摘要
目的：采用抗体蛋白芯片技术观察针刺督脉经(穴)对缺血损伤脑组织修复过程中相关信号蛋白磷酸化的变化,找出与上述过程关系最为密切的信号分子及通路,采用免疫印迹(Western-Blotting)法对该通路中关键蛋白的磷酸化水平进行检测,探讨针刺督脉经(穴)对脑组织缺血性损伤可能的修复机制。
方法：32只SD大鼠随机抽取8只作为假手术组,其他24只采用线栓法制备大脑中动脉阻塞(MCAO)大鼠模型后,随机分为3组,即模型组、针刺对照点组、针刺穴位组,8只／组。本研究分为三个部分,第一部分：6次治疗后先行神经功能缺损评分再处死,对大鼠部分脑组织采用HE染色行脑组织细胞形态学观察。第二部分：提取缺血脑组织细胞,采用720磷酸化抗体蛋白芯片技术检测针刺治疗后的脑组织损伤修复相关信号蛋白磷酸化的变化,筛选出各组差异性表达的蛋白。第三部分：应用Western-Blotting方法检测大鼠缺血脑组织细胞磷酸化的丝裂原活化的蛋白激酶激酶的激酶(MAPKKK即Raf)蛋白(p-Rafl)和磷酸化细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)蛋白(p-ERK)的表达。
结果：
1.针刺穴位可明显降低MCAO大鼠神经功能缺损评分、改善缺血损伤脑组织形态：模型组、针刺对照点组、针刺穴位组神经功能缺损评分明显高于假手术组(P<0.01),且三组的神经细胞及脑组织结构较假手术组均有不同程度的结构破坏、变性坏死；针刺对照点组神经功能缺损评分低于模型组(P<0.05),且神经细胞的结构破坏、脑组织的变性坏死较模型组减少；而针刺穴位评分明显低于模型组(P<0.01)且低于针刺对照点组(P<0.05),且针刺穴位组神经细胞的结构破坏、脑组织的变性坏死较模型组与针刺对照点组均减少。
2.缺血脑组织细胞720抗体蛋白芯片检测结果显示：与假手术组比较,模型组的蛋白磷酸化水平变化大于1.5倍的蛋白中有48种出现上调,29种出现下调；与模型组比较,针刺对照点组蛋白磷酸化水平变化大于1.5倍的蛋白中有35种出现上调,24种出现下调：针刺穴位组中有28种出现上调,51种出现下调；与针刺对照点组比较,针刺穴位组中出现调整的蛋白在数量、功能种类与信号转导通路的归属上均明显优于针刺对照组。且MAPKS信号传导途径中关键蛋白均被激活。
3.与假手术组比较：模型组和针刺对照点组的p-Rafl含量有升高的趋势,针刺穴位组p-Rafl含量有降低的趋势,但均无统计学差异(P>0.05)；与模型组比较：针刺对照点组的p-Rafl含量增高,而针刺穴位组的p-Rafl含量降低,均无统计学差异(P>0.05)；与针刺对照点组比较：针刺穴位组的p-Rafl含量降低,且低于假手术组和模型组,但无统计学差异(P>0.05)。
4.与假手术组比较：模型组和针刺对照点组的p-ERK含量有升高的趋势,针刺穴位组p-ERK含量有降低的趋势,但均无统计学差异(P>0.05)；与模型组比较：针刺对照点组和针刺穴位组的p-ERK都低于模型组,均无统计学差异(P>0.05)；与针刺对照点组比较：针刺穴位组的p-ERK含量低于针刺对照点组,且低于假手术组和模型组,但无统计学差异(P>0.05)。
结论：
1.针刺大椎、百会、人中(穴)能明显改善MCAO大鼠的神经功能缺损和缺血损伤脑组织形态。提示针刺大椎、百会、人中(穴)对缺血损伤脑组织具有一定的保护作用。
2.针刺大椎、百会、人中(穴)可调整造模后失调的与凋亡有关蛋白的磷酸化水平,此外,还可调整多个其它功能种类的蛋白,且涉及到的信号转导通路归属于细胞周期信号转导通路、MAPKS信号途径、细胞凋亡信号途径、黏附分子信号途径、非受体酪氨酸激酶信号途径、受体酪氨酸激酶信号途径等多条信号转导通路。提示多靶点、多条信号转导通路的激活是针刺大椎、百会、人中(穴)促缺血损伤脑组织修复的重要特征。
3.各通路中只有MAPKS信号传导途径中的关键蛋白被激活,提示MAPKS信号转导通路与针刺大椎、百会、人中(穴)对缺血损伤脑组织修复密切相关。
4.针刺大椎、百会、人中(穴)能降低p-Raf1和p-ERK在缺血损伤脑组织中的表达,对缺血性脑损伤起到一定的修复作用。
Objective:To study the effect of the Stimulation with Acupuncture (SA) on the injury repairing of Ischemic Brain Tissues. By observing the changes of related signal protein phosphorylation under SA, the related signal molecule and pathway were revealed. Detecting the phosphorylation levels of key protein with Western-Blotting in the most relevant pathway and from the proteome to analysis the potential mechanism of injury repairing of Ischemic Brain Tissues.
Methods:32healthy SD rats were used for this experiment.8of these rats were randomly chosen as sham-operation group(sham group). And the rest24rats were used to establish the middle cerebral artery occlusion (MCAO) model by using the improved thread embolism method, which then divided into3groups, each group8rats randomly:model group, control group, and treatment group.The research was divided into three parts:Part1:After6treatments, the animals were sacrificed and the ischemic brain tissues were isolated. The scores of neurological functions defic-its were evaluated before sacrifice. Part of the brain tissue were used to do cell morphology by HE staining. Part2:The Ischemic Brain Tissues of the rats per group was extracted. The changes of related signal protein phos-phorylation of Ischemic Brain Tissues injury repairing with Acupuncture were detected by720kinds of phosphorylation of antibody protein chip tech. And the differential expressed phosphorylation signal protein in eac-h group was screened, then further revealed the related pathway to which the differential expressed phosphorylation signal protein belong. Part3:The express of p-Rafl and p-ERK in the Ischemic Brain Tissues were detected with Western-Blotting.
Results:
1. The scores of neurological functions deficits and the cell morphology of the ischemic brain tissues can be improved greatly by acupuncture. Compared with sham group:the Scores of neurological functions deficits in model group, control group and treatment group were remarkable higher(P<0.01),the degeneration and necrosis of ischemic brain tissues in these three groups are serious than in sham group; the scores in control group were lower compared with model group(P<0.05),and the degeneration and necrosis of ischemic brain tissues in control group is not so severely; the neurological functions deficits scores of treatment group were remarkable lower compared with model group(P<0.01) and lower than control group (P<0.05),and the structure of ischemic brain tissues is more complete than other groups.
2.720kinds of phosphorylation of antibody protein chip tech detection shows:compared with model group,24kinds of protein were down regulated and35kinds were up-regulated more than1.5folds in contrast group; while in treatment group,51kinds were down-regulated and28were up-regulated; To conclude, the number, function type and signal transduction pathway of phosphorylation signal protein in the treatment group were more than that of contrast group.
3. Compared with the sham group:the level of p-Rafl in model and control group were higher but lower in treatment group, however, there was no statistically significant,(P>0.05).Compared with the model group: the level of p-Rafl in control group was higher but lower in treatment group,(P>0.05). The level of p-Rafl in treatment group was lower than others, however, there was no statistically significant,(P>0.05).
4. Compared with the sham group:the level of p-ERK in model and control group were higher but lower in treatment group, however, there was no statistically significant,(P>0.05).Compared with the model group: the level of p-ERK in control and treatment group were lower(P>0.05). The level of p-ERK in treatment group was lower than others, however, there was no statistically significant,(P>0.05).
Conclusion:
1. The neurological functions deficits and cell morphology of the ischemic brain tissues in treatment group can be obviously improved, which indicates that Acupuncturing DA ZHUI, BAI HUI,SHUI GOU (ADBS) can obviously repair the Ischemic Brain Tissues;
2. ADBS can not only adjust the disordered level of protein phosphorylation that associated with the proliferation after making model, but also adjust those apoptosis-related disordered level of protein phosphorylation,and can regulate the proteins of model group which involves various functional types and belongs to multiple signal transduction pathways which include the cell cycle regulation of signal transduction pathway, MAPKS signaling pathways, apoptosis signaling pathways, adhesion molecule signaling pathways, non-receptor tyrosine kinase signaling pathways, receptor tyrosine kinase signaling pathways such as signal transduction pathways. It indicates multi-target and multiple-way is the important characteristic of ADBS to repair the Ischemic Brain Tissues.
3.The key protein of the signal transduction pathway-MAPKS were activated which indicates MAPKS signal transduction pathway is close related with repairing of Acupuncture treating group to promote the repairing of Ischemic Brain Tissues.
4. ADBS can decrease the expression of p-Rafl and p-ERK in the ischemic brain tissues, playing a certain role in repair of ischemic injury in brain tissue.
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