| 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 screeningSequential 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 screeningAccording 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 HPLCComparing 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 vivoFocal 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 H2O2We established an apoptotic model induced by H2O2 in PC12 cells and assessed the effectof NECG on it. PC12 cells were pre-treated with NECG and then incubated with 200μmol/L H2O2. 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 H2O2. 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 azideWe 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 dysfunctionsIn the present study, we investigated the capacity of NECG to protect brain mitochondriafrom oxidative damage in vitro. Oxidative damage of brain mitochondria was induced byFe2+/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. ConclusionsThe 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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