新生大鼠缺氧缺血性脑损伤后NPTX1表达及葛根素脑保护作用
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摘要
目的：研究新生大鼠缺氧缺血性脑损伤(HIBD)后神经元五聚体蛋白1表达变化,探讨葛根素脑保护作用的部分机制,监测新生大鼠HIBD后应用葛根素对血糖变化的进一步影响,以期为临床治疗新生儿HIBD提供一定参考。
方法
1.参照Rice法制备HIBD模型：新生7日龄SD大鼠行颈部正中切口,分离结扎左侧颈总动脉后,予0.5L/min的速度输入湿化8%氧气和92%氮气混合气体处理2小时,同时观察新生大鼠的行为能力变化。
2.行为能力检查：分别于手术前、缺氧处理后、第一次处理后24h、48h对每只新生大鼠进行翻身能力、夹尾左旋测试。
3.脑大体形态检查：分别于处死时观察每只新生大鼠脑组织的大体形态。
4.NPTX1基因表达测定：48只新生7日龄SD大鼠随机分为假手术组、模型组及葛根素组。Rice法制备新生大鼠HIBD模型,缺氧缺血2h后,葛根素组100mg/kg腹腔内注射给药,假手术组、HIBD模型组给予等容量生理盐水,间隔24h、48h各组再次给予相同处理,各时间点最后处理2h后各组断头取脑,脑组织放于corning冻存管中,放入液氮罐,后于-80℃冰箱中保存,用于荧光定量PCR检测NPTX1。
5.血糖监测：分别于术前、模型制作后、第一次处理后4h及第二次处理后4h监测新生大鼠血糖变化。
结果：
1.行为能力检查：实验中对新生大鼠进行四次行为能力评估：HIBD模型制作前、HIBD模型制作后、第一次处理后24h、48h。术前每只新生大鼠均做翻身能力、夹尾左旋测试,48只全部正常。经历缺氧缺血的32只大鼠,其中12只翻身不能,5只出现夹尾左旋,15只表现为翻身不能与夹尾左旋同时存在。第一次处理后24h行为评估：葛根素应用组16例有10例翻正反射恢复；模型组16例8例翻正反射恢复。葛根素应用-48h后对新生大鼠进行翻正反射检测：HIBD组、葛根素组翻正反射恢复,但所需时间HIBD组较长,7.7±1.7秒,葛根素组3.8±1.1秒,差异有统计学意义。应用葛根素可使HIBD新生大鼠翻正反射所需时间缩短。假手术组在各时间段行为评估均正常。
2.脑大体形态检查：新生大鼠HIBD模型组左侧结扎半球脑组织受损严重,24h时苍白、水肿、体积大于对侧;48h可看见结扎半球局部淤血、坏死；葛根素应用组上述改变轻微,水肿消退明显；sham组大鼠大脑半球左右对称,未见明显大体形态变化。
3. NPTX1基因表达检测结果：新生大鼠HIBD后NPTX1表达增强,HIBD模型组及假手术组NPTX1相对表达量24h中位数及四分位数间距分别为247.03(138.28-271.55)与4.60(3.16-14.07)：48h为87.03(79.45-94.83)与1.49(1.00-1.89)。新生大鼠HIBD后NPTX1表达量随时间呈下降趋势。葛根素可降低缺血区脑组织NPTX1mRNA表达,24h为45.70(44.41-56.28)：48h为24.08(23.09-25.50)(P均<0.05)。
4.新生大鼠HIBD后短期应用葛根素对血糖变化影响：模型制作前各组血糖差异无统计学意义,血糖均在正常范围内;模型制作1h后各组血糖值差异虽无统计学意义,但HIBD组、葛根素组高血糖例数均为6例,低血糖各1例,而假手术组血糖全部在正常范围。第一次处理后4h,各组血糖值差异具有统计学意义。HIBD组7例高血糖,1例正常；葛根素组6例高血糖,2例正常。第二次处理后4h各组血糖值差异不再具有统计学意义,三组血糖分别为5.87±0.95mmol/L；6.76±0.40mmol/L；6.82±1.02mmol-L F3.19(P>0.05)。HIBD组与葛根素组均为2例高血糖,6例正常。新生大鼠HIBD后主要发生高血糖,低血糖发生较少,与临床研究相近,说明HIBD模型制备与临床新生儿HIBD发病损伤程度接近。新生大鼠血糖正常值范围为5mmol/L～7mmol/L。
结论：
1.新生大鼠HIBD模型制作成功。
2. NPTX1参与了新生大鼠HIBD发病机制,葛根素可通过降低缺血区脑组织NPTX1表达发挥其脑保护作用。
3.短期应用葛根素可使HIBD新生大鼠翻正反射时间缩短。
4.短期应用葛根素不会对新生大鼠HIBD后血糖变化产生进一步影响。
Objective To investigate the effects of puerarin on the expression of NPTX1in neonatal rats with HIBD and explore its possible protective mechanism of brain.at the same time,detect the changes of blood glucose of neonatal rats with HIBD after applying with Puerarin in order to provide some references on studing and researching in the relative investigations.
Methods
1. Establish HIBD model according to Rice:Neonatal Seven-day-old Sprague-Dawley (SD) rats were subjected to a middle anterior incision.The left common carotid artery was isolated and ligated, pups were then exposed to hypoxic environment, with constant flow0.5L/min of humidified8%O2balanced with92%N2for2hour, and the behavior of the rats was observed during hypoxia.
2. Evaluate neurofunction of the rats:righting reflex and levorotary ability when their tails were gripped were observed before the experiment,1h after HI and24h、48h after the first i.p. therapy.
3. General examination of the brain:observe of the brain after HI.
4. Assay the expression of NPTX1using real-time RT PCR.48Seven-day-old Sprague-Dawley (SD) rats were divided randomly into sham control. HIBD and puerarin-treated group. Puerarin100mg/kg was intraperitoneally injected to puerarin group1h、24h and48h after HI respectively,equal volum NS was injected to the other groups. The rats were sacrificed24h and48h after operation. mRNA expression of NPTX1were evaluated by real time RT-PCR analysis.
5. Detect the changes of blood glucose of neonatal rats before experiment,1h after HI,4h after the first treatment and4h after the second treatment.
Results
1. Neurofunction evaluation of the rats:before the experiment all rats were healthy.the rats in sham group were healthy at the other evaluation point,while the righting reflexs of12rats were positive,5rats became levorotary when their tails were gripped and15rats exhibited both behavior problems in the32rats which bared HI injury. After the first treatment, righting reflex of10out of16in puerarin group and8out of16in HIBD group recovered. Applying puerarin for a short time after HIBD could shorten the time of righting reflex.
2. General examination of the brain:The ligated brain hemisphere of HIBD group showed pallor and edema at24h and necrosis lesion at48h after HI. Edema was not serious and disappeared quickly in the Puerarin-treated group.The two brain hemispheres of sham group were symmetric and showed no significant change.
3. The expression of NPTX1were evaluated by real time RT-PCR analysis Compared with sham control, the expression of NPTX1in ischemic brain tissue after HIBD was markedly enhanced. Median and25th-75th percentile:24h247.03(138.28-271.55)vs4.60(3.16-14.07);48h87.03(79.45-94.83)vsl.49(1.00-1.89). Puerarin significantly reduced the expression of NPTX1in ischemic brain tissue after HIBD. Median and25th-75th percentile:24h45.70(44.41-56.28);48h24.08(23.09-25.50)(P＜0.05).
4. There were no statistic differences in blood glucose among groups before experiment, the blood glucose of all rats were in normal range.The differences of blood glucose among groups1h after HI had no statistic significance, but there were6hyperglycemia and1hypoglycemia in puerarin group and HIBD group respectively,blood glucose of rats in sham group were all normal. Statistic differences were existed among groups4h after the first treatment.7out of8in HIBD group and6out of8in puerarin group were hyperglycemia. There were no statistic differences among groups4h after the second treatment.The blood glucose reference of neonatal rats was:5mml/L～7mmol/L.
Conclusions
1. The HIBD model was established successfully.
2. NPTX1played an important role in HIBD, Puerarin attenuate HIBD by down-regulation the expression of NPTX1in ischemic brain tissue.
3. Applying puerarin after HIBD could shorten-the-time-of righting reflex
4. Applying puerarin for a short time after HIBD could not affect the changes of blood glucose further.
方法
1.参照Rice法制备HIBD模型：新生7日龄SD大鼠行颈部正中切口,分离结扎左侧颈总动脉后,予0.5L/min的速度输入湿化8%氧气和92%氮气混合气体处理2小时,同时观察新生大鼠的行为能力变化。
2.行为能力检查：分别于手术前、缺氧处理后、第一次处理后24h、48h对每只新生大鼠进行翻身能力、夹尾左旋测试。
3.脑大体形态检查：分别于处死时观察每只新生大鼠脑组织的大体形态。
4.NPTX1基因表达测定：48只新生7日龄SD大鼠随机分为假手术组、模型组及葛根素组。Rice法制备新生大鼠HIBD模型,缺氧缺血2h后,葛根素组100mg/kg腹腔内注射给药,假手术组、HIBD模型组给予等容量生理盐水,间隔24h、48h各组再次给予相同处理,各时间点最后处理2h后各组断头取脑,脑组织放于corning冻存管中,放入液氮罐,后于-80℃冰箱中保存,用于荧光定量PCR检测NPTX1。
5.血糖监测：分别于术前、模型制作后、第一次处理后4h及第二次处理后4h监测新生大鼠血糖变化。
结果：
1.行为能力检查：实验中对新生大鼠进行四次行为能力评估：HIBD模型制作前、HIBD模型制作后、第一次处理后24h、48h。术前每只新生大鼠均做翻身能力、夹尾左旋测试,48只全部正常。经历缺氧缺血的32只大鼠,其中12只翻身不能,5只出现夹尾左旋,15只表现为翻身不能与夹尾左旋同时存在。第一次处理后24h行为评估：葛根素应用组16例有10例翻正反射恢复；模型组16例8例翻正反射恢复。葛根素应用-48h后对新生大鼠进行翻正反射检测：HIBD组、葛根素组翻正反射恢复,但所需时间HIBD组较长,7.7±1.7秒,葛根素组3.8±1.1秒,差异有统计学意义。应用葛根素可使HIBD新生大鼠翻正反射所需时间缩短。假手术组在各时间段行为评估均正常。
2.脑大体形态检查：新生大鼠HIBD模型组左侧结扎半球脑组织受损严重,24h时苍白、水肿、体积大于对侧;48h可看见结扎半球局部淤血、坏死；葛根素应用组上述改变轻微,水肿消退明显；sham组大鼠大脑半球左右对称,未见明显大体形态变化。
3. NPTX1基因表达检测结果：新生大鼠HIBD后NPTX1表达增强,HIBD模型组及假手术组NPTX1相对表达量24h中位数及四分位数间距分别为247.03(138.28-271.55)与4.60(3.16-14.07)：48h为87.03(79.45-94.83)与1.49(1.00-1.89)。新生大鼠HIBD后NPTX1表达量随时间呈下降趋势。葛根素可降低缺血区脑组织NPTX1mRNA表达,24h为45.70(44.41-56.28)：48h为24.08(23.09-25.50)(P均<0.05)。
4.新生大鼠HIBD后短期应用葛根素对血糖变化影响：模型制作前各组血糖差异无统计学意义,血糖均在正常范围内;模型制作1h后各组血糖值差异虽无统计学意义,但HIBD组、葛根素组高血糖例数均为6例,低血糖各1例,而假手术组血糖全部在正常范围。第一次处理后4h,各组血糖值差异具有统计学意义。HIBD组7例高血糖,1例正常；葛根素组6例高血糖,2例正常。第二次处理后4h各组血糖值差异不再具有统计学意义,三组血糖分别为5.87±0.95mmol/L；6.76±0.40mmol/L；6.82±1.02mmol-L F3.19(P>0.05)。HIBD组与葛根素组均为2例高血糖,6例正常。新生大鼠HIBD后主要发生高血糖,低血糖发生较少,与临床研究相近,说明HIBD模型制备与临床新生儿HIBD发病损伤程度接近。新生大鼠血糖正常值范围为5mmol/L～7mmol/L。
结论：
1.新生大鼠HIBD模型制作成功。
2. NPTX1参与了新生大鼠HIBD发病机制,葛根素可通过降低缺血区脑组织NPTX1表达发挥其脑保护作用。
3.短期应用葛根素可使HIBD新生大鼠翻正反射时间缩短。
4.短期应用葛根素不会对新生大鼠HIBD后血糖变化产生进一步影响。
Objective To investigate the effects of puerarin on the expression of NPTX1in neonatal rats with HIBD and explore its possible protective mechanism of brain.at the same time,detect the changes of blood glucose of neonatal rats with HIBD after applying with Puerarin in order to provide some references on studing and researching in the relative investigations.
Methods
1. Establish HIBD model according to Rice:Neonatal Seven-day-old Sprague-Dawley (SD) rats were subjected to a middle anterior incision.The left common carotid artery was isolated and ligated, pups were then exposed to hypoxic environment, with constant flow0.5L/min of humidified8%O2balanced with92%N2for2hour, and the behavior of the rats was observed during hypoxia.
2. Evaluate neurofunction of the rats:righting reflex and levorotary ability when their tails were gripped were observed before the experiment,1h after HI and24h、48h after the first i.p. therapy.
3. General examination of the brain:observe of the brain after HI.
4. Assay the expression of NPTX1using real-time RT PCR.48Seven-day-old Sprague-Dawley (SD) rats were divided randomly into sham control. HIBD and puerarin-treated group. Puerarin100mg/kg was intraperitoneally injected to puerarin group1h、24h and48h after HI respectively,equal volum NS was injected to the other groups. The rats were sacrificed24h and48h after operation. mRNA expression of NPTX1were evaluated by real time RT-PCR analysis.
5. Detect the changes of blood glucose of neonatal rats before experiment,1h after HI,4h after the first treatment and4h after the second treatment.
Results
1. Neurofunction evaluation of the rats:before the experiment all rats were healthy.the rats in sham group were healthy at the other evaluation point,while the righting reflexs of12rats were positive,5rats became levorotary when their tails were gripped and15rats exhibited both behavior problems in the32rats which bared HI injury. After the first treatment, righting reflex of10out of16in puerarin group and8out of16in HIBD group recovered. Applying puerarin for a short time after HIBD could shorten the time of righting reflex.
2. General examination of the brain:The ligated brain hemisphere of HIBD group showed pallor and edema at24h and necrosis lesion at48h after HI. Edema was not serious and disappeared quickly in the Puerarin-treated group.The two brain hemispheres of sham group were symmetric and showed no significant change.
3. The expression of NPTX1were evaluated by real time RT-PCR analysis Compared with sham control, the expression of NPTX1in ischemic brain tissue after HIBD was markedly enhanced. Median and25th-75th percentile:24h247.03(138.28-271.55)vs4.60(3.16-14.07);48h87.03(79.45-94.83)vsl.49(1.00-1.89). Puerarin significantly reduced the expression of NPTX1in ischemic brain tissue after HIBD. Median and25th-75th percentile:24h45.70(44.41-56.28);48h24.08(23.09-25.50)(P＜0.05).
4. There were no statistic differences in blood glucose among groups before experiment, the blood glucose of all rats were in normal range.The differences of blood glucose among groups1h after HI had no statistic significance, but there were6hyperglycemia and1hypoglycemia in puerarin group and HIBD group respectively,blood glucose of rats in sham group were all normal. Statistic differences were existed among groups4h after the first treatment.7out of8in HIBD group and6out of8in puerarin group were hyperglycemia. There were no statistic differences among groups4h after the second treatment.The blood glucose reference of neonatal rats was:5mml/L～7mmol/L.
Conclusions
1. The HIBD model was established successfully.
2. NPTX1played an important role in HIBD, Puerarin attenuate HIBD by down-regulation the expression of NPTX1in ischemic brain tissue.
3. Applying puerarin after HIBD could shorten-the-time-of righting reflex
4. Applying puerarin for a short time after HIBD could not affect the changes of blood glucose further.
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