本文選題：硫氧還蛋白-1 + 甲醛　； 參考：《昆明理工大學(xué)》2012年博士論文

【摘要】：硫氧還蛋白1(Trx-1)是分子量為12KDa的多功能蛋白質(zhì),含有一個(gè)氧化還原的活性位點(diǎn)：-Cys-Gly-Pro-Cys-。硫氧還蛋白具有多種生物學(xué)功能：調(diào)節(jié)氧化還原、調(diào)節(jié)轉(zhuǎn)錄因子活性、抑制細(xì)胞凋亡以及調(diào)節(jié)炎癥反應(yīng)等。Trx-1能被許多應(yīng)激誘導(dǎo)產(chǎn)生,如過氧化氫、有絲分裂原、多環(huán)芳烴、紫外線、病毒感染和缺血再灌注等。Trx-1具有神經(jīng)營(yíng)養(yǎng)因子樣活性,在神經(jīng)生長(zhǎng)因子誘導(dǎo)的神經(jīng)分化和突出生長(zhǎng)其重要的作用。Trx-1高表達(dá)轉(zhuǎn)基因小鼠與野生型小鼠相比,壽命顯著地延長(zhǎng),而且對(duì)缺血再灌注損傷、糖尿病以及外源環(huán)境應(yīng)激引起的毒害具有明顯的抵抗作用。Trx-1與內(nèi)質(zhì)網(wǎng)應(yīng)激密切相關(guān),具有抵抗內(nèi)質(zhì)網(wǎng)應(yīng)激損傷的作用。目前研究發(fā)現(xiàn)：替普瑞酮(GGA)、蘿卜硫素、神經(jīng)妥樂和泰莫普利等都可以通過誘導(dǎo)Trx-1的表達(dá)水平起細(xì)胞保護(hù)作用。其中,替普瑞酮的研究應(yīng)用最為深入和透徹。替普瑞酮(GGA)是臨床上廣泛用于胃潰瘍治療的藥物,能在多種細(xì)胞中誘導(dǎo)Trx-1的表達(dá),對(duì)胃黏膜、肝臟和心臟等組織起保護(hù)作用。GGA還是一種脂溶性物質(zhì),能透過血腦屏障對(duì)神經(jīng)組織起重要的保護(hù)作用。調(diào)控Trx-1的表達(dá)已成為一個(gè)新的疾病治療策略,無毒性的Trx-1誘導(dǎo)物能促進(jìn)神經(jīng)再生,對(duì)氧化應(yīng)激相關(guān)的神經(jīng)系統(tǒng)疾病具有預(yù)防作用。因此,本論文擬從云南省的天然藥物資源出發(fā),尋找無毒性的Trx-1誘導(dǎo)物用于疾病的治療研究。 甲醛是一種常見的環(huán)境污染物,廣泛存在于香煙煙霧、家具、汽車尾氣、醫(yī)療和工業(yè)用品中。研究表明,室內(nèi)甲醛濃度的增加是導(dǎo)致嚴(yán)重性建筑綜合癥的一個(gè)重要原因。甲醛對(duì)神經(jīng)系統(tǒng)具有毒害作用,已被認(rèn)為是一種潛在的“神經(jīng)毒害物”。長(zhǎng)期暴露于甲醛會(huì)造成神經(jīng)毒害,引發(fā)神經(jīng)退行性疾病,同時(shí),甲醛能改變大鼠大腦的形態(tài)特征,導(dǎo)致行為異常和記憶功能障礙。因此,甲醛已經(jīng)成為危害身體健康的一個(gè)重要因素。為了有效預(yù)防和治療甲醛對(duì)人體的毒性,深入研究甲醛毒害的分子機(jī)制具有重要的意義。 內(nèi)質(zhì)網(wǎng)是蛋白質(zhì)合成和加工的主要場(chǎng)所,在維持細(xì)胞內(nèi)鈣離子濃度和蛋白糖基化具有重要的調(diào)節(jié)作用。當(dāng)內(nèi)質(zhì)網(wǎng)功能紊亂以及未折疊/錯(cuò)誤折疊蛋白過量累積時(shí),會(huì)導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激。內(nèi)質(zhì)網(wǎng)中含有許多分子伴侶,當(dāng)內(nèi)質(zhì)網(wǎng)應(yīng)激發(fā)生時(shí),糖調(diào)節(jié)蛋白78(GRP78)的表達(dá)水平會(huì)發(fā)生顯著增加,以增強(qiáng)內(nèi)質(zhì)網(wǎng)的蛋白質(zhì)折疊能力。內(nèi)質(zhì)網(wǎng)應(yīng)激程度較低時(shí),激活抗凋亡的分子伴侶；但當(dāng)內(nèi)質(zhì)網(wǎng)應(yīng)激過于嚴(yán)重和持久時(shí),則激活JNK、CHOP和caspase-12,導(dǎo)致細(xì)胞凋亡,并且內(nèi)質(zhì)網(wǎng)應(yīng)激與神經(jīng)細(xì)胞死亡密切相關(guān)。甲醛是一種交聯(lián)劑,能與巰基和氨基酸基團(tuán)相互作用,導(dǎo)致蛋白質(zhì)的多聚化,同時(shí)甲醛會(huì)導(dǎo)致蛋白質(zhì)錯(cuò)誤折疊和累積。甲醛與p淀粉樣蛋白相互作用,產(chǎn)生具有神經(jīng)毒性的淀粉樣蛋白復(fù)合物。甲醛還會(huì)導(dǎo)致tau蛋白聚集,誘導(dǎo)人神經(jīng)母細(xì)胞瘤和海馬神經(jīng)細(xì)胞的凋亡。然而,內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的凋亡途徑與甲醛所致神經(jīng)細(xì)胞死亡的相關(guān)研究還未見報(bào)道。 嗎啡濫用已經(jīng)成為全球嚴(yán)重的公共衛(wèi)生和社會(huì)問題。長(zhǎng)期反復(fù)使用嗎啡會(huì)導(dǎo)致軀體依賴和精神依賴的發(fā)生,這限制了它的臨床用途(治療各種急性和慢性疼痛)。嗎啡成癮的主要表現(xiàn)是強(qiáng)迫性用藥,即對(duì)藥物的攝取和尋覓失去控制。成癮的原因主要由于正性強(qiáng)化因素(欣快感和獎(jiǎng)賞效應(yīng))和負(fù)性強(qiáng)化因素(減輕戒斷癥狀)。中腦腹側(cè)被蓋區(qū)、伏隔核、前額葉皮層、杏仁核和海馬等腦區(qū)與嗎啡成癮的過程相關(guān)。除了神經(jīng)系統(tǒng)的表現(xiàn)外,嗎啡在肝臟和腎臟中會(huì)導(dǎo)致氧化應(yīng)激和細(xì)胞凋亡。長(zhǎng)期使用阿片類藥物會(huì)導(dǎo)致肝臟、腎臟損傷,表現(xiàn)有急性肝炎、慢性肝炎等肝臟損傷,以及腎病綜合癥、急性腎小球腎炎、間質(zhì)性腎炎和腎功能衰竭。因此,在藥物成癮治療的過程中需要注意阿片類藥物所致的肝臟和腎臟損傷。尋找保護(hù)嗎啡所致的肝臟和腎臟損傷的藥物對(duì)于降低嗎啡的副作用及更廣泛地應(yīng)用嗎啡具有重要的意義。而且,目前還沒有理想的藥物和方法用于嗎啡成癮的治療。 甲醛和嗎啡都可以導(dǎo)致氧化應(yīng)激損傷、細(xì)胞凋亡和神經(jīng)營(yíng)養(yǎng)因子下調(diào),但其作用方式各不相同；而Trx-1具有抗氧化、抗凋亡和神經(jīng)營(yíng)養(yǎng)因子活性,與甲醛和嗎啡的毒害機(jī)制存在交叉。這就提出了兩個(gè)問題：Trx-1誘導(dǎo)物是否可以抵抗甲醛的毒害作用；Trx-1誘導(dǎo)物是否可以抵抗嗎啡成癮和嗎啡所致肝腎損傷。因此,本論文將研究這兩方面的內(nèi)容。 主要的研究結(jié)果如下： (1)三七總皂甙(PNS)是三七的主要成分之一,以其為主要成分的藥物被廣泛應(yīng)用于臨床中。三七三醇組皂甙(PTS)是PNS的主要成分之一,PTS的主要成分是人參皂甙Rg1、R1和Re(占80%),而其中人參皂甙Rg1的含量高達(dá)60%以上。PTS以劑量依賴和時(shí)間依賴的方式增加PC12細(xì)胞內(nèi)Trx-1的表達(dá)水平。體內(nèi)動(dòng)物實(shí)驗(yàn)結(jié)果：與對(duì)照組小鼠相比,PTS顯著誘導(dǎo)小鼠大腦皮層、海馬和紋狀體區(qū)Trx-1的表達(dá)水平。人參皂甙Rg1是PTS的主要活性成分。我們研究發(fā)現(xiàn)：人參皂甙Rg1顯著性地誘導(dǎo)PC12細(xì)胞內(nèi)Trx-1的表達(dá)水平。 (2)內(nèi)質(zhì)網(wǎng)應(yīng)激與神經(jīng)細(xì)胞凋亡相關(guān)。在本論文中,我們研究發(fā)現(xiàn)：甲醛顯著地誘導(dǎo)GRP78的表達(dá)水平；甲醛處理PC12細(xì)胞12小時(shí)后,能增加CHOP的表達(dá)；在甲醛作用24小時(shí)后pro-caspase-12的表達(dá)水平顯著下降。這些結(jié)果表明：甲醛在一定程度上是通過內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的凋亡途徑導(dǎo)致神經(jīng)毒害。 (3)氧化應(yīng)激是甲醛導(dǎo)致細(xì)胞毒害的一個(gè)重要原因。Trx-1是一種重要的抗氧化物質(zhì),能被氧化應(yīng)激誘導(dǎo)產(chǎn)生。本論文研究發(fā)現(xiàn)：甲醛作用PC12細(xì)胞12小時(shí)后,能誘導(dǎo)Trx-1的表達(dá)；但在甲醛作用24小時(shí)后會(huì)降低Trx-1的表達(dá)。采用siRNA干擾下調(diào)Trx-1的表達(dá)水平,則增加PC12細(xì)胞對(duì)甲醛的敏感性。這些結(jié)果表明：甲醛的神經(jīng)毒害可能與其降低Trx-1的表達(dá)水平有關(guān)。 (4)Trx-1的高表達(dá)可以抵抗氧化應(yīng)激和內(nèi)質(zhì)網(wǎng)應(yīng)激所致的損傷。人參皂甙Rgl對(duì)PC12細(xì)胞的存活活力無影響,但是人參皂甙Rg1的預(yù)處理可以抵抗甲醛對(duì)PC12細(xì)胞的神經(jīng)毒害,并抑制甲醛所致酪氨酸羥化酶表達(dá)水平的降低。人參皂甙Rgl可以恢復(fù)甲醛所致Trx-1表達(dá)水平的降低。而且,人參皂甙Rg1還可以抵抗甲醛所致的內(nèi)質(zhì)網(wǎng)應(yīng)激：抑制甲醛所致GRP78和CHOP表達(dá)水平的增加,抑制甲醛所致pro-caspase-12表達(dá)水平的下降。這些研究結(jié)果表明：Trx-1誘導(dǎo)劑可以抵抗甲醛的神經(jīng)毒害作用。 (5)嗎啡反復(fù)作用會(huì)增加小鼠大腦皮層、海馬、中腦腹側(cè)被蓋區(qū)和伏隔核內(nèi)Trx-1和Hsp70的表達(dá)水平。這些結(jié)果表明：?jiǎn)岱茸饔煤蠹?xì)胞防御機(jī)制會(huì)被激活。本論文研究證實(shí),GGA能誘導(dǎo)小鼠大腦內(nèi)Trx-1和Hsp70的表達(dá),減弱嗎啡急性處理所致小鼠活動(dòng)增強(qiáng)的效應(yīng),阻止嗎啡引起的條件性位置偏愛,緩解嗎啡戒斷癥狀。GGA還可以抑制嗎啡所致CREB活性的增強(qiáng),以及ΔFosB和Cdk5表達(dá)的增加。在NAc區(qū)中,GGA能增強(qiáng)嗎啡所致Trx-1和Hsp70表達(dá)的增加。這些結(jié)果表明：使用無細(xì)胞毒性的誘導(dǎo)物增強(qiáng)Trx-1的表達(dá)是嗎啡成癮治療的新策略；GGA可用于嗎啡成癮的防治。 (6)GGA在肝臟和腎臟中能抑制嗎啡所致MDA含量的增加,阻止嗎啡所致caspase-9和caspase-3的激活。嗎啡能通過線粒體介導(dǎo)的凋亡途徑誘導(dǎo)肝臟和腎臟細(xì)胞的細(xì)胞凋亡,而不是通過內(nèi)質(zhì)網(wǎng)介導(dǎo)的途徑。更重要的是,GGA還能增強(qiáng)嗎啡對(duì)Trx-1和Hsp70的誘導(dǎo)作用。這些結(jié)果表明：GGA可用于嗎啡所致肝腎損傷的防治。 以上結(jié)果表明：內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的凋亡途徑與甲醛所致神經(jīng)細(xì)胞凋亡存在相關(guān)性；人參皂甙Rg1是一種新的Trx-1誘導(dǎo)物,人參皂甙Rg1可以抵抗甲醛所致的神經(jīng)毒害；Trx-1誘導(dǎo)物GGA可以抵抗嗎啡成癮和嗎啡所致的肝腎損傷。這些研究結(jié)果可為甲醛和嗎啡的毒害防治及其藥物開發(fā)提供基礎(chǔ)理論依據(jù)和新靶點(diǎn)。
[Abstract]:Thioredoxin 1 (Trx-1) is a multifunctional protein with molecular weight 12KDa and contains a redox active site: -Cys-Gly-Pro-Cys-. thioredoxin has a variety of biological functions: regulation of redox, regulation of transcription factor activity, inhibition of apoptosis, and regulation of inflammation, such as.Trx-1 can be induced by many stress, such as Hydrogen peroxide, mitogen, polycyclic aromatic hydrocarbons, ultraviolet, viral infection, and ischemia-reperfusion.Trx-1 have neurotrophic factor like activity, and the important role of neural differentiation and protruding growth induced by nerve growth factor is the important effect of.Trx-1 high expression transgenic mice. The toxic effects of injur, diabetes and exogenous environmental stress have obvious resistance to.Trx-1, which is closely related to endoplasmic reticulum stress and is resistant to endoplasmic reticulum stress damage. Current studies have found that tepreone (GGA), sulforaphane, neurotoxin and temopri can all play a role in inducing the expression level of Trx-1. The most widely used and thorough research of teprinone. Teprinone (GGA) is a clinically widely used drug for the treatment of gastric ulcers. It can induce the expression of Trx-1 in a variety of cells, protect the gastric mucosa, liver and heart and other tissues,.GGA or a fat soluble substance, which can penetrate the blood brain barrier to the nerve tissue. The regulation of the expression of Trx-1 has become a new treatment strategy for disease. Nontoxic Trx-1 inducers can promote nerve regeneration and have a preventive effect on nervous system diseases related to oxidative stress. Therefore, this paper is intended to develop from natural drug resources in Yunnan and seek non-toxic Trx-1 inducers for disease. The study of treatment.
Formaldehyde is a common environmental pollutant, widely found in cigarette smoke, furniture, automobile exhaust, medical and industrial supplies. Research shows that the increase in indoor formaldehyde concentration is an important cause of severe construction syndrome. Formaldehyde is toxic to the nervous system and has been considered as a potential "nerve poison". The long-term exposure to formaldehyde can cause nerve toxicity and cause neurodegenerative diseases. At the same time, formaldehyde can change the morphological characteristics of the brain and lead to abnormal behavior and memory dysfunction. Therefore, formaldehyde has become an important factor to harm the health of the body. In order to effectively prevent and treat the toxicity of formaldehyde to the human body, formaldehyde has been studied in depth. The molecular mechanism of toxicity is of great significance.
Endoplasmic reticulum (endoplasmic reticulum) is a major site for protein synthesis and processing. It plays an important regulatory role in maintaining intracellular calcium concentration and protein glycosylation. When endoplasmic reticulum dysfunction and excessive accumulation of unfolded / misfolded protein, endoplasmic reticulum stress is induced. The endoplasmic reticulum contains a number of molecular chaperones, when endoplasmic reticulum stress occurs, sugar The expression level of regulatory protein 78 (GRP78) increases significantly to enhance the protein folding ability of the endoplasmic reticulum. When the endoplasmic reticulum stress is low, it activates the anti apoptotic molecular chaperones; however, when endoplasmic reticulum stress is too severe and persistent, it activates JNK, CHOP and caspase-12, leading to cell apoptosis and endoplasmic reticulum stress and nerve cells. Death is closely related. Formaldehyde is a crosslinker, which can interact with the sulfhydryl and amino acid groups, resulting in protein polycondensation, and formaldehyde causes the error of protein folding and accumulation. Formaldehyde and P amyloid interact to produce a neurotoxic amyloid protein complex. Formaldehyde will also lead to the aggregation of tau protein. The apoptosis of human neuroblastoma and hippocampal neurons, however, the study of the relationship between endoplasmic reticulum stress mediated apoptosis pathway and formaldehyde induced neuronal death has not yet been reported.
Morphine abuse has become a serious public health and social problem worldwide. Long-term use of morphine can lead to the occurrence of body dependence and mental dependence, which restricts its clinical use (treatment of various acute and chronic pain). The main manifestation of morphine addiction is the compulsive drug use, that is, drug intake and loss of control. Addiction. The reasons are mainly due to positive intensifying factors (euphoria and reward effects) and negative intensifying factors (alleviating withdrawal symptoms). The ventral tegmental area of the midbrain, nucleus accumbens, prefrontal cortex, amygdala and hippocampus are related to the process of morphine addiction. In addition to the nervous system, morphine causes oxidative stress and finer in the liver and kidney. Apoptosis. Long-term use of opioids can lead to liver, kidney damage, acute hepatitis, chronic hepatitis and other liver injuries, as well as nephrotic syndrome, acute glomerulonephritis, interstitial nephritis and renal failure. Therefore, attention should be paid to the liver and kidney damage caused by opioid drugs in the process of drug addiction treatment. Drugs for the protection of morphine induced liver and kidney damage are important for reducing the side effects of morphine and more widely using morphine. Moreover, there are no ideal drugs and methods for the treatment of morphine addiction.
Both formaldehyde and morphine can cause oxidative stress damage, apoptosis and neurotrophic factors are down, but their ways of action are different, but Trx-1 has anti oxidation, anti apoptosis and neurotrophic factor activity, and the toxic mechanism of formaldehyde and morphine intersect. This puts forward two questions: whether Trx-1 inducer can resist formaldehyde Whether the Trx-1 inducers can resist morphine addiction or morphine induced liver and kidney injury, this paper will study these two aspects.
The main results are as follows:
(1) 37 total saponins (PNS) are one of the main components of 37, and their main components are widely used in clinical. 373 alcohol group saponins (PTS) are one of the main components of PNS. The main components of PTS are ginsenoside Rg1, R1 and Re (80%), and the content of the saponins Rg1 is above 60%.PTS in dose dependent and time dependent manner. The expression level of Trx-1 in PC12 cells was increased. In vivo animal experiment results: compared with the control group, PTS significantly induced the expression level of Trx-1 in the cerebral cortex, hippocampus and striatum of mice. Ginsenoside Rg1 was the main active component of PTS. We found that ginsenoside Rg1 significantly induces Trx-1 in PC12 cells. Expression level.
(2) endoplasmic reticulum stress is associated with neuronal apoptosis. In this paper, we found that formaldehyde significantly induces the expression level of GRP78; formaldehyde can increase the expression of CHOP after 12 hours of treatment with formaldehyde; the expression level of pro-caspase-12 decreases significantly after 24 hours of formaldehyde action. These results suggest that formaldehyde is to a certain extent. Neurotoxicity is caused by endoplasmic reticulum stress mediated apoptosis pathway.
(3) oxidative stress is an important cause of formaldehyde causing cell toxicity,.Trx-1 is an important antioxidant, which can be induced by oxidative stress. The study found that formaldehyde can induce the expression of Trx-1 after the action of PC12 cells for 12 hours, but the expression of Trx-1 will be reduced after the action of formaldehyde for 24 hours. SiRNA interference is used to reduce Trx-1. The expression level increased the sensitivity of PC12 cells to formaldehyde. These results indicate that the neurotoxicity of formaldehyde may be related to the reduction of Trx-1 expression level.
(4) the high expression of Trx-1 can resist the damage caused by oxidative stress and endoplasmic reticulum stress. Ginsenoside Rgl has no effect on the viability of PC12 cells, but the pretreatment of ginsenoside Rg1 can resist formaldehyde toxicity to PC12 cells and inhibit the decrease of tyrosine hydroxylase expression level induced by formaldehyde. Ginsenoside Rgl can be recovered. The expression level of Trx-1 is reduced by formaldehyde. Furthermore, ginsenoside Rg1 can also resist the endoplasmic reticulum stress induced by formaldehyde: inhibition of the increase of GRP78 and CHOP expression levels caused by formaldehyde and the decrease of pro-caspase-12 expression level caused by formaldehyde. These results suggest that Trx-1 inducers can resist the toxic effects of formaldehyde.
(5) repeated morphine can increase the expression level of Trx-1 and Hsp70 in the cerebral cortex, hippocampus, ventral tegmental area and nucleus accumbens in the mice. These results show that the cellular defense mechanism of morphine can be activated. This study proves that GGA can induce the expression of Trx-1 and Hsp70 in the brain of mice and reduce the activity of mice induced by morphine acute treatment. Dynamic enhancement, blocking morphine induced conditioned place preference, alleviating morphine withdrawal symptoms.GGA can also inhibit the enhancement of CREB activity induced by morphine, as well as the increase in the expression of delta FosB and Cdk5. In NAc region, GGA can enhance the expression of Trx-1 and Hsp70 induced by morphine. These results indicate that the use of cytotoxic inducers to enhance T Rx-1 expression is a new strategy for the treatment of morphine addiction, and GGA can be used for the prevention and treatment of morphine addiction.
(6) GGA can inhibit the increase of MDA content induced by morphine in the liver and kidney, and prevent the activation of caspase-9 and caspase-3 induced by morphine. Morphine can induce apoptosis in liver and kidney cells through mitochondrial mediated apoptosis pathway, rather than through endoplasmic reticulum mediated pathway. More importantly, GGA can also enhance morphine to Trx-1 and Hsp70 These results indicate that GGA can be used for the prevention and treatment of liver and kidney injury induced by morphine.
These results suggest that the apoptosis pathway mediated by endoplasmic reticulum stress is related to the neuronal apoptosis induced by formaldehyde; ginsenoside Rg1 is a new Trx-1 inducer, and ginsenoside Rg1 can resist formaldehyde induced neurotoxicity; Trx-1 inducer GGA can resist morphine addiction and morphine induced liver and kidney injury. The results provide theoretical basis and new targets for the prevention and control of formaldehyde and morphine.

【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114;X503.1

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