| Cerebrovascular disease (stroke) is a common disease which harms human health. It is the third leading cause of death in the world, with the characteristics of high morbidity, high mortality and high disability. In low and middle-income countries, the mortality of stroke is serious (accounting for85.5%of global deaths). The lose of Quality Adjusted Life Years (QALYs) due to disability of stroke is seven times more than the high-income countries, so it is a hot topic to find effective treatment and explain its mechanism in brain science research. Study confirmed that the ischemic core neuronal death was characterized by necrosis, the neuronal death in ischemic surrounding penumbra zone is related with apoptosis, and how to save the damaged neurons in the ischemic penumbra is a noteworthy topics, therefore, study of apoptosis induced signal transduction mechanism may provide new prospects for cerebral ischemia.JAK-STAT system is an important signal transduction pathway in the inflammatory response after cerebral ischemia, is also an important factor to determine the neuron apoptosis in the lesion. JAKs kinase as an important part of the JAK-STAT signal transduction system, is the signal transducer of many cytokines and growth factors. As well known that acupuncture has a unique effect on cerebral ischemic diseases, with the characteristics of multi-link, multi-level, multi-target, and the role of acupuncture on JAKs kinase mechanism in the brain after ischemia is not yet clear. Therefore, this article study on the acupuncture effect of the JAKs kinase and its downstream effectors STATs, and neuron apoptosis, so as to further reveal the mechanism of ischemic stroke treated by acupuncture and provide a solid foundation for the clinical treatment. The paper is divided into three parts:literature, experimental studies and summary. 1. Literature researchThere is no shortage of brilliant discussions about stroke in the ancient Chinese medical classics. Development of the etiology and pathogenesis of stroke have experienced the theories of exterior wind, interior wind and non-wind. It is summed up in the wind, fire, phlegm, qi, deficiency six aspects. The six aspects often influence each other and interact together. In order to discuss the rule of acupoint selection for treating stroke in ancient acupuncture classics, this paper retrieved the12literatures of stroke treated by acupuncture from the Jin Dynasty to the Qing Dynasty. Through the search terms of stroke, database creation, frequency analysis and the comparison of support degree, we summarized the total acupoints selection frequency of stroke, the acupoints selection frequency of hemiplegia, facial distortion and slurred speech of stroke and the relation of meridians and acupoints. In modern literature, this article reviewed the pathophysiology of ischemic stroke, acupuncture mechanism, JAKs kinases and JAK-STAT signaling transduction pathway. This article considered that cerebral ischemia can lead to the abnormal activation of the JAKs kinase, but the relationship between its abnormal activation and neuron surnival, apoptosis due to ischemia is not yet clear. Therefore, to further explore the JAKs kinase and the JAK-STAT signal transduction in the regulatory mechanism of ischemic brain injury and acupuncture intervention role would provide useful clues for the prevention and treatment of ischemic brain injury.2. Experiments researchObjective:The electroacupuncture effects on neuron pathology, apoptosis, tyrosine protein kinases JAKs and its downstream effectors STATs in ischemic cortex of rat model with focal cerebral ischemia in different time groups were observed, so as to reveal the possible mechanism of cerebral ischemic disease treated by acupuncture.Methods:300SPF male SD rats were divided into five groups according to the randomized block method:sham surgery group, model group, electroacupuncture (EA) group, AG490group and EA+AG490group, and each group was divided into post-ischemia2h,1d,3d,7d, and21d different periods subgroups. In the sham surgery group, the middle cerebral artery was exposed without occlusion; Model group was established the focal cerebral ischemia model by occluding the cerebral middle artery; EA group accepted acupuncture treatment after model establishment; AG490group was occluded the cerebral middle artery; The middle cerebral artery of the AG490group and EA+AG490group was occluded for20minutes following intracerebroventricular infusion, than EA+AG490group accepted acupuncture treatment. Observed indicators are as follows:(1) The neurological deficit scores, HE staining and observation of the pathological changes of cerebral cortex ischemic lesion surrounding area by transmission electron microscopy of rats in each group.(2) TUNEL apoptosis staining for observing the different time of neuronal apoptosis in the cerebral cortex ischemic lesions surrounding area of rats.(3) P-JAK1protein and mRNA expression in the cerebral cortex ischemic lesions surrounding area of rats were detected by immunofluorescence staining, Western Blot and in situ hybridization assay.(4) P-JAK2protein and mRNA expression in the cerebral cortex ischemic lesions surrounding area of rats were detected by immunofluorescence staining, Western Blot and in situ hybridization assay.(5) JAKs downstream effectors p-STAT3and p-STAT5expression were observed by immunofluorescence staining and laser confocal microscopy.(6) The correlation between the mean fluorescence intensity values of p-JAK1, p-JAK2, p-STAT3and p-STAT5in2h,1d,3d,7d,21d cerebral ischemia model group and neuron apoptosis were analyzed by two-tailed Pearson/Spearman correlation test.Results:(1) The ischemic rats in early stage displayed the symptoms of neurological deficit. The neurological deficit peaked at1d post-ischemia, and the score of neurological behavior had reduced with the ischemia time, the21d group had decreased most. Except for21d group, compared to the model group in the same time, the neurological deficit scores of the intervention groups had reduced (P<0.05). According to HE staining, electron microscopy, the development of pathological changes and neurological deficit trend was basically consistent. It indicated that the focal cerebral ischemia model by occluding the cerebral middle artery was successful, and the electroacupuncture treatment and AG490intervention can improve the nerve defect signs, promote the recovery of cortical neurons after cerebral ischemia.(2) The results of TUNEL apoptosis showed that there were some apoptotic neurons in ischemic cortex in2h post-ischemia and the expression had reached the peak in Id post-ischemia. After3d of cerebral ischemia, the positive apoptotic neurons were in still higher levels. With the extension of ischemia time, the number of apoptotic cells had gradually decreased, and the21d group reduced the most. Compared to the model group, the different time of apoptosis in EA group reached, especially in1d,3d,7d groups (P<0.01). The trend of apoptosis change of ischemic cortex in AG490group was consistent with the EA group.In EA+AG490group, both of the application can better inhibit the apoptosis in2h post-ischemia (P<0.05), but in1d,3d,7d groups, the difference among the EA+AG490, EA group and AG490group was not significant(P>0.05).(3) The JAK2phosphorylated protein and mRNA expression of rats with focal cerebral ischemia were detected by immunoflourescence staining, western blot and in situ hybridization. There was few reaction of p-JAK1in sham surgery groups, but it can be detected a considerable number of JAK1phosphorylation expression, and had reached the peak in1d post-ischemia. At7d model group, the p-JAK1expression had decreaed continuously, until the21d after ischemia, there was still a small amount of expression. The p-JAK1in Id,3d EA group was significantly higher than that in the same time of model group (P<0.01).(4) P-JAK2expression was minimal in the cortex of sham surgery group, it indicated that the JAK2in the brain tissued under normal physiological condition expressed the non-phophorylated form. There was few p-JAK2expression in ischemic cerebral cortex of rats in2h model group. There was the most expression in Id group, and the3d model group began to decline, but p-JAK2expression in21d group could be detected. Except for the2h,21d groups, the p-JAK2of EA group was less than the same time of the model group, especially in1d,3d group (P<0.01). The intervention to p-JAK2of AG490group was more obvious than EA group, this advantage was mainly reflected in the peak period of cerebral ischemia and neurological deficit (1d post-ischeia). The difference between Ald group and Zld group was significant (P<0.05). Electroacupuncture treatment with AG490can inhibit the JAK2phosphorylation levels (P<0.05). In addition, the E+Ald and E1d, E+A3d and E3d were statistically significant (P<0.05). It indicated that the both treatment can reduce the p-JAK2overexpression, and it was more advantageous than the simple electroacupunture intervention.(5) The p-STAT3, p-STAT5protein expressions in different time of group were observed by immunofluorescence staining. The results showed that:â‘ P-STAT3expression in Id group had increased and reached the peak,3d group began to decline, expression in21d was significantly decreased. The expression of p-STAT3immunoreactive cells and change of p-JAK2and apoptotic cells were consistent in the spatial and temporal distribution. Compared with the modelgroup, electroacupuncture can significantly reduce the p-STAT3expression in Id,3d post-ischemia.â‘¡The model group can be detected a large number of p-STAT5positive cells after2h of cerebral ischemia, it expressed the highest value at Id, the p-STAT5level of model group began to decline in3d post-ischemia. Until7d,21d ischemia, p-STAT5decreased significantly. In the peak period of cerebral ischemia (1d post-ischemia), electroacupuncture can up-regulate the phosphrylation of STAT5; in3d of cerebral ischemia, electroacupuncture can promote the p-STAT5(P<0.01). In7d,21d post-ischemia, the EA group compared with the model group, although the difference was not statistically significant, but the data had certain advantages.(6) The correlation analysis of JAK/STAT signal transduction pathway and neuronal apoptosis after cerebral ischemia:JAK1, JAK2, STAT3phosphorylation expressions were related to the neuronal apoptosis in ischemic peak period (1d,3d after cerebral ischemia), but they were not so obvious in the chronic phase of cerebral ischemia. In addition, p-JAKl protein expression and TUNEL apoptosis was a negative correlation, p-JAK2, p-STAT3protein expression and TUNEL apoptosis was positively correlated, and p-STAT5expression and TUNEL apoptosis without obvious correlation.Conclusion:(1) After cerebral ischemia, the development trend of neurological impairment, pathological changes and apoptosis were improved, it suggested that brain had the self-plasticity regulatory function after injury. Electroapuncutre can improve the damaged nerve dysfunction, and promote the repair of neurons, inhibit the neuronal apoptosis after cerebral ischemia.(2) The intervention of electroacupuncture treatment can significantly increase the JAK1activity in the cerebral ischemic cortex at the early stage of ischemia, and stimulate the body's self-protection mechanism, so as to reduce the cerebral ischemic injury and induce this protective effect to maintain a longer period of time and participate the repair of the organization.(3) The overexpression of JAK2and its downstream effectors STAT3phosphorylation and the abnormal activation of JAK2-STAT3pathway can aggravate the apoptosis. The mechanism of electroacupuncture treatment of ischemic cerebrovascular disease maybe via the activation the response of the somatic nervous system by the acupuncture signal and start the signal transduction pathway. In other words, electroacupuncture can inhibit the abnormal activation of JAK2-STAT3, partially block or reduce intracellular signal transduction of inflammatory cytokines in order to reduce the occurrence of apoptosis.(4) The intervention of AG490can significantly inhibit the expression of JAK2phosphorylation and neuronal apoptosis, and it was more obvious synergies with electroacupuncture. It demonstrated that the repair of AG490to neurons in the ischemic cerebral cortex by acting on the JAK/STAT signal transduction system.(5) EA could enhance the p-STAT5expression in ischemic cortex, the mechanism may be through the activation of certain cytokine pathway which induced by STAT5, and improve the injury of cerebral ischemia, and improve the ability to repair damaged neurons.(6) According to the experimental results, we believed that acupuncture starts the JAK/STAT signal transduction pathway to promote the self-regulation of steady nerve state, it maybe the the important mechanism for the ischemic brain injury treated by acupuncture. |
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