補陽還五湯對大鼠腦缺血再灌注Connexin43表達的影響及機制研究
發(fā)布時間：2018-07-23 12:24
【摘要】：目的:縫隙連接蛋白43(connexin43,Cx43)是大腦星形膠質細胞之間進行物質交換、信息傳遞的重要通道,腦缺血之后,大腦抗缺血損傷以及修復的作用和縫隙連接蛋白43表達的改變有關。本文主要研究大鼠腦缺血再灌注后補陽還五湯對大腦抗損傷以及修復作用。實驗檢測大鼠大腦海馬體Cx43表達,觀察補陽還五湯(Buyang Huanwu Decoction,BYWD)是否能通過影響Cx43的表達對缺血后大腦起保護作用。本實驗對闡釋星形膠質細胞在中醫(yī)藥治療腦缺血的作用及其機制具有一定的創(chuàng)新意義。同時,本文旨在揭示補陽還五湯治療腦缺血的獨特優(yōu)勢(根據(jù)需要針對性地調節(jié)),朝著用現(xiàn)代科學技術對中醫(yī)理論加以闡述和證明的目標前進了一小步,對全世界能更好地理解、接受和應用中醫(yī)中藥具有積極的意義。方法:運用線栓法建立大鼠大腦中動脈栓塞(middle cerebral artery occlusion,MCA0)模型。1.補陽還五湯對腦缺血再灌注后大鼠海馬Cx43影響檢測。大鼠造模、給藥1天、7天后取材,使用免疫組織化學方法以及western blot(WB)法檢測,檢測各組Cx43表達的改變。2.補陽還五湯對腦缺血后大鼠海馬Cx43影響的機制探討。大鼠造模、給藥1天后取材。使用ELISA法檢測細胞因子(白介素-1beta,IL-1 β和白介素-10,IL-10)的表達。補陽還五湯聯(lián)合使用NF-κB激動劑PMA增強炎癥反應后,比較各組海馬CA1區(qū)Cx43的表達同時使用HE染色觀察細胞形態(tài)學改變;大鼠造模7天后取材。免疫組織化學染色法觀察各組海馬CA1區(qū)嗜堿性成纖維細胞生長因子(Basic Fibroblast growth factor,bFGF)的表達情況。補陽還五湯聯(lián)合使用bFGF中和抗體后,比較各組海馬CA1區(qū)Cx43的表達同時HE染色觀察細胞形態(tài)學改變。3.干預connexin43表達后補陽還五湯抗腦缺血作用實驗。大鼠造模、給藥1天后取材。實驗檢測海馬丙二醛(Malondialdehyde,MDA)、超氧化物歧化酶(Superoxide Dismutase,SOD)、一氧化氮(Nitrogen Monoxide,NO)。補陽還五湯配合 Cx43 激動劑治療后,實驗觀察Cx43表達被干預后是否影響補陽還五湯對缺血再灌注后大腦的保護作用。結果:1.大鼠腦缺血1天,與假手術組比較,模型組Cx43的表達明顯上升,與模型組比較,BYHWD組Cx43的表達減少(P0.05);大鼠腦缺血7天,與假手術比較,模型組Cx43的表達上升,與模型組比較,BYHWD組Cx43表達增加(P0.05)。2.大鼠腦缺血1天,同模型組相比,補陽還五湯組IL-1β減少、IL-10增加(P0.05);補陽還五湯聯(lián)合NF-κB激動劑給藥1天后,補陽還五湯對海馬CA1區(qū)Cx43的下調作用被抑制(P0.05),HE染色顯示聯(lián)合組較補陽還五湯組神經(jīng)元損傷加重。腦缺血7天時補陽還五湯增強bFGF和Cx43的表達;補陽還五湯聯(lián)合bFGF中和抗體給藥7天后,海馬CA1區(qū)Cx43的表達較補陽還五湯組降低(P0.05)。HE染色顯示同補陽還五湯組比,神經(jīng)元損傷加重。3.腦缺血1天后,與假手術組比較,模型組MDA、NO上升,SOD下降(P0.05);與假手術組比較,BYHWD組MDA、NO下調,SOD上升,BYHWD聯(lián)合Cx43激動劑GAP-134后,BYHWD對腦缺血后NO、SOD影響減弱。結論:1.補陽還五湯在大腦缺血早期對海馬星形膠質細胞Cx43表達起抑制作用,在大腦缺血晚期則增加Cx43的表達。2.在缺血早期,補陽還五湯通過炎癥因子I1-1 β以及Il-10介導調節(jié)大腦海馬體Cx43的表達;在缺血晚期,補陽還五湯通過神經(jīng)營養(yǎng)因子bFGF介導調節(jié)大腦海馬體Cx43的表達。3.在缺血早期,補陽還五湯通過調節(jié)Cx43表達改變大腦缺血后SOD、NO的含量,對大腦起保護作用。
[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.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5
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