丙酮酸乙酯对新生大鼠脑缺血缺氧白质损伤的保护作用研究

发布时间：2018-05-30 20:07

本文选题：缺血缺氧 + 白质损伤　；参考：《山东大学》2013年博士论文

【摘要】：目的：早产儿脑白质损伤(white matter damage, WMD)是目前早产儿脑损伤的最主要类型,是早产儿死亡及运动智能发育障碍的主要原因之一,严重影响患儿的生存生活质量。脑白质的主要成分为神经纤维及神经胶质细胞,包括少突胶质细胞(oligodendrocyte, OL)、星形胶质细胞、小胶质细胞。孕龄24-32周是WMD发生的高峰期,此时晚期OL前体细胞和未成熟OL是脑室周围白质的主要细胞群,对各种损伤性攻击具有明显易损性,研究证实晚期OL前体是白质损伤发生的主要靶细胞。缺血和感染是导致新生儿WMD的两大主要原因,最终病理结果均为白质OL的损伤与丢失。对新生儿缺血缺氧性脑损伤的治疗,目前国内外尚无统-有效的治疗方案,探寻更有效的治疗方法已成为围产医学中的重要课题。丙酮酸乙酯(ethyl pyruvate, EP)是一种安全、稳定且极易被细胞摄取的物质,已在动物实验中被证实具有较强的抗炎和自由基清除作用。研究表明,丙酮酸乙酯具有一定的神经保护作用,但有关于其在白质损伤中的作用及机制研究尚无报道。本试验在未成熟大鼠制备的脑缺血缺氧损伤动物模型基础上,研究丙酮酸乙酯对缺血缺氧所致白质损伤的保护作用,并探讨其可能的作用机制。方法：将3日龄(P3)SD未成熟新生大鼠随机分为三组：对照组、缺血缺氧组(HI组)、丙酮酸乙酯干预组(HI+EP组)。对照组仅切开颈部皮肤；缺血缺氧组结扎右侧颈总动脉伴缺氧环境(6%O2)60min;丙酮酸乙酯干预组：建立新生大鼠脑缺血缺氧模型,于缺血缺氧后10min、1h、24h分别三次给予丙酮酸乙酯50mg/kg腹腔注射,体内评估丙酮酸乙酯对白质损伤的保护作用。 1.新生大鼠脑缺血缺氧白质损伤及丙酮酸乙酯的干预作用研究：对照组、HI组、HI+EP组分别于术前、术后1天、3天、5天、7天、10天、14天固定时间点测量幼鼠体重,比较各组幼鼠的生长发育情况；在模型建立后2天、6天、14天,通过组织切片HE染色观察新生大鼠缺血缺氧后脑白质损伤的病理变化；应用免疫组化方法对不同发育阶段的晚期少突胶质前体、未成熟OL、成熟OL标志物进行检测,观察缺血缺氧后OL的损伤情况及丙酮酸乙酯的作用。 2.丙酮酸乙酯对新生大鼠缺血缺氧后脑内炎症反应的作用研究：缺血缺氧后2天、6天,应用免疫组化分别对星形胶质细胞标志物、小胶质细胞标志物进行检测,观察两种炎性细胞的激活情况；缺血缺氧后6小时、24小时、2天、6天,分别提取各组新生大鼠脑组织蛋白,应用酶联免疫吸附实验(Elisa)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)的表达情况。 3.丙酮酸乙酯在新生大鼠脑缺血缺氧白质损伤保护中的抗凋亡作用：缺血缺氧脑损伤后48h,应用免疫荧光双标检测幼鼠脑内少突胶质细胞凋亡情况,应用Western blot检测各组幼鼠脑室周围白质内cleaved caspase-3、Bax、Bcl-2的蛋白表达水平,并进行相关性分析。结果： 1.丙酮酸乙酯对新生大鼠脑缺血缺氧白质损伤具有显著保护作用 1)丙酮酸乙酯提高了新生大鼠缺血缺氧后的成活率：对照组幼鼠饮食、活动均正常；HI组新生大鼠死亡率28.32%,出现不同程度的异常反应；丙酮酸乙酯干预组死亡率降为16.84%。 2)丙酮酸乙酯改善了幼鼠缺血缺氧脑损伤后的生长发育情况：术前各组新生大鼠体重无显著性差异(P＞0.05),缺血缺氧后1天至2周幼鼠体重增长速度较对照组明显缓慢(P＜0.05),丙酮酸乙酯干预使幼鼠的体重生长速度显著增快(P＜0.01)。 3)不同时间点组织切片HE染色可见：对照组脑白质结构正常,染色清晰；缺血缺氧后新生大鼠脑白质细胞水肿、结构疏松、纤维紊乱,后期炎性胶质细胞增生活化,脑室扩大明显,可出现软化灶。丙酮酸乙酯显著改善了缺血缺氧后脑白质病理变化、抑制了侧脑室的扩大程度。 4)晚期OL前体、未成熟OL在缺血缺氧后明显损伤,数量较对照组显著减少(P＜0.01),同时髓鞘相关蛋白表达亦显著减少(P＜0.01),出现低髓鞘化。丙酮酸乙酯干预显著减轻了缺血缺氧导致的不同发育阶段OL的损伤与丢失,改善了白质区域的髓鞘化程度(P＜0.01)。 2.丙酮酸乙酯减轻了新生大鼠缺血缺氧后脑内的炎症反应水平 1)新生大鼠缺血缺氧后2天、6天脑内小胶质细胞、星形胶质细胞显著激活,活化细胞数量明显增多(P＜0.01),丙酮酸乙酯有效抑制了不同时间点两种炎症反应细胞的活化增生(P＜0.01)。 2)对照组新生大鼠脑内存在少量的TNF-α、IL-1β表达；缺血缺氧后6小时新生大鼠脑内TNF-α表达水平即开始上调,2天后较对照组有更显著升高,直到缺血缺氧后6天仍维持在较高的水平,较同日龄对照组比较差异均有显著意义(P＜0.01),丙酮酸乙酯显著降低了幼鼠脑内不同时间点TNF-α的表达水平(P＜0.05)；IL-1β的表达水平于缺血缺氧后6小时、24小时、2天、6天亦呈逐渐升高趋势(P＜0.01),丙酮酸乙酯使各时间点IL-1β的表达显著下降(P＜0.01)。 3.丙酮酸乙酯在新生大鼠脑缺血缺氧白质损伤保护中的抗凋亡作用 1)免疫荧光双标结果显示,缺血缺氧后2天脑室周围白质少突胶质细胞前体存在明显凋亡,凋亡细胞数量较对照组明显增多(P＜0.01),丙酮酸乙酯使凋亡细胞数量显著减少(P＜0.01)。 2)新生大鼠缺血缺氧脑损伤后白质内凋亡因子caspase-3显著激活(P＜0.01),腹腔注射丙酮酸乙酯降低了caspase-3的激活水平(P＜0.01)。 3)丙酮酸乙酯对促凋亡蛋白Bax的作用新生大鼠脑缺血缺氧损伤后白质Bax蛋白表达水平较对照组明显升高(P＜0.01),丙酮酸乙酯干预使Bax表达水平显著降低(P＜0.01)。 4)丙酮酸乙酯对抗凋亡蛋白Bcl-2的作用新生大鼠脑缺血缺氧后白质Bcl-2表达水平显著降低(P＜0.01),丙酮酸乙酯明显升高了脑损伤后Bcl-2的表达水平(P＜0.01)。 5)Bax/Bcl-2值与OL凋亡呈正相关相关性分析显示：同一时间点,缺血缺氧后新生大鼠脑白质内Bax/Bcl-2值增高与OL凋亡增多成正相关性。丙酮酸乙酯显著降低了缺血缺氧后Bax/Bcl-2值,对OL细胞凋亡起保护作用。结论： 1.本实验成功建立了3日龄未成熟新生大鼠脑缺血缺氧白质损伤模型,该模型以少突胶质细胞前体损伤为主、与人类早产儿脑室周围白质损伤病理相似。 2.丙酮酸乙酯对未成熟大鼠缺血缺氧所致白质损伤具有显著的保护作用,能改善缺血缺氧后幼鼠的生长发育情况。 3.丙酮酸乙酯抑制脑缺血缺氧后炎症反应细胞的激活、降低其调控的炎症因子的表达水平,抗炎作用参与了丙酮酸乙酯的白质损伤保护作用。 4.凋亡是缺血缺氧少突胶质细胞损伤的重要机制,丙酮酸乙酯具有显著的抗凋亡作用,可抑制促凋亡因子的产生,升高抗凋亡因子的表达水平。 5.该研究为临床治疗早产儿缺血缺氧性白质损伤提供丰富的动物实验信息及依据,为进一步研究早产儿白质损伤的防治提供了可能的干预手段和理论依据。
[Abstract]:Purpose :

Cerebral white matter damage ( WMD ) is one of the main causes of brain injury in preterm infants , which is one of the main causes of premature infant death and intellectual development disturbance .

Method :

Three - day - old ( P3 ) SD immature neonatal rats were randomly divided into three groups : control group , ischemia - hypoxia group ( HI group ) and ethyl pyruvate intervention group ( HI + EP group ) .
The protective effect of ethyl pyruvate on white matter injury was assessed by intraperitoneal injection of ethyl pyruvate at 50 mg / kg for 10 min , 1 h and 24 h after ischemia and hypoxia .

1 . The effects of cerebral ischemia - reperfusion injury and ethyl pyruvate were studied in neonatal rats : control group , HI group , HI + EP group before operation , 1 day after operation , 3 days , 5 days , 7 days , 10 days and 14 days respectively .
After 2 days , 6 days and 14 days after the model was established , the pathological changes of the neonatal rats were observed by HE staining .
Immunohistochemical method was used for the detection of late Oligodendrocyte precursor , immature OL and mature OL marker in different developmental stages . The damage of OL and the effect of ethyl pyruvate after ischemia and hypoxia were observed .

2 . The effect of ethyl pyruvate on the inflammatory response of neonatal rats after ischemia and hypoxia : 2 days and 6 days after ischemia and hypoxia , the astrocyte markers and microglial cell markers were detected by immunohistochemistry , respectively , and the activation of the two inflammatory cells was observed .
The expression of tumor necrosis factor - 伪 ( TNF - 伪 ) and interleukin - 1尾 ( IL - 1尾 ) was detected by enzyme - linked immunosorbent assay ( ELISA ) in 6 hours , 24 hours , 2 days and 6 days after ischemia and hypoxia .

3 . The anti - apoptotic effect of ethyl pyruvate in the protection of cerebral ischemia and hypoxia in neonatal rats : 48h after hypoxic - ischemic brain injury , the expression level of caspase - 3 , Bax and Bcl - 2 in the periventricular white matter of each group was detected by Western blot , and the correlation analysis was carried out .

Results :

1 . Ethyl Pyruvate has a significant protective effect on cerebral ischemia and hypoxia in neonatal rats .

1 ) Ethyl Pyruvate improves the survival rate of neonatal rats after ischemia and hypoxia : the diet and activity of the young rats in the control group are normal ;
The mortality of neonatal rats in HI group was 28.32 % , and abnormal reaction was observed .
The mortality rate in the intervention group of ethyl pyruvate was 16.84 % .

2 ) Ethyl Pyruvate improved the growth and development of neonatal rats following hypoxic - ischemic brain injury : there was no significant difference in the weight of neonatal rats ( P > 0.05 ) before and after operation , and the body weight of young rats increased significantly ( P < 0 . 05 ) from 1 day to 2 weeks after ischemia and hypoxia ( P < 0 . 01 ) .

3 ) HE staining of tissue sections at different time points shows that the brain white matter in the control group is normal and the staining is clear ;
The cerebral white matter cell edema , structural osteoporosis , fiber disorder , late inflammatory glial cell increase activation and cerebral ventricle enlargement are obvious in the neonatal rat after ischemia and hypoxia , and the softening range can be seen in the cerebral ventricle .

4 ) Compared with the control group ( P & lt ; 0.01 ) , the expression of myelin - related protein decreased significantly ( P & lt ; 0.01 ) , and the expression of myelin - related protein decreased significantly ( P & lt ; 0.01 ) , and the degree of myelination in the white matter region was improved ( P & lt ; 0.01 ) .

2 . Ethyl Pyruvate reduces the level of inflammatory response in the brain after ischemia and hypoxia in neonatal rats

1 ) After 2 days after ischemia and hypoxia in neonatal rats , astrocytes and astrocytes were significantly activated in 6 days , and the number of activated cells increased significantly ( P & lt ; 0.01 ) . Ethyl pyruvate effectively inhibited the activation and proliferation of two inflammatory reaction cells at different time points ( P < 0.01 ) .

2 ) There was a small amount of TNF - 伪 and IL - 1尾 in the brain of the neonatal rats in the control group .
The levels of TNF - 伪 in the brain of neonatal rats were up - regulated in 6 hours after ischemia and hypoxia , and the levels of TNF - 伪 in the brain were significantly higher than those in the control group after 2 days ( P < 0.01 ) , and the level of TNF - 伪 in the brain of the young rats was significantly decreased ( P < 0.05 ) .
The expression level of IL - 1尾 increased gradually in 6 hours , 24 hours , 2 days and 6 days after ischemia and hypoxia ( P & lt ; 0.01 ) , and the expression of IL - 1尾 in each time point was significantly decreased ( P & lt ; 0.01 ) .

3 . Anti - apoptosis effect of ethyl pyruvate in the protection of neonatal rat cerebral ischemia and hypoxia

1 ) Compared with control group , the number of apoptotic cells increased significantly ( P & lt ; 0.01 ) , and the number of apoptotic cells decreased significantly ( P & lt ; 0.01 ) .

2 ) The caspase - 3 was significantly activated after hypoxic - ischemic brain injury in neonatal rats ( P & lt ; 0.01 ) , and the activation level of caspase - 3 was decreased by intraperitoneal injection of ethyl pyruvate ( P & lt ; 0.01 ) .

3 ) The expression level of Bax protein was significantly higher than that of the control group ( P < 0.01 ) , and the expression level of Bax was significantly decreased ( P < 0.01 ) .

4 ) The expression level of Bcl - 2 was significantly decreased after cerebral ischemia and hypoxia in neonatal rats ( P < 0.01 ) , and the expression level of Bcl - 2 after brain injury was significantly increased ( P < 0.01 ) .

5 ) The positive correlation between Bax / Bcl - 2 and OL apoptosis revealed that the increase of Bax / Bcl - 2 and OL apoptosis were positively correlated with the increase of Bax / Bcl - 2 in neonatal rats at the same time . Ethyl pyruvate significantly reduced the Bax / Bcl - 2 value after ischemia and hypoxia , and had a protective effect on the apoptosis of OL cells .

Conclusion :

1 . The model of cerebral ischemia - reperfusion injury in 3 - day - old neonatal rats was successfully established in this experiment . The model was mainly damaged by the precursor injury of microglial cells , which was similar to the pathological changes of white matter around the brain of premature infants .

2 . Ethyl Pyruvate has a protective effect on white matter injury caused by ischemia and hypoxia in immature rats , and can improve the growth and development of young rats after ischemia and hypoxia .

3 . Ethyl Pyruvate can inhibit the activation of inflammatory reaction cells after cerebral ischemia and hypoxia , reduce the expression level of inflammatory cytokines and participate in the protective effect of ethyl pyruvate .

4 . Apoptosis is an important mechanism of ischemia and hypoxia , which can inhibit the production of pro - apoptotic factor and increase the level of anti - apoptotic factor .

5 . This study provides abundant animal experiment information and basis for the clinical treatment of hypoxic white matter damage in preterm infants , which provides the possible means and theoretical basis for further study on the prevention and treatment of white matter damage in preterm infants .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R722.1

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