硫化氢对甲醛损伤PC12细胞的保护作用及其机制

发布时间：2018-04-23 01:21

本文选题：硫化氢 + 甲醛　；参考：《南华大学》2011年硕士论文

【摘要】：【研究背景与目的】甲醛(formaldehyde, FA)是一种具有挥发性的有毒物质,其神经毒性越来越受到人们的关注。甲醛暴露可直接造成中枢神经系统的损害。同时,人类机体自身也可产生甲醛,内源性甲醛慢性损伤可能是神经退行性疾病发病的一个重要机制。因此,深入探讨甲醛神经毒性的防治措施具有重要意义。硫化氢(hydrogen sulfide, H_2S)广泛参与机体多种生理和病理过程,被认为是继一氧化氮和一氧化碳之后的第三类气体信号分子。近年来,内源性H_2S对神经功能的调节作用已得到公认,特别是其抗氧化、神经保护的功能倍受关注。越来越多的证据显示氧化应激是甲醛暴露时产生的最关键效应。是否可以用H_2S来防治甲醛的神经毒性呢?值得深入研究。为此,本研究将以甲醛损害PC12细胞为甲醛神经毒性的细胞模型,探讨H_2S对甲醛神经毒性的拮抗作用及其机制。【方法】胎盘蓝拒染法观察PC12细胞的存活率;Elisa法检测细胞上清液中乳酸脱氢酶(lactate dehydrogenase, LDH)的含量,评价PC12细胞的损伤程度;Hoechst 33258染色后,荧光显微镜下观察PC12细胞核染色体形态改变及PI染色流式细胞仪(flow cytometry, FCM)检测细胞凋亡;罗丹明123 (Rhodamine123, Rh123)染色后,FCM检测细胞线粒体膜电位(mitochondrial membrane potential, MMP);JC-1染色后,荧光显微镜下观察细胞内MMP变化; DCFH-DA染色后,荧光显微镜及FCM检测细胞内活性氧(reactive oxydren species, ROS)水平;Western Blot检测PC12细胞内Bcl-2和对氧磷酶-1 (Paraoxonase-1, PON-1)的蛋白表达状况以及细胞色素c (cytochrome c, Cyt-c)的释放;分光光度计法检测PC12细胞内PON-1活性。【结果】 1.甲醛对PC12细胞的毒性作用及机制甲醛(60、120、240μmol/L)处理PC12细胞24 h后,能浓度依赖性地抑制PC12细胞的活力、促进细胞LDH的释放。PC12细胞经120μmol/L甲醛处理24 h后,细胞变成圆形或椭圆形,而且可见部分细胞游离于孔壁呈悬浮状态,并有大量的胞核呈现为强荧光的凋亡细胞;PI染色FCM定量分析亦表明甲醛(120μmol/L)作用24 h可显著诱导PC12细胞凋亡。上述结果表明甲醛对PC12细胞具有毒性作用。 120μmol/L甲醛作用9 h后,细胞MMP下降;PC12细胞经60、120、240μmol/L甲醛作用24 h后,Bcl-2蛋白表达水平呈浓度依赖性地降低,Cyt-c的释放呈浓度依赖性地增强,表明甲醛可通过激活细胞线粒体凋亡通路诱导细胞凋亡和神经毒性。 120μmol/L甲醛处理PC12细胞9 h后,细胞内ROS显著升高;甲醛(60、120、240μmol/L)作用24 h后,PC12细胞PON-1的蛋白表达及其酶活性呈浓度依赖性地降低,提示甲醛的神经毒性与其抑制PON-1蛋白表达及其酶活性,进而促进ROS积累有关。 2.硫化氢对甲醛损伤PC12细胞的保护作用及机制 200μmol/L H_2S预处理30 min可明显升高120、240μmol/L甲醛作用24 h后的PC12细胞活力,200、400μmol/L H_2S预处理30 min可明显升高120μmol/L甲醛作用24 h后的PC12细胞活力。200μmol/L H_2S预处理PC12细胞30 min能显著降低120μmol/L甲醛处理24 h后对细胞LDH释放的促进作用、对细胞形态的损害作用、对细胞凋亡的诱导作用。这些结果表明H_2S对甲醛损伤PC12细胞具有拮抗作用。 200μmol/L H_2S预处理30 min可显著抑制120μmol/L甲醛对PC12细胞MMP的降低作用、对Bcl-2表达的下调作用和对Cyt-c释放的促进作用,表明H_2S可阻止甲醛激活细胞线粒体凋亡通路。 200μmol/L H_2S预处理30 min可显著减轻120μmol/L甲醛对PC12细胞内ROS积累的诱导作用、对细胞PON-1表达及其活性的抑制作用,而且200μmol/L H_2S可显著增强PON-1的活性,表明H_2S可抑制ROS积累,阻止甲醛抑制PON-1的表达和活性,并能上调PON-1的活性。 200μmol/L PON-1的特异性抑制剂二羟基喹啉(2-Hydroxy-4-methylquinoline, 2-HQ)预处理30 min可显著降低200μmol/L H_2S对甲醛(120μmol/L, 24h)促进PC12细胞内ROS积累、诱导细胞凋亡、降低PC12细胞活力的拮抗作用,表明H_2S可通过阻止甲醛抑制PON-1的表达和活性、增强PON-1的活性而降低细胞ROS的积累,进而阻止甲醛激活细胞线粒体凋亡通路而实现其抗甲醛神经毒性作用。【结论】 1.甲醛对PC12细胞具有细胞毒性作用,其机理可能与其抑制细胞PON-1蛋白的表达和活性,从而促进细胞内ROS的积累,进而激活细胞线粒体凋亡通路有关。 2.硫化氢可拮抗甲醛对PC12细胞的毒性作用,其机理可能与其增强细胞PON-1活性、减轻甲醛对PON-1蛋白表达和活性的抑制作用,从而降低细胞内ROS含量,并进一步抑制甲醛激活细胞线粒体凋亡通路有关。
[Abstract]:[research background and purpose]
Formaldehyde (FA) is a volatile toxic substance, and its neurotoxicity is attracting more and more attention. Formaldehyde exposure can directly cause damage to the central nervous system. At the same time, the human body can also produce formaldehyde. The chronic damage of endogenous formaldehyde may be an important mechanism for the pathogenesis of neurodegenerative disease. Therefore, it is of great significance to explore the prevention and cure measures of formaldehyde neurotoxicity.
Hydrogen sulfide (H_2S) is widely involved in various physiological and pathological processes of the body. It is considered to be the third kind of gas signal after nitric oxide and carbon monoxide. In recent years, the regulation of endogenous H_2S has been recognized, especially its anti oxidation, and the function of neuroprotection has attracted much attention.
More and more evidence shows that oxidative stress is the most important effect of formaldehyde exposure. Is it possible to use H_2S to prevent the neurotoxicity of formaldehyde? It is worth further study. Therefore, this study will explore the antagonism and mechanism of H_2S to formaldehyde neurotoxicity by using formaldehyde to damage the PC12 cell as a formaldehyde neurotoxicity.
[method]
The survival rate of PC12 cells was observed by placental blue staining; the content of lactate dehydrogenase (LDH) in cell supernatant was detected by Elisa method and the damage degree of PC12 cells was evaluated. After Hoechst 33258 staining, the morphological changes of chromosomes in PC12 and PI staining flow cytometer (flow cytometry, FCM) were observed under the fluorescence microscope. After staining with Luo Danming 123 (Rhodamine123, Rh123), the mitochondrial membrane potential (mitochondrial membrane potential, MMP) was detected by FCM. After JC-1 was stained, the MMP changes in the cells were observed under the fluorescence microscope. After DCFH-DA staining, the fluorescence microscope and FCM detected the intracellular reactive oxygen species. T detected the expression of Bcl-2 in PC12 cells and the expression of -1 (Paraoxonase-1, PON-1) and the release of cytochrome c (cytochrome c, Cyt-c), and the spectrophotometer was used to detect the PON-1 activity in the PC12 cells.
[results]
The toxic effect of 1. formaldehyde on PC12 cells and its mechanism
After the treatment of 24 h by formaldehyde (60120240 mol/L), the activity of PC12 cells can be inhibited in a concentration dependent manner, which promotes the release of.PC12 cells from LDH by 120 mu mol/L formaldehyde to treat 24 h, and the cells become round or oval, and some cells are suspended in the wall of the pore, and a large number of nuclei appear to be strong fluorescent apoptosis. PI staining FCM quantitative analysis also showed that formaldehyde (120 u mol/L) action of 24 h could significantly induce apoptosis of PC12 cells. The above results showed that formaldehyde had toxic effects on PC12 cells.
After the action of 120 mu mol/L formaldehyde for 9 h, the cell MMP decreased, and the expression level of Bcl-2 protein decreased in a concentration dependent manner after the 24 h action of 60120240 mu mol/L formaldehyde. The release of Cyt-c increased in a concentration dependent manner, indicating that formaldehyde could induce apoptosis and neurotoxicity by activating the apoptosis pathway of mitochondria.
After 120 mu mol/L formaldehyde treated PC12 cells for 9 h, the intracellular ROS increased significantly. After 24 h of formaldehyde (60120240 mu mol/L), the protein expression and enzyme activity of PON-1 in PC12 cells decreased in a concentration dependent manner. It suggested that the neurotoxicity of formaldehyde was related to the inhibition of the expression of PON-1 protein and the activity of the enzyme, thus promoting the accumulation of ROS.
Protective effect and mechanism of hydrogen sulfide on formaldehyde injured PC12 cells 2.
200 mu mol/L H_2S pretreatment 30 min can significantly increase the activity of PC12 cells after 120240 u mol/L formaldehyde effect 24 h, 200400 mu mol/L H_2S pretreatment 30 min can obviously increase the activity of 120 mu mol/L after 24 h PC12 cells 30 These results indicate that H_2S has an antagonistic effect on formaldehyde induced PC12 cells.
200 mol/L H_2S pretreatment 30 min could significantly inhibit the decrease of 120 mu mol/L on PC12 cell MMP, the down regulation of Bcl-2 expression and the promotion of Cyt-c release, indicating that H_2S can prevent the apoptosis pathway of formaldehyde activated mitochondria.
200 mu mol/L H_2S pretreatment 30 min could significantly reduce the induction of ROS accumulation in PC12 cells by 120 mu mol/L, and inhibit the expression and activity of PON-1 in cell, and 200 u mol/L H_2S significantly enhanced the activity of PON-1, indicating that H_2S could inhibit the accumulation of ROS, prevent the expression and activity of formaldehyde inhibited and up regulate the activity of PON-1.
The pre treatment of two hydroxyquinoline (2-Hydroxy-4-methylquinoline, 2-HQ), a specific inhibitor of 200 mol/L PON-1, could significantly reduce the inhibitory effect of 200 mu H_2S on the accumulation of formaldehyde (120 mu mol/L, 24h) in PC12 cells, inducing cell apoptosis and reducing the activity of PC12 cells. Activity can enhance the activity of PON-1 and decrease the accumulation of ROS, thereby preventing formaldehyde from activating mitochondrial apoptosis pathway and achieving its neuroprotective effect against formaldehyde.
[Conclusion]
1. formaldehyde has cytotoxic effect on PC12 cells. The mechanism may be related to the inhibition of the expression and activity of PON-1 protein in cell, thus promoting the accumulation of ROS in cells and activating the apoptosis pathway of cell mitochondria.
2. hydrogen sulfide can antagonize the toxic effect of formaldehyde on PC12 cells. Its mechanism may enhance the activity of PON-1, reduce the inhibitory effect of formaldehyde on the expression and activity of PON-1 protein, thus reduce the intracellular ROS content, and further inhibit the activation of the mitochondrial apoptosis pathway by formaldehyde.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

【参考文献】