本文選題：N-乙酰半胱氨酸 + 腎缺血再灌注��； 參考：《山東大學(xué)》2014年博士論文

【摘要】：研究背景： 急性腎功能損害是臨床上常見(jiàn)的病理現(xiàn)象,以急性腎臟功能突然喪失,腎小球?yàn)V過(guò)率的急劇下降和血液中含氮廢物的增多為特征,伴隨電解質(zhì)、酸堿平衡失調(diào)及尿量異常。缺血再灌注損傷是引起急性腎功能衰竭的常見(jiàn)原因之一。直視下心臟手術(shù)、腎移植、腎上腺腫瘤、藥物引起的腎病以及休克、嚴(yán)重腹瀉等,都可以導(dǎo)致缺血再灌注損傷,是常見(jiàn)的引起急性腎功能損傷的因素。近來(lái)文獻(xiàn)報(bào)道,腎臟缺血再灌注損傷與細(xì)胞凋亡密切相關(guān),且氧自由基是損害腎臟結(jié)構(gòu)和功能的關(guān)鍵因素,腎缺血再灌注時(shí)產(chǎn)生的過(guò)量氧自由基通過(guò)對(duì)核酸、脂質(zhì)、蛋白質(zhì)等大分子物質(zhì)的過(guò)氧化作用直接誘發(fā)腎小管上皮細(xì)胞凋亡,而腎小管上皮細(xì)胞的功能性損傷則引起腎臟間質(zhì)炎癥、壞死最終引發(fā)腎功能損傷甚至腎功能衰竭。 轉(zhuǎn)錄因子NF-E2相關(guān)因子2(Nrf2)在氧化應(yīng)激發(fā)生時(shí)可與抗氧化反應(yīng)元件(ARE)結(jié)合,調(diào)節(jié)各種抗氧化基因的表達(dá)。目前認(rèn)為Nrf2是調(diào)控細(xì)胞對(duì)抗外來(lái)異物和氧化損傷的關(guān)鍵轉(zhuǎn)錄因子。Nrf2缺失或激活障礙,會(huì)引起細(xì)胞對(duì)應(yīng)激源的敏感性增高。Nrf2-ARE通路是迄今為止發(fā)現(xiàn)的最為重要的內(nèi)源性抗氧化應(yīng)激通路。生理狀態(tài)下,Nrf2在細(xì)胞質(zhì)中與它的抑制蛋白keap1結(jié)合,并通過(guò)keap1蛋白將其錨定于由肌動(dòng)蛋白構(gòu)成的細(xì)胞骨架上,從而無(wú)法進(jìn)入細(xì)胞核發(fā)揮轉(zhuǎn)錄活性,并促進(jìn)Nrf2泛素化繼而被蛋白酶體降解。當(dāng)發(fā)生ROS的刺激時(shí),首先Nrf2與keap1解偶聯(lián),繼而Nrf2轉(zhuǎn)移進(jìn)入細(xì)胞核,與基因中的Maf蛋白結(jié)合成異二聚體后識(shí)別并結(jié)合ARE,繼而啟動(dòng)下游保護(hù)性蛋白基因的轉(zhuǎn)錄,提高細(xì)胞抗氧化應(yīng)激能力。 N-乙酰半胱氨酸(NAC)已被用來(lái)作為抗氧化劑的前體作用于谷胱甘肽。有報(bào)道顯示,NAC具有抗生物氧化的作用,對(duì)缺血再灌注損傷的大鼠腎組織起到保護(hù)作用。然而,目前對(duì)于NAC所誘導(dǎo)的細(xì)胞保護(hù)作用的作用機(jī)制研究不詳,本研究的目的是探討NAC是否可以通過(guò)Nrf2信號(hào)通路發(fā)揮防止腎缺血再灌注損傷的作用。 研究目的： 1.探討N-乙酰半胱氨酸對(duì)腎缺血再灌注損傷后大鼠腎功能的影響。 2.探討N-乙酰半胱氨酸對(duì)腎缺血再灌注損傷誘導(dǎo)的細(xì)胞凋亡的影響。 3.探討N-乙酰半胱氨酸對(duì)腎缺血再灌注損傷誘發(fā)細(xì)胞凋亡保護(hù)作用的分子機(jī)制。 研究方法： 1.雄性SD大鼠,體重約200-260g,將所有實(shí)驗(yàn)動(dòng)物隨機(jī)分為4組,每組10只：1)假手術(shù)組(Sham組)；2)缺血再灌注組(I/R組)；3)缺血再灌注+生理鹽水組(I/R+saline組)；4)缺血再灌注+NAC組(I/R+NAC組)。應(yīng)用水合氯醛(300mg/kg)作為麻醉劑對(duì)大鼠進(jìn)行腹膜內(nèi)注射,采用無(wú)創(chuàng)傷血管鉗夾閉雙側(cè)大鼠腎臟動(dòng)靜脈,引發(fā)腎缺血45min后再灌注的方法制備腎缺血/再灌注模型。造模時(shí)大鼠直腸溫度維持在37℃左右。NAC和生理鹽水處理組的大鼠在缺血前三天腹膜內(nèi)注射溶于0.9%生理鹽水的NAC (150mg/kg)或等體積的生理鹽水。假手術(shù)組使用相同的手術(shù)暴露程序進(jìn)行手術(shù)。去除夾閉腎動(dòng)靜脈的血管鉗以實(shí)現(xiàn)再灌注。 2.缺血/再灌注完成后處死大鼠,從腔靜脈處收集血液樣品,采用生化分析儀檢測(cè)血清BUN和cr含量,依據(jù)試劑盒說(shuō)明檢測(cè)丙二醛(MDA)含量。 3.取出的腎臟立即在液氮中冷凍或固定于多聚甲醛中TUNEL染色,檢測(cè)腎小管上皮細(xì)胞凋亡情況。 4.免疫印跡檢測(cè)Nrf2、P53及pro-caspase3表達(dá)情況。 5.免疫印跡檢測(cè)血紅素氧合酶1(HO-1)表達(dá)情況。 研究結(jié)果： 1.腎缺血再灌注后NAC對(duì)腎功能的影響。實(shí)驗(yàn)發(fā)現(xiàn),恢復(fù)血供后的24h,腎功能指標(biāo)之一的血肌酐在缺血再灌注組迅速升到159μmol/L。而NAC預(yù)處理組其血肌酐水平恢復(fù)到正常。同時(shí),代表腎功能的另一個(gè)指標(biāo)血尿素氮,在缺血再灌注組迅速升高到22mmol/L,而NAC預(yù)處理組其血肌酐水平也降低并恢復(fù)到正常。 2.腎缺血再灌注后NAC對(duì)脂質(zhì)過(guò)氧化的影響。MDA,脂質(zhì)過(guò)氧化的最終產(chǎn)物,是自由基和氧應(yīng)激介導(dǎo)損傷的重要標(biāo)志。與I/R組相比,NAC處理組其MDA的水平也是顯著下降的。 3.NAC預(yù)處理對(duì)腎缺血再灌注誘導(dǎo)的細(xì)胞凋亡的保護(hù)作用。復(fù)灌后24h時(shí),取大鼠腎臟組織用多聚甲醛固定并行TUNEL染色處理。結(jié)果顯示缺血復(fù)灌后TUNEL陽(yáng)性凋亡細(xì)胞主要分布在外髓質(zhì)遠(yuǎn)端小管,在皮質(zhì)腎小管分布很少,有些凋亡細(xì)胞脫落進(jìn)入腎小管的管腔內(nèi)。幾乎未有TUNEL陽(yáng)性細(xì)胞出現(xiàn)在假手術(shù)組。與saline組和I/R組相比,NAC預(yù)處理后能顯著降低TUNEL陽(yáng)性細(xì)胞。 4.腎缺血再灌注NAC預(yù)處理后Nrf2、HO-1和p53的表達(dá)情況。實(shí)驗(yàn)結(jié)果顯示,與單純I/R組相比,NAC預(yù)處理后Nrf2核匯聚顯著增多。其下游基因HO-1也顯著上高。同時(shí),因缺血再灌注引發(fā)的cleaved pro-caspase3增多也顯著被抑制。但是,在同樣的情況下,saline組的Nrf2、HO-1和pro-caspase3的表達(dá)無(wú)明顯改變。而且,NAC能顯著抑制由缺血再灌注引發(fā)的p53增高。 研究結(jié)論： 1.NAC可以抑制Nrf2的泛素化降解。 2.NAC可增加Nrf2的表達(dá)及促其核轉(zhuǎn)位。 3.NAC激活Nrf2下游抗氧化基因的表達(dá)從而減輕腎缺血再灌注損傷。 綜上所述,NAC具有腎保護(hù)作用,能改善大鼠腎缺血再灌注后引發(fā)的腎臟損傷,其機(jī)制可能是通過(guò)調(diào)控Nrf2信號(hào)通路從而發(fā)揮其抗凋亡的作用。 論文主要?jiǎng)?chuàng)新點(diǎn)：在實(shí)驗(yàn)中我們首次證明了在體內(nèi)模型中誘導(dǎo)Nrf2的激活和Nrf2的依賴性的抗氧化基因的表達(dá)。因此我們推測(cè),特定的激活Nrf2抗氧化途徑可以為機(jī)體減輕正在進(jìn)行的和隨后而至的氧化損傷導(dǎo)致的IRI。我們還提出了NAC特異性激活Nrf2的參與的證據(jù)。本研究支持NAC預(yù)處理的結(jié)果作為一個(gè)保護(hù)腎臟和其它器官的缺血再灌注損傷的新策略。對(duì)于Nrf2信號(hào)途徑參與這種反應(yīng)的進(jìn)一步的研究可以幫助我們未來(lái)充分地認(rèn)識(shí)到NAC預(yù)處理所具有的對(duì)于減少臟器缺血再灌注損傷的巨大治療潛力。
[Abstract]:Research background:
Acute renal impairment is a common pathological phenomenon in the clinic, characterized by sudden loss of acute renal function, rapid decline of glomerular filtration rate and the increase of nitrogen containing waste in the blood, accompanied by electrolytes, acid-base imbalance and abnormal urine volume. Ischemia reperfusion injury is one of the common causes of acute renal failure. Cardiac surgery, renal transplantation, adrenal tumor, nephrosis caused by drugs, shock and severe diarrhea can lead to ischemia reperfusion injury. It is a common cause of acute renal impairment. Recently, renal ischemia reperfusion injury is closely related to cell apoptosis, and oxygen free radicals are harmful to the structure and function of the kidney. The key factor, the excess oxygen free radicals produced in renal ischemia and reperfusion, directly induces apoptosis of renal tubular epithelial cells through the peroxidation of macromolecular substances such as nucleic acids, lipids and proteins, and the functional damage of renal tubular epithelial cells causes renal interstitial inflammation, and necrosis eventually leads to renal function damage and even renal failure.
The transcription factor NF-E2 related factor 2 (Nrf2) can be combined with antioxidant response element (ARE) during oxidative stress to regulate the expression of various antioxidant genes. Currently, Nrf2 is considered to be the key transcription factor to regulate cell resistance to foreign foreign bodies and oxidative damage,.Nrf2 deletion or activation obstacle, which will cause the sensitivity of cell response to the source of.Nrf2-. ARE pathway is the most important endogenous antioxidant stress pathway found so far. In physiological state, Nrf2 is combined with its inhibitory protein Keap1 in the cytoplasm and anchored to actin cytoskeleton through Keap1 protein, thus unable to enter the nucleus to play the transcriptional activity and promote Nrf2 ubiquitination. When ROS is stimulated, Nrf2 and Keap1 are uncoupled first, then Nrf2 is transferred into the nucleus, and the Maf protein in the gene is combined into a hetero two polymer to recognize and combine with ARE, then the transcription of the downstream protective protein gene is started, and the antioxidant activity of the cells is enhanced.
N- acetylcysteine (NAC) has been used as a precursor of antioxidants in glutathione. It is reported that NAC has a protective effect on the renal tissue of rats with ischemia-reperfusion injury. However, the mechanism of the action of NAC induced cell protection is unknown. The purpose of this study is To investigate whether NAC can play a role in preventing renal ischemia-reperfusion injury through Nrf2 signaling pathway.
The purpose of the study is:
1. to investigate the effect of N- acetylcysteine on renal function after renal ischemia-reperfusion injury in rats.
2. to investigate the effect of N- acetylcysteine on apoptosis induced by renal ischemia-reperfusion injury.
3. to explore the molecular mechanism of protective effect of N- acetylcysteine on apoptosis induced by renal ischemia-reperfusion injury.
Research methods:
1. male SD rats, weighing about 200-260g, randomly divided all experimental animals into 4 groups, 10 rats in each group, 1) sham operation group (group Sham), 2) ischemia reperfusion group (group I/R), 3) ischemia reperfusion + physiological saline group (group I/R+saline); 4) ischemia reperfusion +NAC group (group I/R+NAC). The use of chloral chloral (300mg/kg) as an anesthetic in the peritoneum of rats. The renal ischemia / reperfusion model was prepared by injecting non traumatic vascular clamp and clamping the renal artery and vein in bilateral rats to induce renal ischemia after 45min reperfusion. The rectal temperature of rats was maintained at about 37.NAC and the rats in the saline treatment group were injected with 0.9% physiological saline NAC (150mg/kg) within the peritoneum before the ischemia. The same surgical exposure procedure was used in the sham operation group to remove the clamp of the renal artery and vein to achieve reperfusion.
2. the rats were killed after ischemia / reperfusion. The blood samples were collected from the vena cava. The content of serum BUN and Cr was detected by biochemical analyzer, and the content of malondialdehyde (MDA) was detected according to the kit.
3. the kidneys removed were immediately frozen or immobilized in paraformaldehyde in liquid nitrogen, and TUNEL staining was used to detect the apoptosis of renal tubular epithelial cells.
4. Western blotting was used to detect the expression of Nrf2, P53 and pro-caspase3.
5. the expression of heme oxygenase 1 (HO-1) was detected by Western blotting.
The results of the study:
1. the effect of NAC on renal function after ischemia-reperfusion. It was found that the blood creatinine, one of the indexes of renal function, was rapidly increased to 159 mol/L. in the ischemia reperfusion group and the blood creatinine level of the NAC preconditioning group was restored to normal in the ischemic reperfusion group, and the blood urea nitrogen, the other indicator of the renal function, was rapidly increased in the ischemia-reperfusion group. At 22mmol/L, the serum creatinine level in NAC preconditioning group also decreased and returned to normal.
The effect of NAC on lipid peroxidation after renal ischemia and reperfusion (2.).MDA, the final product of lipid peroxidation, is an important marker of free radical and oxygen stress mediated damage. Compared with group I/R, the level of MDA in the NAC treatment group is also significantly decreased.
The protective effect of 3.NAC preconditioning on the apoptosis induced by renal ischemia-reperfusion. At 24h after reperfusion, the rat kidney tissues were treated with paraformaldehyde and TUNEL staining. The results showed that the TUNEL positive apoptotic cells were mainly distributed in the distal medulla tubules after ischemia reperfusion, and the distribution of some of the apoptotic cells in the cortex and renal tubules was very small. There were almost no TUNEL positive cells in the sham operation group. Compared with the saline group and the I/R group, the TUNEL positive cells were significantly reduced after the NAC preconditioning.
4. the expression of Nrf2, HO-1 and p53 after NAC preconditioning in the renal ischemia reperfusion. The experimental results showed that the aggregation of Nrf2 nuclei increased significantly after NAC preconditioning. The downstream gene HO-1 was also significantly higher. At the same time, the increase of cleaved pro-caspase3 caused by ischemia-reperfusion was also significantly inhibited. But, in the same case, saline The expression of Nrf2, HO-1 and pro-caspase3 in the group did not change significantly, and NAC significantly inhibited the increase of p53 induced by ischemia-reperfusion.
The conclusions are as follows:
1.NAC can inhibit the ubiquitination of Nrf2.
2.NAC can increase the expression of Nrf2 and promote its nuclear transposition.
3.NAC activates the expression of antioxidant genes downstream of Nrf2, thereby alleviating renal ischemia-reperfusion injury.
To sum up, NAC has the role of renal protection and can improve renal injury induced by renal ischemia-reperfusion in rats. The mechanism may be to regulate the anti apoptosis effect by regulating the Nrf2 signaling pathway.
The main innovation in this paper is that in the experiment we first demonstrated the activation of Nrf2 in the body model and the expression of Nrf2 dependent antioxidant genes. Therefore, we speculate that specific activation of the Nrf2 antioxidant pathway can also help the body to reduce the IRI. caused by the ongoing and subsequent oxygenation damage. We also put forward NAC. The evidence of the participation of the heterosexual activation of Nrf2. This study supports the results of NAC preconditioning as a new strategy to protect the ischemia-reperfusion injury of the kidney and other organs. Further research on the involvement of the Nrf2 signaling pathway may help us to fully recognize that NAC preconditioning has the effect of reducing organ ischemia. The great therapeutic potential of reperfusion injury.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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3 唐鐵龍;缺血后適應(yīng)對(duì)大鼠腎臟熱缺血再灌注早期損傷的保護(hù)作用及其機(jī)制研究[D];四川大學(xué);2006年

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7 劉鳳超;急性肝衰竭模型鼠肝細(xì)胞凋亡機(jī)制研究[D];華中科技大學(xué);2009年

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10 秦曉凌;膽紅素對(duì)帕金森病的神經(jīng)保護(hù)作用[D];蘇州大學(xué);2013年

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