本文選題：梓醇 + 腦缺血再灌注損傷��； 參考：《鄭州大學(xué)》2014年博士論文

【摘要】：背景：臨床上對(duì)腦缺血再灌注(cerebral ischemia/reperfusion, CI/R)損傷仍缺乏有效的防治措施,尋找適合于缺血再灌注的神經(jīng)保護(hù)藥物,闡明其作用機(jī)制和作用靶點(diǎn),是當(dāng)今醫(yī)學(xué)的研究熱點(diǎn)。目前天然藥物組分對(duì)CI/R的保護(hù)作用引起廣泛關(guān)注。梓醇是地黃的主要藥效成分,已有研究顯示梓醇具有一定的神經(jīng)保護(hù)作用,但機(jī)制尚未完全闡明。目的：本研究主要采用沙土鼠CUR模型,從行為學(xué)、體內(nèi)研究、體外研究3個(gè)方面,探討梓醇預(yù)處理對(duì)缺血性腦損傷的神經(jīng)保護(hù)作用機(jī)制。方法：1.行為學(xué)評(píng)估1.1 采用結(jié)扎雙側(cè)頸總動(dòng)脈缺血10 min再灌注6 h,建立沙土鼠CI/R模型,分為假手術(shù)組、模型組、梓醇三個(gè)劑量組(C5、C10、C20,劑量分別為5、10、20mg/kg),每組10只。治療組術(shù)前3天及術(shù)前30min,分別腹腔注射(ip)給藥1次。觀察再灌注6 h內(nèi)神經(jīng)癥狀,計(jì)算卒中指數(shù)。1.2采用永久性雙側(cè)頸總動(dòng)脈結(jié)扎建立大鼠腦灌注不足模型,分組同上。采用ip給藥,術(shù)前3天,及術(shù)后2周內(nèi)每天1次。通過Morris水迷宮評(píng)價(jià)2周后大鼠學(xué)習(xí)記憶能力的改變,及HE染色觀察大鼠皮層病理改變。2. 在體實(shí)驗(yàn)研究應(yīng)用沙土鼠CI/R模型,分組同上,術(shù)前3天及術(shù)前30 min,分別ip給藥1次。采用黃嘌呤氧化酶法測(cè)定SOD活力,硫代巴比妥酸法測(cè)定MDA含量,放射免疫法測(cè)定ET和CGRP含量,定磷法測(cè)定ATP酶活性,高效液相色譜法測(cè)定谷氨酸(Glu)和天冬氨酸(Asp)含量,ELISA法測(cè)定TNF-a和IL-1β的表達(dá)水平。3.離體實(shí)驗(yàn)研究采用原代培養(yǎng)大鼠大腦皮層星形膠質(zhì)細(xì)胞,建立糖氧剝奪再灌注(OGD/R)模型。分為正常組,模型組,梓醇預(yù)處理組(濃度：25、50、100 μmol/l)。從星形膠質(zhì)細(xì)胞存活率、LDH活性、SOD活力、MDA含量、ATP酶活性等方面探討梓醇對(duì)大鼠大腦皮層星形膠質(zhì)細(xì)胞損傷的保護(hù)作用機(jī)制。結(jié)果：1.行為學(xué)評(píng)估1.1 模型組卒中指數(shù)明顯高于假手術(shù)組(P0.01)。C5、C10和C20組的卒中指數(shù)均降低,與模型組比較有顯著性差異(P0.05),提示梓醇預(yù)處理對(duì)CI/R沙土鼠神經(jīng)功能的改善作用。1.2與假手術(shù)組比較,模型組的潛伏期明顯延長(zhǎng)(P0.01),表明造模后大鼠學(xué)習(xí)記憶能力明顯受損。治療組大鼠與模型組比較,潛伏期明顯縮短(P0.05),表明梓醇干預(yù)使大鼠學(xué)習(xí)記憶能力得到改善。2.在體實(shí)驗(yàn)研究2.1梓醇對(duì)氧化應(yīng)激指標(biāo)的影響模型組沙土鼠腦組織勻漿的SOD活性較假手術(shù)組降低,MDA含量較假手術(shù)組升高(P0.01)。C5、C10和C20組的SOD活性均顯著高于模型組(P0.05)；C5、C10和C20組MDA含量均較模型組降低,其中C10和C20組與模型組比較有顯著性差異(P0.01),提示梓醇預(yù)處理增強(qiáng)CI/R沙土鼠抗氧化能力,抑制脂質(zhì)過氧化反應(yīng)。2.2梓醇對(duì)神經(jīng)肽類指標(biāo)的影響模型組沙土鼠血漿中ET水平顯著高于假手術(shù)組(P0.01),CGRP水平則顯著低于假手術(shù)組(P0.05)。C5、C10和C20組均可降低血漿ET水平,與模型組比較有顯著性差異(P0.05)；C5、C10和C20組均有升高血漿CGRP水平的趨勢(shì),但與模型組比較并無顯著性差異(P0.05),表明梓醇預(yù)處理可通過抑制內(nèi)源性神經(jīng)肽ET的產(chǎn)生發(fā)揮作用。2.3梓醇對(duì)能量代謝指標(biāo)的影響模型組沙土鼠腦組織勻漿Na+-K+-ATP酶、Ca2+-ATP酶和MG2+-ATP酶的活性較假手術(shù)組均顯著降低(P0.01)。C5、C10和C20組對(duì)ATP酶活性有不同程度的影響,與模型組比較,C10和C20組可顯著升高Na+-K+-ATP酶活性(P0.05),C5、C10和C20組均可顯著升高Ca2+-ATP酶活性(P0.05)；各組對(duì)Mg2+-ATP酶活性則無明顯影響(P0.05),提示梓醇預(yù)處理對(duì)ATP酶的活性的影響,且主要為Na+-K+-ATP酶和Ca2+-ATP酶。2.4梓醇對(duì)EAA指標(biāo)的影響模型組沙土鼠腦組織勻漿中Glu含量顯著高于假手術(shù)組(P0.01),Asp含量也顯著高于假手術(shù)組(P0.05)。C5、C10和C20組均能降低CI/R沙土鼠腦組織的Glu含量,與模型組比較有統(tǒng)計(jì)學(xué)意義(P0.05),對(duì)Asp含量則無明顯影響(P0.05),提示梓醇預(yù)處理對(duì)CI/R沙土鼠興奮性氨基酸毒性的抑制作用。2.5梓醇對(duì)炎癥因子指標(biāo)的影響模型組腦組織勻漿中TNF-α和IL-1β水平均顯著高于假手術(shù)組(P0.01)。梓醇預(yù)處理后,各劑量組(C5、C10和C20)均使沙土鼠腦組織中的TNF-α含量明顯減低(P0.01)。同時(shí),梓醇3個(gè)劑量組(C5、C10和C20)均明顯降低了腦組織中IL-1β的表達(dá)水平(P0.05),提示梓醇預(yù)處理可減輕CI/R后的炎癥反應(yīng)。3.離體實(shí)驗(yàn)研究3.1梓醇對(duì)OGD/R損傷星形膠質(zhì)細(xì)胞存活率的影響缺氧缺糖3h再灌注24 h后,腦星形膠質(zhì)細(xì)胞存活率顯著降低至50.3%,梓醇各濃度(25.50.100μmol/l)預(yù)處理可顯著提高細(xì)胞存活率至62.2%、70.7%、71.5%,與模型組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。3.2梓醇對(duì)OGD/R損傷星形膠質(zhì)細(xì)胞培養(yǎng)液LDH活性的影響模型組星形膠質(zhì)細(xì)胞培養(yǎng)液中LDH活性明顯升高(P0.01),表明細(xì)胞膜出現(xiàn)嚴(yán)重破壞；梓醇各濃度組(25、50、100 μmol/l) LDH活性顯著降低,與模型組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05),表明梓醇預(yù)處理可減輕細(xì)胞膜損傷的程度。3.3梓醇對(duì)OGD/R損傷星形膠質(zhì)細(xì)胞SOD活力和MDA含量的影響與正常對(duì)照組比較,模型組SOD活力顯著降低,MDA含量明顯升高(P0.01),表明細(xì)胞受到自由基氧化損傷,同時(shí)自身抗氧化能力降低。梓醇(25、50、100μmol/1)預(yù)處理可明顯增強(qiáng)星形膠質(zhì)細(xì)胞SOD活力(P0.01),且呈現(xiàn)一定的濃度依賴性。此外,梓醇各濃度均可顯著降低星形膠質(zhì)細(xì)胞內(nèi)MDA水平(P0.05),提示梓醇預(yù)處理可改善損傷星形膠質(zhì)細(xì)胞的抗氧化能力,減輕脂質(zhì)過氧化損傷。3.4梓醇對(duì)OGD/R損傷星形膠質(zhì)細(xì)胞ATP酶活性的影響與正常對(duì)照組比較,模型組星形膠質(zhì)細(xì)胞內(nèi)的ATP酶活性明顯降低(P0.01),表明星形膠質(zhì)細(xì)胞有氧氧化能力降低、無氧酵解程度升高。梓醇(25、50、100μmol/l)預(yù)處理可顯著提高ATP酶活性,與模型組比較,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),表明梓醇可改善OGD/R損傷后星形膠質(zhì)細(xì)胞的能量代謝。結(jié)論：1.梓醇預(yù)處理能降低CI/R沙土鼠的卒中指數(shù),改善其神經(jīng)功能。2.梓醇預(yù)處理可以改善腦灌注不足大鼠的學(xué)習(xí)記憶能力。3.梓醇預(yù)處理對(duì)沙土鼠CI/R損傷的保護(hù)作用是通過減少腦組織自由基的產(chǎn)生、抑制脂質(zhì)過氧化反應(yīng)、抑制內(nèi)源性神經(jīng)肽ET的產(chǎn)生、保護(hù)細(xì)胞膜ATP酶的活性、降低EAA毒性、抑制TNF-α和IL-1β等炎癥因子的表達(dá),減輕腦組織的炎癥反應(yīng)等途徑實(shí)現(xiàn)的。4.梓醇預(yù)處理對(duì)大鼠大腦皮層星形膠質(zhì)細(xì)胞OGD/R損傷的保護(hù)作用是通過增強(qiáng)細(xì)胞抗氧化能力、改善能量代謝等途徑實(shí)現(xiàn)的。
[Abstract]:Background: there is still a lack of effective prevention and control measures for cerebral ischemia/reperfusion (CI/R) injury in clinical. It is a hot topic in modern medicine to find neuroprotective drugs suitable for ischemia-reperfusion and clarify its mechanism and target target. At present, the protective effect of Tian ran drug component on CI/R attracts extensive attention. Catalpol is the main effective component of rehmannia. Research has shown that Catalpol has a certain neuroprotective effect, but the mechanism has not been fully elucidated. Objective: This study mainly used the CUR model of gerbils, from 3 aspects of behavioral, in vivo and in vitro studies, and discussed the mechanism of the neuroprotective effect of Catalpol pretreatment on ischemic brain injury. 1. behavioral assessment (1.1) by ligating bilateral common carotid artery ischemia 10 min reperfusion 6 h, and establishing CI/R model of gerbils, divided into sham operation group, model group, Catalpol three dose group (C5, C10, C20, dosage 5,10,20mg/kg respectively), 10 in each group. The treatment group was given 1 times by intraperitoneal injection (IP), respectively, 3 days before and before operation, and 6 h in 6 h. After symptoms, the stroke index.1.2 was established by permanent bilateral common carotid artery ligation to establish a model of cerebral perfusion deficiency in rats. IP was used, 3 days before operation, and 1 times a day in 2 weeks after operation. The changes of learning and memory ability of rats were evaluated by Morris water maze after 2 weeks, and HE staining was used to observe the pathological changes of.2. in the body of rats. The CI/R model of gerbil was applied to the same group, 3 days before operation and 30 min before operation, IP was given 1 times respectively. The activity of SOD was determined by xanthine oxidase method, the content of MDA was determined by thiobarbituric acid method, the content of ET and CGRP was determined by radioimmunoassay, the activity of ATP enzyme was determined by the method of radioimmunoassay, and the content of Glu and aspartic acid (Asp) was determined by high performance liquid chromatography The ELISA method was used to determine the expression level of TNF-a and IL-1 beta in.3. in vitro. The primary cultured rat cerebral cortex astrocytes were cultured and the model of oxygen deprivation reperfusion (OGD/R) was established. The model group was divided into normal group, model group, Catalpol preconditioning group (concentration: 25,50100 mu mol/l). The survival rate of astrocytes, LDH activity, SOD activity, MDA content, ATP. The protective effect of Catalpol on astrocyte injury in the cerebral cortex of rats was investigated. Results: the stroke index of 1. behavioral assessment 1.1 model group was significantly higher than that of sham operation group (P0.01).C5, and the stroke index in group C10 and C20 decreased significantly (P0.05), suggesting Catalpol pretreatment on CI/R Sandy soil The improvement of neural function of rat.1.2 compared with the sham operation group, the incubation period of the model group was obviously prolonged (P0.01), indicating that the learning and memory ability of the rats was significantly impaired after the model group. The incubation period of the rats in the treatment group was significantly shorter than that in the model group (P0.05), indicating that the intervention of Catalpol could improve the learning and memory ability of the rats by.2. in the study of 2.1 Catalpol. The SOD activity of the brain homogenate of the model group was lower than that of the sham group, and the content of MDA was higher than that of the sham group (P0.01).C5, and the SOD activity in the C10 and C20 groups was significantly higher than that of the model group (P0.05), and the MDA content of the group of C10 and C20 was lower than that of the model group. .01), it was suggested that Catalpol pretreatment enhanced the antioxidant capacity of CI/R gerbils, inhibited lipid peroxidation and inhibited.2.2 Catalpol effect on neuropeptides. The level of ET in the rat plasma was significantly higher than that of the sham group (P0.01), and the level of CGRP was significantly lower than that of the sham group (P0.05).C5, and the C10 and C20 groups could reduce the ET level of plasma, compared with the model group. There was a significant difference (P0.05); C5, C10 and C20 had a tendency to increase the level of plasma CGRP, but there was no significant difference compared with the model group (P0.05), indicating that Catalpol pretreatment could inhibit the production of endogenous neuropeptide ET and the effect of.2.3 Catalpol on the energy metabolism index in the model group of rat brain homogenate Na+-K+-ATP enzyme, C, C. The activity of a2+-ATP and MG2+-ATP decreased significantly (P0.01).C5, and C10 and C20 groups had different effects on the activity of ATP enzyme. Compared with the model group, C10 and C20 groups could significantly increase the activity of Na+-K+-ATP enzyme (P0.05). Influence (P0.05), the effect of Catalpol pretreatment on the activity of ATP enzyme, and the effect of Na+-K+-ATP enzyme and Ca2+-ATP enzyme.2.4 Catalpol on the EAA index, the content of Glu in the homogenate of gerbils was significantly higher than that of the sham group (P0.01), and the content of Asp was significantly higher than that of the artificial group (P0.05).C5. Both C10 and the group could reduce the brain of gerbils. The Glu content of the tissue was statistically significant (P0.05) and no significant effect on the Asp content (P0.05). The inhibitory effect of Catalpol pretreatment on the toxicity of excitatory amino acids in CI/R gerbils.2.5 Catalpol effect on the inflammatory factors in the model group, the level of TNF- A and IL-1 beta in the homogenate of the brain tissue was significantly higher than that of the sham operation group (P0.01). After pre treatment of Catalpol, the content of C5, C10 and C20 in every dose group reduced the content of TNF- alpha in the brain tissue of the gerbils (P0.01). At the same time, 3 dose groups of Catalpol (C5, C10 and C20) significantly reduced the expression level of IL-1 beta in the brain tissue (P0.05), suggesting that Catalpol pretreatment could reduce the inflammatory response in CI/R after the experiment of 3.1 Catalpol. The survival rate of astrocytes was significantly reduced to 50.3% after 24 h reperfusion, and the survival rate of Catalpol (25.50.100 mu mol/l) could be significantly increased to 62.2%, 70.7%, 71.5%, compared with the model group (P0.05).3.2 Catalpol was starlike to OGD/R damage. The activity of LDH in glial culture medium increased significantly (P0.01) in the model group of astrocyte culture medium (P0.01), indicating that the cell membrane was seriously damaged, and the activity of 25,50100 mol/l LDH decreased significantly, and the difference was statistically significant (P0.05) compared with the model group (P0.05), indicating that Catalpol pretreatment could reduce the cell membrane loss. The effect of.3.3 Catalpol on the activity of SOD and the content of MDA in OGD/R damaged astrocytes was significantly lower than that of the normal control group. The SOD activity of the model group was significantly reduced, the content of MDA increased significantly (P0.01), indicating that the cells were damaged by free radical oxidation and their own antioxidant capacity decreased. The pre treatment of Catalpol (25,50100 u mol/1) could obviously enhance the star stellate. The SOD activity (P0.01) of glial cells showed a certain concentration dependence. In addition, the concentration of Catalpol could significantly reduce the MDA level in astrocytes (P0.05), suggesting that Catalpol pretreatment could improve the antioxidant capacity of damaged astrocytes and reduce the activity of.3.4 Catalpol against OGD/R damaged astrocyte ATP enzyme in OGD/R damaged astrocytes. Compared with the normal control group, the activity of ATP enzyme in the astrocytes of the model group decreased significantly (P0.01), the oxygen oxidation ability of the star glial cells decreased and the degree of anaerobic glycolysis increased. The ATP enzyme activity of Catalpol (25,50100 mu mol/l) pretreatment could be significantly improved. The difference was statistically significant (P0.05) and indicated Catalpol compared with the model group (P0.05). The energy metabolism of astrocytes after OGD/R injury can be improved. Conclusion: 1. the preconditioning of Catalpol can reduce the stroke index of CI/R gerbils and improve the neural function.2. Catalpol preconditioning can improve the learning and memory ability of the rats with cerebral perfusion.3.. The protective effect of Catalpol preconditioning on the CI/R damage of gerbils is by reducing the brain tissue freedom Base production, inhibiting the lipid peroxidation, inhibiting the production of endogenous neuropeptide ET, protecting the activity of ATP enzyme in the cell membrane, reducing the toxicity of EAA, inhibiting the expression of TNF- A and IL-1 beta and other inflammatory factors and alleviating the inflammatory reaction of brain tissue, the protective effect of.4. Catalpol preconditioning on the OGD/R damage in the astrocytes of the rat cerebral cortex is protected. It is achieved by enhancing cell antioxidant capacity and improving energy metabolism.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743
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本文編號(hào)：1894072