| Ischemic stroke is one of the life-threatening diseases featured by high morbidity and mortality. Treatment in the acute stage of stroke is important for the prognosis, which is already become the hotspot in international study. Study has showed that, excitotoxicity, oxidative stress, Ca2+ overload, inflammation and apoptosis are serially triggered by ischemic stroke, and then become pathological basis of neuron injury. In the cascade, excitotoxicity is the pivotal link. After stroke, energy deprivation and membrane depolarization accelerate excitatory amino acid release, represented by glutamate, which over-activate NMDA receptor and lead to Ca2+ overload. Function disorder of mitochondria and endoplasmic reticulum are induced by oxidative stress, therefore, apoptosis is initiated and finally death of neurons. In this process, oxidative stress is very important, since NO and O2- not only directly attack neurons, but also activate other injury mechanism. Recent research show that, protein interaction within NR2B-PSD95-nNOS is the main molecular mechanism of NO production, linked with activation of nNOS mediated by excitotoxicity. To interfere the protein interaction within NR2B-PSD95-nNOS is a new target in treatment of ischemic stroke. Brennan has report his research on Nature Neuroscience, he found that NADPH oxidase is central mechanism in O2- production mediated by NMDA receptor. O2- may activate production of superoxide in mitochondria.Tradition Chinese Medicine (TCM) plays an important role in treatment of ischemic stroke during the acute stage. TCM practitioner hold that poison is one of the pathogenic factors in ischemic stroke, so clearing heat and detoxicating method is one of the effective treatment on cerebral ischemic stroke. Author collected 39 randomized controlled trial (RCT) of treatment cases and reviewed by meta-analysis, result demonstrated that the effectiveness rate of treatment based on clearing heat and detoxicating method show significant difference compared with control group. Therefore, this dissertation choose the typical Chinese medicine QingKaiLing(QKL), which represents clearing heat and detoxicating medicine, for further research. However, QKL confronts high rate of adverse reaction and restrained clinical use. Based on such condition, our team put forward a secondary development peinciple of famous Chinese medicine formula "selective substitution, partial optimization, quality stabilization and efficacy improvement", refine QKL into JZQKL. Follow these guidelines, this paper optimized the prescription of JZQKLAbove these basis, we considered that:first, the mechanism after ischemic stroke is very complicated, so multi-targeted treatment maybe more effective than single targeted-treatment; second, through macro- and micro-analysis, excitotoxicity and poison in TCM might linked significantly. So clearing heat and detoxicating medicine JZQKL, could it be effective on multi-targeted treatment on excitotoxicity?Guided by system biology, Our team evaluated efficacy of JZQKL on model animal and its effect on multi-targets, focused on excitotoxicity induced overall and dynamic changes, to explain molecular mechanism of its anti-ischemia and anti-excitotoxicity effects.This research mainly centered 3 subtopics:1, Pharmacodynamic study of JZQKL on cerebral ischemia. This part includes comparing neurological deficit rating methods; screening and optimizing JZQKL prescription; evaluating pharmacodynamic effect of JZQKL and distinguishing its treatment time window on cerebral ischemia.2, JZQKL interfere with cascade induced by excitotoxicity. This part includes observe effect of JZQKL on Glu abnormal release, Ca2+ overload, oxidative stress, mitochondria energy failure, endoplasmic reticulum stress and apoptosis.3, Molecular mechanism of JZQKL on excitotoxicity. This part analyze two pathways of oxidative stress, respectively NR2B-PSD95-nNOS and NADPH oxidase activation.1. JZQKL injection is an effective anti-ischemia medicine with broad time window.We studied several animal models to evaluate anti-ischemia effect of JZQKL, endpoint criteria are focused on infarction lesion and neurological function. Firstly, we compared Bederson, Julio H and Clark neurological function rating system, found latter two have higher correlated with cerebral infarction volume, so Julio H and Clark rating system are used to evaluate neurological injury degree. Secondly, we employed pMCAO and tMCAO rats models to study pharmacological effect of JZQKL (6,3,1.5ml/Kg), under 3ml/Kg QKL as control. JZQKL showed significant therapeutic effect, and the pharmacological effect is dosage-dependent; as well, JZQKL is effective for BCCAO mice. We explored time window for JZQKL on animals 1.5,3,6,9h after cerebral ischemia, administration within 6h after ischemia is significantly effective to protect brain from infarction and neurological deficit. Broad time window is an important adventage to a clinically widely used medicine.2. JZQKL regulates cascade reactions induced by excitotoxicity after cerebral ischemiaAimed at suppression of excitotoxicity, we found JZQKL exert multi-targeted effect on the whole process. JZQKL not only inhibited Glu over-release in MCAO rats'brain,but also inhibited Ca2+ concentration in neurons 2h after cerebral ischemia significantly. ROS and MDA concentration in mice brain were significantly lowered by JZQKL 24h after ischemia, and SOD activity was increased,. Caspase12 activation and P-elF2a expression in mice brain homogenate were inhibited, as well, positive neuronal cell number were decreased by JZQKL. All there above reslults implied JZQKL is effective to inhibit Glu over-release and Ca2+ overload, it possess anti-oxidative and anti-apoptosis effect, and also effective on endoplasmic reticulum stress.3. One of pharmacological target of JZQKL on molecular level is to intervene protein reaction between PSD95-nNOS to downregulate nNOS and NADPH oxidase activation.Recent research demonstrate that protein reaction within NR2B-PSD95-nNOS is key molecular mechanism of NO production mediated by excitotoxicity induced nNOS activation. The reaction between PSD95-nNOS was studied by co-immunoprecitation. nNOS:PSD95 ratio in ischemic forehead cortex was significantly increased after 1.5h ischemia and 0.5h reperfusion; JZQKL inhibited nNOS:PSD95 ratio increase, in another words, JZQKL inhibited PSD95-nNOS protein reaction after ischemia. We also studied NADPH oxidase acitivity by observing its activated protein NOX2 expression, JZQKL suppressed NADPH oxidase over-activity and downregulated NOX2 expression both in hippocampus and cortex in ischemic mice brain after 1.5h ischemia and 22.5h reperfusion. In a word, JZQKL might inhibit oxidative stress induced by excitotoxicity through intervening PSD95-nNOS and NADPH oxidase.This study also observed that nNOS phosphorylation is upregulated by JZQKL. nNOS phosphorylation is alternative mechanism for organism to inhibit over-activation of nNOS. JZQKL increased nNOS phosphorylation might be another pharmacological target to inhibit nNOS activation, which need deeper research to explain.All above is a multi-level, and multi-perspective study on clearing heat and detoxicating prescription JZQKL. Data showed JZQKL possess remarkable anti-ischemia effect with a broad time window. All the pharmacological efficacy are related to its comprehensive effect on excitotoxicity cascade mechanism, especially on NADPH oxidase and reaction between PSD95-nNOS.Data supported our early hypothesis powerfully, clearing heat and detoxicating prescription JZQKL indeed exert its multi-targeted effect on excitotoxicity cascade process, and finally anti-cerebral ischemia. Study on TCM prescription should analyze complicated condition under TCM principle on one side, differentiate modern diseased with their phathophysiological characters on the other side. Meanwhile, tracing the newest research development, and integrate two sides under system biological strategy to put forward new scientific hypothesis. Then experimental methods are used to testify the hypothesis, this might suggest a new way for future Chinese medicine modernization process.In brief, this study on JZQKL was guided by system biological strategy. Character of Traditional Chinese medicine is combined with modern pharmacological development to explore comprehensive effect of inhibition on excitotoxicity cascade injury of JZQKL, two possible target of JZQKL were elaborated on molecular level, which will be a solid research basis for further development on anti-ischemia significance of JZQKL. |
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