5-HMF抗氧化损伤作用及其作用机制的初步探讨

发布时间：2018-05-13 07:27

本文选题：5-HMF + 抗氧化损伤　；参考：《中国人民解放军军事医学科学院》2009年硕士论文

【摘要】： 氧化应激与过氧化物积聚是导致多种疾病(如脑缺血、Alzheimer’s病(AD)、脑动脉粥样硬化等)的根本原因。H_2O_2是氧化应激反应密切相关的活性氧之一,其主要产物OH-具有很强的氧化性并可自由进入细胞,破坏细胞内活性氧和抗氧化代谢的平衡,使抗氧化酶等活性下降,同时会损伤体内脂质、蛋白质和DNA等生物大分子,造成细胞内酶活力变化、代谢紊乱、DNA损伤和细胞调亡。机体自身具有清除或修复氧化损伤的能力,例如超氧化物歧化酶(SOD)和过氧化物酶(POD)是机体内重要的抗氧化酶系之一,其作用在于清除体内的自由基;无嘌呤/无嘧啶核酸内切酶(APE/Ref-1, apurinic/apyrimidinic endonuclease/redox factor-1)则在碱基切除修复(BER, base excise repair)中起关键作用,是参与DNA氧化损伤修复的重要抗氧化酶。氧化损伤时这些酶的表达及活性降低,因此,提高机体氧化损伤时抗氧化酶的表达/活性将有利于对上述疾病的预防及治疗。 APE/Ref-1是个具有多种生物学功能的蛋白质,在许多生物过程中均发挥着重要作用。APE/Ref-1蛋白由318个氨基酸组成,其C末端功能区具有核酸内切酶活性,当DNA分子受氧化损伤时,APE/Ref-1发挥核酸内切酶活性参与DNA的碱基切除修复:由糖基化酶(OGG1)移除受损碱基后,APE/Ref-1在无嘌呤/无嘧啶(apurinic/apyrimidinic, AP)部位的5′端切断DNA链,DNA聚合酶以其外切酶作用切除AP部位的核苷酸,并根据碱基互补原则插入新的核苷酸进行修复;APE/Ref-1蛋白N末端具氧化还原功能,在众多转录因子的激活中起重要作用,如维持Fos、Jun、HIF-1等转录因子的DNA结合域内半胱氨酸处于还原状态,促进转录因子的DNA结合功能。 APE/Ref-1对生物体的存活起着至关重要的作用,APE/Ref-1敲除小鼠在胚胎期E3.5时即发生死亡,APE/Ref-1的缺失使培养的细胞也难以存活。以上提示APE/Ref-1表达降低是细胞氧化损伤以至死亡的共同通路。因此,我们有理由相信以APE/Ref-1为靶点,定向增加其蛋白表达,或抑制其在氧化损伤过程中表达下调来防治氧化损伤性疾病的药物研究将具有重要的科学意义及应用价值。 5-羟甲基糠醛(5-hydroxymethyl-2-furaldehyde, 5-HMF)是广泛存在含糖类植物中的一种小分子单体化合物,主要由己糖经加热分解产生。近年来研究表明5-HMF具有抗氧化、改善血液流变学等对人体有利的作用。本实验室最近研究发现5-HMF能延长小鼠在严重低氧条件下的存活时间,提高细胞存活率,但其抗氧化损伤作用及机制尚不明确。本实验用H_2O_2诱导PC12细胞氧化损伤,研究5-HMF的抗氧化损伤作用及其作用机制。研究内容主要包括两大部分:(1)5-HMF的抗氧化损伤作用;(2)5-HMF的抗氧化损伤作用机制的初步探讨。主要研究APE/Ref-1是否参与了5-HMF的抗氧化作用。实验结果如下: 1. 5-HMF抗氧化损伤作用研究以不同浓度的5-HMF处理细胞,在不同的时间点检测细胞存活率,寻找到合适浓度的5-HMF。再用此浓度的5-HMF预处理PC12细胞,以H_2O_2处理细胞后检测细胞存活,细胞形态学变化及DNA氧化损伤情况,研究发现5-HMF具有明显的抗H_2O_2引起的氧化损伤作用。 1.1细胞存活检测 1.1.1不同浓度5-HMF对细胞存活率的影响用50μg/ml、100μg/ml、200μg /ml、400μg /ml的5-HMF分别处理PC12细胞2小时、6小时和24小时后,用MTT检测细胞存活率,发现200μg /ml、400μg /ml的5-HMF作用6小时和24小时后,细胞存活率明显下降,而50μg/ml、100μg/ml的5-HMF在作用24小时后细胞存活率仍未受影响,因此选用这两个浓度的5-HMF,研究其在抗氧化损伤中的作用。 1.1.2 H_2O_2对细胞存活的影响及5-HMF的作用分别用50μg/ml、100μg/ml 5-HMF预处理细胞30分钟后,再用400μM H_2O_2处理1小时,MTT检测细胞存活率,结果表明H_2O_2处理后细胞存活率降低,50μg/ml和100μg/ml 5-HMF则能明显提高H_2O_2氧化损伤后的细胞存活率。 1.2细胞形态学观察进一步用倒置相差显微镜观察细胞形态学上的变化,发现400μM H_2O_2处理后细胞皱缩变小,细胞数目明显减少,50μg/ml和100μg/ml 5-HMF预处理后能减缓H_2O_2引发的形态学改变。 1.3 DNA氧化损伤检测为确认H_2O_2引起的DNA氧化损伤以及5-HMF的作用,实验采用免疫细胞化学方法检测DNA氧化损伤标志物8-oxo-dG的表达变化情况。结果发现,400μM H_2O_2处理PC12细胞1小时后8-oxo-dG表达增强,而50μg/ml和100μg/ml 5-HMF明显抑制了H_2O_2对8-oxo-dG的诱导作用,该结果表明5-HMF具有抗H_2O_2引起的DNA氧化损伤作用。 2. 5-HMF抗氧化损伤作用机制的初步研究由于APE/Ref-1在DNA氧化损伤的碱基切除修复中具有重要作用,它很有可能是5-HMF抗氧化作用的靶点,为此,我们着重探讨了APE/Ref-1在5-HMF抗氧化损伤中的作用。 2.1 5-HMF对APE/Ref-1表达变化的影响 2.1.1 5-HMF对APE/Ref-1蛋白表达的影响免疫细胞化学结果显示:400μM H_2O_2导致APE/Ref-1表达明显下调,5-HMF处理本身并未增加APE/Ref-1表达,但却抑制了APE/Ref-1在H_2O_2处理后的表达下调。为确认5-HMF对APE/Ref-1蛋白表达的影响,实验进一步用Western blot方法检验了APE/Ref-1的表达情况,经验证与免疫细胞化学检测的结果一致。 2.1.2 5-HMF对APE/Ref-1 mRNA表达的影响 5-HMF对APE/Ref-1蛋白表达的影响是否与其对APE/Ref-1 mRNA水平的调控有关呢?为此我们以RT-PCR检测了APE/Ref-1 mRNA表达情况,结果显示H_2O_2处理未改变APE/Ref-1 mRNA水平,亦不受5-HMF的影响。以上实验表明,5-HMF能抑制H_2O_2引起的APE/Ref-1蛋白表达下调,且与其对APE/Ref-1的转录调节无关。此外,APE/Ref-1作为核酸内切酶,其活性是否受参与5-HMF的抗氧化损伤作用尚不清楚,因此接下来我们采用APE/Ref-1抑制剂MX抑制其酶活性后,进一步检测了5-HMF的抗氧化损伤作用。 2.2 APE/Ref-1酶活性介导了5-HMF的抗氧化损伤作用 2.2 .1检测不同浓度MX在不同时间点对PC12细胞存活率的影响以1 mM、2.5 mM、5 mM、7.5 mM和10 mM MX分别处理PC12细胞2小时、16小时和24小时,MTT检测细胞存活率,发现7.5 mM和10 mM MX作用2小时细胞生长受到抑制,而此时1 mM、2.5 mM和5 mM MX对细胞存活基本没有影响,结合文献报道我们最后选用5 mM MX抑制APE/Ref-1酶活性。 2.2.2 MX处理后对5-HMF抗氧化损伤作用的影响用5 mM MX抑制APE/Ref-1核酸内切酶活性,检测5-HMF的抗氧化损伤作用。结果显示,APE/Ref-1酶活性受抑制后,5-HMF原有的提高氧化损伤时细胞存活能力降低,并且其抗DNA氧化损伤作用也被减弱。这些结果暗示5-HMF的抗氧化损伤作用是通过APE/Ref-1的酶活性来介导的。综上所述,5-HMF具有明显抗H_2O_2引起的氧化损伤作用,且其作用是通过稳定APE/Ref-1蛋白表达,使其发挥核酸内切酶活性来实现的。5-HMF如何抑制APE/Ref-1在氧化损伤时的降解,以及APE/Ref-1的氧化还原活性-即对转录因子的调节功能是否参与了5-HMF的抗氧化损伤作用还需进一步深入研究。
[Abstract]:Oxidative stress and peroxide accumulation are the fundamental causes of a variety of diseases (such as cerebral ischemia, Alzheimer 's disease (AD), and cerebral atherosclerosis)..H_2O_2 is one of the reactive oxygen species closely related to oxidative stress. Its main product, OH-, is highly oxidizing and can be derived from the cells and destroys intracellular reactive oxygen species and antioxidant metabolism. The balance, which reduces the activity of antioxidant enzymes, and damages biological macromolecules such as lipid, protein and DNA in the body, causes changes in enzyme activity, metabolic disorder, DNA damage and cell death. The body itself has the ability to remove or repair oxidative damage, such as superoxide dismutase (SOD) and peroxidase (POD), which are internal weight of the body. One of the antioxidant enzymes required is to scavenge free radicals, and purine / apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1, endonuclease/redox factor-1) plays a key role in base excision repair (BER, base excise repair), and is an important antioxidant involved in the repair of DNA oxidative damage. The expression and activity of these enzymes decrease. Therefore, improving the expression and activity of antioxidant enzymes in oxidative damage of the body will be beneficial to the prevention and treatment of these diseases.
APE/Ref-1 is a protein with many biological functions and plays an important role in many biological processes..APE/Ref-1 protein is composed of 318 amino acids. Its C terminal functional region has endonuclease activity. When DNA molecules are damaged by oxidation, APE/Ref-1 plays the activity of endonuclease in nucleic acid radical resection and repair of DNA, which is derived from glycosyl group. After the enzyme (OGG1) removal of the damaged base, APE/Ref-1 cuts the DNA chain at the 5 'end of the purine / pyrimidine (apurinic/apyrimidinic, AP) site. The DNA polymerase excuses the nucleotide of the AP site by its external enzyme action, and inserts the new nucleotide according to the principle of base complementarity. The N terminal of APE/Ref-1 protein has the redox function, in many turns. The activation of transcription factors plays an important role, such as maintaining the reduction state of cysteine in the DNA binding domain of the transcription factors such as Fos, Jun, HIF-1 and other transcription factors, and promoting the DNA binding function of the transcription factors.
APE/Ref-1 plays a vital role in the survival of the organism. APE/Ref-1 knocks off mice at the embryonic stage of E3.5, and the absence of APE/Ref-1 makes the cultured cells difficult to survive. These suggest that the decrease in APE/Ref-1 expression is the common pathway of cell oxidative damage and even death. For this reason, we have reason to believe that APE/Ref-1 is the target, It is of great scientific significance and application value to increase the expression of its protein or inhibit its regulation of down regulation in the process of oxidative damage to prevent oxidative damage.
5- hydroxymethyl furfural (5-hydroxymethyl-2-furaldehyde, 5-HMF) is a kind of small molecular monomer compound widely existed in sugar containing plants, which is mainly decomposed by hexose. In recent years, studies have shown that 5-HMF has antioxidation and improves blood rheology and other beneficial effects on human body. Recently, the study found that 5-HMF can prolong the small size of 5-HMF. The survival time of rat under severe hypoxic conditions increased the survival rate of cells, but the effect and mechanism of antioxidant damage were not clear. This experiment used H_2O_2 to induce oxidative damage in PC12 cells and study the antioxidant effects and mechanism of 5-HMF. The main contents of the study include two parts: (1) the antioxidant effect of 5-HMF; (2) the resistance of 5-HMF. A preliminary study on the mechanism of oxidative damage. We mainly studied whether APE/Ref-1 was involved in the antioxidant effect of 5-HMF.
Study on the effect of 1. 5-HMF on antioxidant injury
The cells were treated with different concentrations of 5-HMF, and the cell survival rate was detected at different time points. The suitable concentration of 5-HMF. was found and then the PC12 cells were pretreated with this concentration of 5-HMF, and the cells survived, the cell morphology and the DNA oxidative damage were detected after H_2O_2 treatment. It was found that 5-HMF has obvious oxidation resistance to H_2O_2. Damage effect.
1.1 cell survival detection
The effect of different concentrations of 5-HMF on cell survival rate of 1.1.1
After 2 hours, 6 hours and 24 hours of PC12 cells treated with 50 mu g/ml, 100 mu g/ml, 200 mu g /ml and 400 mu g /ml, the cell survival rate was detected by MTT, and 200 micron g /ml. After 6 hours and 24 hours of 6 hours and 24 hours, the survival rate of cell viability was still not affected by 50 mu. Therefore, we chose these two concentrations of 5-HMF to study its role in antioxidant damage.
The effect of 1.1.2 H_2O_2 on cell survival and the role of 5-HMF
The cells were pretreated with 50 g/ml and 100 g/ml 5-HMF for 30 minutes respectively, then 400 M H_2O_2 was treated for 1 hours. The survival rate of cells was detected by MTT. The results showed that the survival rate of cells decreased after H_2O_2 treatment, and the survival rate of cells after H_2O_2 oxidative damage was obviously improved.
Morphological observation of 1.2 cells
The morphological changes of cells were observed by inverted phase contrast microscope. It was found that the cell shrinkage was smaller and the number of cells decreased significantly after 400 M H_2O_2 treatment. The morphological changes caused by H_2O_2 could be slowed down after 50 g/ml and 100 mu g/ml 5-HMF pretreatment.
1.3 DNA oxidative damage detection
In order to confirm the oxidative damage of DNA caused by H_2O_2 and the effect of 5-HMF, the expression of 8-oxo-dG was detected by immunocytochemistry. The results showed that the expression of 8-oxo-dG was enhanced after 1 hours of PC12 cells treated with 400 u M H_2O_2, while 50 mu g/ml and 100 mu g /ml significantly inhibited the induction of PC12. The results indicate that 5-HMF has anti H_2O_2 induced oxidative damage to DNA.
Preliminary study on the mechanism of anti oxidative damage of 2. 5-HMF
As APE/Ref-1 plays an important role in the alkali radical resection and repair of DNA oxidative damage, it is likely to be the target of the antioxidant activity of 5-HMF. Therefore, we have focused on the role of APE/Ref-1 in the oxidative damage of 5-HMF.
The effect of 2.1 5-HMF on the changes of APE/Ref-1 expression
The effect of 2.1.1 5-HMF on the expression of APE/Ref-1 protein
The immunocytochemical results showed that the expression of APE/Ref-1 was obviously downregulated by 400 M H_2O_2, and the 5-HMF treatment itself did not increase the expression of APE/Ref-1, but it inhibited the downregulation of APE/Ref-1 expression after H_2O_2 treatment. To confirm the effect of 5-HMF on the expression of APE/Ref-1 protein, the experiment further tested the expression of APE/Ref-1 by Western blot method. The results were consistent with the results of immunocytochemistry.
The effect of 2.1.2 5-HMF on the expression of APE/Ref-1 mRNA
Is the effect of 5-HMF on the expression of APE/Ref-1 protein related to the regulation of the level of APE/Ref-1 mRNA? For this reason, we detected the expression of APE/Ref-1 mRNA by RT-PCR. The results showed that H_2O_2 treatment did not change the APE/Ref-1 mRNA level and was not affected by 5-HMF.
The above experiments show that 5-HMF can inhibit the downregulation of APE/Ref-1 protein expression caused by H_2O_2 and is not related to the transcriptional regulation of APE/Ref-1. In addition, it is not clear whether APE/Ref-1 acts as an endonuclease, and its activity is not known to be involved in the antioxidant damage of 5-HMF, so we then use the APE/Ref-1 inhibitor MX to inhibit its enzyme activity. The anti oxidative damage effect of 5-HMF was detected step by step.
2.2 APE/Ref-1 enzyme activity mediates the antioxidant damage of 5-HMF.
2.2.1 detection of different concentrations of MX at different time points on the survival rate of PC12 cells.
With 1 mM, 2.5 mM, 5 mM, 7.5 mM and 10 mM MX respectively, PC12 cells were treated for 2 hours, 16 hours and 24 hours, MTT detected cell viability, and the growth of 7.5 mM and 10 mM MX was inhibited in 2 hours. Activity.
Effect of 2.2.2 MX treatment on antioxidant damage of 5-HMF
The inhibitory activity of APE/Ref-1 endonuclease with 5 mM MX was used to detect the antioxidant effect of 5-HMF. The results showed that after the activity of APE/Ref-1 enzyme was inhibited, the survival ability of 5-HMF was reduced when the oxidation damage was raised, and the anti DNA oxidative damage was also weakened. These results suggest that the antioxidant effect of 5-HMF is through APE/Ref-1. The enzyme activity is mediated.
To sum up, 5-HMF has a significant effect on oxidative damage induced by H_2O_2, and its effect is by stabilizing the expression of APE/Ref-1 protein to make the.5-HMF inhibit the degradation of APE/Ref-1 during oxidative damage and the redox activity of APE/Ref-1, namely, whether the regulatory function of the transcription factor is involved. The antioxidant effect of 5-HMF needs further study.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

【参考文献】