硫化氢上调SIRT1拮抗同型半胱氨酸诱导PC12细胞内质网应激

发布时间：2018-05-12 16:30

本文选题：硫化氢 + 同型半胱氨酸　；参考：《南华大学》2014年硕士论文

【摘要】：【研究背景与目的】同型半胱氨酸(Homocysteine, Hcy)具有神经毒性，是老年性痴呆的独立危险因子。我们以往的研究结果显示硫化氢(Hydrogen sulfide, H2S)具有抗Hcy神经毒性作用，但作用机制有待深入阐明。过度的内质网(Endoplasmic reticulum, ER)应激可导致神经凋亡。沉默信息调节因子(Silent informationation regulator1, SIRT1)对神经保护具有重要的调节作用。我们推测H2S通过调控SIRT1的表达拮抗Hcy所致的ER应激而发挥其抗Hcy神经毒性作用。为此，我们以Hcy损伤PC12细胞为Hcy神经毒性的细胞模型，探讨H2S对Hcy诱导PC12细胞ER应激的拮抗作用以及SIRT1对H2S抗ER应激和抗Hcy神经毒性作用的介导效应。【方法】 Cell Counting Kit-8(CCK-8)法检测PC12细胞存活率，碘化丙啶(propidiumiodide, PI)染色流式细胞仪(Flow cytometry, FCM)法检测PC12细胞凋亡率，蛋白质印迹法(Western blot)法检测ER应激相关蛋白(Glucose-regulated protein78, GRP78;Cleaved caspase-12, Cleaved caspase12)及SIRT1的表达情况。【结果】 1. Hcy可促进PC12细胞ER应激并下调PC12细胞SIRT1表达以1.25,2.5,5mM的Hcy分别处理PC12细胞24h后，PC12细胞内GRP78、Cleaved caspase12表达呈浓度依赖性上升，提示Hcy可诱导PC12细胞ER应激而发挥其神经毒性。以1.25,2.5,5mM的Hcy分别处理PC12细胞24h后，PC12细胞内SIRT1的表达呈浓度依赖性下降，提示Hcy诱导PC12细胞ER应激可能与其下调SIRT1的表达有关。 2. H2S拮抗Hcy诱导PC12细胞ER应激 200和400μM的NaHS (H2S供体)合用5mM的Hcy处理PC12细胞24h后，PC12细胞内GRP78和Cleaved caspase12的表达水平较Hcy (5mM,24h)单用时明显下降，表明H2S可拮抗Hcy诱导的PC12细胞ER应激。 3. SIRT1介导H2S的抗Hcy诱导PC12细胞内质网应激和神经毒性的作用 3.1H2S能上调PC12细胞SIRT1的表达 100、200和400μM的NaHS处理PC12细胞24h，PC12细胞SIRT1表达水平显著增加，表明H2S能促进PC12细胞高表达SIRT1。 3.2H2S能减轻Hcy对SIRT1表达的抑制作用 400μM的NaHS合用5mM的Hcy处理PC12细胞24h后，Hcy对PC12细胞SIRT1表达的抑制作用明显减轻。 3.3Sirtinol (SIRT1抑制剂)能阻断H2S对Hcy诱导PC12细胞ER应激的拮抗作用 20nM的Sirtinol预处理PC12细胞2h后，H2S对Hcy诱导PC12细胞GRP78和Cleaved caspase12表达的抑制作用被阻断，表明H2S可通过上调SIRT1的表达拮抗Hcy诱导PC12细胞ER应激。 3.4Sirtinol (SIRT1抑制剂)能阻断H2S对Hcy诱导PC12细胞毒性和凋亡的拮抗作用 20nM的Sirtinol预处理PC12细胞2h后，H2S对Hcy诱导PC12细胞活力下降和凋亡增加的拮抗作用被阻断，表明H2S可通过上调SIRT1的表达拮抗Hcy的神经毒性。【结论】 1. Hcy可通过下调SIRT1表达诱导PC12细胞ER应激； 2. H2S可通过上调SIRT1的表达拮抗Hcy诱导PC12细胞ER应激。
[Abstract]:[background and purpose of the study] Homocysteine (Hcyine) has neurotoxicity and is an independent risk factor for Alzheimer's disease. Our previous studies have shown that hydrogen sulfide Hydrogen sulfide (H _ 2S) has an anti-neurotoxic effect on Hcy, but the mechanism of action needs to be further elucidated. Excessive endoplasmic reticulum stress (ERS) may lead to neuronal apoptosis. Silent informationation regulator 1 (SIRT1) plays an important role in neuroprotection. We speculate that H2S can inhibit Hcy neurotoxicity by regulating the expression of SIRT1 and antagonizing ER stress induced by Hcy. Therefore, we used Hcy to damage PC12 cells as a cell model of Hcy neurotoxicity, to investigate the antagonistic effect of H2S on ER stress induced by Hcy and the mediated effect of SIRT1 on anti-ER stress and anti-Hcy neurotoxicity of PC12 cells induced by H2S. [methods] The survival rate of PC12 cells was detected by Cell Counting Kit-8 CCK-8 method, the apoptosis rate of PC12 cells was detected by flow cytometry, and the expression of ER stress-related proteins Glucose-regulated protein 78, GRP78Cleaved caspase-12, Cleaved caspase12 and SIRT1 were detected by Western blot. [results] 1. Hcy promoted ER stress in PC12 cells and down-regulated SIRT1 expression in PC12 cells treated with Hcy of 1.25 ~ 2.5mm for 24 h. The expression of GRP78-Cleaved caspase12 in PC12 cells increased in a concentration-dependent manner, suggesting that Hcy could induce ER stress in PC12 cells and exert its neurotoxicity. The expression of SIRT1 in PC12 cells decreased in a concentration-dependent manner after treated with 1.25 ~ 2.5mm Hcy for 24 h, suggesting that ER stress induced by Hcy might be related to the down-regulation of SIRT1 expression in PC12 cells. 2. H _ 2S antagonizes ER stress induced by Hcy in PC12 cells The expression levels of GRP78 and Cleaved caspase12 in PC12 cells treated with 200 渭 M and 400 渭 M NaHS / H2S + 5mM Hcy for 24 h were significantly lower than those in Hcy 5 mm M2s alone, indicating that H2S could antagonize ER stress induced by Hcy in PC12 cells. 3. Effects of SIRT1 mediated H2S on endoplasmic reticulum stress and neurotoxicity induced by Hcy in PC12 cells 3.1H2S can up-regulate the expression of SIRT1 in PC12 cells 100200 and 400 渭 M NaHS treatment increased the expression of SIRT1 in PC12 cells for 24 h, suggesting that H2S could promote the overexpression of SIRT1 in PC12 cells. 3.2H2S can attenuate the inhibitory effect of Hcy on the expression of SIRT1 After treated with 400 渭 M NaHS and 5mM Hcy for 24 h, the inhibitory effect of Hcy on SIRT1 expression in PC12 cells was significantly alleviated. 3.3Sirtinol inhibitor SIRT1 can block the antagonistic effect of H 2S on ER stress induced by Hcy in PC12 cells. After pretreatment of PC12 cells with Sirtinol of 20nM for 2 h, the inhibitory effect of H2S on GRP78 and Cleaved caspase12 expression induced by Hcy was blocked, which indicated that H2S could antagonize ER stress induced by Hcy by up-regulating SIRT1 expression. 3.4Sirtinol inhibitor SIRT1 can block the antagonistic effect of H 2S on the cytotoxicity and apoptosis of PC12 cells induced by Hcy After pretreatment of PC12 cells with Sirtinol of 20nM for 2 h, the antagonistic effect of H 2S on the decrease of PC12 cell viability and increase of apoptosis induced by Hcy was blocked, which indicated that H2S could antagonize the neurotoxicity of Hcy by up-regulating the expression of SIRT1. [conclusion] 1. Hcy can induce ER stress in PC12 cells by down-regulation of SIRT1 expression. 2. H _ 2S can antagonize ER stress of PC12 cells induced by Hcy by up-regulating the expression of SIRT1.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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