中药复方脑得生的有效成分组研究
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摘要
缺血性中风属中医学“中风”范畴，是一种严重影响人类健康的常见病。其发病机制复杂，是一种多因素多途径相互作用的疾病，涉及到大量的生物活性物质，目前对其发病机制的研究尚未完全阐明。其病理机制主要有脑组织的能量代谢紊乱、自由基损伤、炎症反应、兴奋性氨基酸神经毒性作用、钙超载、凋亡相关基因的表达等。近年来，脑缺血的治疗主要是针对神经元的保护作用，减少梗塞面积，减少神经元的迟发性死亡等。本文以其发病机制为依据，对复方脑得生有效成分组的神经保护作用及其机制进行了深入研究。
一、适合高通量筛选样品的制备
本实验采用现代植物化学分离技术，制定规范的操作程序，将提取分离和层析等技术相结合，用不同溶剂对组方中原料药进行提取，将复方脑得生中的全部成分分为170个独立组分。其中，对乙醇部分进行硅胶柱层析，共计得到90个组分；对石油醚部分进行硅胶柱层析，共计得到80个组分。所有样品进行浓缩供下一步的筛选及研究用。
二、对样品进行高通量筛选
建立与脑得生功效主治相关的高通量筛选模型。根据脑得生的功效主治寻找与现代医学共同点，在细胞和分子水平上建立能够反映作用机理的筛选模型。缺血性脑损伤的发生主要与过氧化损伤、钙超载、兴奋毒性作用、炎症反应以及线粒体功能的受损等相关。因此，本研究中我们根据上述发病机制分别建立了相应的损伤模型，并对所得的170个组分进行了筛选。最后，将以上研究结果进行汇总，发现部分样品在不同模型上作用吻合，可能是脑得生发挥作用的活性物质，另外一些样品在多个模型作用强度较弱甚至相反，可能是影响脑得生疗效的部分。通过综合分析，将作用符合我们要求的组分按一定比例混合组成脑得生的有效成分组(2-11，16-21，25-29，31-35，38-41，47-52，59-73，95-99，116-119，122-127，131-136，167-170)，进行高效液相成分分析，找出适合大规模生产的有效成分组。
三、应用HPLC分析高通量筛选结果，实现生产工艺的转换
分别将硅胶柱分离的醇溶部分和石油醚部分中的抗脑缺血有效成分，按其编号从其DMSO溶液中取出，按照各自得率，有机混合在一起作为有效成分组供检测用。分别将大孔树脂分离的30％、60％、90％乙醇沈脱物部分与抗脑缺血有效成分组做对比，通过HPLC分析寻找适合大规模生产的目的组分。由液相色谱图可以看出，脑得生有效成分主要分布在30％和60％乙醇洗脱物中。因此，我们将30％和60％乙醇洗脱物按比例混合作为新的复方脑得生的有效成分组(NECG)，进行下一步的整体动物实验，对其疗效进行验证，并进一步进行机制研究。
四、利用动物实验进行有效成分组的验证
采用栓线法造成大鼠大脑中动脉阻断模型，通过预防性以及治疗性两种给药方式，从脑梗塞体积、行为学、脑组织能量代谢、病理学改变、炎症反应以及凋亡等多方面指标进行观察，全面评价了我们所确证的有效成分组的神经保护作用。
实验结果表明，治疗性给药时NECG大剂量组(0.07 g／kg)可显著降低脑梗死体积、改善神经功能，并且可抑制炎症反应，以及凋亡的发生和发展；NECG中剂量组(0.02 g／kg)和小剂量组(0.007 g／kg)也有改善作用，但某些指标与模型组相比，没有显著性差异(P＞0.05)。造模前先连续给予7天药物时，与模型组以及治疗给药的实验结果比，药物的保护作用则更加明显，尤其NECG中剂量组表现出了明显的保护作用。另外，我们同时与原方的疗效进行了对照研究，实验结果说明NECG的疗效明显优于原方。
五、体外对其作用机制进一步探讨
实验结果表明，NECG对神经细胞的保护作用机理不是单一的，其药效是多种机理综合作用的结果。
1、NECG能抑制过氧化氢诱导的PC12细胞凋亡。实验结果表明，NECG可显著改善过氧化氢引起的细胞存活率的降低；流式细胞仪分析表明100μg／ml和10μg／ml的NECG均能显著改善过氧化氢引起的凋亡；Western Blotting的分析结果显示，100μg／ml和10μg／ml NECG能够明显抑制过氧化氢诱导的Bcl-2蛋白表达的下调和Bax蛋白表达的上调，使Bcl-2／Bax比值上升，还能明显抑制细胞色素C从线粒体的释放以及p53的活化；同时，预先加入NECG还可明显抑制过氧化氢损伤引起的caspase-3的活化，使其活性降低。
2、NECG能够抑制叠氮钠诱导的PC12细胞凋亡。我们的实验结果首先证明了叠氮钠诱导的凋亡伴随有细胞内ROS时间依赖性的产生增多，并且与凋亡的线粒体途径改变密切相关。同时，我们的实验结果也提示，NECG可剂量依赖性的显著改善上述改变：NECG可改善细胞存活率和凋亡率，降低细胞内ROS的增多，100μg／ml和10μg／ml的NECG能够抑制叠氮钠诱导的Bcl-2表达的下调和Bax蛋白表达的上调，使Bcl-2和Bax比值上调，预先加入NECG能明显抑制叠氮钠损伤引起的细胞色素C自线粒体的释放以及caspase-3的活化，从而有效的降低了细胞凋亡的发生。
3、NECG有改善线粒体功能的作用。NECG体外能够抑制50μmoL／L FeSO_4／500μmoL／L半胱胺酸损伤体系诱导的线粒体MDA生成的增加，降低脂质过氧化的发生率，具有明显的保护线粒体免遭氧化损伤的作用；可减轻线粒体肿胀度，维持线粒体的膜电位，抑制H~+-ATP酶活性的降低，从而保护了线粒体的结构和功能；NECG还可抑制GSH含量的降低，保护了线粒体内源性抗氧化物质，从而使氧自由基的生成与清除保持平衡，使线粒体膜免遭损伤。
因此，NECG抗细胞凋亡的作用机制一方面和清除氧自由基有关，另一方面NECG可能通过直接影响p53的表达，细胞色素C的释放、caspase-3活性或者通过调控Bcl-2、Bax的蛋白表达间接影响线粒体依赖的caspase-3途径来抑制细胞凋亡。
六、结论
研究结果表明，中药复方脑得生对脑缺血有显著的防治作用，这种作用的物质基础是其中多种有效成分的有机组合，这些成分通过不同的作用途径和相互影响，产生综合作用，实现对脑缺血疾病的预防和治疗作用。药效成分组的作用机制研究证明中药有效成分组通过不同的成分从不同的途径发挥作用，如抗过氧化损伤、抑制细胞凋亡以及减轻炎症反应等。结果证明有效成分组可以比较合理的代表了中药的整体作用，反映了中药复方的作用特点。本实验结果也证明，应用高通量筛选方法研究中药复方具有一定的技术优势，也是行之有效的方法。
Ischemic stroke is a common disease, which affects the health of human seriously. Themechanism is complex, and could be regulated by many factors, including the activationsof many kinds of biologically active materials, and so on. At present, the details have notbeen well established, and the possible mechanisms include the dysfunctions of energymetabolism, oxidative stress, inflammation responses, glutamate excitotoxic damage,calcium overload, and the expressiones of some apoptosis-related proteins. In recent years,the main targets to treat brain ischemia are to protect the neurons, decrease the infarctionvolumes, and extenuate the delayed neuronal death. According to this context, weinvestigated the neuroprotective effect and the possible mechanism of the NECG ofNaoDeSheng (NDS) in present study.
1. The preparation of NDS samples for high throughput screening
Sequential components of NDS samples were prepared by the morden seperationtechniques. In brief, the powdered herbs were extracted with petroleum ether, 95％alcoholand aqueous in turn, and both of 95％alcohol extract fraction and petroleum ether extractfraction were further separated, respectively. The natural samples were collected on fixedinterval, and we obtained 170 sequential components in total, including 90 sequentialcomponents of 95％alcohol extract fraction and 80 sequential components of petroleumether extract fraction. All of these samples were concentrated for the further screening andresearching.
2. Bioactivities of NDS samples tested by the method of High-throughput screening
According to the mechanism of ischemic stroke, we established ischemia-related injurymodels, including chemical hypoxia model, mitochondfial injury model, inflammatorymodels and the models induced by hydrogen peroxide and glutamate, and evaluated theeffects of 170 sequential components (L1-L90, A91-A170) of NDS on them, respectively.Finally, we combined all of these screening results, and found some of samples alwaysexert effective roles even in different models, which were defined as the effective components group (ECG), some of samples have somewhat weak effect, and others evenhave an contrary effect. By comprehensive analysis of these results, we regarded thefollowing parts as the ECG of NDS (2-11,16-21, 25-29,31-35,38-41,47-52,59-73, 95-99,116-119, 122-127, 131-136, 167-170). In the following experiment, we further analysed theECG by high performance liquid chromatography (HPLC) to find the NDS's ECG (NECG),which is appropriate for producing Chinese herbal medicinal prescription in a commercialscale.
3. Analysis of the ECG of NDS samples by HPLC
Comparing the ECG with the extraction of 30％, 60％, and 90％EtOH fraction by HPLC,we found that the mixture of 30％and 60％EtOH fractions of NaoDeSheng could beregarded as the NECG of NaoDeSheng. Furthermore, we will confirm the pharmalogicaleffects in the following in vivo experiment and will researched its mechanism deeply invitro.
4. The protective effect of NECG on the MCAO in vivo
Focal brain ischemia was induced by the intraluminal suture MCA occlusion method(MCAO) with minor modifications, NECG was administrated prior to or after MCAO, andthe neuroprotections of NECG were evaluated comprehensively, including themeasurement of infarct volume, alterations in the neurological deficits, oxidative stress,inflammatory responses, and the occurrence of apoptosis. Our results indicated that the highdose of NECG (0.07 g/kg) administrated after the occlusion of MCAO could reduce theinfarct area, extenuate neurological deficient. This protective effects of NECG might bepartially due to its ability to extenuate the oxidative stress and the inflammatory responses.Our results also strongly suggested that NECG exerted its antiapoptotic effect viaregulating the expressions of Bax and caspase-3 proteins. In addition, both of the middledose of NECG (0.02 g/kg) and the low dose (0.007 g/kg) have the protective effects,but there is no statistically significant, compared to the sham-operated control. The effectsof NECG are much better when it is administrated 7 days before the MCAO, compared toadministration after the MCAO.
5. The effects of NECG on neuronal injury and the mechanism in vitro
Our results indicated that NECG exerted its neuroprotection through muti-mechanisms.
5.1. The effect of NECG on PC12 cells apoptosis induced by H_2O_2
We established an apoptotic model induced by H_2O_2 in PC12 cells and assessed the effectof NECG on it. PC12 cells were pre-treated with NECG and then incubated with 200μmol/L H_2O_2. Neuronal apoptosis were examined by flow cytometry. The Western blottingresults showed that NECG (100μg/ml and 10μg/ml) could effectively attenuate neuronalapoptosis and antagonized the up-regulation of Bax and the down-regulation of Bcl-2,inhibited the release of cytochrome C and the activations of P53 and caspase-3, all of whichwere closely associated with the occurrence of apoptosis induced by H_2O_2. We concludedthat NECG could significantly attenuate neuronal apoptosis, which may result from itsability to alter the expression of apoptosis-related genes.
5.2. The effect of NECG on PC12 cells apoptosis induced by sodium azide
We firstly documented the connection between the generation of ROS and the activity ofcaspase-3 in sodium azide-induced apoptosis in PC12 cells. Secondly, we investigated themolecular mechanism of sodium azide-induced apoptosis, and the protective effects ofNECG against it. The results showed that there was an increase of the production of ROS insodium azide-induced apoptosis, which is associated with the activity of caspase-3, and theapoptosis was followed by the decrease of the ration of bcl-2/bax and the activation ofcaspase cascades. Concomitantly, our results indicated that NECG could attenuate PC12cells apoptosis induced by sodium azide, inhibit caspase-3 activity and extenuate thedecrease of the ratio of Bcl-2/Bax, which suggested that the effects of NECG on intrinsiccaspase-3 pathway might be a downstream event of regulation of the expression of Bcl-2family genes.
5.3. The protective effects of NECG on mitochondrial dysfunctions
In the present study, we investigated the capacity of NECG to protect brain mitochondriafrom oxidative damage in vitro. Oxidative damage of brain mitochondria was induced byFe~(2+)/Cys. Our results showed that NECG markedly decreased mitochondrial swelling,inhibited lipid peroxidation, prevented the decrease of GSH, protected the mitochondrialH~+-ATPase activity and maintained the mitochondrial membrane potential. It might be concluded that NECG could protect mitochondria from oxidative injury and have potentialsfor treating diseases mediated by ROS.
6. Conclusions
The results showed that NaoDeSheng has an effective protections against brainischemia, whichi is based on the combination of many effective components, and thesecomponents extert an additive effect to prevente and treat of brain ischemia by differentpathways and mutual influence between them. The studies on the mechanism of its actionconfirmed that NECG take its effects by different components and pathways, includinganti-oxidative stress, inhibiting neuronal apoptosis, attenuating inflammation responses,and so on. All of the data indicated that NECG could represent the overall effects ofNaoDeSheng rationally, and reflected the character of Traditional Chinese Medicine (TCM).Our results also indicated that effective components group-guided methodology is afeasible tool to improve the neuoroprotective properties of Traditional Chinese Medicineprescription NDS in rat focal cerebral ischemia.
一、适合高通量筛选样品的制备
本实验采用现代植物化学分离技术，制定规范的操作程序，将提取分离和层析等技术相结合，用不同溶剂对组方中原料药进行提取，将复方脑得生中的全部成分分为170个独立组分。其中，对乙醇部分进行硅胶柱层析，共计得到90个组分；对石油醚部分进行硅胶柱层析，共计得到80个组分。所有样品进行浓缩供下一步的筛选及研究用。
二、对样品进行高通量筛选
建立与脑得生功效主治相关的高通量筛选模型。根据脑得生的功效主治寻找与现代医学共同点，在细胞和分子水平上建立能够反映作用机理的筛选模型。缺血性脑损伤的发生主要与过氧化损伤、钙超载、兴奋毒性作用、炎症反应以及线粒体功能的受损等相关。因此，本研究中我们根据上述发病机制分别建立了相应的损伤模型，并对所得的170个组分进行了筛选。最后，将以上研究结果进行汇总，发现部分样品在不同模型上作用吻合，可能是脑得生发挥作用的活性物质，另外一些样品在多个模型作用强度较弱甚至相反，可能是影响脑得生疗效的部分。通过综合分析，将作用符合我们要求的组分按一定比例混合组成脑得生的有效成分组(2-11，16-21，25-29，31-35，38-41，47-52，59-73，95-99，116-119，122-127，131-136，167-170)，进行高效液相成分分析，找出适合大规模生产的有效成分组。
三、应用HPLC分析高通量筛选结果，实现生产工艺的转换
分别将硅胶柱分离的醇溶部分和石油醚部分中的抗脑缺血有效成分，按其编号从其DMSO溶液中取出，按照各自得率，有机混合在一起作为有效成分组供检测用。分别将大孔树脂分离的30％、60％、90％乙醇沈脱物部分与抗脑缺血有效成分组做对比，通过HPLC分析寻找适合大规模生产的目的组分。由液相色谱图可以看出，脑得生有效成分主要分布在30％和60％乙醇洗脱物中。因此，我们将30％和60％乙醇洗脱物按比例混合作为新的复方脑得生的有效成分组(NECG)，进行下一步的整体动物实验，对其疗效进行验证，并进一步进行机制研究。
四、利用动物实验进行有效成分组的验证
采用栓线法造成大鼠大脑中动脉阻断模型，通过预防性以及治疗性两种给药方式，从脑梗塞体积、行为学、脑组织能量代谢、病理学改变、炎症反应以及凋亡等多方面指标进行观察，全面评价了我们所确证的有效成分组的神经保护作用。
实验结果表明，治疗性给药时NECG大剂量组(0.07 g／kg)可显著降低脑梗死体积、改善神经功能，并且可抑制炎症反应，以及凋亡的发生和发展；NECG中剂量组(0.02 g／kg)和小剂量组(0.007 g／kg)也有改善作用，但某些指标与模型组相比，没有显著性差异(P＞0.05)。造模前先连续给予7天药物时，与模型组以及治疗给药的实验结果比，药物的保护作用则更加明显，尤其NECG中剂量组表现出了明显的保护作用。另外，我们同时与原方的疗效进行了对照研究，实验结果说明NECG的疗效明显优于原方。
五、体外对其作用机制进一步探讨
实验结果表明，NECG对神经细胞的保护作用机理不是单一的，其药效是多种机理综合作用的结果。
1、NECG能抑制过氧化氢诱导的PC12细胞凋亡。实验结果表明，NECG可显著改善过氧化氢引起的细胞存活率的降低；流式细胞仪分析表明100μg／ml和10μg／ml的NECG均能显著改善过氧化氢引起的凋亡；Western Blotting的分析结果显示，100μg／ml和10μg／ml NECG能够明显抑制过氧化氢诱导的Bcl-2蛋白表达的下调和Bax蛋白表达的上调，使Bcl-2／Bax比值上升，还能明显抑制细胞色素C从线粒体的释放以及p53的活化；同时，预先加入NECG还可明显抑制过氧化氢损伤引起的caspase-3的活化，使其活性降低。
2、NECG能够抑制叠氮钠诱导的PC12细胞凋亡。我们的实验结果首先证明了叠氮钠诱导的凋亡伴随有细胞内ROS时间依赖性的产生增多，并且与凋亡的线粒体途径改变密切相关。同时，我们的实验结果也提示，NECG可剂量依赖性的显著改善上述改变：NECG可改善细胞存活率和凋亡率，降低细胞内ROS的增多，100μg／ml和10μg／ml的NECG能够抑制叠氮钠诱导的Bcl-2表达的下调和Bax蛋白表达的上调，使Bcl-2和Bax比值上调，预先加入NECG能明显抑制叠氮钠损伤引起的细胞色素C自线粒体的释放以及caspase-3的活化，从而有效的降低了细胞凋亡的发生。
3、NECG有改善线粒体功能的作用。NECG体外能够抑制50μmoL／L FeSO_4／500μmoL／L半胱胺酸损伤体系诱导的线粒体MDA生成的增加，降低脂质过氧化的发生率，具有明显的保护线粒体免遭氧化损伤的作用；可减轻线粒体肿胀度，维持线粒体的膜电位，抑制H~+-ATP酶活性的降低，从而保护了线粒体的结构和功能；NECG还可抑制GSH含量的降低，保护了线粒体内源性抗氧化物质，从而使氧自由基的生成与清除保持平衡，使线粒体膜免遭损伤。
因此，NECG抗细胞凋亡的作用机制一方面和清除氧自由基有关，另一方面NECG可能通过直接影响p53的表达，细胞色素C的释放、caspase-3活性或者通过调控Bcl-2、Bax的蛋白表达间接影响线粒体依赖的caspase-3途径来抑制细胞凋亡。
六、结论
研究结果表明，中药复方脑得生对脑缺血有显著的防治作用，这种作用的物质基础是其中多种有效成分的有机组合，这些成分通过不同的作用途径和相互影响，产生综合作用，实现对脑缺血疾病的预防和治疗作用。药效成分组的作用机制研究证明中药有效成分组通过不同的成分从不同的途径发挥作用，如抗过氧化损伤、抑制细胞凋亡以及减轻炎症反应等。结果证明有效成分组可以比较合理的代表了中药的整体作用，反映了中药复方的作用特点。本实验结果也证明，应用高通量筛选方法研究中药复方具有一定的技术优势，也是行之有效的方法。
Ischemic stroke is a common disease, which affects the health of human seriously. Themechanism is complex, and could be regulated by many factors, including the activationsof many kinds of biologically active materials, and so on. At present, the details have notbeen well established, and the possible mechanisms include the dysfunctions of energymetabolism, oxidative stress, inflammation responses, glutamate excitotoxic damage,calcium overload, and the expressiones of some apoptosis-related proteins. In recent years,the main targets to treat brain ischemia are to protect the neurons, decrease the infarctionvolumes, and extenuate the delayed neuronal death. According to this context, weinvestigated the neuroprotective effect and the possible mechanism of the NECG ofNaoDeSheng (NDS) in present study.
1. The preparation of NDS samples for high throughput screening
Sequential components of NDS samples were prepared by the morden seperationtechniques. In brief, the powdered herbs were extracted with petroleum ether, 95％alcoholand aqueous in turn, and both of 95％alcohol extract fraction and petroleum ether extractfraction were further separated, respectively. The natural samples were collected on fixedinterval, and we obtained 170 sequential components in total, including 90 sequentialcomponents of 95％alcohol extract fraction and 80 sequential components of petroleumether extract fraction. All of these samples were concentrated for the further screening andresearching.
2. Bioactivities of NDS samples tested by the method of High-throughput screening
According to the mechanism of ischemic stroke, we established ischemia-related injurymodels, including chemical hypoxia model, mitochondfial injury model, inflammatorymodels and the models induced by hydrogen peroxide and glutamate, and evaluated theeffects of 170 sequential components (L1-L90, A91-A170) of NDS on them, respectively.Finally, we combined all of these screening results, and found some of samples alwaysexert effective roles even in different models, which were defined as the effective components group (ECG), some of samples have somewhat weak effect, and others evenhave an contrary effect. By comprehensive analysis of these results, we regarded thefollowing parts as the ECG of NDS (2-11,16-21, 25-29,31-35,38-41,47-52,59-73, 95-99,116-119, 122-127, 131-136, 167-170). In the following experiment, we further analysed theECG by high performance liquid chromatography (HPLC) to find the NDS's ECG (NECG),which is appropriate for producing Chinese herbal medicinal prescription in a commercialscale.
3. Analysis of the ECG of NDS samples by HPLC
Comparing the ECG with the extraction of 30％, 60％, and 90％EtOH fraction by HPLC,we found that the mixture of 30％and 60％EtOH fractions of NaoDeSheng could beregarded as the NECG of NaoDeSheng. Furthermore, we will confirm the pharmalogicaleffects in the following in vivo experiment and will researched its mechanism deeply invitro.
4. The protective effect of NECG on the MCAO in vivo
Focal brain ischemia was induced by the intraluminal suture MCA occlusion method(MCAO) with minor modifications, NECG was administrated prior to or after MCAO, andthe neuroprotections of NECG were evaluated comprehensively, including themeasurement of infarct volume, alterations in the neurological deficits, oxidative stress,inflammatory responses, and the occurrence of apoptosis. Our results indicated that the highdose of NECG (0.07 g/kg) administrated after the occlusion of MCAO could reduce theinfarct area, extenuate neurological deficient. This protective effects of NECG might bepartially due to its ability to extenuate the oxidative stress and the inflammatory responses.Our results also strongly suggested that NECG exerted its antiapoptotic effect viaregulating the expressions of Bax and caspase-3 proteins. In addition, both of the middledose of NECG (0.02 g/kg) and the low dose (0.007 g/kg) have the protective effects,but there is no statistically significant, compared to the sham-operated control. The effectsof NECG are much better when it is administrated 7 days before the MCAO, compared toadministration after the MCAO.
5. The effects of NECG on neuronal injury and the mechanism in vitro
Our results indicated that NECG exerted its neuroprotection through muti-mechanisms.
5.1. The effect of NECG on PC12 cells apoptosis induced by H_2O_2
We established an apoptotic model induced by H_2O_2 in PC12 cells and assessed the effectof NECG on it. PC12 cells were pre-treated with NECG and then incubated with 200μmol/L H_2O_2. Neuronal apoptosis were examined by flow cytometry. The Western blottingresults showed that NECG (100μg/ml and 10μg/ml) could effectively attenuate neuronalapoptosis and antagonized the up-regulation of Bax and the down-regulation of Bcl-2,inhibited the release of cytochrome C and the activations of P53 and caspase-3, all of whichwere closely associated with the occurrence of apoptosis induced by H_2O_2. We concludedthat NECG could significantly attenuate neuronal apoptosis, which may result from itsability to alter the expression of apoptosis-related genes.
5.2. The effect of NECG on PC12 cells apoptosis induced by sodium azide
We firstly documented the connection between the generation of ROS and the activity ofcaspase-3 in sodium azide-induced apoptosis in PC12 cells. Secondly, we investigated themolecular mechanism of sodium azide-induced apoptosis, and the protective effects ofNECG against it. The results showed that there was an increase of the production of ROS insodium azide-induced apoptosis, which is associated with the activity of caspase-3, and theapoptosis was followed by the decrease of the ration of bcl-2/bax and the activation ofcaspase cascades. Concomitantly, our results indicated that NECG could attenuate PC12cells apoptosis induced by sodium azide, inhibit caspase-3 activity and extenuate thedecrease of the ratio of Bcl-2/Bax, which suggested that the effects of NECG on intrinsiccaspase-3 pathway might be a downstream event of regulation of the expression of Bcl-2family genes.
5.3. The protective effects of NECG on mitochondrial dysfunctions
In the present study, we investigated the capacity of NECG to protect brain mitochondriafrom oxidative damage in vitro. Oxidative damage of brain mitochondria was induced byFe~(2+)/Cys. Our results showed that NECG markedly decreased mitochondrial swelling,inhibited lipid peroxidation, prevented the decrease of GSH, protected the mitochondrialH~+-ATPase activity and maintained the mitochondrial membrane potential. It might be concluded that NECG could protect mitochondria from oxidative injury and have potentialsfor treating diseases mediated by ROS.
6. Conclusions
The results showed that NaoDeSheng has an effective protections against brainischemia, whichi is based on the combination of many effective components, and thesecomponents extert an additive effect to prevente and treat of brain ischemia by differentpathways and mutual influence between them. The studies on the mechanism of its actionconfirmed that NECG take its effects by different components and pathways, includinganti-oxidative stress, inhibiting neuronal apoptosis, attenuating inflammation responses,and so on. All of the data indicated that NECG could represent the overall effects ofNaoDeSheng rationally, and reflected the character of Traditional Chinese Medicine (TCM).Our results also indicated that effective components group-guided methodology is afeasible tool to improve the neuoroprotective properties of Traditional Chinese Medicineprescription NDS in rat focal cerebral ischemia.
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