大鼠腦缺血再灌注損傷Cathepsin D介導(dǎo)的細(xì)胞凋亡與MLK3的關(guān)系
發(fā)布時(shí)間:2018-12-14 22:11
【摘要】:目的 本實(shí)驗(yàn)通過建立大鼠大腦中動(dòng)脈腦缺血再灌注模型,并運(yùn)用Cathepsin D抑制劑pepstatin A對(duì)其進(jìn)行干預(yù),檢測(cè)Cathepsin D和pMLK3的蛋白表達(dá)變化,探討Cathepsin D與MLK3在大鼠腦缺血再灌注損傷過程中神經(jīng)細(xì)胞凋亡的關(guān)系,為研究溶酶體參與腦缺血再灌注損傷神經(jīng)細(xì)胞凋亡提供新的探索,為缺血性腦血管病提供新的治療靶點(diǎn)。 方法 67只清潔級(jí)健康雄性Sprague-Dawley大鼠,體重280±20g,隨機(jī)分為3組:假手術(shù)組(sham,n=7),模型組(M,n=30),干預(yù)組(I,n=30):于再灌注前及再灌注后每12h腹腔注射溶酶體Cathepsin D抑制劑pepstatin A2ml/100g(而假手術(shù)組及模型組于相應(yīng)時(shí)間腹腔注射生理鹽水2ml/100g)。其中模型組和干預(yù)組又分為2小時(shí)組(M-2h,n=6;I-2h,n=6),6小時(shí)組(M-6h,n=6;I-6h,n=6),24小時(shí)組(M-24h,n=6;I-24h,n=6),48小時(shí)組(M-48h,n=12;I-48h,n=12)。應(yīng)用改良的Long線栓法制作大鼠大腦中動(dòng)脈腦缺血再灌注模型,缺血2小時(shí)后恢復(fù)再灌注,使用Longa’s的5級(jí)評(píng)分法評(píng)價(jià)神經(jīng)功能;使用TTC染色法、TUNEL凋亡檢測(cè)法及免疫組織化學(xué)方法,分別檢測(cè)腦梗死體積、細(xì)胞凋亡數(shù)量、CathepsinD與pMLK3的表達(dá)變化。 結(jié)果 1.模型成功率76.14%;模型組48小時(shí)相對(duì)腦梗死體積為37.36±1.44%,干預(yù)組48小時(shí)相對(duì)腦梗死體積為27.49±2.30%。 2.假手術(shù)組腦組織未見梗死灶,,模型組和干預(yù)組缺血側(cè)皮質(zhì)及皮質(zhì)下可見白色梗死灶,但干預(yù)組48小時(shí)相對(duì)梗死體積較模型組減少(p=0.000),且干預(yù)組神經(jīng)功能缺損評(píng)分較模型組也得到明顯改善(p0.05)。 3.大鼠腦缺血側(cè)皮質(zhì)區(qū),假手術(shù)組凋亡細(xì)胞很少,模型組2小時(shí)可以觀察到凋亡細(xì)胞,6小時(shí)、24小時(shí)、48小時(shí)呈上升趨勢(shì)(p0.001);干預(yù)組2小時(shí)凋亡細(xì)胞較模型組2小時(shí)無顯著改變(p=0.127),干預(yù)組6小時(shí)、24小時(shí)、48小時(shí)觀察到的凋亡細(xì)胞較模型組明顯減少(p0.05)。 4.大鼠腦缺血側(cè)皮質(zhì)區(qū),假手術(shù)組可見少量Cathepsin D的表達(dá),模型組Cathepsin D的表達(dá)在再灌注2小時(shí)、6小時(shí)、24小時(shí)呈上升趨勢(shì),24小時(shí)達(dá)高峰,48h組較24h組有所下降,但仍居高水平,均比假手術(shù)組高(p=0.000);干預(yù)組各時(shí)間點(diǎn)Cathepsin D的表達(dá)較模型組明顯減少(p=0.000)。 5.大鼠腦缺血側(cè)皮質(zhì)區(qū),假手術(shù)組pMLK3少量表達(dá),模型組pMLK3的表達(dá)在再灌注2小時(shí)即有大量表達(dá),于6小時(shí)達(dá)高峰,之后逐漸下降,均高于假手術(shù)組(p=0.000);干預(yù)組各時(shí)間點(diǎn)pMLK3的表達(dá)均較模型組明顯減少(p0.05)。 結(jié)論 1.大鼠腦缺血再灌注損傷過程中溶酶體Cathepsin D可能通過上調(diào)MLK3的磷酸化而介導(dǎo)神經(jīng)細(xì)胞凋亡。 2.Pepstatin A可能通過抑制Cathepsin D而下調(diào)MLK3的磷酸化活性表達(dá),從而減少細(xì)胞凋亡,減少腦梗死體積,起到神經(jīng)保護(hù)作用。
[Abstract]:Objective to investigate the changes of Cathepsin D and pMLK3 protein expression in middle cerebral artery (MCAA) ischemia-reperfusion model of rats by using pepstatin A, a Cathepsin D inhibitor, to establish a rat model of cerebral ischemia-reperfusion. To investigate the relationship between Cathepsin D and MLK3 in the process of cerebral ischemia-reperfusion injury in rats, and to explore the role of lysosome in neuronal apoptosis during cerebral ischemia-reperfusion injury, and to provide a new therapeutic target for ischemic cerebrovascular disease. Methods Sixty-seven clean grade healthy male Sprague-Dawley rats, weighing 280 鹵20 g, were randomly divided into three groups: sham operation group (sham,n=7), model group (Mannan 30), intervention group (I), and control group (I). Nongma 30: lysosomal Cathepsin D inhibitor pepstatin A2ml/100g was injected intraperitoneally every 12 hours before and after reperfusion (while normal saline 2ml/100g was injected intraperitoneally in sham operation group and model group at the corresponding time). Among them, the model group and the intervention group were divided into two hours group (M-2hnntir 6L), 6-hour group (M-6hnntir 6hnc1hnc6), 24-hour group (M-24hntir 6I-24hncnc6), and 48-hour group (M-48hntrol 12), the model group and the intervention group were divided into three groups: the model group and the intervention group, respectively. I-48h ~ (12). The rat model of middle cerebral artery ischemia reperfusion was established by modified Long thread occlusion method, and the reperfusion was resumed 2 hours after ischemia. The nerve function was evaluated by Longa's 5 grade scoring method. TTC staining, TUNEL apoptosis detection and immunohistochemistry were used to detect the volume of cerebral infarction, the number of apoptosis and the expression of CathepsinD and pMLK3. Result 1. The relative volume of cerebral infarction was 37.36 鹵1.44 in the model group and 27.49 鹵2.30 in the intervention group at 48 hours. 2. No infarct was found in the cerebral tissue of sham-operation group, and white infarct was found in ischemic cortex and subcortical of model group and intervention group, but the relative infarct volume of 48 hours in intervention group was lower than that in model group (p0. 000). The neurological deficit score in the intervention group was significantly improved than that in the model group (p0.05). 3. There were few apoptotic cells in the cerebral ischemic cortex of rats in the sham-operation group. The apoptotic cells were observed in the model group at 2 hours, and increased at 6 hours, 24 hours and 48 hours (p0.001). There was no significant change in apoptotic cells in the intervention group at 2 hours compared with that in the model group (p0. 127). The apoptotic cells in the intervention group at 6 hours, 24 hours and 48 hours were significantly lower than those in the model group (p0.05). 4. A small amount of Cathepsin D expression was observed in the sham-operated group. The expression of Cathepsin D in the model group increased at 2 hours, 6 hours and 24 hours after reperfusion, and reached its peak at 24 hours. The expression of Cathepsin D in the 48 h group was lower than that in the 24 h group, but it was still at a high level. All of them were higher than those of sham operation group (p0. 000). The expression of Cathepsin D in the intervention group was significantly lower than that in the model group (P < 0. 000). 5. There was a little expression of pMLK3 in the cerebral ischemic cortex and sham-operation group. The expression of pMLK3 in the model group reached its peak at 6 hours after reperfusion and was higher than that in the sham-operation group (p0. 000). The expression of pMLK3 in the model group was higher than that in the sham operation group (P < 0. 000). The expression of pMLK3 in the intervention group was significantly lower than that in the model group at each time point (p0. 05). Conclusion 1. Lysosomal Cathepsin D may induce neuronal apoptosis by up-regulating the phosphorylation of MLK3 during cerebral ischemia-reperfusion injury in rats. 2.Pepstatin A may down-regulate the expression of phosphorylation of MLK3 by inhibiting Cathepsin D, thus reducing apoptosis and cerebral infarct volume, thus playing a neuroprotective role.
【學(xué)位授予單位】:南華大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2014
【分類號(hào)】:R743.3
本文編號(hào):2379406
[Abstract]:Objective to investigate the changes of Cathepsin D and pMLK3 protein expression in middle cerebral artery (MCAA) ischemia-reperfusion model of rats by using pepstatin A, a Cathepsin D inhibitor, to establish a rat model of cerebral ischemia-reperfusion. To investigate the relationship between Cathepsin D and MLK3 in the process of cerebral ischemia-reperfusion injury in rats, and to explore the role of lysosome in neuronal apoptosis during cerebral ischemia-reperfusion injury, and to provide a new therapeutic target for ischemic cerebrovascular disease. Methods Sixty-seven clean grade healthy male Sprague-Dawley rats, weighing 280 鹵20 g, were randomly divided into three groups: sham operation group (sham,n=7), model group (Mannan 30), intervention group (I), and control group (I). Nongma 30: lysosomal Cathepsin D inhibitor pepstatin A2ml/100g was injected intraperitoneally every 12 hours before and after reperfusion (while normal saline 2ml/100g was injected intraperitoneally in sham operation group and model group at the corresponding time). Among them, the model group and the intervention group were divided into two hours group (M-2hnntir 6L), 6-hour group (M-6hnntir 6hnc1hnc6), 24-hour group (M-24hntir 6I-24hncnc6), and 48-hour group (M-48hntrol 12), the model group and the intervention group were divided into three groups: the model group and the intervention group, respectively. I-48h ~ (12). The rat model of middle cerebral artery ischemia reperfusion was established by modified Long thread occlusion method, and the reperfusion was resumed 2 hours after ischemia. The nerve function was evaluated by Longa's 5 grade scoring method. TTC staining, TUNEL apoptosis detection and immunohistochemistry were used to detect the volume of cerebral infarction, the number of apoptosis and the expression of CathepsinD and pMLK3. Result 1. The relative volume of cerebral infarction was 37.36 鹵1.44 in the model group and 27.49 鹵2.30 in the intervention group at 48 hours. 2. No infarct was found in the cerebral tissue of sham-operation group, and white infarct was found in ischemic cortex and subcortical of model group and intervention group, but the relative infarct volume of 48 hours in intervention group was lower than that in model group (p0. 000). The neurological deficit score in the intervention group was significantly improved than that in the model group (p0.05). 3. There were few apoptotic cells in the cerebral ischemic cortex of rats in the sham-operation group. The apoptotic cells were observed in the model group at 2 hours, and increased at 6 hours, 24 hours and 48 hours (p0.001). There was no significant change in apoptotic cells in the intervention group at 2 hours compared with that in the model group (p0. 127). The apoptotic cells in the intervention group at 6 hours, 24 hours and 48 hours were significantly lower than those in the model group (p0.05). 4. A small amount of Cathepsin D expression was observed in the sham-operated group. The expression of Cathepsin D in the model group increased at 2 hours, 6 hours and 24 hours after reperfusion, and reached its peak at 24 hours. The expression of Cathepsin D in the 48 h group was lower than that in the 24 h group, but it was still at a high level. All of them were higher than those of sham operation group (p0. 000). The expression of Cathepsin D in the intervention group was significantly lower than that in the model group (P < 0. 000). 5. There was a little expression of pMLK3 in the cerebral ischemic cortex and sham-operation group. The expression of pMLK3 in the model group reached its peak at 6 hours after reperfusion and was higher than that in the sham-operation group (p0. 000). The expression of pMLK3 in the model group was higher than that in the sham operation group (P < 0. 000). The expression of pMLK3 in the intervention group was significantly lower than that in the model group at each time point (p0. 05). Conclusion 1. Lysosomal Cathepsin D may induce neuronal apoptosis by up-regulating the phosphorylation of MLK3 during cerebral ischemia-reperfusion injury in rats. 2.Pepstatin A may down-regulate the expression of phosphorylation of MLK3 by inhibiting Cathepsin D, thus reducing apoptosis and cerebral infarct volume, thus playing a neuroprotective role.
【學(xué)位授予單位】:南華大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2014
【分類號(hào)】:R743.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 ;HBx activates FasL and mediates HepG2 cell apoptosis through MLK3-MKK7-JNKs signal module[J];World Journal of Gastroenterology;2012年13期
本文編號(hào):2379406
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