植酸对缺血再灌注小鼠脑组织的保护作用及对p-Akt，NF-κB，MMP-9及claudin-5调节作用的研究

发布时间：2018-08-19 13:51

【摘要】：目的：脑血管病是造成我国居民死亡和残疾的首位原因，其中以缺血性脑血管病最为常见。尽管缺血性脑血管病的病理、生理学机制已经取得了一些研究成果，但是寻找积极有效的药物治疗仍是当前缺血性脑卒中的难点。炎症和凋亡损伤在脑缺血的发病机制中起着重要的作用，因此可作为治疗缺血后继发性脑损伤的靶点。试验表明，脑缺血后位于缺血半暗带的神经元可以存活数小时，甚至数天。这种延迟的组织损伤过程包括细胞凋亡和炎症。细胞凋亡是细胞死亡的一种特殊形式，继发于脑缺血后，取决于细胞凋亡信号和生存信号之间的平衡。研究证实凋亡出现在外围的缺血半暗带，并且磷脂酰肌醇3激酶/蛋白激酶B（PI3K/Akt）通路可以介导脑缺血和再灌注后神经细胞的生存。Akt激活后可以通过磷酸化作用灭活凋亡效应分子，如糖原合成酶激酶3（glycogen synth ase kinase，GSK-3β）。GSK3β通过调控核转录因子kappaB（NF-κB）p65亚基的活化调控炎症反应。NF-κB是一种大量存在于组织中的转录因子，调节参与炎症，细胞生存和凋亡的基因表达。在脑缺血再灌注后，特别是神经元中，NF-κB被激活。同时有研究证明，在永久性脑缺血模型中抑制NF-κB可以减少梗死体积，减轻脑损伤，特别是NF-κB可以调节某些前炎介质的表达，如金属基质蛋白-9（MMP-9）。过多的金属基质蛋白是有害的，直接从脑组织中注射MMP-9可以引起细胞死亡和炎症。植酸（Inositol hexaphosphate，IP6）是从谷物中发现的一种有机磷酸类的物质，具有抗炎、抗氧化应激、抗肿瘤等作用，尤其植酸对心脏再灌注损伤有保护作用，因此受到了人们的广泛关注。但是植酸是否对脑缺血再灌注的脑组织有保护作用及其作用机制如何，有待于我们的进一步研究。方法：采用成年健康雄性CD1小鼠，应用改良后的Longa线栓法建立短暂小鼠右侧缺血再灌注（tMCAO）模型。实验动物随机分为假手术组（Sham组）、缺血再灌注组（tMCAO组）、植酸低剂量组（IP6-L group，tMCAO+Inositol hexaphosphate75mg/kg）、植酸高剂量组（IP6-H group，tMCAO+Inositol hexaphosphate125mg/kg）。植酸干预组于缺血再灌注后立即予小鼠腹腔注射植酸溶液，Sham组和tMCAO组则腹腔注射等容量生理盐水。术后24h对小鼠进行神经功能评分，评分完毕后将小鼠断头处死，用干湿重法测定脑组织含水量，用2％2,3,5-三苯基四唑氮红（triphenyltetrazolium chloride, TTC）染色法测定脑梗死体积，用免疫组化，免疫印记（Western blot）和实时荧光定量聚合酶链式反应（RT-qPCR）测定p-Akt、NF-κB、MMP-9和claudin-5的表达，用尼氏染色（Nissl's staining）测定细胞凋亡。结果： 1神经功能评分：小鼠缺血再灌注24小时后采用改良的5分法进行神经功能行为学评分，每组24只小鼠。与Sham组相比，tMCAO组的行为学评分明显高于Sham组。与tMCAO组相比，IP6-L组和IP6-H组明显改善了神经功能学评分（IP6-L vs. tMCAO:3.16±0.70vs.3.67±0.87, P 0.05; IP6-H vs. tMCAO:2.67±0.87vs.3.67±0.87, P0.01），IP6-L组与IP6-H组相比，IP6-H组行为学评分更少（IP6-L vs. IP6-H:3.16±0.70vs.2.67±0.87, P0.05）。 2脑梗死体积（%HLV）的测定：Sham组没有观察到脑梗死体积，而tMCAO组可以在皮层观察到白色的梗死区域。IP6-L组与tMCAO组相比，IP6-L组梗死体积明显减小，差异有统计学意义（IP6-L vs. tMCAO:47.20%±6.16%vs.62.45%±9.41%P 0.01）。IP6-H组与tMCAO组相比，梗死体积有所减小，差异有统计学意义（IP6-H vs. tMCAO:62.45%±9.41%vs.34.59%±7.37%P 0.01）。IP6-L组与IP6-H组相比，IP6-H组梗死体积减小的更为明显，差异具有统计学意义（IP6-L vs. IP6-H:47.20%±6.16%vs.34.59%±7.37%P 0.05）。 3脑组织含水量测定：Sham组同侧脑组织含水量为79.80%±0.98%。与tMCAO组相比，IP6-L组和IP6-H组脑组织含水量均降低并具有统计学意义（tMCAO vs. IP6-L:85.12%±1.67%vs.82.73%±1.62%, P 0.05;tMCAO vs. IP6-H:85.12%±1.67%vs.81.13%±0.72%, P 0.05），IP6-L组与IP6-H组相比，IP6-H组更低，，差异有统计学意义（IP6-L vs. IP6-H:82.73%±1.62%vs.81.13%±0.72%, P 0.05）。 4植酸对p-Akt，NF-κB，MMP-9和claudin-5表达的影响：免疫组化的结果显示，IP6-L组和IP6-H组极大的提高了p-Akt的阳性细胞数，植酸治疗组明显减少了NF-κB和MMP-9的阳性细胞的表达。在蛋白水平，植酸上调了p-Akt和claudin-5的表达，下调了NF-κB和MMP-9的表达。在mRNA水平，IP6-L组与IP6-H组的NF-κB和MMP-9表达水平明显降低。 5植酸减少了细胞凋亡：尼氏染色在tMCAO模型上显示了植酸干预后细胞延迟死亡的特征。Sham组未出现细胞死亡，tMCAO组出现细胞的减少，神经元的损伤和轴突的扭曲。与tMCAO组相比，IP6-L组和IP6-H组则减少了上述细胞的死亡。 6植酸可以改善血脑屏障的通透性：MMP-9和claudin-5与血脑屏障的完整性相关。与tMCAO组相比，脑缺血再灌注后24小时植酸低剂量和高剂量组claudin-5的表达明显上调，MMP-9的表达明显下调。WesternBlot及RT-qPCR均可证实。结论：植酸改善神经功能评分、减轻脑水肿及梗死体积，对脑缺血再灌注损伤有保护作用，其作用机制可能与植酸可以上调p-Akt、claudin-5的表达水平，同时降低NF-κB和MMP-9的表达及改善血脑屏障的通透性有关。
[Abstract]:Objective: Cerebrovascular disease is the leading cause of death and disability in Chinese residents, among which ischemic cerebrovascular disease is the most common. Although the pathological and physiological mechanisms of ischemic cerebrovascular disease have made some research results, it is still difficult to find active and effective drug treatment for ischemic stroke.
Inflammation and apoptotic damage play important roles in the pathogenesis of cerebral ischemia, so they can be used as targets for the treatment of secondary brain damage after ischemia. The experiments show that neurons located in the ischemic penumbra can survive for several hours, even days after cerebral ischemia. Apoptosis occurs in the peripheral ischemic penumbra, and the phosphoinositide 3 kinase/protein kinase B (PI3K/Akt) pathway mediates neuronal survival after cerebral ischemia and reperfusion. Glycogen synth ASE kinase 3 (GSK-3beta) can inactivate apoptosis effector molecules by phosphorylation. GSK3beta regulates inflammatory response by regulating the activation of nuclear transcription factor kappa B (NF-kappa B) p65 subunit. NF-kappa B is a transcription factor that exists in a large number of tissues and regulates the bases involved in inflammation, cell survival and apoptosis. NF-kappa B is activated after cerebral ischemia and reperfusion, especially in neurons. It has also been shown that inhibition of NF-kappa B in permanent cerebral ischemia model can reduce infarct size and reduce brain injury, especially NF-kappa B can regulate the expression of some pro-inflammatory mediators, such as matrix metalloproteinase-9 (MMP-9). Injecting MMP-9 directly from brain tissue can cause cell death and inflammation.
Inositol hexaphosphate (IP6) is a kind of organic phosphoric acid found in cereals. It has anti-inflammatory, anti-oxidative stress, anti-tumor and other effects. In particular, phytic acid has protective effects on heart reperfusion injury, so it has been widely concerned. But whether phytic acid has protective effects on brain tissue after cerebral ischemia-reperfusion and its effects. The mechanism of action remains to be further studied.
Methods: Adult healthy male CD1 mice were randomly divided into sham operation group (Sham group), ischemia reperfusion group (tMCAO group), low dose phytic acid group (IP6-L group, tMCAO + Inositol hexaphosphate 75mg/kg) and high dose phytic acid group (IP6-H group). Up, tMCAO + Inositol hexaphosphate 125 mg / kg). The mice in the phytic acid intervention group were injected with phytic acid solution into the abdominal cavity immediately after ischemia-reperfusion, while the mice in the Sham group and the tMCAO group were injected with normal saline of equal volume. The neurological function of the mice was scored 24 hours after operation, and the mice were decapitated and the brain water content was measured by dry-wet weight method, and 2% of the brain water content was used. The volume of cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The expressions of p-Akt, NF-kappa B, MMP-9 and claudin-5 were detected by immunohistochemistry, Western blot and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and apoptosis was detected by Nissl's staining.
Result:
Neurological Function Score: Modified 5-point method was used to evaluate the neurobehavioral function of 24 mice in each group 24 hours after ischemia-reperfusion. Compared with Sham group, the behavioral score of tMCAO group was significantly higher than that of Sham group. Compared with tMCAO group, IP6-L group and IP6-H group significantly improved the neurological function score (IP6-L vs. tMCAO: 3.16 + 0.70 vs. 3.67). IP6-L vs. IP6-H vs. 3.16 + 0.70 vs. 2.67 + 0.87, P 0.05; IP6-H vs. tMCAO: 2.67 + 0.87 vs. 3.67 + 0.87, P 0.01; IP6-L vs. IP6-H: 3.16 + 0.70 vs. 2.67 + 0.87, P 0.05).
The infarct volume of IP6-L group was significantly smaller than that of tMCAO group (IP6-L vs. tMCAO: 47.20% + 6.16% vs. 62.45% + 9.41% P 0.01). The infarct volume of IP6-L group was significantly smaller than that of tMCAO group (IP6-L vs. tMCAO: 47.20% + 6.16% vs. 62.45% + 9.41% P 0.01). The infarct volume of IP6-H vs. tMCAO: 62.45% + 9.41% vs. 34.59% + 7.37% P 0.01). Compared with IP6-H group, the infarct volume of IP6-H group decreased more significantly (IP6-L vs. IP6-H: 47.20% + 6.16% vs. 34.59% + 7.37% P 0.05).
3. Measurements of brain tissue water content: Sham group ipsilateral brain tissue water content was 79.80% + 0.98%. Compared with tMCAO group, IP6-L group and IP6-H group brain tissue water content were decreased and statistically significant (tMCAO vs. IP6-L: 85.12% + 1.67% vs. 82.73% + 1.62%, P 0.05; tMCAO vs. IP6-H: 85.12% + 1.67% vs. 81.13% + 0.72%, P 0.05), IP6-L group compared with IPH group. The IP6-H group was lower, and the difference was statistically significant (IP6-L vs. IP6-H:82.73% + 1.62%vs.81.13% + 0.72%, P 0.05).
4 The effect of phytic acid on the expression of p-Akt, NF-kappa B, MMP-9 and claudin-5: Immunohistochemical results showed that IP6-L and IP6-H groups significantly increased the number of p-Akt positive cells, and phytic acid treatment group significantly decreased the expression of NF-kappa B and MMP-9 positive cells. At protein level, phytic acid increased the expression of p-Akt and claudin-5, and decreased the expression of NF-kappa B and MMP-9. At the level of mRNA, the expression level of NF- B and MMP-9 in group IP6-L and IP6-H decreased significantly.
Phytic acid reduced apoptosis: Nissl staining showed delayed cell death after phytic acid intervention in the tMCAO model. No cell death was observed in the Sham group, but decreased cells, neuronal damage and axonal distortion were observed in the tMCAO group.
6 Phytic acid can improve the permeability of BBB: MMP-9 and claudin-5 are related to the integrity of BBB. Compared with tMCAO group, the expression of claudin-5 in low dose and high dose phytic acid groups increased significantly 24 hours after cerebral ischemia-reperfusion, and the expression of MMP-9 was significantly down-regulated. Both Western Blot and RT-qPCR can confirm this.
CONCLUSION: Phytic acid can improve neurological function score, reduce cerebral edema and infarct volume, and protect cerebral ischemia-reperfusion injury. The mechanism may be related to the up-regulation of p-Akt and claudin-5 expression by phytic acid, the down-regulation of NF-kappa B and MMP-9 expression and the improvement of blood-brain barrier permeability.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.31

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