补阳还五汤对大鼠脑缺血再灌注Connexin43表达的影响及机制研究
发布时间：2018-07-23 12:24
【摘要】：目的:缝隙连接蛋白43(connexin43,Cx43)是大脑星形胶质细胞之间进行物质交换、信息传递的重要通道,脑缺血之后,大脑抗缺血损伤以及修复的作用和缝隙连接蛋白43表达的改变有关。本文主要研究大鼠脑缺血再灌注后补阳还五汤对大脑抗损伤以及修复作用。实验检测大鼠大脑海马体Cx43表达,观察补阳还五汤(Buyang Huanwu Decoction,BYWD)是否能通过影响Cx43的表达对缺血后大脑起保护作用。本实验对阐释星形胶质细胞在中医药治疗脑缺血的作用及其机制具有一定的创新意义。同时,本文旨在揭示补阳还五汤治疗脑缺血的独特优势(根据需要针对性地调节),朝着用现代科学技术对中医理论加以阐述和证明的目标前进了一小步,对全世界能更好地理解、接受和应用中医中药具有积极的意义。方法:运用线栓法建立大鼠大脑中动脉栓塞(middle cerebral artery occlusion,MCA0)模型。1.补阳还五汤对脑缺血再灌注后大鼠海马Cx43影响检测。大鼠造模、给药1天、7天后取材,使用免疫组织化学方法以及western blot(WB)法检测,检测各组Cx43表达的改变。2.补阳还五汤对脑缺血后大鼠海马Cx43影响的机制探讨。大鼠造模、给药1天后取材。使用ELISA法检测细胞因子(白介素-1beta,IL-1 β和白介素-10,IL-10)的表达。补阳还五汤联合使用NF-κB激动剂PMA增强炎症反应后,比较各组海马CA1区Cx43的表达同时使用HE染色观察细胞形态学改变;大鼠造模7天后取材。免疫组织化学染色法观察各组海马CA1区嗜碱性成纤维细胞生长因子(Basic Fibroblast growth factor,bFGF)的表达情况。补阳还五汤联合使用bFGF中和抗体后,比较各组海马CA1区Cx43的表达同时HE染色观察细胞形态学改变。3.干预connexin43表达后补阳还五汤抗脑缺血作用实验。大鼠造模、给药1天后取材。实验检测海马丙二醛(Malondialdehyde,MDA)、超氧化物歧化酶(Superoxide Dismutase,SOD)、一氧化氮(Nitrogen Monoxide,NO)。补阳还五汤配合 Cx43 激动剂治疗后,实验观察Cx43表达被干预后是否影响补阳还五汤对缺血再灌注后大脑的保护作用。结果:1.大鼠脑缺血1天,与假手术组比较,模型组Cx43的表达明显上升,与模型组比较,BYHWD组Cx43的表达减少(P0.05);大鼠脑缺血7天,与假手术比较,模型组Cx43的表达上升,与模型组比较,BYHWD组Cx43表达增加(P0.05)。2.大鼠脑缺血1天,同模型组相比,补阳还五汤组IL-1β减少、IL-10增加(P0.05);补阳还五汤联合NF-κB激动剂给药1天后,补阳还五汤对海马CA1区Cx43的下调作用被抑制(P0.05),HE染色显示联合组较补阳还五汤组神经元损伤加重。脑缺血7天时补阳还五汤增强bFGF和Cx43的表达;补阳还五汤联合bFGF中和抗体给药7天后,海马CA1区Cx43的表达较补阳还五汤组降低(P0.05)。HE染色显示同补阳还五汤组比,神经元损伤加重。3.脑缺血1天后,与假手术组比较,模型组MDA、NO上升,SOD下降(P0.05);与假手术组比较,BYHWD组MDA、NO下调,SOD上升,BYHWD联合Cx43激动剂GAP-134后,BYHWD对脑缺血后NO、SOD影响减弱。结论:1.补阳还五汤在大脑缺血早期对海马星形胶质细胞Cx43表达起抑制作用,在大脑缺血晚期则增加Cx43的表达。2.在缺血早期,补阳还五汤通过炎症因子I1-1 β以及Il-10介导调节大脑海马体Cx43的表达;在缺血晚期,补阳还五汤通过神经营养因子bFGF介导调节大脑海马体Cx43的表达。3.在缺血早期,补阳还五汤通过调节Cx43表达改变大脑缺血后SOD、NO的含量,对大脑起保护作用。
[Abstract]:Objective: gap connexin 43 (connexin43, Cx43) is an important channel for material exchange and information transfer between astrocytes in the brain. After cerebral ischemia, the role of cerebral ischemia injury and repair is related to the change of the expression of gap junction protein 43. Damage and repair effect. Test the expression of Cx43 in the rat's hippocampus, and observe whether the Buyang Huanwu Decoction (BYWD) can protect the brain after the expression of Cx43. This experiment has some innovative significance to explain the role and mechanism of astrocytes in the treatment of cerebral ischemia in Chinese medicine. At the same time, the purpose of this paper is to reveal the unique advantage of Buyang Huanhui Five Decoction in the treatment of cerebral ischemia (according to the need to adjust), and forward a small step towards the goal of explaining and proving the theory of traditional Chinese medicine with modern science and technology. It has a positive significance for the whole world to understand better, accept and apply traditional Chinese medicine. The effect of middle cerebral artery occlusion (MCA0) model.1. Buyang Huanyang Five Decoction on hippocampal Cx43 in rats after cerebral ischemia reperfusion was established. Rats were built for 1 days and 7 days after administration. Immunohistochemistry and Western blot (WB) method were used to detect the Cx43 expression of.2. tonifying yang and five soup. The mechanism of the effect of Cx43 on hippocampal Cx43 after cerebral ischemia. Rat model was made, and the material was obtained after 1 days. ELISA was used to detect the expression of cytokines (interleukin -1beta, IL-1 beta and interleukin -10, IL-10). After the combined use of NF- kappa B agonist PMA to improve the inflammatory reaction, the expression of HE staining was compared with Cx43 expression in the CA1 region of hippocampus of each group. Observe the morphological changes of the cells. The rat model was made after 7 days. The expression of Basic Fibroblast growth factor (bFGF) in the hippocampal CA1 region was observed by immunohistochemical staining. The expression of Cx43 in the hippocampus CA1 area was observed by HE staining compared with the combined use of bFGF neutralization antibody and the combined use of bFGF neutralization antibody. Cell morphological changes.3. intervention connexin43 expression after the anti cerebral ischemia experiment of Buyang Huanwu Five Decoction. Rats were built for 1 days after administration. Experimental detection of hippocampal malondialdehyde (Malondialdehyde, MDA), superoxide dismutase (SOD), nitric oxide (Nitrogen Monoxide, NO). Supplementing Yang five soup with Cx43 agonist after treatment The effect of Cx43 expression on the protective effect of Buyang Huanhui Five Decoction on cerebral ischemia reperfusion was observed. Results: 1. rats had cerebral ischemia for 1 days. Compared with the sham group, the expression of Cx43 in the model group increased obviously. Compared with the model group, the expression of Cx43 in the group BYHWD decreased (P0.05); the rat cerebral ischemia was compared with the sham operation, and the model group was Cx43. Compared with the model group, the expression of Cx43 in BYHWD group increased (P0.05).2. rats cerebral ischemia for 1 days. Compared with the model group, IL-1 beta decreased and IL-10 increased (P0.05). After 1 days of supplementing Yang Huanwu Decoction Combined with NF- kappa B agonist, the effect of Bu Yang five soup on Cx43 in hippocampus CA1 region was inhibited (P0.05). After 7 days of cerebral ischemia, the expression of bFGF and Cx43 was enhanced by tonifying yang and five soup. The expression of Cx43 in the hippocampus CA1 region was lower than that of Buyang Huanwu Decoction group (P0.05).HE staining showed the ratio of the group of tonifying yang to the five Soup for 1 days, and the neuron injury aggravated.3. brain ischemia for 1 days. Compared with the operation group, the model group MDA, NO increased, and SOD decreased (P0.05). Compared with the sham group, BYHWD group MDA, NO down-regulation, SOD rise, BYHWD combined Cx43 agonist GAP-134 after the brain ischemia, the effect weakened. Conclusion: 1. tonifying yang and five soup inhibits the expression of hippocampus astrocytes in the early cerebral ischemia, in the late cerebral ischemia The expression of Cx43 was increased in the early stage of ischemia, and the expression of Cx43 in the hippocampus was regulated by Buyang Huanwu five soup through the inflammatory factor I1-1 beta and Il-10. In the late stage of ischemia, the tonifying yang five soup mediated the expression of.3. in the hippocampus through the neurotrophic factor bFGF to regulate the expression of.3. in the hippocampus of the hippocampus in the early stage of ischemia, and the tonifying yang and five soup changed the expression of Cx43 by regulating the expression of Cx43. After cerebral ischemia, the contents of SOD and NO protect the brain.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
[Abstract]:Objective: gap connexin 43 (connexin43, Cx43) is an important channel for material exchange and information transfer between astrocytes in the brain. After cerebral ischemia, the role of cerebral ischemia injury and repair is related to the change of the expression of gap junction protein 43. Damage and repair effect. Test the expression of Cx43 in the rat's hippocampus, and observe whether the Buyang Huanwu Decoction (BYWD) can protect the brain after the expression of Cx43. This experiment has some innovative significance to explain the role and mechanism of astrocytes in the treatment of cerebral ischemia in Chinese medicine. At the same time, the purpose of this paper is to reveal the unique advantage of Buyang Huanhui Five Decoction in the treatment of cerebral ischemia (according to the need to adjust), and forward a small step towards the goal of explaining and proving the theory of traditional Chinese medicine with modern science and technology. It has a positive significance for the whole world to understand better, accept and apply traditional Chinese medicine. The effect of middle cerebral artery occlusion (MCA0) model.1. Buyang Huanyang Five Decoction on hippocampal Cx43 in rats after cerebral ischemia reperfusion was established. Rats were built for 1 days and 7 days after administration. Immunohistochemistry and Western blot (WB) method were used to detect the Cx43 expression of.2. tonifying yang and five soup. The mechanism of the effect of Cx43 on hippocampal Cx43 after cerebral ischemia. Rat model was made, and the material was obtained after 1 days. ELISA was used to detect the expression of cytokines (interleukin -1beta, IL-1 beta and interleukin -10, IL-10). After the combined use of NF- kappa B agonist PMA to improve the inflammatory reaction, the expression of HE staining was compared with Cx43 expression in the CA1 region of hippocampus of each group. Observe the morphological changes of the cells. The rat model was made after 7 days. The expression of Basic Fibroblast growth factor (bFGF) in the hippocampal CA1 region was observed by immunohistochemical staining. The expression of Cx43 in the hippocampus CA1 area was observed by HE staining compared with the combined use of bFGF neutralization antibody and the combined use of bFGF neutralization antibody. Cell morphological changes.3. intervention connexin43 expression after the anti cerebral ischemia experiment of Buyang Huanwu Five Decoction. Rats were built for 1 days after administration. Experimental detection of hippocampal malondialdehyde (Malondialdehyde, MDA), superoxide dismutase (SOD), nitric oxide (Nitrogen Monoxide, NO). Supplementing Yang five soup with Cx43 agonist after treatment The effect of Cx43 expression on the protective effect of Buyang Huanhui Five Decoction on cerebral ischemia reperfusion was observed. Results: 1. rats had cerebral ischemia for 1 days. Compared with the sham group, the expression of Cx43 in the model group increased obviously. Compared with the model group, the expression of Cx43 in the group BYHWD decreased (P0.05); the rat cerebral ischemia was compared with the sham operation, and the model group was Cx43. Compared with the model group, the expression of Cx43 in BYHWD group increased (P0.05).2. rats cerebral ischemia for 1 days. Compared with the model group, IL-1 beta decreased and IL-10 increased (P0.05). After 1 days of supplementing Yang Huanwu Decoction Combined with NF- kappa B agonist, the effect of Bu Yang five soup on Cx43 in hippocampus CA1 region was inhibited (P0.05). After 7 days of cerebral ischemia, the expression of bFGF and Cx43 was enhanced by tonifying yang and five soup. The expression of Cx43 in the hippocampus CA1 region was lower than that of Buyang Huanwu Decoction group (P0.05).HE staining showed the ratio of the group of tonifying yang to the five Soup for 1 days, and the neuron injury aggravated.3. brain ischemia for 1 days. Compared with the operation group, the model group MDA, NO increased, and SOD decreased (P0.05). Compared with the sham group, BYHWD group MDA, NO down-regulation, SOD rise, BYHWD combined Cx43 agonist GAP-134 after the brain ischemia, the effect weakened. Conclusion: 1. tonifying yang and five soup inhibits the expression of hippocampus astrocytes in the early cerebral ischemia, in the late cerebral ischemia The expression of Cx43 was increased in the early stage of ischemia, and the expression of Cx43 in the hippocampus was regulated by Buyang Huanwu five soup through the inflammatory factor I1-1 beta and Il-10. In the late stage of ischemia, the tonifying yang five soup mediated the expression of.3. in the hippocampus through the neurotrophic factor bFGF to regulate the expression of.3. in the hippocampus of the hippocampus in the early stage of ischemia, and the tonifying yang and five soup changed the expression of Cx43 by regulating the expression of Cx43. After cerebral ischemia, the contents of SOD and NO protect the brain.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
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