植酸對缺血再灌注小鼠腦組織的保護(hù)作用及對p-Akt，NF-κB，MMP-9及claudin-5調(diào)節(jié)作用的研究
發(fā)布時間：2018-08-19 13:51
【摘要】：目的：腦血管病是造成我國居民死亡和殘疾的首位原因，其中以缺血性腦血管病最為常見。盡管缺血性腦血管病的病理、生理學(xué)機(jī)制已經(jīng)取得了一些研究成果，但是尋找積極有效的藥物治療仍是當(dāng)前缺血性腦卒中的難點(diǎn)。 炎癥和凋亡損傷在腦缺血的發(fā)病機(jī)制中起著重要的作用，因此可作為治療缺血后繼發(fā)性腦損傷的靶點(diǎn)。試驗(yàn)表明，腦缺血后位于缺血半暗帶的神經(jīng)元可以存活數(shù)小時，甚至數(shù)天。這種延遲的組織損傷過程包括細(xì)胞凋亡和炎癥。細(xì)胞凋亡是細(xì)胞死亡的一種特殊形式，繼發(fā)于腦缺血后，取決于細(xì)胞凋亡信號和生存信號之間的平衡。研究證實(shí)凋亡出現(xiàn)在外圍的缺血半暗帶，并且磷脂酰肌醇3激酶/蛋白激酶B（PI3K/Akt）通路可以介導(dǎo)腦缺血和再灌注后神經(jīng)細(xì)胞的生存。Akt激活后可以通過磷酸化作用滅活凋亡效應(yīng)分子，如糖原合成酶激酶3（glycogen synth ase kinase，GSK-3β）。GSK3β通過調(diào)控核轉(zhuǎn)錄因子kappaB（NF-κB）p65亞基的活化調(diào)控炎癥反應(yīng)。NF-κB是一種大量存在于組織中的轉(zhuǎn)錄因子，調(diào)節(jié)參與炎癥，細(xì)胞生存和凋亡的基因表達(dá)。在腦缺血再灌注后，特別是神經(jīng)元中，NF-κB被激活。同時有研究證明，在永久性腦缺血模型中抑制NF-κB可以減少梗死體積，減輕腦損傷，特別是NF-κB可以調(diào)節(jié)某些前炎介質(zhì)的表達(dá)，如金屬基質(zhì)蛋白-9（MMP-9）。過多的金屬基質(zhì)蛋白是有害的，直接從腦組織中注射MMP-9可以引起細(xì)胞死亡和炎癥。 植酸（Inositol hexaphosphate，IP6）是從谷物中發(fā)現(xiàn)的一種有機(jī)磷酸類的物質(zhì)，具有抗炎、抗氧化應(yīng)激、抗腫瘤等作用，尤其植酸對心臟再灌注損傷有保護(hù)作用，因此受到了人們的廣泛關(guān)注。但是植酸是否對腦缺血再灌注的腦組織有保護(hù)作用及其作用機(jī)制如何，有待于我們的進(jìn)一步研究。 方法：采用成年健康雄性CD1小鼠，應(yīng)用改良后的Longa線栓法建立短暫小鼠右側(cè)缺血再灌注（tMCAO）模型。實(shí)驗(yàn)動物隨機(jī)分為假手術(shù)組（Sham組）、缺血再灌注組（tMCAO組）、植酸低劑量組（IP6-L group，tMCAO+Inositol hexaphosphate75mg/kg）、植酸高劑量組（IP6-H group，tMCAO+Inositol hexaphosphate125mg/kg）。植酸干預(yù)組于缺血再灌注后立即予小鼠腹腔注射植酸溶液，Sham組和tMCAO組則腹腔注射等容量生理鹽水。術(shù)后24h對小鼠進(jìn)行神經(jīng)功能評分，評分完畢后將小鼠斷頭處死，用干濕重法測定腦組織含水量，用2％2,3,5-三苯基四唑氮紅（triphenyltetrazolium chloride, TTC）染色法測定腦梗死體積，用免疫組化，免疫印記（Western blot）和實(shí)時熒光定量聚合酶鏈?zhǔn)椒磻?yīng)（RT-qPCR）測定p-Akt、NF-κB、MMP-9和claudin-5的表達(dá)，用尼氏染色（Nissl's staining）測定細(xì)胞凋亡。 結(jié)果： 1神經(jīng)功能評分：小鼠缺血再灌注24小時后采用改良的5分法進(jìn)行神經(jīng)功能行為學(xué)評分，每組24只小鼠。與Sham組相比，tMCAO組的行為學(xué)評分明顯高于Sham組。與tMCAO組相比，IP6-L組和IP6-H組明顯改善了神經(jīng)功能學(xué)評分（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組與IP6-H組相比，IP6-H組行為學(xué)評分更少（IP6-L vs. IP6-H:3.16±0.70vs.2.67±0.87, P0.05）。 2腦梗死體積（%HLV）的測定：Sham組沒有觀察到腦梗死體積，而tMCAO組可以在皮層觀察到白色的梗死區(qū)域。IP6-L組與tMCAO組相比，IP6-L組梗死體積明顯減小，差異有統(tǒng)計學(xué)意義（IP6-L vs. tMCAO:47.20%±6.16%vs.62.45%±9.41%P 0.01）。IP6-H組與tMCAO組相比，梗死體積有所減小，差異有統(tǒng)計學(xué)意義（IP6-H vs. tMCAO:62.45%±9.41%vs.34.59%±7.37%P 0.01）。IP6-L組與IP6-H組相比，IP6-H組梗死體積減小的更為明顯，差異具有統(tǒng)計學(xué)意義（IP6-L vs. IP6-H:47.20%±6.16%vs.34.59%±7.37%P 0.05）。 3腦組織含水量測定：Sham組同側(cè)腦組織含水量為79.80%±0.98%。與tMCAO組相比，IP6-L組和IP6-H組腦組織含水量均降低并具有統(tǒng)計學(xué)意義（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組與IP6-H組相比，IP6-H組更低，，差異有統(tǒng)計學(xué)意義（IP6-L vs. IP6-H:82.73%±1.62%vs.81.13%±0.72%, P 0.05）。 4植酸對p-Akt，NF-κB，MMP-9和claudin-5表達(dá)的影響：免疫組化的結(jié)果顯示，IP6-L組和IP6-H組極大的提高了p-Akt的陽性細(xì)胞數(shù)，植酸治療組明顯減少了NF-κB和MMP-9的陽性細(xì)胞的表達(dá)。在蛋白水平，植酸上調(diào)了p-Akt和claudin-5的表達(dá)，下調(diào)了NF-κB和MMP-9的表達(dá)。在mRNA水平，IP6-L組與IP6-H組的NF-κB和MMP-9表達(dá)水平明顯降低。 5植酸減少了細(xì)胞凋亡：尼氏染色在tMCAO模型上顯示了植酸干預(yù)后細(xì)胞延遲死亡的特征。Sham組未出現(xiàn)細(xì)胞死亡，tMCAO組出現(xiàn)細(xì)胞的減少，神經(jīng)元的損傷和軸突的扭曲。與tMCAO組相比，IP6-L組和IP6-H組則減少了上述細(xì)胞的死亡。 6植酸可以改善血腦屏障的通透性：MMP-9和claudin-5與血腦屏障的完整性相關(guān)。與tMCAO組相比，腦缺血再灌注后24小時植酸低劑量和高劑量組claudin-5的表達(dá)明顯上調(diào)，MMP-9的表達(dá)明顯下調(diào)。WesternBlot及RT-qPCR均可證實(shí)。 結(jié)論：植酸改善神經(jīng)功能評分、減輕腦水腫及梗死體積，對腦缺血再灌注損傷有保護(hù)作用，其作用機(jī)制可能與植酸可以上調(diào)p-Akt、claudin-5的表達(dá)水平，同時降低NF-κB和MMP-9的表達(dá)及改善血腦屏障的通透性有關(guān)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.31
本文編號：2191846
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.31
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