BDNF-TrkB通路通过下调ALDH2介导硫化氢的抗甲醛神经毒性作用

发布时间：2018-02-24 04:32

本文关键词： 硫化氢甲醛神经毒性脑源性神经营养因子乙醛脱氢酶-2　出处：《南华大学》2013年硕士论文　论文类型：学位论文

【摘要】：【研究背景与目的】甲醛(Formaldehyde, FA)具有神经毒性作用。我们研究发现新型气体信号分子硫化氢(Hydrogen sulfide, H2S)可拮抗甲醛的神经细胞毒性作用。但作用机理未阐明。脑源性神经营养因子(Brain derived neurophic factor, BDNF)是机体内一种重要的神经保护因子。乙醛脱氢酶-2(Aldehyde-dehydrogenase2,ALDH2)是机体重要的醛类代谢酶。我们推测H2S可通过调节BDNF和/或ALDH2实现其抗甲醛神经细胞毒性作用。为此，我们以甲醛损伤PC12细胞为甲醛神经毒性的细胞模型，从BDNF和ALDH2等方面探讨H2S抗甲醛神经细胞毒性的作用机制。【方法】 Cell Counting Kit-8(CCK-8)法检测PC12细胞活力；PI染色流式细胞术检测细胞凋亡；NBT还原法检测细胞内活性氧累积水平；ALDH2活性试剂盒检测PC12细胞ALDH2活性；Western Blot检测PC12细胞BDNF、ALDH2、Bax和Bcl-2的表达；Elisa法检测细胞内Caspase-3活力以及MDA和4-HNE的含量。【结果】 1. BDNF-TrkB通路介导H2S的抗甲醛损伤PC12细胞作用 1.1H2S可上调PC12细胞BDNF的表达 100,200和400μM的NaHS处理PC12细胞24h后，细胞BDNF的表达呈浓度依赖性地上升；提前30min加入200μM的H2S，，可抑制FA (120μM,24h)对PC12细胞BDNF表达的下调作用，提示H2S的抗甲醛神经毒性作用可能与其上调BDNF的表达相关。 1.2阻断BDNF-TrkB通路可逆转H2S对甲醛损伤PC12细胞的保护作用 PC12细胞先提前给予BDNF-TrkB通路阻断剂K252a (10nM)处理30min后，然后给予200μM H2S处理30min，最后加入120μM FA共处理细胞24h。结果显示K252a可逆转H2S对甲醛降低PC12细胞活力的抑制作用、对甲醛诱导PC1细胞凋亡的拮抗作用和对甲醛促进PC12细胞活性醛4-羟基壬烯醛(4-Hydroxy-2-nonenal,4-HNE)和丙二醛(Malondialdehyde, MDA)以及活性氧(Reactive oxygenspecies, ROS)积累的抑制作用，还可逆转H2S对甲醛下调PC12细胞Bcl-2表达和上调Bax表达的拮抗作用和对甲醛激活caspase-3的降低作用。这些结果显示K5252a阻断BDNF受体TrkB可取消H2S的抗甲醛神经细胞毒性作用。上述结果表明BDNF-TrkB通路介导了H2S的抗甲醛神经细胞毒性作用。 2. H2S可通过下调ALDH2拮抗甲醛损伤PC12细胞 2.1甲醛可上调PC12细胞ALDH2的表达和活性 60,120和240μM的FA处理PC12细胞24h后，细胞ALDH2的表达和活性呈浓度依赖性地增加。 2.2抑制ALDH2的活性可拮抗甲醛对PC12细胞的损伤作用 PC12细胞用ALDH2抑制剂Dadzin (10μM)预处理30min后，再加入甲醛(120μM)共处理24h， Daidzin可减轻甲醛对细胞活力的抑制作用和对细胞凋亡的诱导作用、可降低甲醛对PC12细胞MDA、4-HNE和活性氧的累积作用，表明抑制ALDH2的表达和活性可拮抗甲醛神经细胞毒性。 2.3H2S可抑制PC12细胞ALDH2的表达 100、200和400μM的NaHS处理PC12细胞24h后，细胞ALDH2的表达则呈浓度依赖性下降。 2.4H2S可抑制甲醛对PC12细胞ALDH2表达和活性的上调作用 PC12细胞经H2S (200μM)预处理30min，再合用FA(120μmol/L)24h后，FA (120μM)对PC12细胞ALDH2表达和活性的上调作用明显下降。上述结果表明H2S可通过下调ALDH2拮抗甲醛的神经细胞毒性作用。 3. BDNF-TrkB通路参与了H2S对PC12细胞ALDH2的下调作用 10nM K252a (TrkB的阻断剂)预处理PC12细胞30min后加入200μMH2S，30min后加入120μM的FA共处理细胞24h，H2S对甲醛上调ALDH-2表达的抑制作用明显减轻，表明BDNF-TrkB通路参与了H2S对PC12细胞ALDH2的下调作用。【结论】 H2S通过上调BDNF-TrkB通路、进而下调ALDH2而实现其抗甲醛神经细胞毒性作用。
[Abstract]:[research background and purpose]
Formaldehyde (Formaldehyde, FA) has neurotoxic effects. We found that hydrogen sulfide (Hydrogen sulfide, H2S) nerve cells can antagonize toxicity of formaldehyde. But the mechanism is not clear. The brain-derived neurotrophic factor (Brain derived, neurophic factor, BDNF) is considered as an important neuroprotective factor. Aldehyde dehydrogenase -2 (Aldehyde-dehydrogenase2, ALDH2) is an important metabolic enzymes in the body of aldehydes. We speculated that H2S may regulate BDNF and / or ALDH2 to realize the anti formaldehyde nerve cell toxicity.
Therefore, we use formaldehyde to damage PC12 cells to form a formaldehyde neurotoxic cell model, and discuss the mechanism of H2S anti formaldehyde cytotoxicity from BDNF and ALDH2.
[method]
Cell Counting Kit-8 (CCK-8) method to detect PC12 cell activity; PI staining and flow cytometry; active oxygen reduction method in the detection of NBT cell accumulation level; PC12 cell ALDH2 activity ALDH2 activity detection kit; Western Blot detection of PC12 cell BDNF, ALDH2, expression of Bax and Bcl-2; Elisa was detected by Caspase-3 activity and MDA and 4-HNE content.
[results]
1. BDNF-TrkB pathway mediates the effect of H2S on the anti formaldehyde damage of PC12 cells
1.1H2S can increase the expression of BDNF in PC12 cells
NaHS treatment of PC12 cells with 24h 100200 and 400 M, the expression of BDNF cells in a concentration dependent manner in 30min rise; add 200 mu M H2S, inhibited FA (120 M, 24h) on the expression of PC12 BDNF cells down regulated expression, suggesting that anti formaldehyde neurotoxicity of H2S and its regulation BDNF.
1.2 blocking BDNF-TrkB pathway can reverse the protective effect of H2S on formaldehyde damaged PC12 cells
PC12 cells give BDNF-TrkB pathway inhibitor K252a (10nM) after 30min treatment, then given 200 M H2S 30min, 120 M FA were added to the end of treatment of 24h. cells showed that K252a can inhibit the formaldehyde reducing PC12 reversed the effect of H2S on cell viability, antagonistic effects on apoptosis of PC1 cells induced by formaldehyde and formaldehyde to promote the PC12 cell activity of aldehyde 4- hydroxy 2-nonenal (4-Hydroxy-2-nonenal, 4-HNE) and malondialdehyde (Malondialdehyde, MDA) and reactive oxygen species (Reactive OXYGENSPECIES, ROS) inhibited the accumulation, but also can antagonize the expression of H2S reversed the effect of PC12 cell Bcl-2 expression and up regulation of Bax on formaldehyde under the activation of caspase-3 and reduce the role of formaldehyde. These results indicate that K5252a blockade of BDNF receptor TrkB to suppress anti formaldehyde nerve cell toxicity of H2S.
These results suggest that BDNF-TrkB pathway mediates the toxic effect of H2S on anti formaldehyde nerve cells.
2. H2S can antagonize formaldehyde by down regulation of ALDH2 to damage PC12 cells
2.1 formaldehyde can increase the expression and activity of ALDH2 in PC12 cells
After PC12 cell 24h was treated with 60120 and 240 M FA, the expression and activity of ALDH2 increased in a concentration dependent manner.
2.2 inhibition of ALDH2 activity can antagonize the damage of formaldehyde to PC12 cells
PC12 cells with ALDH2 inhibitor Dadzin (10 M) 30min after pretreatment, adding formaldehyde (120 M) were 24h, Daidzin can reduce the inhibitory effect of formaldehyde on cell viability and induce apoptosis of the PC12 cells, can reduce the formaldehyde on MDA, 4-HNE and ROS accumulation effect that inhibition of ALDH2 expression and activity can be nerve cell toxicity induced by formaldehyde.
2.3H2S inhibits the expression of ALDH2 in PC12 cells
After PC12 cell 24h was treated with 100200 and 400 M NaHS, the expression of ALDH2 in cells decreased in a concentration dependent manner.
2.4H2S inhibits the up-regulated effect of formaldehyde on the expression and activity of ALDH2 in PC12 cells
After the PC12 cells were pretreated with H2S (200 u M) for 30min and then FA (120 mol/L) 24h, FA (120 u M) decreased the ALDH2 expression and activity of PC12 cells.
These results suggest that H2S can antagonize the neurotoxicity of formaldehyde by down regulation of ALDH2.
3. BDNF-TrkB pathway participates in the downregulation of H2S on PC12 cell ALDH2
10nM K252a (TrkB blocker) pretreated PC12 cells 30min, added 200 MH2S, 30min added 120 M M FA 24h, H2S inhibited the inhibition of formaldehyde upregulated expression of the colon significantly, indicating that the pathway is involved in the downregulation of the cells.
[Conclusion]
The toxic effect of H2S was achieved by up regulation of BDNF-TrkB pathway and then down-regulation of ALDH2.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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