夏天无对脑梗死大鼠NOS、iNOS和NSCs、SYN表达影响的实验研究
摘要
目的：本实验旨在通过建立大鼠局灶性脑梗死模型,检测大鼠血清中一氧化氮合酶(NOS),诱导型一氧化氮合酶(iNOS)的表达,常规HE染色观察大鼠海马区的形态学改变,免疫组织化学法检测神经干细胞(NSCs)和突触素(SYN)阳性表达,评价夏天无对脑梗死大鼠模型的改善情况,探讨夏天无对大鼠脑梗死后脑可塑性的影响,以期为临床提供实验依据。
方法：(1)造模：参照文献采用栓线法制作大鼠永久性大脑中动脉闭塞模型。(2)分组：造模成功的大鼠采用随机数字表法分为4组,分别为模型组,夏天无低剂量组,夏天无高剂量组,维脑路通组,另设空白组及假手术组。(3)给药：夏天无低剂量组给予夏天无2.4g.kg-1.d-1灌胃,夏天无高剂量组给予夏天无9.6g.kg-1.d-1灌胃,维脑路通组予维脑路通144mg.kg-1.d-1灌胃。模型组、空白组及假手术组予生理盐水6ml.kg-1.d-1灌胃,连续7天。(4)5—溴脱氧尿嘧啶(BrdU)标记：各组大鼠造模成功12H后1-7d给予腹腔注射BrdU生理盐水(10mg/ml),30 mg.kg-1.d-1,每日2次,间隔8H。(5)指标测定：紫外分光光度仪测大鼠血清中NOS、iNOS的吸光度,计算血清NOS、iNOS活力;常规HE染色观察大鼠海马区的形态学改变,免疫组织化学法检测BrdU标记的NSCs和SYN阳性表达。
结果：(1)HE检测病理变化：空白组、假手术组未发现明显病理变化;模型组可见结构松散,神经细胞变形,胞核固缩;各治疗组大部分神经细胞形态及结构接近正常,偶见细胞胞体变小,胞核固缩。(2)NOS、iNOS检测：与空白组、假手术组相比,模型组NOS、iNOS活性均明显增强(P<0.05),各组治疗组与模型组相比,NOS、iNOS活性均降低,有差异显著性(P<0.05),治疗组间比较差异无统计学意义(P>0.05)。(4)脑可塑性相关检测：NSCs和SYN。空白组和假手术组NSCs、SYN有少量表达；与空白组及假手术组比较,模型组NSCs、SYN表达变化显著(P<0.05)；各治疗组较模型组NSCs、SYN表达显著增强(P<0.05)；各治疗组间差异无统计学意义(P>0.05)。
结论：(1)采用栓线法制作大鼠局灶性脑梗死模型,有较好的可操作性和稳定性,是理想的造模方法之一。(2)夏天无可改善脑梗死模型大鼠脑组织的病理变化,提示夏天无可能具有防治脑梗死的作用。(3)夏天无可抑脑梗死大鼠NOS、iNOS的活性,提示夏天无可干扰损伤级联反应,并间接减少NO神经毒性,抑制神经细胞凋亡或死亡,为神经修复提供基础。(4)夏天无增强NSCs的阳性表达,抑制SYN下降,促进神经功能的恢复及突触的重建和联系,可能是夏天无促进脑可塑的机制之一。(5)夏天无与维脑路通对脑梗死的干预作用等效,且无剂量依赖性。
Objective:Detecting the expression of nitric oxide synthase(NOS)、inducible nitric oxide synthase (iNOS) in blood serum of rats and the pathological of the hippocampus by hematoxylin and eosin (HE) and content of neural stem cells(NSCs)、synaptophysin (SYN) in the hippocampus tissue of rats that corydalis decumbens improved the situation of middle cerebral artery occlusion(MCAO) model in rats. Exploring the effects that corydalis decumbens whether could contribute to the Brain Plasticity in rats. Provide the experimental basis for corydalis decumbens clinically.
Methods:(1)Model:cerebral ischemic(CI) model was formed by adopting the MCAO method using intralumina thread in health male Whister rats. (2)Groop:According to the literature using random number table this model success were randomly divided into four groups:model group, high dose of Corydalis decumbens group, low dose of Corydalis decumbens group group, and Hydroxyethylrutin group ; setting up a blank group and sham model group. (3) Delivery methods:For 7 days, blank group、sham model group and group model group were intragastrically given to rats by a dose of 6ml/kg/d physiological saline. While the rats in low dose of Corydalis decumbens group, high dose of Corydalis decumbens group group, and Hydroxyethylrutin group were intragastically given different medicine:to. 2.4g/kg/d、9.6g/kg/d Corydalis decumbens and 144mg/kg/d Hydroxyethylrutin.. (4)5—bromodeoxyuridine(BrdU) labeled:Every groups rats was injected intraperitoneally BrdU physiological saline (30mg/kg/d) 2 times at 8—hour intervals on day 7. (5)Detect indicators: Measured blood serum NOS、iNOS absorbance of NOS、iNOS activity was by spectrophotometry. HE staining of pathological changes of the hippocampus. 5—bromodeoxyuridine labeling protocols and immunohistochemistry detection of rat hippocampal BrdU and SYN protein expression was observed.
Results:(1) HE pathological of changes:blank group and sham model group had no obvious pathologica changes; Model group has loose framework, never cells cytomorphosis, karyopycnosis; Three therapeutics groups has a bulk of normal morphous and formation of never cells,small cells cytomorphosis and karyopycnosis. (2) NOS and iNOS detected:blank group and sham model group can reduce the activity of NOS and iNOS compared with the model group(P <0.05); treatment groups can reduce the activity of NOS and iNOS compared with mode group(P< 0.05), but no difference between the three treatment groups significantly (P>0.05). (3) About the Brain Plasticity detected:NSCs和SYN. blank group and sham model group had rarely NSCs和SYN expression, Another groups more NSCs和SYN differences expression(P< 0.05).Three reatment groups had more significant rises in NSCs和SYN expression (P<0.05). but no difference among treatment group significantly (P>0.05)
Conclusions:(1) MCAO method using intralumina thread had well operation and stability.It was a perfectly model for CI model.(2) Corydalis decumbens improved the pathological change of the hippocampus of the CI model. Corydalis decumbens can prevent and treatment CI model. (3) Corydalis decumbens can reduce the activity of NOS and iNOS, to decrease NO.It can be see that Corydalis decumbens can lesse neurotoxicity by NO, blocking cascade reaction to protect nevers cytoprotection. It was problely that Corydalis decumbens should enhance the cerebral plasticity. (4) Corydalis decumbens can increase the NSCs and hold-back the declined expression of SYN in hippocampus. It could promote the synaptic re-establishment and connection. Corydalis decumbens may contribute to the cerebral plasticity. (5) Corydalis as well as Hydroxyethylrutin can contribute to the cerebral ischemic. It'cure independency of dose.
方法：(1)造模：参照文献采用栓线法制作大鼠永久性大脑中动脉闭塞模型。(2)分组：造模成功的大鼠采用随机数字表法分为4组,分别为模型组,夏天无低剂量组,夏天无高剂量组,维脑路通组,另设空白组及假手术组。(3)给药：夏天无低剂量组给予夏天无2.4g.kg-1.d-1灌胃,夏天无高剂量组给予夏天无9.6g.kg-1.d-1灌胃,维脑路通组予维脑路通144mg.kg-1.d-1灌胃。模型组、空白组及假手术组予生理盐水6ml.kg-1.d-1灌胃,连续7天。(4)5—溴脱氧尿嘧啶(BrdU)标记：各组大鼠造模成功12H后1-7d给予腹腔注射BrdU生理盐水(10mg/ml),30 mg.kg-1.d-1,每日2次,间隔8H。(5)指标测定：紫外分光光度仪测大鼠血清中NOS、iNOS的吸光度,计算血清NOS、iNOS活力;常规HE染色观察大鼠海马区的形态学改变,免疫组织化学法检测BrdU标记的NSCs和SYN阳性表达。
结果：(1)HE检测病理变化：空白组、假手术组未发现明显病理变化;模型组可见结构松散,神经细胞变形,胞核固缩;各治疗组大部分神经细胞形态及结构接近正常,偶见细胞胞体变小,胞核固缩。(2)NOS、iNOS检测：与空白组、假手术组相比,模型组NOS、iNOS活性均明显增强(P<0.05),各组治疗组与模型组相比,NOS、iNOS活性均降低,有差异显著性(P<0.05),治疗组间比较差异无统计学意义(P>0.05)。(4)脑可塑性相关检测：NSCs和SYN。空白组和假手术组NSCs、SYN有少量表达；与空白组及假手术组比较,模型组NSCs、SYN表达变化显著(P<0.05)；各治疗组较模型组NSCs、SYN表达显著增强(P<0.05)；各治疗组间差异无统计学意义(P>0.05)。
结论：(1)采用栓线法制作大鼠局灶性脑梗死模型,有较好的可操作性和稳定性,是理想的造模方法之一。(2)夏天无可改善脑梗死模型大鼠脑组织的病理变化,提示夏天无可能具有防治脑梗死的作用。(3)夏天无可抑脑梗死大鼠NOS、iNOS的活性,提示夏天无可干扰损伤级联反应,并间接减少NO神经毒性,抑制神经细胞凋亡或死亡,为神经修复提供基础。(4)夏天无增强NSCs的阳性表达,抑制SYN下降,促进神经功能的恢复及突触的重建和联系,可能是夏天无促进脑可塑的机制之一。(5)夏天无与维脑路通对脑梗死的干预作用等效,且无剂量依赖性。
Objective:Detecting the expression of nitric oxide synthase(NOS)、inducible nitric oxide synthase (iNOS) in blood serum of rats and the pathological of the hippocampus by hematoxylin and eosin (HE) and content of neural stem cells(NSCs)、synaptophysin (SYN) in the hippocampus tissue of rats that corydalis decumbens improved the situation of middle cerebral artery occlusion(MCAO) model in rats. Exploring the effects that corydalis decumbens whether could contribute to the Brain Plasticity in rats. Provide the experimental basis for corydalis decumbens clinically.
Methods:(1)Model:cerebral ischemic(CI) model was formed by adopting the MCAO method using intralumina thread in health male Whister rats. (2)Groop:According to the literature using random number table this model success were randomly divided into four groups:model group, high dose of Corydalis decumbens group, low dose of Corydalis decumbens group group, and Hydroxyethylrutin group ; setting up a blank group and sham model group. (3) Delivery methods:For 7 days, blank group、sham model group and group model group were intragastrically given to rats by a dose of 6ml/kg/d physiological saline. While the rats in low dose of Corydalis decumbens group, high dose of Corydalis decumbens group group, and Hydroxyethylrutin group were intragastically given different medicine:to. 2.4g/kg/d、9.6g/kg/d Corydalis decumbens and 144mg/kg/d Hydroxyethylrutin.. (4)5—bromodeoxyuridine(BrdU) labeled:Every groups rats was injected intraperitoneally BrdU physiological saline (30mg/kg/d) 2 times at 8—hour intervals on day 7. (5)Detect indicators: Measured blood serum NOS、iNOS absorbance of NOS、iNOS activity was by spectrophotometry. HE staining of pathological changes of the hippocampus. 5—bromodeoxyuridine labeling protocols and immunohistochemistry detection of rat hippocampal BrdU and SYN protein expression was observed.
Results:(1) HE pathological of changes:blank group and sham model group had no obvious pathologica changes; Model group has loose framework, never cells cytomorphosis, karyopycnosis; Three therapeutics groups has a bulk of normal morphous and formation of never cells,small cells cytomorphosis and karyopycnosis. (2) NOS and iNOS detected:blank group and sham model group can reduce the activity of NOS and iNOS compared with the model group(P <0.05); treatment groups can reduce the activity of NOS and iNOS compared with mode group(P< 0.05), but no difference between the three treatment groups significantly (P>0.05). (3) About the Brain Plasticity detected:NSCs和SYN. blank group and sham model group had rarely NSCs和SYN expression, Another groups more NSCs和SYN differences expression(P< 0.05).Three reatment groups had more significant rises in NSCs和SYN expression (P<0.05). but no difference among treatment group significantly (P>0.05)
Conclusions:(1) MCAO method using intralumina thread had well operation and stability.It was a perfectly model for CI model.(2) Corydalis decumbens improved the pathological change of the hippocampus of the CI model. Corydalis decumbens can prevent and treatment CI model. (3) Corydalis decumbens can reduce the activity of NOS and iNOS, to decrease NO.It can be see that Corydalis decumbens can lesse neurotoxicity by NO, blocking cascade reaction to protect nevers cytoprotection. It was problely that Corydalis decumbens should enhance the cerebral plasticity. (4) Corydalis decumbens can increase the NSCs and hold-back the declined expression of SYN in hippocampus. It could promote the synaptic re-establishment and connection. Corydalis decumbens may contribute to the cerebral plasticity. (5) Corydalis as well as Hydroxyethylrutin can contribute to the cerebral ischemic. It'cure independency of dose.
引文
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[9]邓湘平,顾振纶,谢梅林.夏天无总生物碱对东莨菪碱及D-半乳糖所致大鼠学习记忆障碍的影响.中草药.2003.34(4)：350～352
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[1]Davenport R, Dennis M. Neurological emegrencies:acute stroke. J Neurol Neurosurg Psychiaty 2000.68(3):277-288
[2]马国义,张子昭,陈植和.普鲁托品对家兔血小板超微结构的影响.昆明医学院学报.1995,16(4)：1-4
[3]高健,王天佑,何相好,等.夏天无总碱抑制血小板聚集作用的研究.苏州大学学报(医学版).2004,24(2)：137-140
[4]高健,顾振纶,王天佑.夏天无总碱对大鼠实验性脑血栓形成的影响.中国血液流变学杂志.2003.13(4)325～326
[5]Nawashiro H,Martin D, Hallenbeck J M, et al Neuroprotective effects of TNF binding protein in focal cerebral ischemia. Brain Res.1997.778(2):265～267
[6]张国玺,石京山,余丽梅,等.夏天无注射液对大鼠脑缺血再灌注损伤及TNF-α及L-6mRNA表达影响.中成药.2008.30(8)：1222-1224
[7]何晓南,周俐,胡晓,等.夏天无注射液抗炎实验研究.赣南医学院学报.1998.18.(6)：103-105
[8]余丽梅,于爽,龚其海,等.夏天无注射液对血管性痴呆大鼠海马血管生成素-1表达的影响.中成药.2006.28(6)：839～842
[9]邓湘平,顾振纶,谢梅林.夏天无总生物碱对东莨菪碱及D-半乳糖所致大鼠学习记忆障碍的影响.中草药.2003.34(4)：350～352
[10]张熠,顾振纶,蒋小岗.夏天无总碱提取物对痴呆大鼠脑内5-HT和DA含量的影响.苏州大学学报：医学版.2004,24(2)：134-136
[11]张慧灵,顾振纶,曹奕.夏天无总生物碱对痴呆大鼠学习记忆障碍及中枢胆碱能神经系统功能的影响.中国药理学通报.2004.20(10)：1158-1160
[12]盛瑞,顾振纶,蒋航,等.夏天无对小鼠学习及脑内乙酰胆碱脂酶的影.中草药,2003.34(6).543-545
[13]余丽梅,文国容,邓江等.夏天无注射液对小鼠脑缺血和神经元保护作用的实验研究.上海中医杂志.2006.40(9)
[14]吴二兵,胡雪勇,孙安盛,等.夏天无、钩藤生物碱抗脑细胞凋亡作用及急性毒性.遵义医学院学报.2004,27(5)：444-446
[15]杨旭明.复方夏天无片治疗缺血性脑血管病30例临床疗效观察.国际医药卫生导报.2006,12,(19)：61-63
[16]周华.针药结合治疗脑血管意外后遗症178例.吉林中医药.1994,(2)：19
[17]徐新初.穴位注射治疗动脉硬化性脑梗塞64例疗效观察.中国针灸.1989,9(2)：10.
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