p-Akt和caspase-3參與遠程創(chuàng)傷預(yù)處理腦保護作用
發(fā)布時間：2019-02-21 08:56
【摘要】：目的：采用大鼠局灶性腦缺血再灌注模型探討遠程創(chuàng)傷預(yù)處理(remote preconditioning of trauma, RPCT)對腦缺血再灌注損傷的影響,研究p-Akt和caspase-3在遠程創(chuàng)傷預(yù)處理腦保護機制中的作用。 方法：(一)70只健康雄性SD大鼠,體重250-300g,隨機分成七組(n=10)：假手術(shù)組(Sham組)、缺血再灌注組(I/R組)、RPCT0.5h組、RPCT1h組、RPCT2h組、RPCT4h組和RPCT24h組。用以測定腦梗死容積及神經(jīng)功能缺損評分。(二)40只健康雄性SD大鼠隨機分成四組(n=10):Sham、I/R組、RPCT1h組和RPCT24h組。用以組織形態(tài)學(xué)、免疫組化及Western Blot法檢測。①Sham組：僅分離大鼠頸部血管,不插入拴線阻塞大腦中動脈。②I/R組：采用Longa線栓法制作MCAO模型方法建立局灶性腦缺血再灌注模型。③RPCT組：在MCAO開始前分別0.5h、1h、2h、4h和24h做2cm腹部正中切口并切開腹壁全層,隨即縫合傷口。各組大鼠均于缺血再灌注后觀察24h,在24h對大鼠行神經(jīng)功能缺損評分；行TTC染色法測定腦梗死容積；HE染色法觀察腦組織形態(tài)；Nissl染色法觀察神經(jīng)細胞存活情況；免疫組化及Western Blot法觀察并測定缺血半影區(qū)Akt、p-Akt及caspase-3蛋白的表達。 結(jié)果：①TTC染色：Sham組未見腦梗死；與I/R組比較,RPCT1h組、RPCT2h組、RPCT4h組及RPCT24h組腦梗死容積顯著減小(P0.05),RPCT0.5h組與I/R組腦梗死容積無顯著差異(P0.05)。②神經(jīng)功能缺損評分：RPCT1h組、RPCT2h組、RPCT4h組和RPCT24h組Garcia評分較I/R組顯著提高(P0.05),RPCT0.5h組與I/R組Garcia評分無顯著差異；各組Longa五分制評分無顯著差異(P0.05)。③HE染色：Sham組大鼠大腦皮質(zhì)神經(jīng)元形態(tài)正常；與I/R組相比,RPCT1h組及RPCT24h組缺血半影區(qū)神經(jīng)元數(shù)目較多,層次較清楚,細胞形態(tài)較完整,細胞間質(zhì)水腫減輕；I/R組、RPCT1h組及RPCT24h組缺血中心區(qū)均出現(xiàn)細胞壞死,神經(jīng)元數(shù)目減少,核固縮深染,組織疏松。④Nissl染色：Sham組大腦皮質(zhì)神經(jīng)元形態(tài)正常；與I/R組相比,RPCT1h組及RPCT24h組缺血半影區(qū)存活神經(jīng)元數(shù)量顯著增加(P0.05)。⑤免疫組化及Western Blot結(jié)果：Akt在缺血半影區(qū)表達穩(wěn)定,各組間無統(tǒng)計學(xué)差異(P0.05)；I/R組、RPCT1h組及RPCT24h組p-Akt蛋白和caspase-3蛋白在缺血半影區(qū)的表達均顯著高于Sham組(P0.05)；與I/R組相比,RPCT1h組及RPCT24h組缺血半影區(qū)的p-Akt表達顯著增加(P0.05),而caspase-3表達顯著減少(P0.05)。 結(jié)論：遠程創(chuàng)傷預(yù)處理能夠減輕大鼠局灶性腦缺血再灌注損傷,其機制可能與激活A(yù)kt蛋白、抑制caspase-3表達有關(guān)。
[Abstract]:Aim: to investigate the effects of remote preconditioning (remote preconditioning of trauma, RPCT) on cerebral ischemia-reperfusion injury in rats with focal cerebral ischemia-reperfusion, and to investigate the role of p-Akt and caspase-3 in the protective mechanism of remote traumatic preconditioning (RIP). Methods: (1) 70 healthy male SD rats, weighing 250-300 g, were randomly divided into seven groups: sham operation group (Sham group), ischemia reperfusion group (I / R group), RPCT0.5h group, RPCT1h group, RPCT2h group, RPCT4h group and RPCT24h group. To determine the volume of cerebral infarction and neurological impairment score. (2) 40 healthy male SD rats were randomly divided into four groups: Sham,I/R group, RPCT1h group and RPCT24h group. For histomorphology, immunohistochemistry and Western Blot detection. 1Sham group: isolated only the cervical vessels of rats, Middle cerebral artery occlusion was not inserted into the middle cerebral artery. 2I/R group: the model of focal cerebral ischemia-reperfusion was established by using Longa thread occlusion method. The 3RPCT group: 0.5 h before the start of MCAO, 1 h and 2 h, respectively, in the 3RPCT group, the model of focal cerebral ischemia and reperfusion was established by using the method of Longa thread embolization. 2cm median abdominal incision was performed at 4 h and 24 h, and the whole abdominal wall was cut. Then the wound was sutured. All the rats in each group were observed for 24 hours after ischemia and reperfusion. The neurological deficit score was evaluated at 24 hours; the volume of cerebral infarction was measured by TTC staining; the morphology of brain tissue was observed by HE staining; the survival of nerve cells was observed by Nissl staining. The expression of Akt,p-Akt and caspase-3 in ischemic penumbra was observed by immunohistochemistry and Western Blot method. Results: 1TTC staining: no cerebral infarction was found in Sham group. Compared with I / R group, cerebral infarction volume in RPCT1h group, RPCT2h group, RPCT4h group and RPCT24h group was significantly decreased (P0.05), but there was no significant difference in cerebral infarction volume between RPCT0.5h group and I / R group (P0.05). The Garcia score of RPCT4h and RPCT24h group was significantly higher than that of I / R group (P0.05). There was no significant difference in Garcia score between RPCT0.5h group and I / R group. There was no significant difference in Longa score in each group (P0.05). 3HE staining: the cerebral cortex neurons in Sham group were normal. Compared with I / R group, RPCT1h group and RPCT24h group had more neurons in ischemic penumbra, more clear layers, more complete cell morphology and less interstitial edema. In I / R group, RPCT1h group and RPCT24h group, there were cell necrosis, decreased number of neurons, deep staining of nucleus pyknosis and loose tissue. 4Nissl staining: the morphology of cerebral cortex neurons in Sham group was normal. Compared with I / R group, the number of surviving neurons in ischemic penumbra of RPCT1h group and RPCT24h group was significantly increased (P0.05). 5 the results of immunohistochemistry and Western Blot showed that the expression of Akt was stable in ischemic penumbra area, and there was no statistical difference among the groups (P0.05). The expression of p-Akt protein and caspase-3 protein in I / R group, RPCT1h group and RPCT24h group were significantly higher than those in Sham group (P0.05). Compared with I / R group, the expression of p-Akt in ischemic penumbra of RPCT1h group and RPCT24h group was significantly increased (P0.05), while the expression of caspase-3 was significantly decreased (P0.05). Conclusion: remote trauma preconditioning can attenuate focal cerebral ischemia-reperfusion injury in rats, and its mechanism may be related to activation of Akt protein and inhibition of caspase-3 expression.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3
本文編號：2427405
[Abstract]:Aim: to investigate the effects of remote preconditioning (remote preconditioning of trauma, RPCT) on cerebral ischemia-reperfusion injury in rats with focal cerebral ischemia-reperfusion, and to investigate the role of p-Akt and caspase-3 in the protective mechanism of remote traumatic preconditioning (RIP). Methods: (1) 70 healthy male SD rats, weighing 250-300 g, were randomly divided into seven groups: sham operation group (Sham group), ischemia reperfusion group (I / R group), RPCT0.5h group, RPCT1h group, RPCT2h group, RPCT4h group and RPCT24h group. To determine the volume of cerebral infarction and neurological impairment score. (2) 40 healthy male SD rats were randomly divided into four groups: Sham,I/R group, RPCT1h group and RPCT24h group. For histomorphology, immunohistochemistry and Western Blot detection. 1Sham group: isolated only the cervical vessels of rats, Middle cerebral artery occlusion was not inserted into the middle cerebral artery. 2I/R group: the model of focal cerebral ischemia-reperfusion was established by using Longa thread occlusion method. The 3RPCT group: 0.5 h before the start of MCAO, 1 h and 2 h, respectively, in the 3RPCT group, the model of focal cerebral ischemia and reperfusion was established by using the method of Longa thread embolization. 2cm median abdominal incision was performed at 4 h and 24 h, and the whole abdominal wall was cut. Then the wound was sutured. All the rats in each group were observed for 24 hours after ischemia and reperfusion. The neurological deficit score was evaluated at 24 hours; the volume of cerebral infarction was measured by TTC staining; the morphology of brain tissue was observed by HE staining; the survival of nerve cells was observed by Nissl staining. The expression of Akt,p-Akt and caspase-3 in ischemic penumbra was observed by immunohistochemistry and Western Blot method. Results: 1TTC staining: no cerebral infarction was found in Sham group. Compared with I / R group, cerebral infarction volume in RPCT1h group, RPCT2h group, RPCT4h group and RPCT24h group was significantly decreased (P0.05), but there was no significant difference in cerebral infarction volume between RPCT0.5h group and I / R group (P0.05). The Garcia score of RPCT4h and RPCT24h group was significantly higher than that of I / R group (P0.05). There was no significant difference in Garcia score between RPCT0.5h group and I / R group. There was no significant difference in Longa score in each group (P0.05). 3HE staining: the cerebral cortex neurons in Sham group were normal. Compared with I / R group, RPCT1h group and RPCT24h group had more neurons in ischemic penumbra, more clear layers, more complete cell morphology and less interstitial edema. In I / R group, RPCT1h group and RPCT24h group, there were cell necrosis, decreased number of neurons, deep staining of nucleus pyknosis and loose tissue. 4Nissl staining: the morphology of cerebral cortex neurons in Sham group was normal. Compared with I / R group, the number of surviving neurons in ischemic penumbra of RPCT1h group and RPCT24h group was significantly increased (P0.05). 5 the results of immunohistochemistry and Western Blot showed that the expression of Akt was stable in ischemic penumbra area, and there was no statistical difference among the groups (P0.05). The expression of p-Akt protein and caspase-3 protein in I / R group, RPCT1h group and RPCT24h group were significantly higher than those in Sham group (P0.05). Compared with I / R group, the expression of p-Akt in ischemic penumbra of RPCT1h group and RPCT24h group was significantly increased (P0.05), while the expression of caspase-3 was significantly decreased (P0.05). Conclusion: remote trauma preconditioning can attenuate focal cerebral ischemia-reperfusion injury in rats, and its mechanism may be related to activation of Akt protein and inhibition of caspase-3 expression.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3
【參考文獻】
相關(guān)期刊論文 前2條
1 ;Greater stress protein expression enhanced by combined prostaglandin A_1 and lithium in a rat model of focal ischemia[J];Acta Pharmacologica Sinica;2007年08期
2 Uwe Grutzner;Melanie Keller;Michael Bach;Alexandra K Kiemer;Herbert Meissner;Manfred Bilzer;Stefan Zahler;Alexander L Gerbes;Angelika M Vollmar;;PI 3-kinase pathway is responsible for antiapoptotic effects of atrial natriuretic peptide in rat liver transplantation[J];World Journal of Gastroenterology;2006年07期
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