DHA和远志皂苷对丙泊酚重复麻醉大鼠学习记忆损伤的干预作用

发布时间：2018-07-04 06:58

本文选题：DHA + 丙泊酚麻醉　；参考：《山东大学》2016年博士论文

【摘要】：第一部分 DHA对丙泊酚重复麻醉大鼠学习记忆损伤的干预作用目的研究不同剂量的DHA对丙泊酚重复麻醉SD大鼠引起的学习记忆功能损伤的干预作用、量效关系及作用机制,以期为临床使用丙泊酚重复麻醉产生的副作用提供改善解决的方案。方法将60只SD大鼠随机分为6组,每组10只,生理盐水对照组(A组),溶剂脂肪乳对照组(B组),丙泊酚组(C组),低剂量DHA+丙泊酚组(D组),中剂量DHA+丙泊酚组(E组),高剂量DHA+丙泊酚组(F组)。丙泊酚组(C组)连续腹腔内注射丙泊酚5天,每天1次,剂量为75 mg/kg/day;A组、B组与C组方法相同,B组剂量为10 ml/kg/day的脂肪乳；A组剂量为10 ml/kg/day的0.9%生理盐水；F组在给予丙泊酚前10天开始每天灌胃给予3g/kg/day的DHA,然后继续给予5天DHA的同时每天腹腔注射75 mg/kg/day的丙泊酚；E组、D组方法同F组。E组、D组分别为1g/kg/day和0.3g/kg/day的DHA。所有大鼠在第1-5天的训练前腹腔注射丙泊酚或脂肪乳或0.9%生理盐水,在第6天的测试前不给予任何药物(注：丙泊酚麻醉大鼠用新生儿血氧饱和度监测,排除因缺氧导致的脑损伤,注射丙泊酚麻醉的大鼠在翻正反射恢复后20分钟进行训练)。定位航行实验：SD大鼠在首次游泳时,一般不易发现隐藏于水下的站台,在120秒内若大鼠仍未发现水池中的站台或者无法登上站台时,将动物引导置于站台上停留30秒,将大鼠从站台上拿下擦干,休息60秒后进行下一次训练,每天固定时间训练4次,连续训练5天,大鼠当日的学习成绩以大鼠当日4次训练潜伏期的平均值来计算。空间探索实验：大鼠进行定位航行试验完毕后24小时,将站台撤走,将大鼠在某个随机选取的相同的入水点放入水中,记录大鼠在120s内的游泳线路,把大鼠在目标象限停留的时间以及在目标象限穿越的次数记录下来,探索大鼠的空间学习规律。定位航行试验结束后将所有大鼠解剖取海马组织并分成两份,一部分用高效液相色谱荧光法检测氨基酸神经递质谷氨酸(GLU)、γ-氨基丁酸(GABA),另一部分用ELISA法进行脑源性神经营养因子(BNDF)、丙二醛(MDA)、超氧化物歧化酶(SOD)与谷胱甘肽过氧化物酶(GSH-Px)检测。结果实验期间各组大鼠皮毛颜色正常,能够自由进食和饮水,未见明显异常病理状态；C组大鼠的活动量相比其它各组有明显的减少,表现为行动迟缓、精神萎靡；各DHA组大鼠也有不同程度的活动量减少。A组与B组大鼠逃逸潜伏期及穿越平台的次数无显著差异(P0.05),与A组大鼠相比,C组大鼠逃逸潜伏期明显延长(P0.01),穿越平台的次数明显减少(P0.01),有显著差异；与C组大鼠相比,E组(P0.05)与F组(P0.01)大鼠的逃逸潜伏期明显缩短(P0.01),并且穿越平台的次数明显增加(P0.01)。显然,随着DHA剂量的增加,D组、E组、F组大鼠的逃逸潜伏期呈下降的趋势,而穿越平台的次数则逐渐增加。A组与B组大鼠无论是谷氨酸和γ-氨基丁酸的含量还是Glu/GABA比值均无显著差异(P0.05)。与A组大鼠相比,C组大鼠谷氨酸含量和Glu/GABA比值显著增加(P0.01),而γ-氨基丁酸的含量显著减少(P0.05)。与C组大鼠相比较, D组(P0.05)、E组(P0.05)与F组(P0.01)大鼠的谷氨酸含量显著降低而γ-氨基丁酸的含量升高(P0.05)。随着DHA剂量的增加,谷氨酸含量逐渐降低而γ-氨基丁酸含量逐渐升高,表现出了一定的剂量依赖性。A组与B组大鼠S0D和GSH-Px的活力无显著差异(P0.05)。与A、B对照组相比,C组大鼠海马组织内SOD和GSH-Px的活力分别下降了35.1%(P0.01)、39.8%(P0.01)。而E组(P0.05)与F组(P0.01)则使S0D与GSH-Px的活力明显增强且随着DHA剂量的增加,DHA的作用明显增强。A组与B组大鼠的MDA浓度无显著差异(P0.05),与A、B对照组大鼠相比,经丙泊酚重复麻醉处理的各组大鼠MDA浓度明显升高(P0.01)。D组(P0.05)、E组(P0.05)、F组(P0.01)的MDA浓度与C组大鼠相比显著降低。A组与B组大鼠的BDNF浓度无显著差异(P0.05),与A组大鼠相比,C组大鼠的BDNF浓度下降了34.7%(P0.05),有显著差异。与C组大鼠相比,D组、E组、F组大鼠给予DHA使得BDNF的浓度分别升高了11.3%(P0.05)、33.9%(P0.05)、47.8%(P0.01),差异有显著性,说明随着DHA剂量的增加,其干预作用逐渐增强。结论DHA通过调节G1u与GABA的浓度平衡,降低氧化应激损伤和增加BDNF的水平从而改善丙泊酚重复麻醉引起的学习与记忆障碍,而且DHA剂量增加,干预作用增强。基于上述研究结果,我们建议DHA可有效的用于丙泊酚重复麻醉导致的学习与记忆障碍的术后治疗,但是需要更多的研究支持这一假设。第二部分远志皂苷对丙泊酚重复麻醉大鼠学习记忆损伤的干预作用目的研究不同剂量远志皂苷对丙泊酚重复麻醉大鼠所导致学习记忆损伤的干预作用及作用机制,为临床使用丙泊酚重复麻醉产生的副作用提供更多的解决方案。方法将60只SD大鼠随机分为6组,生理盐水对照组(A组),溶剂脂肪乳对照组(B组),丙泊酚组(C组),低剂量组(50mg/kg/day)远志皂苷+丙泊酚组(D组),中剂量组(200mg/kg/day)远志皂苷+丙泊酚组(E组),高剂量组(500mg/kg/day)远志皂苷+丙泊酚组(F组)。C组于实验开始第11～15天连续腹腔内注射5天丙泊酚注射液,每天1次,剂量为75mg/kg/day;B组与C组方法相同,给药剂量为10ml/kg/day的脂肪乳；A组同上,连续注射10ml/kg/day的0.9%生理盐水；F组在实验第1-10天(即给予丙泊酚麻醉前10天)开始每天灌胃远志皂苷,然后继续给予5天远志皂苷的同时每天腹腔注射75 mg/kg/day的丙洎酚；E组、D组给予方法同F组。各组大鼠在第16～20天的水迷宫测试前腹腔内注射丙泊酚或脂肪乳或生理盐水,在第21天的测试前不给予任何药物(注：丙泊酚麻醉大鼠用新生儿血氧饱和度监测,排除因缺氧导致的脑损伤,注射丙泊酚麻醉的大鼠在翻正反射恢复后20分钟进行训练)。实验期间观察动物日常状况,并每周称量体重；大鼠的学习记忆情况采用Morris Water Maze (MWM)即水迷宫测定；解剖动物观察脑系数并对海马组织进行组织病理学检查；测定脑细胞MDA、SOD、GSH的活性,观察细胞损伤的程度；提取各组大鼠海马细胞DNA,进行片断化检测；检测大鼠海马组织细胞凋亡相关因子Bcl-2和Caspase-3表达量的情况,观察细胞凋亡情况。结果1.各组大鼠皮毛颜色正常,能够自由进食和饮水,无明显异常病理状态；C组大鼠的活动量相比于A组、B组有明显的减少,表现为行动迟缓、精神萎靡；D组、E组、F组大鼠也有不同程度的活动量减少。各组大鼠3周内的体重逐渐增长,C组、D组、E组、F组与A、B对照组比较未见明显统计学差异(P0.05),但C组大鼠的体重增长有缓慢的趋势。2.定位航行实验和空间探索实验发现,A组与B组大鼠逃逸潜伏期及穿越平台的次数以及目标象限停留时间均无显著差异(P0.05)。与A组比较,C组、D组、E组、F组与A组之间存在显著差异(P0.05),与C组比较,D组、E组、F组差异有显著性(P0.05),提示随着远志皂苷剂量的增加,其潜伏期逐渐缩短,穿越平台的次数增加以及目标象限停留时间增长。3.A组与B组大鼠各氧化损伤指标无显著差异(P0.05)。与A组、B组比较,其余各组大鼠MDA、SOD、GSH有明显差异(P0.05)。与A组比较,C组MDA含量明显增加,SOD含量与GSH浓度显著降低,说明C组大鼠脑细胞氧化损伤严重；与C组比较,D组、E组、F组MDA含量逐渐降低,E组、F组有显著差异(P0.05)；与C组比较,D组、E组、F组SOD、GSH旨标逐渐升高,F组有显著差异(P0.05)。4.A组与B组大鼠脑系数无显著差异(P0.05)。与A组比较,C组大鼠脑系数有明显减小(P0.05),表明C组大鼠脑细胞有一定程度的萎缩；远志皂苷各剂量组大鼠脑系数有少量减少,E组有显著差异(P0.05)。与C组比较,远志皂苷各剂量组脑系数有一定程度的增加,有显著差异(P0.05),F组高于E组与D组。表明低、中、高剂量的远志皂苷均能够一定程度改善丙泊酚重复麻醉大鼠的脑功能,但是仍然低于对照组。5.组织病理学检查发现,A组和B组大鼠海马组织结构清晰,神经细胞排列紧密,细胞染色均匀,其形态完整无水肿；细胞质染色清晰；细胞核呈蓝紫色,核仁清晰可见,细胞核结构完整。C组大鼠海马组织的神经细胞数目明显减少且排列不规整,细胞呈萎缩状态,有浓染现象；部分细胞核核仁不明显,细胞核溶解固缩,大量空泡物质出现在细胞核周围；细胞的细胞质染色不均；在神经细胞周围可见散在的大量炎性细胞。远志皂苷各剂量组与C组比较,海马组织有所改善,可见细胞染色均匀,仅少量细胞形态固缩,排列不规整；细胞核核仁不明显,且细胞核体积增大；细胞质染色尚清晰；在细胞周围可见散在的少量炎性细胞,F组优于E组与D组。6.A组与B组海马细胞DNA电泳都是单条带,表明DNA结构完整；而C组海马细胞DNA电泳出现明显的梯状带表明DNA出现断裂；远志皂苷D、E、F剂量组出现梯状带的数量依次减少,但其数量都低于丙泊酚组。7. ELISA结果显示：A组与B组大鼠凋亡因子表达无显著差异(P0.05)。与A组比较,C组Bcl-2蛋白表达量显著降低(P0.05),Caspase-3表达量显著升高(P0.05)。与C组比较,D组、E组、F组Bc1-2蛋白表达量不同程度的逐渐升高,Caspase-3表达量不同程度的逐渐降低,有显著差异(P0.05)。结论远志皂苷通过降低氧化应激损伤,调控凋亡因子Bcl-2和Caspase-3的表达,减少了细胞凋亡的发生,减轻了海马组织的损伤,增强了大鼠的空间学习记忆能力从而对丙泊酚重复麻醉所导致大鼠学习记忆损伤具有保护作用。
[Abstract]:In this study , 60 SD rats were randomly divided into 6 groups , 10 rats in each group , normal saline control group ( group A ) , solvent fat emulsion control group ( group B ) , propofol group ( group C ) , low - dose DHA + propofol group ( group D ) , middle - dose DHA + propofol group ( group C ) , low - dose DHA + propofol group ( group D ) , middle - dose DHA + propofol group ( group E ) , high - dose DHA + propofol group ( group F ) .
Group A was 0.9 % normal saline at 10 ml / kg / day ;
Group F was given 3 g / kg / day of DHA per day prior to administration of propofol for 10 days , followed by continued administration of 5 days of DHA and 75 mg / kg / day of propofol per day ;
Group E and Group D were treated with propofol or fat emulsion or 0.9 % normal saline at 1 - 5 days . All rats received propofol or fat emulsion or 0.9 % normal saline before training on Day 1 - 5 . No drugs were administered before the 6 - day test ( note : propofol anesthesia rats were trained with neonatal blood oxygen saturation , excluding brain injury due to hypoxia , and 20 minutes after the normal reflex recovery rats were injected ) . The rats were placed on the platform for 30 seconds , the rats were guided on the platform for 30 seconds , the rats were placed on the platform for 30 seconds , the rats were trained 4 times daily , and the rats were trained for 5 days .
Compared with other groups , the activity of the rats in group C decreased significantly compared with other groups .
Compared with group A , the latency of escape latency of group A and group B was significantly prolonged ( P0.01 ) .
Compared with group A , group D ( P0.05 ) , group E ( P0.05 ) and group F ( P0.01 ) showed no significant difference ( P0.05 ) .
In group A , 0.9 % normal saline was injected continuously for 10 ml / kg / day .
Group F was administered daily gavage of hyperoside saponins on day 1 to 10 of the experiment ( i.e . 10 days prior to administration of propofol ) , followed by continued administration of 75 mg / kg / day of propofol per day while continuing to give 5 days of apogenin ;
Group E and Group D were given the same method as group F . Each group received propofol or fat emulsion or physiological saline in the abdominal cavity before the water maze test on Days 16 to 20 . No drug was administered before the first day of test ( note : propofol anesthesia rats used neonatal blood oxygen saturation monitoring to eliminate brain injury due to hypoxia , and rats injected with propofol anesthesia were trained 20 minutes after normal reflex recovery ) . The daily status of animals was observed during the experiment and the body weight was weighed weekly ;
The nucleolus of some nuclei is not obvious , the nucleus dissolves and shrinks , and a lot of vacuoles appear around the nucleus ;
The brain coefficients were observed in the dissected animals and the histopathological examination was performed on the hippocampus .
The activity of MDA , SOD and GSH in brain cells was measured and the extent of cell damage was observed .
extracting the DNA of each group of rat hippocampal cellular DNA and performing fragment detection ;
The expression of apoptosis - related factors Bcl - 2 and Caspase - 3 in hippocampus of rats was detected , and the apoptosis was observed .
Compared with group A and group B , the activity of group C was significantly decreased compared with group A and group B .
Compared with group A , group C , group D , E group , F group and group A had no significant difference ( P0.05 ) . Compared with group A , group C , group D , E group , F group had no significant difference ( P0.05 ) . Compared with group A , group C , group D , E group , F group had no significant difference ( P0.05 ) . Compared with group A , group C , group D , E group , F group had no significant difference ( P0.05 ) . Compared with group A , group C , group D , group E , group F had no significant difference ( P0.05 ) . Compared with group A , group C , group D , group E , group F had no significant difference ( P0.05 ) .
Compared with group C , the content of MDA in group D , E group and F group decreased gradually , and there was significant difference between group E and F ( P0.05 ) .
Compared with group C , there was no significant difference between group D , group E , SOD and GSH of group F ( P0.05 ) . There was no significant difference between group A and group B ( P0.05 ) . Compared with group A , the brain coefficients of group C were significantly decreased ( P0.05 ) .
Compared with group C , there was a significant difference in the brain coefficients ( P0.05 ) and the F group was higher than that in group E and D .
the cytoplasm dyeing is clear ;
The nucleus was blue - purple , the nucleolus was clearly visible , the nucleus structure was intact , the number of nerve cells in the hippocampus of group C was obviously decreased and the arrangement was not regular , the cells were in atrophy state , and there was strong staining phenomenon ;
The learning and memory of rats was determined by Morris Water Maze ( MWM ) , the water maze ;
cell cytoplasm staining was not uniform ;
There was a large amount of inflammatory cells scattered around the nerve cells . Compared with group C , the hippocampal tissue was improved , the cells were stained uniformly , only a small amount of cells were fixed , and the arrangement was irregular ;
Nuclear nucleolus is not obvious , and the nucleus volume increases ;
The cytoplasm dyeing is still clear .
There was a small amount of inflammatory cells scattered around the cells , the F group was superior to that of group E and D . 6 . DNA electrophoresis of hippocampal cells in group B and group B was a single band , indicating that the DNA structure was intact ;
In group C , DNA electrophoresis showed that the DNA appeared to be fractured .
The number of ladder - like bands in D , E and F groups decreased in turn , but the number was lower than that of propofol group . Compared with group A , the expressions of Bcl - 2 and Caspase - 3 in group C and group C were significantly decreased ( P0.05 ) .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R614

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