梓醇预处理对脑缺血再灌注损伤的神经保护作用研究

发布时间：2018-05-15 21:39

  本文选题：梓醇 + 脑缺血再灌注损伤　； 参考：《郑州大学》2014年博士论文

【摘要】：背景：临床上对脑缺血再灌注(cerebral ischemia/reperfusion, CI/R)损伤仍缺乏有效的防治措施,寻找适合于缺血再灌注的神经保护药物,阐明其作用机制和作用靶点,是当今医学的研究热点。目前天然药物组分对CI/R的保护作用引起广泛关注。梓醇是地黄的主要药效成分,已有研究显示梓醇具有一定的神经保护作用,但机制尚未完全阐明。目的：本研究主要采用沙土鼠CUR模型,从行为学、体内研究、体外研究3个方面,探讨梓醇预处理对缺血性脑损伤的神经保护作用机制。方法：1.行为学评估1.1 采用结扎双侧颈总动脉缺血10 min再灌注6 h,建立沙土鼠CI/R模型,分为假手术组、模型组、梓醇三个剂量组(C5、C10、C20,剂量分别为5、10、20mg/kg),每组10只。治疗组术前3天及术前30min,分别腹腔注射(ip)给药1次。观察再灌注6 h内神经症状,计算卒中指数。1.2采用永久性双侧颈总动脉结扎建立大鼠脑灌注不足模型,分组同上。采用ip给药,术前3天,及术后2周内每天1次。通过Morris水迷宫评价2周后大鼠学习记忆能力的改变,及HE染色观察大鼠皮层病理改变。2. 在体实验研究应用沙土鼠CI/R模型,分组同上,术前3天及术前30 min,分别ip给药1次。采用黄嘌呤氧化酶法测定SOD活力,硫代巴比妥酸法测定MDA含量,放射免疫法测定ET和CGRP含量,定磷法测定ATP酶活性,高效液相色谱法测定谷氨酸(Glu)和天冬氨酸(Asp)含量,ELISA法测定TNF-a和IL-1β的表达水平。3.离体实验研究采用原代培养大鼠大脑皮层星形胶质细胞,建立糖氧剥夺再灌注(OGD/R)模型。分为正常组,模型组,梓醇预处理组(浓度：25、50、100 μmol/l)。从星形胶质细胞存活率、LDH活性、SOD活力、MDA含量、ATP酶活性等方面探讨梓醇对大鼠大脑皮层星形胶质细胞损伤的保护作用机制。结果：1.行为学评估1.1 模型组卒中指数明显高于假手术组(P0.01)。C5、C10和C20组的卒中指数均降低,与模型组比较有显著性差异(P0.05),提示梓醇预处理对CI/R沙土鼠神经功能的改善作用。1.2与假手术组比较,模型组的潜伏期明显延长(P0.01),表明造模后大鼠学习记忆能力明显受损。治疗组大鼠与模型组比较,潜伏期明显缩短(P0.05),表明梓醇干预使大鼠学习记忆能力得到改善。2.在体实验研究2.1梓醇对氧化应激指标的影响模型组沙土鼠脑组织匀浆的SOD活性较假手术组降低,MDA含量较假手术组升高(P0.01)。C5、C10和C20组的SOD活性均显著高于模型组(P0.05)；C5、C10和C20组MDA含量均较模型组降低,其中C10和C20组与模型组比较有显著性差异(P0.01),提示梓醇预处理增强CI/R沙土鼠抗氧化能力,抑制脂质过氧化反应。2.2梓醇对神经肽类指标的影响模型组沙土鼠血浆中ET水平显著高于假手术组(P0.01),CGRP水平则显著低于假手术组(P0.05)。C5、C10和C20组均可降低血浆ET水平,与模型组比较有显著性差异(P0.05)；C5、C10和C20组均有升高血浆CGRP水平的趋势,但与模型组比较并无显著性差异(P0.05),表明梓醇预处理可通过抑制内源性神经肽ET的产生发挥作用。2.3梓醇对能量代谢指标的影响模型组沙土鼠脑组织匀浆Na+-K+-ATP酶、Ca2+-ATP酶和MG2+-ATP酶的活性较假手术组均显著降低(P0.01)。C5、C10和C20组对ATP酶活性有不同程度的影响,与模型组比较,C10和C20组可显著升高Na+-K+-ATP酶活性(P0.05),C5、C10和C20组均可显著升高Ca2+-ATP酶活性(P0.05)；各组对Mg2+-ATP酶活性则无明显影响(P0.05),提示梓醇预处理对ATP酶的活性的影响,且主要为Na+-K+-ATP酶和Ca2+-ATP酶。2.4梓醇对EAA指标的影响模型组沙土鼠脑组织匀浆中Glu含量显著高于假手术组(P0.01),Asp含量也显著高于假手术组(P0.05)。C5、C10和C20组均能降低CI/R沙土鼠脑组织的Glu含量,与模型组比较有统计学意义(P0.05),对Asp含量则无明显影响(P0.05),提示梓醇预处理对CI/R沙土鼠兴奋性氨基酸毒性的抑制作用。2.5梓醇对炎症因子指标的影响模型组脑组织匀浆中TNF-α和IL-1β水平均显著高于假手术组(P0.01)。梓醇预处理后,各剂量组(C5、C10和C20)均使沙土鼠脑组织中的TNF-α含量明显减低(P0.01)。同时,梓醇3个剂量组(C5、C10和C20)均明显降低了脑组织中IL-1β的表达水平(P0.05),提示梓醇预处理可减轻CI/R后的炎症反应。3.离体实验研究3.1梓醇对OGD/R损伤星形胶质细胞存活率的影响缺氧缺糖3h再灌注24 h后,脑星形胶质细胞存活率显著降低至50.3%,梓醇各浓度(25.50.100μmol/l)预处理可显著提高细胞存活率至62.2%、70.7%、71.5%,与模型组比较差异均有统计学意义(P0.05)。3.2梓醇对OGD/R损伤星形胶质细胞培养液LDH活性的影响模型组星形胶质细胞培养液中LDH活性明显升高(P0.01),表明细胞膜出现严重破坏；梓醇各浓度组(25、50、100 μmol/l) LDH活性显著降低,与模型组比较差异均有统计学意义(P0.05),表明梓醇预处理可减轻细胞膜损伤的程度。3.3梓醇对OGD/R损伤星形胶质细胞SOD活力和MDA含量的影响与正常对照组比较,模型组SOD活力显著降低,MDA含量明显升高(P0.01),表明细胞受到自由基氧化损伤,同时自身抗氧化能力降低。梓醇(25、50、100μmol/1)预处理可明显增强星形胶质细胞SOD活力(P0.01),且呈现一定的浓度依赖性。此外,梓醇各浓度均可显著降低星形胶质细胞内MDA水平(P0.05),提示梓醇预处理可改善损伤星形胶质细胞的抗氧化能力,减轻脂质过氧化损伤。3.4梓醇对OGD/R损伤星形胶质细胞ATP酶活性的影响与正常对照组比较,模型组星形胶质细胞内的ATP酶活性明显降低(P0.01),表明星形胶质细胞有氧氧化能力降低、无氧酵解程度升高。梓醇(25、50、100μmol/l)预处理可显著提高ATP酶活性,与模型组比较,差异具有统计学意义(P0.05),表明梓醇可改善OGD/R损伤后星形胶质细胞的能量代谢。结论：1.梓醇预处理能降低CI/R沙土鼠的卒中指数,改善其神经功能。2.梓醇预处理可以改善脑灌注不足大鼠的学习记忆能力。3.梓醇预处理对沙土鼠CI/R损伤的保护作用是通过减少脑组织自由基的产生、抑制脂质过氧化反应、抑制内源性神经肽ET的产生、保护细胞膜ATP酶的活性、降低EAA毒性、抑制TNF-α和IL-1β等炎症因子的表达,减轻脑组织的炎症反应等途径实现的。4.梓醇预处理对大鼠大脑皮层星形胶质细胞OGD/R损伤的保护作用是通过增强细胞抗氧化能力、改善能量代谢等途径实现的。
[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.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743
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