红景天苷类似物对神经细胞的保护作用及其机制探讨
发布时间：2018-07-15 18:18
【摘要】：目的神经退行性疾病是一类复杂的疾病类型,而神经细胞受损是导致该类疾病的一个主要诱因。近年来,红景天苷因其广泛的药理活性而倍受关注,而它在神经细胞保护方面的作用也日益引起人们的重视。红景天苷的药理作用纵然广泛,然而它的药效却始终存在不尽如人意之处,其亲水性太强,不容易透过细胞质膜和血脑屏障,从而影响其发挥作用。现阶段对红景天苷类似物发挥神经细胞保护的报道较少,在前期研究中本实验室合成了一系列红景天苷类似物,本研究通过建立神经细胞损伤模型,模拟体内神经细胞损伤状态并将红景天苷类似物运用至其中,以探明它们在神经细胞保护方面发挥的作用。结果表明,与红景天苷相比较,MADP和化合物6的亲酯性有了很大提高,其细胞保护作用比红景天苷更强,该研究结果为今后进一步开发防治神经系统退行性疾病的新药提供参考依据。方法本研究采用两种细胞损伤模型,在第一种模型中,预先给予海马神经元以MADP(120,240μM)和Sal(120,240μM)预保护24 h,再以125μM谷氨酸作用海马神经元15 min,细胞继续培养24 h后,光镜下观察海马神经元形态变化,通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)及乳酸脱氢酶(LDH)释放量评估细胞活力,同时采用Hoechst 33342、TUNEL、Annexin V-FITC/PI染色检测神经元凋亡情况,通过Fluo-4/AM检测[Ca2+]i动态变化,最后通过QPCR和Western blot实验分别检测与凋亡相关的基因和蛋白的表达情况。在第二种模型中,预先给予PC12细胞以化合物6,7,11,12(75,150,300μg/mL)和Sal(300μg/mL)预保护24 h,再以低糖低血清损伤细胞24 h,通过MTT实验检测细胞活力,光镜观察细胞形态变化,采用Hoechst 33342染色检测细胞凋亡率,最后通过Western blot和Caspase-3活性检测试剂盒分别检测Bcl-2/Bax蛋白的表达和Caspase-3的活性。结果谷氨酸损伤模型中的MTT和LDH释放测定实验结果表明,相同条件下MADP组中细胞活力高于Sal组;Hoechst 33342、TUNEL、Annexin V-FITC/PI染色揭示了MADP组中细胞凋亡率低于Sal组;Fluo-4/AM荧光染色结果显示Sal组中[Ca2+]i荧光强度强于MADP组;Sal组的Caspase-3 mRNA表达量低于MADP组;Western blot结果显示两个类别组中Bcl-2/Bax以及p-ERK蛋白表达量均未发生变化,而MADP组中p-Akt和p-JNK的表达量高于Sal组。在低糖低血清损伤模型中,MTT实验的结果表明大多数受试化合物都发挥了对PC12细胞的保护作用,尤以化合物6的保护作用最强;化合物6的预保护能改善受损的PC12细胞形态;Hoechst 33342染色、Caspase-3活性测定以及Western blot实验结果均揭示了化合物6可剂量依赖性的抑制低糖低血清损伤诱导的PC12细胞凋亡,化合物6在300μg/mL时发挥的保护作用最佳。结论红景天苷类似物MADP和化合物6较红景天苷能更好的发挥对海马神经元和PC12细胞的保护作用。
[Abstract]:Objective Neurodegenerative diseases are a kind of complex diseases, and nerve cell damage is one of the main causes of neurodegenerative diseases. In recent years, salidroside has attracted much attention because of its wide range of pharmacological activities, and its role in the protection of nerve cells has attracted more and more attention. Although salidroside has a wide range of pharmacological effects, its pharmacodynamics is always unsatisfactory, and its hydrophilicity is too strong to penetrate the cytoplasmic membrane and blood-brain barrier. At present, there are few reports that salidroside analogues play a role in nerve cell protection. A series of salidroside analogues were synthesized in our laboratory in previous studies. To study the role of salidroside analogue in neuronal protection by simulating neuronal injury in vivo. The results showed that the lipophilic properties of MADP and compound 6 were greatly improved compared with salidroside, and their cell protection was stronger than salidroside. The results provide a reference for the further development of new drugs for the prevention and treatment of neurodegenerative diseases. Methods in the first model, hippocampal neurons were preprotected with MADP (120240 渭 M) and Sal (120240 渭 M) for 24 h, then treated with 125 渭 M glutamic acid for 15 min. The morphological changes of hippocampal neurons were observed under light microscope. The cell viability was evaluated by the release of 3- (4-dimethylthiazol-2) -2-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH), and the apoptosis of neurons was detected by Hoechst 33342Tunel Annexin V-FITCP-PI staining. The dynamic changes of [Ca 2] I were detected by Fluo-4 / AM, and the expression of apoptosis-related genes and proteins were detected by QPCR and Western blot, respectively. In the second model, PC12 cells were pretreated with compounds 6C71111C12 (75150300 渭 g / mL) and Sal (300 渭 g / mL) for 24 h, then the cells were injured with low glucose and low serum for 24 h. The cell viability was detected by MTT assay, the morphological changes of cells were observed by light microscope, and the apoptosis rate was detected by Hoechst 33342 staining. Finally, the expression of Bcl-2 / Bax protein and the activity of Caspase-3 were detected by Western blot and Caspase-3 assay kit. Results MTT and LDH release assay in glutamate injury model showed that, The cell viability in MADP group was higher than that in Sal group by Hoechst33342Tunel Annexin V-FITC / Pi staining under the same condition. The results showed that the apoptotic rate in MADP group was lower than that in Sal group Fluo-4 / AM fluorescence staining showed that [Ca 2] I fluorescence intensity in Sal group was stronger than that in Sal group and Caspase-3 mRNA expression in Sal group was lower than that in MADP group. The results of Western blot showed that the expression of Bcl-2 / Bax and p-ERK protein did not change in both groups. The expression of p-Akt and p-JNK in MADP group was higher than that in Sal group. The results of MTT assay showed that most of the tested compounds had protective effect on PC12 cells, especially compound 6 had the strongest protective effect on PC12 cells. The preprotection of compound 6 could improve the activity of Caspase-3 in damaged PC12 cells by Hoechst 33342 staining and the results of Western blot assay showed that compound 6 could inhibit apoptosis of PC12 cells induced by low glucose and low serum levels in a dose-dependent manner. Compound 6 had the best protective effect at 300 渭 g / mL. Conclusion salidroside analogues MADP and compound 6 can protect hippocampal neurons and PC12 cells better than salidroside.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
，
本文编号：2124976
[Abstract]:Objective Neurodegenerative diseases are a kind of complex diseases, and nerve cell damage is one of the main causes of neurodegenerative diseases. In recent years, salidroside has attracted much attention because of its wide range of pharmacological activities, and its role in the protection of nerve cells has attracted more and more attention. Although salidroside has a wide range of pharmacological effects, its pharmacodynamics is always unsatisfactory, and its hydrophilicity is too strong to penetrate the cytoplasmic membrane and blood-brain barrier. At present, there are few reports that salidroside analogues play a role in nerve cell protection. A series of salidroside analogues were synthesized in our laboratory in previous studies. To study the role of salidroside analogue in neuronal protection by simulating neuronal injury in vivo. The results showed that the lipophilic properties of MADP and compound 6 were greatly improved compared with salidroside, and their cell protection was stronger than salidroside. The results provide a reference for the further development of new drugs for the prevention and treatment of neurodegenerative diseases. Methods in the first model, hippocampal neurons were preprotected with MADP (120240 渭 M) and Sal (120240 渭 M) for 24 h, then treated with 125 渭 M glutamic acid for 15 min. The morphological changes of hippocampal neurons were observed under light microscope. The cell viability was evaluated by the release of 3- (4-dimethylthiazol-2) -2-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH), and the apoptosis of neurons was detected by Hoechst 33342Tunel Annexin V-FITCP-PI staining. The dynamic changes of [Ca 2] I were detected by Fluo-4 / AM, and the expression of apoptosis-related genes and proteins were detected by QPCR and Western blot, respectively. In the second model, PC12 cells were pretreated with compounds 6C71111C12 (75150300 渭 g / mL) and Sal (300 渭 g / mL) for 24 h, then the cells were injured with low glucose and low serum for 24 h. The cell viability was detected by MTT assay, the morphological changes of cells were observed by light microscope, and the apoptosis rate was detected by Hoechst 33342 staining. Finally, the expression of Bcl-2 / Bax protein and the activity of Caspase-3 were detected by Western blot and Caspase-3 assay kit. Results MTT and LDH release assay in glutamate injury model showed that, The cell viability in MADP group was higher than that in Sal group by Hoechst33342Tunel Annexin V-FITC / Pi staining under the same condition. The results showed that the apoptotic rate in MADP group was lower than that in Sal group Fluo-4 / AM fluorescence staining showed that [Ca 2] I fluorescence intensity in Sal group was stronger than that in Sal group and Caspase-3 mRNA expression in Sal group was lower than that in MADP group. The results of Western blot showed that the expression of Bcl-2 / Bax and p-ERK protein did not change in both groups. The expression of p-Akt and p-JNK in MADP group was higher than that in Sal group. The results of MTT assay showed that most of the tested compounds had protective effect on PC12 cells, especially compound 6 had the strongest protective effect on PC12 cells. The preprotection of compound 6 could improve the activity of Caspase-3 in damaged PC12 cells by Hoechst 33342 staining and the results of Western blot assay showed that compound 6 could inhibit apoptosis of PC12 cells induced by low glucose and low serum levels in a dose-dependent manner. Compound 6 had the best protective effect at 300 渭 g / mL. Conclusion salidroside analogues MADP and compound 6 can protect hippocampal neurons and PC12 cells better than salidroside.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
，
本文编号：2124976
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