本文關(guān)鍵詞： 硫化氫 甲醛 神經(jīng)毒性 腦源性神經(jīng)營養(yǎng)因子 乙醛脫氫酶-2　出處：《南華大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：【研究背景與目的】 甲醛(Formaldehyde, FA)具有神經(jīng)毒性作用。我們研究發(fā)現(xiàn)新型氣體信號分子硫化氫(Hydrogen sulfide, H2S)可拮抗甲醛的神經(jīng)細(xì)胞毒性作用。但作用機(jī)理未闡明。腦源性神經(jīng)營養(yǎng)因子(Brain derived neurophic factor, BDNF)是機(jī)體內(nèi)一種重要的神經(jīng)保護(hù)因子。乙醛脫氫酶-2(Aldehyde-dehydrogenase2,ALDH2)是機(jī)體重要的醛類代謝酶。我們推測H2S可通過調(diào)節(jié)BDNF和/或ALDH2實(shí)現(xiàn)其抗甲醛神經(jīng)細(xì)胞毒性作用。 為此，我們以甲醛損傷PC12細(xì)胞為甲醛神經(jīng)毒性的細(xì)胞模型，從BDNF和ALDH2等方面探討H2S抗甲醛神經(jīng)細(xì)胞毒性的作用機(jī)制。 【方法】 Cell Counting Kit-8(CCK-8)法檢測PC12細(xì)胞活力；PI染色流式細(xì)胞術(shù)檢測細(xì)胞凋亡；NBT還原法檢測細(xì)胞內(nèi)活性氧累積水平；ALDH2活性試劑盒檢測PC12細(xì)胞ALDH2活性；Western Blot檢測PC12細(xì)胞BDNF、ALDH2、Bax和Bcl-2的表達(dá)；Elisa法檢測細(xì)胞內(nèi)Caspase-3活力以及MDA和4-HNE的含量。 【結(jié)果】 1. BDNF-TrkB通路介導(dǎo)H2S的抗甲醛損傷PC12細(xì)胞作用 1.1H2S可上調(diào)PC12細(xì)胞BDNF的表達(dá) 100,200和400μM的NaHS處理PC12細(xì)胞24h后，細(xì)胞BDNF的表達(dá)呈濃度依賴性地上升；提前30min加入200μM的H2S，，可抑制FA (120μM,24h)對PC12細(xì)胞BDNF表達(dá)的下調(diào)作用，提示H2S的抗甲醛神經(jīng)毒性作用可能與其上調(diào)BDNF的表達(dá)相關(guān)。 1.2阻斷BDNF-TrkB通路可逆轉(zhuǎn)H2S對甲醛損傷PC12細(xì)胞的保護(hù)作用 PC12細(xì)胞先提前給予BDNF-TrkB通路阻斷劑K252a (10nM)處理30min后，然后給予200μM H2S處理30min，最后加入120μM FA共處理細(xì)胞24h。結(jié)果顯示K252a可逆轉(zhuǎn)H2S對甲醛降低PC12細(xì)胞活力的抑制作用、對甲醛誘導(dǎo)PC1細(xì)胞凋亡的拮抗作用和對甲醛促進(jìn)PC12細(xì)胞活性醛4-羥基壬烯醛(4-Hydroxy-2-nonenal,4-HNE)和丙二醛(Malondialdehyde, MDA)以及活性氧(Reactive oxygenspecies, ROS)積累的抑制作用，還可逆轉(zhuǎn)H2S對甲醛下調(diào)PC12細(xì)胞Bcl-2表達(dá)和上調(diào)Bax表達(dá)的拮抗作用和對甲醛激活caspase-3的降低作用。這些結(jié)果顯示K5252a阻斷BDNF受體TrkB可取消H2S的抗甲醛神經(jīng)細(xì)胞毒性作用。 上述結(jié)果表明BDNF-TrkB通路介導(dǎo)了H2S的抗甲醛神經(jīng)細(xì)胞毒性作用。 2. H2S可通過下調(diào)ALDH2拮抗甲醛損傷PC12細(xì)胞 2.1甲醛可上調(diào)PC12細(xì)胞ALDH2的表達(dá)和活性 60,120和240μM的FA處理PC12細(xì)胞24h后，細(xì)胞ALDH2的表達(dá)和活性呈濃度依賴性地增加。 2.2抑制ALDH2的活性可拮抗甲醛對PC12細(xì)胞的損傷作用 PC12細(xì)胞用ALDH2抑制劑Dadzin (10μM)預(yù)處理30min后，再加入甲醛(120μM)共處理24h， Daidzin可減輕甲醛對細(xì)胞活力的抑制作用和對細(xì)胞凋亡的誘導(dǎo)作用、可降低甲醛對PC12細(xì)胞MDA、4-HNE和活性氧的累積作用，表明抑制ALDH2的表達(dá)和活性可拮抗甲醛神經(jīng)細(xì)胞毒性。 2.3H2S可抑制PC12細(xì)胞ALDH2的表達(dá) 100、200和400μM的NaHS處理PC12細(xì)胞24h后，細(xì)胞ALDH2的表達(dá)則呈濃度依賴性下降。 2.4H2S可抑制甲醛對PC12細(xì)胞ALDH2表達(dá)和活性的上調(diào)作用 PC12細(xì)胞經(jīng)H2S (200μM)預(yù)處理30min，再合用FA(120μmol/L)24h后，F(xiàn)A (120μM)對PC12細(xì)胞ALDH2表達(dá)和活性的上調(diào)作用明顯下降。 上述結(jié)果表明H2S可通過下調(diào)ALDH2拮抗甲醛的神經(jīng)細(xì)胞毒性作用。 3. BDNF-TrkB通路參與了H2S對PC12細(xì)胞ALDH2的下調(diào)作用 10nM K252a (TrkB的阻斷劑)預(yù)處理PC12細(xì)胞30min后加入200μMH2S，30min后加入120μM的FA共處理細(xì)胞24h，H2S對甲醛上調(diào)ALDH-2表達(dá)的抑制作用明顯減輕，表明BDNF-TrkB通路參與了H2S對PC12細(xì)胞ALDH2的下調(diào)作用。 【結(jié)論】 H2S通過上調(diào)BDNF-TrkB通路、進(jìn)而下調(diào)ALDH2而實(shí)現(xiàn)其抗甲醛神經(jīng)細(xì)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.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R114

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;The protective effect of vitamin E against oxidative damage caused by formaldehyde in the testes of adult rats[J];Asian Journal of Andrology;2006年05期

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