolved mitochondria, endoplasmic reticulumpartly dissolved, cytoplasm height edema, vacuoles were observed, thenumbers of organelles reduced. After ischemia reperfusion12h, karyopy-knosis, swollen nucleus, edge chromatin, a large number of vacuolation incytoplasm were observed, mitochondria had completely dissolved. Afterischemia reperfusion24h, organelles were significantly decreased, vacuolatedmitochondria structure, disappeared outer, apoptosis body, and roughendoplasmic reticulum degranulation were observed. After ischemia reperfu-sion48h, karyopyknosis, nuclear membrane invagination, chromatin clumpsblock edge set under nuclear membrane, height edema cytoplasm, less numberof organelles, different degree of empty mitochondria, and lysosome wereseen. After ischemia reperfusion72h, karyopyknosis, nuclear membranedissolved, chromatin clumps block edge set under nuclear membrane, heightedema cytoplasm, different level of mitochondrial breakage, rough endoplas-mic reticulum degranulation, less number of organelles were seen. The resultsshowed neuronal ultrastructure was changed by CIR in mice. In the treatmentgroups, the chromosome distribution was relatively uniform, and the nuclearmembrane was clear. The mitochondria were reduced to some extent, butswelling of the rough endoplasmic reticulum was mild.
3Chr-lip alleviated histopathological changes: the histopathologicalexamination revealed that there was no obvious damage in the control andsham group. After ischemia reperfusion3h, mild edema in infarcts ofsurrounding brain tissue, mott cell in intercellular space with a small amountof inflammatory cell infiltration, pyknosis degeneration of neurons and glialcells were seen; there was no clear cell necrosis; with the reperfusion6h-12h,the above change gradually aggravated; with the reperfusion24h-48h,karyopyknosis, thick dyeing, condensed chromatin gathered around thenucleus with the change before the apoptosis or apoptosis, significant damage of neuron structure were seen; after ischemia reperfusion72h, cell edemagradually alleviated, edema surrounding part of the nerve cells, inflammatorycell infiltration gradually reduced, gliocyte proliferation were observed. Theresults showed histopathological were changed by CIR in mice. Chr-lipmarkedly alleviated histopathological changes induced by CIR.
4Chr-lip enhanceds antioxidant ability: the content of MDA reached apeak by12h, and was maintaining from24to48hours. Compared with shamgroup, the content of MDA significantly increased at different time points ofreperfusion (P<0.01). The activity of SOD reached a nadir by12h, andincreased from24to72hours. Compared with sham group, the activity ofSOD significantly decreased at different time points of reperfusion (P<0.01).The activity of GSH-PX reached a nadir by24h, and increased from48to72hours. Compared with sham group, the activity of GSH-PX significantlydecreased at different time points of reperfusion (P<0.01). The content of NOreached a peak by6h, and maintained from12to24hours. Compared withsham group, the content of NO significantly increased at different time pointsof reperfusion (P<0.01). The activity of NOS reached a peak by12h, andincreased from48to72hours. Compared with sham group, the activity ofNOS significantly decreased at different time points of reperfusion (P<0.01).The results showed that the antioxidant ability was decreased induced by CIR.Compared with model group, treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)significantly reduced the content of MDA (P<0.05~P<0.01), NO (P<0.05~P<0.01), the activity of NOS (P<0.01, P<0.05, P＞0.05), enhanced theactivity of SOD (P<0.01), GSH-PX (P<0.05~P<0.01).
Conclusion: Neurological deficits, neuronal ultrastructure injury,pathologic histology injury, the activity of SOD, GSH-PX, NOS, the contentof MDA, NO, were dynamic changed, as well as cerebral ischemia reperfusion12to24hours was an important time turning point in the process of cerebralischemic injury. We demonstrated that Chr-lip protected against CIR injury byimproving neurological, neuronal ultrastructure and histological deficits, andthese beneficial effects were associated with inhibition of oxidative stress, such as elevation of SOD and GSH-PX activities, reduction of the activity ofNOS, the content of MDA, NO. So the protective mechanisms of Chr-lipagainst CIR injury might be involved to its anti-oxidant activities.
PartⅢ Effects of Chrysophanol liposome on cereral ischemia-reperfus-ion injury induced apoptosis in mice
Objective: neuronal apoptosis, the expression of Bax, Bcl-2, Cytc,caspase3, were dynamic observed, and then the relation was confirmedbetween oxidative damage with CIR injury. We estimated the apoptosis ofChr-lip on CIR injury in mice and explore its possible mechanisms.
Methods: Kunming mice,24~26g weight, were subjected to middlecerebral artery occlusion. Experiment1: Kunming mice were randomlydivided into the control group, the sham group, the model group. The modelgroup were applied and reperfused after60min, and then the model groupswere divided into6subgroups according to different reperfusion time points,3h,6h,12h,24h,48h. Experiment2: Kunming male mice were randomlydivided into the control group, sham group, model group (reperfusion24h),and Chr-lip treatment (10.0,5.0,0.5mg·kg-1) groups. Chr-lip treatment groupsintraperitoneally injected with Chr-lip (10.0,5.0,0.5mg·kg-1) for threesuccessive days, then subjected to brain ischemia induced by MCAO.According to the different time points, neuronal apoptosis were detected byHoechst33258stainning; Immunohistochemistry was used for measuring thepositive cells of Bax, Bcl-2, Cytc, caspase3. The protein and mRNAexpression of Bax, Bcl-2, Cytc and caspase3were detected by western blotand real-time quantitative PCR.
Results:
1Chr-lip attenuated the neuronal apoptosis: neuronal injuries in theischemic hemispheres were analyzed by Hoechst33258staining. Apoptoticcells were sparsely detected in the control and sham group; after ischemiareperfusion3h, there was a small amount apoptotic cell with chromosomehyperchromatic and pyknosis; with the reperfusion extend, the number ofapoptotic cell with shrink and chromatin agglutination increased; however, after ischemia reperfusion48h, the number of apoptotic cell with nuclearhyperchromatism, pyknosis decreased. Compared with sham group, there wasa significant difference in the number of neurons apoptosis between modelgroups (P<0.05). Neuronal injuries in the ischemic hemispheres were analyzedby Hoechst33258staining. The treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)effectively attenuated the neuronal apoptosis caused by CIR injury, asindicated by significant reduction of apoptotic rate (P<0.05~P<0.01).
2Chr-lip effected on the positive cell of apoptosis-related: the number ofBax, Cytc, caspase3positive cell increased gradually, reached a peak atreperfusion24h, decreased from reperfusion48to72hours; compared withsham group, the number of Bax, Cytc, caspase3positive cell obviouslyincreased at different time points of reperfusion (P<0.01). The number ofBcl-2positive cell decreased gradually from reperfusion3to24hours, thenumber of Bcl-2positive cell reached a nadir at reperfusion24h, increasedfrom reperfusion48to72hours; compared with sham group, the number ofBcl-2positive cell obviously decreased at different time points of reperfusion(P<0.01). The results showed that the number of Bax, Cytc, and caspase3positive cell increased, Bcl-2positive cell obviously decreased after CIR.Compared with model group, treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)significantly reduced the number of Bax positive cell, Cytc positive cell,caspase3positive cell (P<0.05~P<0.01), increased the number of Bcl-2positive cell (P<0.05~P<0.01).
3Chr-lip effected on the proteins of apoptosis-related: the proteins ofBax, Cytc, caspase3increased gradually, reached a peak at reperfusion24h,decreased from reperfusion48to72hours; compared with sham group, theprotein of Bax, Cytc, caspase3levels was obviously up-regulated (P<0.01);the protein of Bcl-2decreased gradually, reached a nadir at reperfusion24h,increased from reperfusion48to72hours; the protein of Bcl-2levels wasobviously down-regulated (P<0.05~P<0.01). The results showed that theproteins of Bax, Cytc, and caspase3increased, Bcl-2obviously decreased afterCIR. Compared with model group, treatment with Chr-lip (10.0,5.0mg·kg-1) significantly reduced the protein of Bax, Cytc, and caspase3(P<0.01),increased the protein of Bcl-2(P<0.01). Treatment with Chr-lip (0.5mg·kg-1)significantly reduced the protein of Bax, Bcl-2, Cytc, caspase3showedsignificant differences (P<0.05~P<0.01).
4Chr-lip effected on the mRNA expression of apoptosis-related:compared with sham group, the mRNA levels of Bax, Cytc, caspase3wasobviously up-regulated (P<0.05~P<0.01), reached a peak at reperfusion24h;the mRNA levels of Bcl-2was obviously down-regulated (P<0.05~P<0.01),reached a nadir at reperfusion24h. The results showed that the mRNA levelsof Bax, Cytc, and caspase3increased, Bcl-2obviously decreased after CIR.Compared with model group, treatment with Chr-lip (10.0,5.0,0.5mg·kg-1)significantly reduced the mRNA expression of Bax, Cytc, and caspase3(P<0.05~P<0.01), increased the mRNA expression of Bcl-2(P<0.01).
Conclusion: Neuronal apoptosis, the expression of Bax, Bcl-2, Cytc andcaspase3, were dynamic changed, as well as cerebral ischemia reperfusion24h was an important time turning point in the process of cerebral ischemicinjury. We demonstrated that Chr-lip protected against CIR injury by reducingneuronal apoptosis, promoting of Bcl-2expression, inhibiting of Baxexpression and Cytc release, and suppressing of caspase3activation. So theprotective mechanisms of Chr-lip against CIR injury might be involved to itsanti-apoptotic.
引文
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