| Background:Cerebrovascular disease is one of the major diseases that presents a serious hazard to human health.Died of cerebral-vascular disease in our country is more than heart disease and cancer,which ranks first in the three leading causes of death.Ischemic cerebrovascular disease is a threat to common and frequently-occurring diseases of people.To find effective treatments and to explain its pathogenesis are still the hot topics in the field of brain science. C-Jun N-terminal kinase(JNK) also calls stress activated protein kinase(SAPK).It is an important member of the MAPK family and one of the main signal transduction pathways in the central nervous system,which belongs to conservative serine/threonine protein kinase on the evolution.JNK signaling pathway can be activated by many factors,such as cytokines, growth factors, stress and so on.It involves in cell proliferation and differentiation, shape maintenance,cytoskeleton building, apoptosis and other biological processes. JNK plays an important role in cellular stress responses,and it can be activated by a variety of extracellular stress signals.Thus JNK also calls stress-activated protein kinases.JNK is a cytoplasmic serine/threonine protein kinase and it contains Dual-phosphorylationdomainThr-Pro-Tyr.It can be combined with c-Jun amino-terminal activation domain and phosphorylate the63rd and73rd serine residues.That’s why we call it JNK.People have found10homeotic proteins made up by different splice forms of JNK1,JNK2and JNK3. There is comprehensive expression on JNK1and JNK2. But JNK3is only expressed in brain, heart and testes.Purpose:This experiment is to explore the impacts of MAPK family signaling pathway in the brain regulation and acupuncture intervention after cerebral ischemia/reperfusion by the scientific methods,and learn more about electro-acupuncture therapy regulatory mechanism for cerebral ischemia/reperfusion brain signaling system of MAPK family beaconage."Acupuncture stimulation-a composite signal regulation-Virtuous cultivation mechanism is one of the key mechanisms to start the mechanisms of ischemia/reperfusion therapy",the scientific hypothesis makes a certain reference value to the study of electro-acupuncture therapy for cerebral ischemia/reperfusion intervention.Methods: 36JNK gene knockout mice were randomly divided into3groups:sham operated group, model group and acusector group.Each group was divided into2h (hour), Id (day),3d(day) three periods.So there are9groups totally,and each group contains4mice. Simulating the syndromes of clinical stroke and choosing the middle cerebral artery oeelusion(MCAO)of knockout mice as models.Treating focal cerebral ischemia by clinical electro-acupuncture combined acupuncture and electro-stimulation is on the efficacy of cerebral ischemia and its sequela. To catch the points about Function and mechanism of MAPK pathway in brain ischemia,the effects of electro-acupuncture on MAPK pathway and protection of electro-acupuncture on hippocampal neurons after cerebral ischemia must be observed from multi-link, multi-level and multi-targeting.And the changes of ischemic brain neurons astrocytes, microgliacyte and vascular basement membrane’s modality and quantitycan be compared by immunohistochemistry. To evaluate the results by neurological impairment scores test on knockout mice modeling and EA intervention.The EA is promoting to the discussion about the protection of cerebral ischemic hippocampal neurons and MAPK pathway, mechanism of action and mechanism of possible in-depth.It provide more precise theoretical basis for further clarifing the neurobiological mechanisms of brain plasticity, as well as for the clinical improvements of the brain function recovery after cerebral ischemia.Results:1. The effects of electro acupuncture working on the cerebral ischemia reperfusion (I/R) JNK gene knocking-out mice neural function defect scale.After molded in each period Groups of mice’s behavior was to be observed and to be scored. All the data was analyzed by spss13.0statistics and the data was normally distributed. Use the LSD test to analyze all the variance. The gene knocking-out mice neural behavior all was normal in control group and each score was0point. Model2h and electro acupuncture2h compared with control group, there were significant difference (P<0.01) and statistically significance, suggesting that knocking-out mice model of cerebral ischemia reperfusion was successful. In the comparison of1d group and3d group in the two groups, the nerve-behavioral score was not only statistically significant, but also was significantly different (P<0.01), suggesting that the electro acupuncture had a therapeutic effect on cerebral ischemia reperfusion injury. Early intervention would work on it, and the expanded effect was more obvious by the therapeutic time. 2. The effects of electro acupuncture working on the cerebral ischemia reperfusion (I/R) JNK gene knocking-out mice Brain tissue morphology (TTC staining)Observe and analyze the images by shooting of the cerebral ischemia reperfusion tissue after TTC staining, and observe them by using the corresponding image signal acquisition and analysis system. After modeled the gene knocking-out mice in Model2h group and electro acupuncture group compared with control group, their brain tissue morphology turned up much change and massive infarction in the early reperfusion. The largest infarction area was in the model group, while the electro acupuncture group’s infarction areas were reduced after1d and3d. The results showed as follow, first, the knocking-out mice model of cerebral ischemia reperfusion of modeled was successful. Second, the injury of brain tissue had great relation with how much the time of cerebral ischemia-reperfusion took. Third, applying electroacuputure therapy was helpful to the protection of cerebral ischemia reperfusion, and how much time the therapy took is related to the protection.3. The effects of electro acupuncture working on the cerebral ischemia reperfusion (I/R) JNK gene knocking-out mice Brain tissue micro-morphologyAfter modeled each group compared to control group, the observation of the micro-morphology group was more intuitive. The most obvious injury of the brain tissue in the slice occurred in day3in each time. And the cellar’s structure and arrangement could be seen disturbed in different degree. Results from each group showed that cerebral cortex’s each layer of cells were roughly normal. The cell arrangement orderliness, the structure was complete. The cerebral cortex’s cells’structure was quite complete and well-organized but a few neurocyte was shrinkage necrosis in the electro acupuncture group, while a lot of neurocyte were shrinking and were in degeneration necrosis contraction.4. The effects of electro acupuncture working on the cerebral ischemia reperfusion (I/R) JNK gene knocking-out mice of neurocyte’s apoptotic index.After the knocking-kout mice had been modeled, seen from the pathological section, model2h group and electro acupuncture2h group compared with control group h2, a large number of nerve cells were apoptotic. Model2h and electro acupuncture2h compared with control group2h, the apoptotic index obviously got high and there was significant difference (P<0.01). Electro acupuncture2h compared with model2h, the index got low and the difference had statistically significance (P<0.05). After ischemia reperfusion1day, the peak of cells apoptosis time appeared. The day3of ischemia reperfusion, cells apoptosis reduced apparently.3d group and Id group in two group had significant difference (P<0.01). The results showed that the model group and the electro acupuncture group compared with control group, the difference was statistically significant (P<0.01), suggesting that after cerebral ischemia reperfusion caused knocking-out mice brain nerve cells apoptosis. The cells apoptosis peak appeared after ischemia reperfusion1d in the electro acupuncture group and model group, which showed that occurrence of apoptosis had certain time window, to a certain extent from the cells were apoptotic and to begin to decline. The electro acupuncture group and control group of each time period had differences, and had statistical differences (P<0.01). it showed that the intervention of electro acupuncture could protect the neurocyte’s apoptotic index effetely after the cerebral ischemia reperfusion. The comparison between each time period among the electro acupuncture group, all had difference and statistical significance (P<0.01), which showed that took intervention earlier, the more nerve cells after cerebral ischemia got protection.5. The effects of electro acupuncture working on the cerebral ischemia reperfusion (I/R) JNK gene knocking-out mice of P-ERK protein expressionThe distribution of P-ERK in brain tissue was tested by immunohistochemical staining. After the mice were modeled, that the p-ERK expression in the brain ischemia lateral cortex and hippocampus CA1area was in certain degree inhibited of positive cells was not much. Compared to the model group, the positive cells of the electro acupuncture group in each time increased markedly (P<0.05). The timing was obvious in1d and3d(p<0.01).The results showed that after modeled, in the mice’s the brain ischemia lateral cortex erea the p-ERK expression was inhabited, compared to control group. The difference was statistically significant (P<0.01). The comparison between electro acupuncture group and control group had statistical differences (P<0.05), which showed that p-ERK’s activity was improved after the electro acupuncture therapy so that it could be contributed to the survival and repair of the brain cells which caused by hydroxy-ischemia.6. The effects of electro acupuncture working on the cerebral ischemia reperfusion (I/R) JNK gene knocking-out mice’s P38MAPKprotein expressionThe distribution of P38MAPK in brain tissue was testes by immunohistochemical staining.After the mice were modeled, that the P38MAPK in the brain ischemia lateral cortex and hippocampus CA1area was increased. Compared to the model group, the positive cells of the electro acupuncture group in each time was reduced markedly (P<0.05). The timing was obvious in1d and3d(p<0.01). The most obvious timing was3d(p<0.01). The results showed that compared to control group, the model group’s difference was statistically significant (P<0.01), which suggesting that being modeled caused to the p38expression of the gene knocking-out mice got up-regulated. Both electro acupuncture group and model group had difference and statistical significance (P<0.05), which showed that the path of p38was inhabited after the cerebral ischemia by early electro acupuncture therapy. The Id group and3d group had marked difference and statistical significance (P<0.05), which suggested that there was bound time-window in cerebral ischemia-reperfusion injury. Therefore, the early electro acupuncture therapy could protect the nerve cells from the injury caused by cerebral ischemia.Conclusions:1. Electro acupuncture of the points ZHIZE ZUSANLI SANYINJIAO could save apparently the nerve function injury and the brain tissue of the JNK gene knocking-out mice’s cerebral ischemia-reperfusion injury and could decrease the cells apoptosis Index. It suggested that Electro acupuncture of the points ZHIZE ZUSANLI SANYINJIAO had certain protection to the ischemia injured brain tissue.2. Electro acupuncture of the points ZHIZE ZUSANLI SANYINJIAO could contribute to up-regulation of the P-E protein expression,which suggested that ERK signal transduction’s activation is the important Characteristic of Electro acupuncture of the points ZHIZE ZUSANLI SANYINJIAO contributing to the repair of ischemic injured brain tissue.3. Electro acupuncture of the points ZHIZE ZUSANLI SANYINJIAO could contribute to down-regulation of the P38MAPK protein expression, which suggested that P38MAPK signal transduction’s Inhibition is the important Characteristic of Electro acupuncture of the points ZHIZE ZUSANLI SANYINJIAO contributing to the repair of ischemic injured brain tissue.4. The effect of electro acupuncture of the points ZHIZE ZUSANLI SANYINJIAO to the nerve function, the brain tissue, the cells apoptosis index and the signal path regulation after the cerebral ischemia-reperfusion was obvious in day3after the cerebral ischemia-reperfusion. It showed that this time is the best treatment time-window to promote the the self-repair.5.The experimental result suggested that the acupuncture-stimulation-activating the MAPK signal regulation-antagonize cell apoptosis was one of the key mechanisms of acupuncture treating the cerebral ischemia-reperfusion.The innovative viewpoint of this topic: 1. It was the first time that applying JNK gene knocking-out mice to the research of cerebral ischemia-reperfusion.2. In the model of cerebral ischemia JNK gene knocking-out mice,the effect of acupuncture intervention to MAPK signal path’s regulation was first systematically observed which provided new theory to clinical treatment for cerebral ischemia disease. |
PDF Full Text Request