电针不同穴组对急性脑缺血大鼠治疗作用比较及相关机理探讨
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摘要
目的:通过观察电针“督脉取穴法”(人中、百会穴),“背俞穴取穴法”(肝俞、肾俞穴),“阳明经取穴法”(曲池、足三里穴)对大鼠大脑中动脉阻塞(MCAO)脑缺血模型神经行为学评分、脑梗死体积,神经元密度的影响,比较三种取穴法的针刺效应,并从一氧化氮(NO)代谢、内皮素(ET)及神经肽降钙素基因相关肽(CGRP)、神经肽Y(NPY)和NPY基因表达的角度探讨电针抗急性脑缺血损伤的作用机理。
方法:将雄性SD大鼠75只随机分为5组,即:假手术组,模型组,人中、百会组,肝俞、肾俞组,曲池、足三里组,每组15只。建立MCAO模型,采用TTC染色法测脑梗死体积;免疫组化及腿染色法测病灶侧海马CA1区、皮层Ⅲ-Ⅳ层神经元密度;硝酸还原酶法测NO;比色法测一氧化氮合酶(NOS);放射免疫法测ET、CGRP、NPY;RT-PCR半定量分析法测NPYmRNA的表达。
结果:1.大鼠MCAO急性脑缺血72h后,与假手术组比较:神经功能障碍进行性加重;出现病灶侧脑梗死,海马CA1区及皮层Ⅲ-Ⅳ层神经元密度降低;血及脑组织NO、NOS、ET、NPY含量明显升高,而CGRP含量明显降低,(P＜0.05或P＜0.01)。
2.大鼠MCAO急性脑缺血72h后,针刺各组与模型组比较:电针人中、百会组及肝俞、肾俞组对行为学评分有不同程度的改善,并在缺血48h后产生效应;脑梗死体积缩小;海马CA1区及皮层Ⅲ～Ⅳ层神经元密度增加(P＜0.05或P＜0.01);血清NO及脑组织NOS含量降低(P＜0.05或P＜0.01),但对血清NOS及脑组织NO无明显影响(P＞0.05);血浆NPY,脑组织NPY及ET含量降低,但对血浆ET含量无明显影响(P＞0.05);血浆及脑组织CGRP含量升高(P＜0.05或P＜0.01)。两组之间上述结果比较,差异无统计学意义(P＞0.05)。而电针曲池、足三里组对上述指标均无明显影响(P＞0.05)。
3.大鼠MCAO急性脑缺血72h后,与假手术组比较:脑组织NPYmRNA相对密度值明显升高(P＜0.01)。针刺各组与模型组比较:电针人中、百会组及肝俞、肾俞组使脑组织NPYmRNA相对密度值明显降低(P＜0.05);两组之间比较,差异无统计学意义(P＞0.05)。而电针曲池、足三里组对其无明显影响(P＞0.05)。
结论:1.大鼠MCAO急性脑缺血72h后,血及脑组织NO、NOS、ET及NPY含量明显升高,而CGRP含量明显降低,提示上述物质的异常变化与急性脑缺血损伤的病理机制有关。
2.电针人中、百会组及肝俞、肾俞组能改善行为学评分(缺血48h后产生效应),缩小脑梗死体积,增加海马CA1区及皮层Ⅲ～Ⅳ层神经元密度,对大鼠MCAO急性脑缺血损伤有保护作用。抑制NO、NOS、ET、NPY及促进CGRP合成、分泌与释放可能是其保护机制之一,且两穴组针刺效应相近。
3.电针曲池、足三里组对大鼠MCAO急性脑缺血损伤无明显影响,可能因“治痿独取阳明”,上述2穴只适合作用于脑缺血损伤的恢复及后遗症期有关,有待进一步验证。
4.抑制NPYmRNA在病灶侧脑组织的表达可能是电针人中、百会组和肝俞、肾俞组对急性脑缺血损伤保护作用的分子机制之一。
Objective:By electro-acupuncturing renzhong,Baihui points of Du Meridian, Ganshu,Shenshu points of Beishu points,Quchi,Zusanli points of Yangrning Meridians,we compared their acupuncturing effects on the volume of cerebral infarction,neuron density of the model rats with the middle cerebral artery occlusion(MCAO),by analyzing NO metabolism,ET and neuropeptide CGRP, NPY and NPY gene express of model rats with MCAO,we explored the mechanism of electro-acupuncturing treating the acute cerebral ischemia injury.
Methods:75 male SD rats were randomly divided five groups.i.e., pseudo-operation group,model group,Renzhong、Baihui group,Ganshu、Shenshu group and Quchi、Zusanli group.Each group had 15 rats.To establish the MCAO model,we used TTC manipulation experience to measure the cerebral infarction's volume,immunohistochemistry and HE manipulation experience to evaluate neuron density of Hippocampus CA1 area and cerebral cortexⅢ-Ⅳ, nitrate reductase to detect NO,colorinetric assay to inspect NOS, radioimmunoassay to check ET、CGRP and NPY;RT-PCR to examine NPYmRNA express.
Results:1.After rats with MCAO had acute cerebral ischemia for 72 hours, compared with pseudo-operation group,we found nerve malfunction progressively accerated,cerebral infarction appeared in the side focus on disease and neuron density of Hippocampus CA1 area and cerebral cortexⅢ-Ⅳdecreased,the contents of NO、NOS、ET and NPY in brood and brain tissue apparently increased, however,the content of CGRP apparently decreased.(p＜0.05 or p＜0.01)
2.After rats with MCAO had acute cerebral ischemia for 72 hours,compared all electro-acupuncturing groups with model group,we found:Electroacupuncturing in Renzhong、Baihui group and Ganshu、Shenshu group could improve the behavior score to some extent,moreover,it took effect after ischemia for 48 hours,meanwhile,the volume cerebral infarction reduced and neuron density of Hippocampus CA1 area and density of cerebral cortexⅢ-Ⅳincreased (p＜0.05 or p＜0.01).Besides the results above,we also found that the content of NO in serum and content of NOS in brain tissue decreased while NO in brain tissue and NOS in serum have no any change(P＞0.05).The contents of NPY in plasma and brain tissue and ET in brain tissue decreased while the content of ET in plasma have no apparent change(p＞0.05).Moreover,the content of CGRP in plasma and brain tissue also increased(p＜0.05 or p＜0.01).If we compared the results of both group,it is not significant(P＞0.05).Electro-acupuncturing in Quchi、Zusanli group has no apparent effect on the parameters above(p＞0.05).
3.After rats with MCAO had acute cerebral ischemia for 72 hours,compared with pseudo-operation group,we found:the relative density of NPYmRNA in brain tissue apparently increased(P＜0.01).Compared all electro-acupuncturing groups with model group,we also found that electro-acupuncturing in the Renzhong、Baihui group,Ganshu、Shenshu group would apparently decreased the relative density of NPYmRNA in brain tissue(P＜0.05).Compared with the results of both groups,it is no significance(P＞0.05).However,elctro-acupuncturing in Quchi、Zusanli group has no apparent effect on the parameters above(P＞0.05).
Conclusions:1.After rats with MCAO had acute cerebral ischemia for 72 hours, the contents of NO、NOS、ET and NPY in brood and brain tissue apparently rise, however,the content of CGRP apparently falls down.It suggests that the abnormal change of the substances above is related to the pathology mechanism of acute cerebral ischemia.
2.Electro-acupuncturing in Renzhong、Baihui group and Ganshu、Shenshu group can stabilize and improve the behavior score to some extent(it takes effect after acute cerebral ischemia for 48 hours),reduce the volume cerebral infarction and increase neuron density of Hippocampus CA1 area and cerebral cortexⅢ-Ⅳ. Moreover,Electro-acupuncturing can also bring the protective effect on rats with MCAO having acute cerebral ischemia.However,To restrain NO、NOS、ET、NPY and To promote the synthesis,excretion and secretion of CGRP are one of possible protective mechanisms.And the effect arisen by electro-acupuncturing is the same in both groups.
3.Electro-acupuncturing in the Quchi、Zusanli group has no apparent effects on acute cerebral ischemia of Rats.This should be attributed to treat the Weizheng only acupuncturing the Yangming meridians.It is possible that electro- acupuncturing on the two points above is suitable to treat cerebral ischemia injury recovery and sequela.We need further validate it.
4.Inhibiting NPYmRNA express of the focus on MCAO rats' brain tissue is probably one of the molecular mechanisms of the protective effect on the acute cerebral ischemia injury by electro-acupuncturing renzhong and Baihui points of Du Meridian,Ganshu and Shenshu points of Beishu points.
方法:将雄性SD大鼠75只随机分为5组,即:假手术组,模型组,人中、百会组,肝俞、肾俞组,曲池、足三里组,每组15只。建立MCAO模型,采用TTC染色法测脑梗死体积;免疫组化及腿染色法测病灶侧海马CA1区、皮层Ⅲ-Ⅳ层神经元密度;硝酸还原酶法测NO;比色法测一氧化氮合酶(NOS);放射免疫法测ET、CGRP、NPY;RT-PCR半定量分析法测NPYmRNA的表达。
结果:1.大鼠MCAO急性脑缺血72h后,与假手术组比较:神经功能障碍进行性加重;出现病灶侧脑梗死,海马CA1区及皮层Ⅲ-Ⅳ层神经元密度降低;血及脑组织NO、NOS、ET、NPY含量明显升高,而CGRP含量明显降低,(P＜0.05或P＜0.01)。
2.大鼠MCAO急性脑缺血72h后,针刺各组与模型组比较:电针人中、百会组及肝俞、肾俞组对行为学评分有不同程度的改善,并在缺血48h后产生效应;脑梗死体积缩小;海马CA1区及皮层Ⅲ～Ⅳ层神经元密度增加(P＜0.05或P＜0.01);血清NO及脑组织NOS含量降低(P＜0.05或P＜0.01),但对血清NOS及脑组织NO无明显影响(P＞0.05);血浆NPY,脑组织NPY及ET含量降低,但对血浆ET含量无明显影响(P＞0.05);血浆及脑组织CGRP含量升高(P＜0.05或P＜0.01)。两组之间上述结果比较,差异无统计学意义(P＞0.05)。而电针曲池、足三里组对上述指标均无明显影响(P＞0.05)。
3.大鼠MCAO急性脑缺血72h后,与假手术组比较:脑组织NPYmRNA相对密度值明显升高(P＜0.01)。针刺各组与模型组比较:电针人中、百会组及肝俞、肾俞组使脑组织NPYmRNA相对密度值明显降低(P＜0.05);两组之间比较,差异无统计学意义(P＞0.05)。而电针曲池、足三里组对其无明显影响(P＞0.05)。
结论:1.大鼠MCAO急性脑缺血72h后,血及脑组织NO、NOS、ET及NPY含量明显升高,而CGRP含量明显降低,提示上述物质的异常变化与急性脑缺血损伤的病理机制有关。
2.电针人中、百会组及肝俞、肾俞组能改善行为学评分(缺血48h后产生效应),缩小脑梗死体积,增加海马CA1区及皮层Ⅲ～Ⅳ层神经元密度,对大鼠MCAO急性脑缺血损伤有保护作用。抑制NO、NOS、ET、NPY及促进CGRP合成、分泌与释放可能是其保护机制之一,且两穴组针刺效应相近。
3.电针曲池、足三里组对大鼠MCAO急性脑缺血损伤无明显影响,可能因“治痿独取阳明”,上述2穴只适合作用于脑缺血损伤的恢复及后遗症期有关,有待进一步验证。
4.抑制NPYmRNA在病灶侧脑组织的表达可能是电针人中、百会组和肝俞、肾俞组对急性脑缺血损伤保护作用的分子机制之一。
Objective:By electro-acupuncturing renzhong,Baihui points of Du Meridian, Ganshu,Shenshu points of Beishu points,Quchi,Zusanli points of Yangrning Meridians,we compared their acupuncturing effects on the volume of cerebral infarction,neuron density of the model rats with the middle cerebral artery occlusion(MCAO),by analyzing NO metabolism,ET and neuropeptide CGRP, NPY and NPY gene express of model rats with MCAO,we explored the mechanism of electro-acupuncturing treating the acute cerebral ischemia injury.
Methods:75 male SD rats were randomly divided five groups.i.e., pseudo-operation group,model group,Renzhong、Baihui group,Ganshu、Shenshu group and Quchi、Zusanli group.Each group had 15 rats.To establish the MCAO model,we used TTC manipulation experience to measure the cerebral infarction's volume,immunohistochemistry and HE manipulation experience to evaluate neuron density of Hippocampus CA1 area and cerebral cortexⅢ-Ⅳ, nitrate reductase to detect NO,colorinetric assay to inspect NOS, radioimmunoassay to check ET、CGRP and NPY;RT-PCR to examine NPYmRNA express.
Results:1.After rats with MCAO had acute cerebral ischemia for 72 hours, compared with pseudo-operation group,we found nerve malfunction progressively accerated,cerebral infarction appeared in the side focus on disease and neuron density of Hippocampus CA1 area and cerebral cortexⅢ-Ⅳdecreased,the contents of NO、NOS、ET and NPY in brood and brain tissue apparently increased, however,the content of CGRP apparently decreased.(p＜0.05 or p＜0.01)
2.After rats with MCAO had acute cerebral ischemia for 72 hours,compared all electro-acupuncturing groups with model group,we found:Electroacupuncturing in Renzhong、Baihui group and Ganshu、Shenshu group could improve the behavior score to some extent,moreover,it took effect after ischemia for 48 hours,meanwhile,the volume cerebral infarction reduced and neuron density of Hippocampus CA1 area and density of cerebral cortexⅢ-Ⅳincreased (p＜0.05 or p＜0.01).Besides the results above,we also found that the content of NO in serum and content of NOS in brain tissue decreased while NO in brain tissue and NOS in serum have no any change(P＞0.05).The contents of NPY in plasma and brain tissue and ET in brain tissue decreased while the content of ET in plasma have no apparent change(p＞0.05).Moreover,the content of CGRP in plasma and brain tissue also increased(p＜0.05 or p＜0.01).If we compared the results of both group,it is not significant(P＞0.05).Electro-acupuncturing in Quchi、Zusanli group has no apparent effect on the parameters above(p＞0.05).
3.After rats with MCAO had acute cerebral ischemia for 72 hours,compared with pseudo-operation group,we found:the relative density of NPYmRNA in brain tissue apparently increased(P＜0.01).Compared all electro-acupuncturing groups with model group,we also found that electro-acupuncturing in the Renzhong、Baihui group,Ganshu、Shenshu group would apparently decreased the relative density of NPYmRNA in brain tissue(P＜0.05).Compared with the results of both groups,it is no significance(P＞0.05).However,elctro-acupuncturing in Quchi、Zusanli group has no apparent effect on the parameters above(P＞0.05).
Conclusions:1.After rats with MCAO had acute cerebral ischemia for 72 hours, the contents of NO、NOS、ET and NPY in brood and brain tissue apparently rise, however,the content of CGRP apparently falls down.It suggests that the abnormal change of the substances above is related to the pathology mechanism of acute cerebral ischemia.
2.Electro-acupuncturing in Renzhong、Baihui group and Ganshu、Shenshu group can stabilize and improve the behavior score to some extent(it takes effect after acute cerebral ischemia for 48 hours),reduce the volume cerebral infarction and increase neuron density of Hippocampus CA1 area and cerebral cortexⅢ-Ⅳ. Moreover,Electro-acupuncturing can also bring the protective effect on rats with MCAO having acute cerebral ischemia.However,To restrain NO、NOS、ET、NPY and To promote the synthesis,excretion and secretion of CGRP are one of possible protective mechanisms.And the effect arisen by electro-acupuncturing is the same in both groups.
3.Electro-acupuncturing in the Quchi、Zusanli group has no apparent effects on acute cerebral ischemia of Rats.This should be attributed to treat the Weizheng only acupuncturing the Yangming meridians.It is possible that electro- acupuncturing on the two points above is suitable to treat cerebral ischemia injury recovery and sequela.We need further validate it.
4.Inhibiting NPYmRNA express of the focus on MCAO rats' brain tissue is probably one of the molecular mechanisms of the protective effect on the acute cerebral ischemia injury by electro-acupuncturing renzhong and Baihui points of Du Meridian,Ganshu and Shenshu points of Beishu points.
引文
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