硫化氢通过上调瘦素信号通路拮抗槟榔碱诱导的PC12细胞毒性

发布时间：2018-10-10 10:14

【摘要】：[研究背景与目的]最近研究发现槟榔中的主要生物碱槟榔碱(Arecoline)具有神经毒性作用,其神经毒性与其诱导细胞凋亡和氧化应激有关。硫化氢(Hydrogen sulfide,H2S)不仅仅是一种新型的中枢系统神经调质,而且是一种具有抗凋亡和抗氧化应激作用的神经保护剂。瘦素(leptin)是由脂肪细胞分泌的一种激素,通过作用于在中枢神经系统(Central nervous system,CNS)广泛分布的长型瘦素受体(leptin receptor,lep Rb)发挥多种生物学功能。越来越多的研究证实leptin信号通路具有神经保护作用。因此,在本研究中,我们拟探讨H2S是否可拮抗Arecoline诱导的神经细胞毒性以及leptin信号通路是否介导H2S拮抗Arecoline诱导的神经细胞毒性。[方法]Cell Counting Kit-8(CCK-8)法检测PC12细胞活力;Hoechst 33258染色检测凋亡细胞形态;Annexin V/PI(propidium iodide)染色流式细胞术检测PC12细胞凋亡率;Elisa法检测PC12细胞内Caspase-3活力和细胞培养上清液中leptin水平;Unisensn H2S微电极检测PC12细胞培养上清液中H2S水平;Western Blot检测PC12细胞内H2S生成酶:胱硫醚-β-合成酶(Cystathionine-bata-synthase,CBS)和3-巯基丙酮酸盐硫(3-mercaptopyruvate sulphurtransferase,3-MST)蛋白的表达、leptin和Lep Rb蛋白的表达、凋亡相关蛋白(Bax和Bcl-2)、以及内质网应激相关蛋白Glucose-regulated protein 78(GRP78)、CCAAT/enhancer binding protein(C/EBP)homologous protein(CHOP)和Cleaved caspase-12 proteins的表达。[结果]Arecoline(0.5、1和2m M)处理PC12细胞24 h能明显降低PC12细胞活力、诱导PC12细胞发生凋亡形态改变以及增加细胞凋亡率、上调细胞内Caspase-3的活性以及促凋亡蛋白Bax的表达、降低抗凋亡蛋白Bcl-2的表达,增加PC12细胞内质网应激相关蛋白(GRP78、CHOP和Cleaved caspase-12)的表达,降低细胞内H2S合酶(CBS和3-MST)蛋白表达以及培养上清液中H2S水平。而预处理Na HS(H2S供体,400和800μM)能明显逆转Arecoline诱导的PC12细胞损伤、凋亡以及内质网应激,并可抑制Arecoline诱导的leptin和lep Rb蛋白表达的降低以及培养上清液中leptin水平的下降,而预处理leptin t A(leptin信号通路抑制剂,50 n M)能取消Na HS对Arecoline诱导的神经细胞毒性的保护作用。[结论]H2S通过上调leptin信号通路实现其抗Arecoline神经细胞毒性作用。
[Abstract]:[background and objective] recent studies have found that arecoline (Arecoline), the main alkaloid in areca nut, has neurotoxic effects, and its neurotoxicity is related to its induction of apoptosis and oxidative stress. Hydrogen sulfide (Hydrogen sulfide,H2S) is not only a new neuromodulator of central nervous system, but also a neuroprotective agent with anti-apoptosis and anti-oxidative stress. Leptin (leptin), a hormone secreted by adipocytes, plays a variety of biological functions by acting on the long leptin receptor (leptin receptor,lep Rb) widely distributed in the central nervous system (Central nervous system,CNS). More and more studies have confirmed the neuroprotective effect of leptin signaling pathway. Therefore, in this study, we intend to investigate whether H2S can antagonize the neuronal toxicity induced by Arecoline and whether the leptin signaling pathway mediates H2S to antagonize the neuronal toxicity induced by Arecoline. [methods] Cell Counting Kit-8 (CCK-8) assay was used to detect the viability of PC12 cells by Hoechst 33258 staining. Apoptosis rate of PC12 cells was detected by flow cytometry with Annexin V/PI (propidium iodide) staining. Caspase-3 activity in PC12 cells and leptin level in supernatant of cell culture were detected by Elisa. The level of H2S in the supernatant of PC12 cells was detected by electrode and the expression of H2S producing enzymes: cystathithione- 尾 -synthase (Cystathionine-bata-synthase,CBS) and 3-mercaptopyruvate sulfur (3-mercaptopyruvate sulphurtransferase,3-MST) protein in PC12 cells were detected by Western Blot. Apoptosis-related proteins (Bax and Bcl-2) and endoplasmic reticulum stress-related protein Glucose-regulated protein 78 (GRP78) expressed CCAATP enhancer binding protein (C/EBP) homologous protein (CHOP) and Cleaved caspase-12 proteins. [results] after treated with Arecoline (0.5 and 2m M) for 24 h, PC12 cells significantly decreased the viability of PC12 cells, induced apoptosis morphologic changes in PC12 cells, increased the rate of apoptosis, up-regulated the activity of Caspase-3 and the expression of pro-apoptotic protein Bax. The expression of anti-apoptotic protein Bcl-2 increased the expression of endoplasmic reticulum stress-related proteins (GRP78,CHOP and Cleaved caspase-12) in PC12 cells decreased the expression of H2S synthase (CBS and 3-MST) and the level of H2S in culture supernatant. Pretreatment with Na HS (H2S donor 400 and 800 渭 M could significantly reverse the PC12 cell damage, apoptosis and endoplasmic reticulum stress induced by Arecoline, and inhibit the decrease of leptin and lep Rb protein expression induced by Arecoline and the decrease of leptin level in culture supernatant. Pretreatment of leptin t A (leptin signaling pathway inhibitor of 50 n M) could cancel the protective effect of Na HS on the neuronal toxicity induced by Arecoline. [conclusion] H _ 2S can inhibit the toxicity of Arecoline neurons by upregulating leptin signaling pathway.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R114

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