The Effect Of Tetramethylpyrazine On Ischemic Stroke In Rats And The Study Of Its Mechanism

Stroke is a common refractory disease and a serious hazard to human health and safety. It is even considered as one of the four incurable diseases including "stroke, consumption, bloating, dysphagia" in Chinese medicine. Due to its characteristics of high incidence, high morbidity and high mortality, it has become a priority for this disease for reducing the incidence of stroke morbidity/mortality, and improving prevention and treatment of stroke. Because85percent of all stroke cases are due to ischemic event, it is becoming more important to explore pathogenesis, mechanisms and counter measures of ischemic stroke in theoretical value and clinical practice.The work of this paper includes two parts, review and experimental research.1. Ischemic stroke and the research progress in the treatment of ischemic stroke with Chinese medicineThis study systemically reviewed the development of the pathogenesis of ischemic stroke, the development of the signal transduction pathways involved in ischemic cerebral stroke from ischemic injury and the research progress in the treatment of ischemic stroke with Chinese medicine. It systemically summarized the obstacle of energy metabolism during ischemic stroke, peri-infarct depolarization, oxidative stress, inflammation and mechanism of apoptosis. It focused on the research progress of the inflammation and oxidative stress-mediated signaling transduction pathways in the pathogenesis of ischemic stroke, the clinical and basic research of tetramethylpyrazine (TMP) progress in ischemic stroke. Clarifying all of these progresses in the research was to lay a solid theoretical foundation for this study and provided preconditions for the experimental design.2Experimental research2.1Research methodsExperimental study using histopathological method, immunohistochemical method, modern molecular biological techniques.2.2Technical manipulationsFlow cytometry, Real-time RT-PCR for gene detection, quantitative immunoblotting, immunofluorescence method and chemical colorimetry technique.2.3Detection index2.3.1Brain injury index:the area of cerebral infarction, blood-brain barrier permeability, cerebral water content. 2.3.2Oxidative stress related factors:MDA, NO, CAT, GSH-Px.2.3.3Immune index2.3.3.1Immune cells, neutrophils and T lymphocytes, macrophage/microglial resting and activated ratio.2.3.3.2Inflammatory mediators:MPO, TNF-α and IL-1β.2.3.4Signal transduction molecule:the immediate early genes c-fos and c-jun, p-c-jun, Fos protein, Jun protein, JNK, AP-1, HO-1, Nrf2.2.4The research results2.4.1Experiment1Effect of Tetramethylpyrazine on experimental neuron-tissue injury and function of ischemic stroke in rats2.4.1.1A reliable Ischemicstroke rat model from experimental preparation:Clear and definite brain tissue injury in experimental rats (cerebral infarction, blood-brain barrier damage and brain edema), and concomitant neurological behavioral changes;2.4.1.2Tetramethylpyrazine has clear and definite pharmacological intervention on ischemic stroke rats:To express the reduction of cerebral infarction area, reduce brain edema and blood-brain barrier damage, improve the abnormal neurological behavior of rat. Suggestion:Tetramethylpyrazine has a neuroprotective effect.2.4.2Experiment2Effect of Tetramethylpyrazine on oxidative stress reaction of rats with ischemic stroke2.4.2.1The peroxidation of permanent cerebral ischemia of ischemic stroke in the rat with damaged cortical tissueThe increase of MDA content, increased content of NO in neutrophils, CAT, GSH-Px activity had increased trend;2.4.2.2Tetramethylpyrazine can reduce peroxidation of damaged cortical tissue of rat with cerebral ischemic stroke:Tetramethylpyrazine may reduce the content of MDA and content of NO in neutrophils.Tetramethylpyrazine may increase the activity of antioxidant CAT, GSH-Px.Suggestion:intervention mechanism of Tetramethylpyrazine on ischemic stroke, inhibiting oxidative stress injury may be one of its mechanisms.2.4.3Experimental3Study of on inflammatory reaction of ischemic stroke2.4.3.1Ischemic stroke of rats produced obvious inflammation:2.4.3.1.1On injury cortex tissue, the number of CD45positive immune cells increased and the number of macrophages/microglia activated increased;2.4.3.1.2On injury cortex tissue, the expression increased of MPO activity and CD68surface antigen protein; 2.4.3.2Tetramethylpyrazine interventing in inflammation of ischemic stroke in rats:To down-regulation of immune cells number and activated macrophages/microglia number.To down-regulation of MPO activity and CD68surface antigen protein expression of impaired cortical tissue caused by cerebral ischemia.Suggestion:intervention mechanism of Tetramethylpyrazine in ischemic stroke, inhibit inflammatory reaction may be one of the important mechanisms.2.4.4Experimental4Experimental study of Tetramethylpyrazine on signal transduction pathway induced and activated by ischemia in rats with ischemic stroke.2.4.4.1Confirmed one of the inflammatory signal transduction pathway initi ated by ischemic brain tissue in rats with ischemic stroke is JNK/AP-1pathway.The anti-inflammatory mechanism of Tetramethylpyrazine intervention of ischemic stroke in rats:the inhibition of AP-1DNA binding activity, restricted or decreased transcription protein c-Fos protein, c-Jun protein and JNK protein phosphorylation.Suggestion:Tetramethylpyrazine block or weaken the JNK/AP-1inflamm ation signal transduction pathway, so as to achieve the effect of anti-inflammati on and protect the brain tissue.2.4.4.2Confirmed one of the oxidative stress signal transduction pathway initiated by ischemic brain tissue in rats with ischemic stroke is Nrf2/ARE pathway.Effect and mechanism of antioxidant stress of Tetramethylpyrazine invent ischemia rats with ischemic stroke:further up regulation of immunofluor escence cells number of HO-1and Nrf2, up-regulating expression of Nrf2and HO-1protein.Suggestion:Tetramethylpyrazine may activate Nrf2/ARE pathway, the phase Ⅱ enzymes of HO-1release, antioxidation and clear the free radical, in order to reduce the injury of brain tissue.3. Innovation of the research3.1It is the first time to study the mechanisms of signal transduction pathway of TMP intervening in ischemic stroke.It confirmed that the pathways of TMP intervening in ischemic stroke are JNK/AP-1and Nrf2/ARE pathway in inflammation and oxidative stress-mediated signal transduction.3.2This study observed the effects of TMP focus on MDA, NO, Catalase and GPx role of damaged cortical tissue of ischemic stroke in rats with injury of oxidative stress as a target. It determined that TMP can reduce peroxidation caused by permanent brain ischemia.3.3This study focused on the effects of TMP on damaged cortical tissue immune cells of permanent ischemic stroke in rat (neutrophils, T lymphocytes, macrophages/microglia) and inflammatory cytokines (TNF-α, IL-1β, MPO, etc.) with inflammatory response of ischemic stroke as a target.It confirmed that TMP anti-inflammatory effect is by down-regulating immune cells and inflammatory cytokines way.