NAC对大鼠肾IRI诱导的肾小管上皮细胞凋亡的保护作用研究

发布时间：2018-05-31 23:39

本文选题：N-乙酰半胱氨酸 + 肾缺血再灌注　；参考：《山东大学》2014年博士论文

【摘要】：研究背景：急性肾功能损害是临床上常见的病理现象,以急性肾脏功能突然丧失,肾小球滤过率的急剧下降和血液中含氮废物的增多为特征,伴随电解质、酸碱平衡失调及尿量异常。缺血再灌注损伤是引起急性肾功能衰竭的常见原因之一。直视下心脏手术、肾移植、肾上腺肿瘤、药物引起的肾病以及休克、严重腹泻等,都可以导致缺血再灌注损伤,是常见的引起急性肾功能损伤的因素。近来文献报道,肾脏缺血再灌注损伤与细胞凋亡密切相关,且氧自由基是损害肾脏结构和功能的关键因素,肾缺血再灌注时产生的过量氧自由基通过对核酸、脂质、蛋白质等大分子物质的过氧化作用直接诱发肾小管上皮细胞凋亡,而肾小管上皮细胞的功能性损伤则引起肾脏间质炎症、坏死最终引发肾功能损伤甚至肾功能衰竭。转录因子NF-E2相关因子2(Nrf2)在氧化应激发生时可与抗氧化反应元件(ARE)结合,调节各种抗氧化基因的表达。目前认为Nrf2是调控细胞对抗外来异物和氧化损伤的关键转录因子。Nrf2缺失或激活障碍,会引起细胞对应激源的敏感性增高。Nrf2-ARE通路是迄今为止发现的最为重要的内源性抗氧化应激通路。生理状态下,Nrf2在细胞质中与它的抑制蛋白keap1结合,并通过keap1蛋白将其锚定于由肌动蛋白构成的细胞骨架上,从而无法进入细胞核发挥转录活性,并促进Nrf2泛素化继而被蛋白酶体降解。当发生ROS的刺激时,首先Nrf2与keap1解偶联,继而Nrf2转移进入细胞核,与基因中的Maf蛋白结合成异二聚体后识别并结合ARE,继而启动下游保护性蛋白基因的转录,提高细胞抗氧化应激能力。 N-乙酰半胱氨酸(NAC)已被用来作为抗氧化剂的前体作用于谷胱甘肽。有报道显示,NAC具有抗生物氧化的作用,对缺血再灌注损伤的大鼠肾组织起到保护作用。然而,目前对于NAC所诱导的细胞保护作用的作用机制研究不详,本研究的目的是探讨NAC是否可以通过Nrf2信号通路发挥防止肾缺血再灌注损伤的作用。研究目的： 1.探讨N-乙酰半胱氨酸对肾缺血再灌注损伤后大鼠肾功能的影响。 2.探讨N-乙酰半胱氨酸对肾缺血再灌注损伤诱导的细胞凋亡的影响。 3.探讨N-乙酰半胱氨酸对肾缺血再灌注损伤诱发细胞凋亡保护作用的分子机制。研究方法： 1.雄性SD大鼠,体重约200-260g,将所有实验动物随机分为4组,每组10只：1)假手术组(Sham组)；2)缺血再灌注组(I/R组)；3)缺血再灌注+生理盐水组(I/R+saline组)；4)缺血再灌注+NAC组(I/R+NAC组)。应用水合氯醛(300mg/kg)作为麻醉剂对大鼠进行腹膜内注射,采用无创伤血管钳夹闭双侧大鼠肾脏动静脉,引发肾缺血45min后再灌注的方法制备肾缺血/再灌注模型。造模时大鼠直肠温度维持在37℃左右。NAC和生理盐水处理组的大鼠在缺血前三天腹膜内注射溶于0.9%生理盐水的NAC (150mg/kg)或等体积的生理盐水。假手术组使用相同的手术暴露程序进行手术。去除夹闭肾动静脉的血管钳以实现再灌注。 2.缺血/再灌注完成后处死大鼠,从腔静脉处收集血液样品,采用生化分析仪检测血清BUN和cr含量,依据试剂盒说明检测丙二醛(MDA)含量。 3.取出的肾脏立即在液氮中冷冻或固定于多聚甲醛中TUNEL染色,检测肾小管上皮细胞凋亡情况。 4.免疫印迹检测Nrf2、P53及pro-caspase3表达情况。 5.免疫印迹检测血红素氧合酶1(HO-1)表达情况。研究结果： 1.肾缺血再灌注后NAC对肾功能的影响。实验发现,恢复血供后的24h,肾功能指标之一的血肌酐在缺血再灌注组迅速升到159μmol/L。而NAC预处理组其血肌酐水平恢复到正常。同时,代表肾功能的另一个指标血尿素氮,在缺血再灌注组迅速升高到22mmol/L,而NAC预处理组其血肌酐水平也降低并恢复到正常。 2.肾缺血再灌注后NAC对脂质过氧化的影响。MDA,脂质过氧化的最终产物,是自由基和氧应激介导损伤的重要标志。与I/R组相比,NAC处理组其MDA的水平也是显著下降的。 3.NAC预处理对肾缺血再灌注诱导的细胞凋亡的保护作用。复灌后24h时,取大鼠肾脏组织用多聚甲醛固定并行TUNEL染色处理。结果显示缺血复灌后TUNEL阳性凋亡细胞主要分布在外髓质远端小管,在皮质肾小管分布很少,有些凋亡细胞脱落进入肾小管的管腔内。几乎未有TUNEL阳性细胞出现在假手术组。与saline组和I/R组相比,NAC预处理后能显著降低TUNEL阳性细胞。 4.肾缺血再灌注NAC预处理后Nrf2、HO-1和p53的表达情况。实验结果显示,与单纯I/R组相比,NAC预处理后Nrf2核汇聚显著增多。其下游基因HO-1也显著上高。同时,因缺血再灌注引发的cleaved pro-caspase3增多也显著被抑制。但是,在同样的情况下,saline组的Nrf2、HO-1和pro-caspase3的表达无明显改变。而且,NAC能显著抑制由缺血再灌注引发的p53增高。研究结论： 1.NAC可以抑制Nrf2的泛素化降解。 2.NAC可增加Nrf2的表达及促其核转位。 3.NAC激活Nrf2下游抗氧化基因的表达从而减轻肾缺血再灌注损伤。综上所述,NAC具有肾保护作用,能改善大鼠肾缺血再灌注后引发的肾脏损伤,其机制可能是通过调控Nrf2信号通路从而发挥其抗凋亡的作用。论文主要创新点：在实验中我们首次证明了在体内模型中诱导Nrf2的激活和Nrf2的依赖性的抗氧化基因的表达。因此我们推测,特定的激活Nrf2抗氧化途径可以为机体减轻正在进行的和随后而至的氧化损伤导致的IRI。我们还提出了NAC特异性激活Nrf2的参与的证据。本研究支持NAC预处理的结果作为一个保护肾脏和其它器官的缺血再灌注损伤的新策略。对于Nrf2信号途径参与这种反应的进一步的研究可以帮助我们未来充分地认识到NAC预处理所具有的对于减少脏器缺血再灌注损伤的巨大治疗潜力。
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

【共引文献】