激活素及其信号通路在异氟醚后处理大鼠氧糖剥夺损伤中的作用

发布时间：2018-08-29 18:41

【摘要】：目的:评价1.5%、3.0%以及4.5%的异氟醚后处理对大鼠海马脑片氧糖剥夺损伤中的神经保护作用;探讨激活素A及其下游因子ERK1/2对异氟醚后处理对抗大鼠海马脑片氧糖剥夺损伤中的保护作用。方法:新生SD大鼠80只,2周龄,体重50~70g,采用随机数字表法,将其分为十组(n=8):正常对照组(Normal组),缺氧无糖损伤组(OGD组),异氟醚后处理组(1.5%、3.0%、4.5%ISPOC组)、激活素A抑制剂组(SB431542组)ERK1/2抑制剂组(U0126组)、异氟醚后处理+抑制剂组(ISPOC+SB431542或U0126组)、溶媒对照组(DMSO组)。抑制剂在不同异氟醚浓度组使用时间为OGD开始之前。HE染色、TTC染色以及PI染色用于测定海马脑片组织的活性。免疫荧光技术、Western blot技术以及实时荧光定量PCR技术用于检测激活素A、Smad2/3、磷酸化Smad2/3(p-Smad2/3)、ERK1/2以及磷酸化ERK1/2(p-ERK1/2)蛋白以及m RNA的表达。结果:与Normal组相比较,OGD组坏死神经元细胞的数量、PI染色的平均荧光强度增加,但甲瓒生成量降低、激活素A以及p-ER强度增加,但甲瓒产量、激活素A、p-Smad2/3以及p-ERK1/2蛋白表达水平以及激活素A m RNA水平有所降低。然而,4.5%ISPOC组中激活素A、p-Smad2/3以及p-ERK1/2蛋白表达水平以及激活素A m RNA水平要高于OGD组,差异具有统计学意义(P0.05);其他实验结果在SB431542组/U0126组与3.0%ISPOC组之间相比较,PI染色的平均荧光强度增加,而激活素A、p-Smad2/3以及K1/2蛋白的表达水平、激活素A m RNA的水平有所降低(P0.05);与OGD组相比较,1.5%ISPOC组和3.0%ISPOC组坏死神经元细胞的数量以及PI染色的平均荧光强度降低,但甲瓒产量、激活素A、p-Smad2/3以及p-ERK1/2蛋白表达水平以及激活素A m RNA水平显著升高(P0.05);4.5%ISPOC组的实验室检查结果与1.5%ISPOC、3.0%ISPOC组结果完全相反,该组中损伤神经元细胞的数量以及平均荧光p-ERK1/2蛋白表达水平降低(P0.05);ERK1/2在所有组之间表达没有统计学意义(p0.05)。结论:该研究证实与1.5%和4.5%异氟醚浓度相比,3.0%的异氟醚后处理能够提供更好的神经保护作用。激活素A、激活素A/Smads以及激活素A/ERK1/2信号调节通路参与了异氟醚后处理诱导的脑保护作用的机制。
[Abstract]:Objective: to evaluate the neuroprotective effect of 1.5% and 4.5% isoflurane on oxygen and glucose deprivation injury in rat hippocampal slices. To investigate the protective effect of activin A and its downstream factor ERK1/2 on the protective effect of isoflurane post-treatment on oxygen-glucose deprivation injury in rat hippocampal slices. Methods: 80 neonatal SD rats, aged 2 weeks and weighing 50 ~ 70g, were randomly divided into two groups. They were divided into 10 groups: normal control group (Normal group), hypoxic and glucose free injury group (OGD group), isoflurane post-treatment group (1.5%), activin A inhibitor group (SB431542 group), ERK1/2 inhibitor group (U0126 group), isoflurane post-treatment inhibitor group (ISPOC SB431542 or U0126 group). Solvent control group (DMSO group). In different isoflurane concentration groups, the activity of hippocampal slices was determined by HE staining and PI staining before the onset of OGD. Western blot and real-time fluorescent quantitative PCR were used to detect the expression of activin A Smad2 / 3, phosphorylated Smad2/3 (p-Smad2/3) / ERK1 / 2, phosphorylated ERK1/2 (p-ERK1/2) protein and mRNA. Results: compared with Normal group, the number of necrotic neurons in OGD group increased with the average fluorescence intensity of Pi staining, but the amount of Gan produced decreased, the intensity of activin A and p-ER increased, but the yield of Jia Zan increased. The expression of activin p-Smad2 / 3, p-ERK1/2 protein and activin A m RNA decreased. However, the levels of activin p-Smad2 / 3 and p-ERK1/2 protein and activin A m RNA in ISPOC group were significantly higher than those in OGD group (P0.05), and the average fluorescence intensity of Pi staining in SB431542 group / U0126 group was higher than that in 3.0%ISPOC group. The expression of activin p-Smad2 / 3 and K1 / 2 protein and the level of activin A m RNA were decreased (P0.05), and the number of necrotic neurons and the average fluorescence intensity of PI staining in OGD group and 3.0%ISPOC group were lower than those in OGD group, but the average fluorescence intensity of PI staining was decreased. The levels of activin p-Smad2 / 3 and p-ERK1/2 protein and activin A m RNA were significantly increased (P0.05). The results of laboratory examination in 4.5 ISPOC group were contrary to those in 1.5 ISPOC3.0 group. The number of injured neuron cells and the expression level of average fluorescent p-ERK1/2 protein decreased (P0.05) the expression of ERK1 / 2 in all groups was not statistically significant (p0.05). Conclusion: compared with 1.5% and 4.5% isoflurane concentrations, 3.0% isoflurane post treatment can provide better neuroprotective effect. Activin A, activin A/Smads and activin A/ERK1/2 signaling pathway are involved in the mechanism of brain protection induced by isoflurane postprocessing.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

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