米糠粗黄酮抑制羟基自由基和Aβ诱导的SH-SY5Y凋亡的作用及机理研究

发布时间：2018-01-22 16:47

  本文关键词： 老年痴呆症 米糠黄酮 提取 铜离子 β-淀粉样多肽　出处：《中南林业科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：老年痴呆症是常见的神经退行性疾病,患者具有大脑萎缩、细胞外出现淀粉样斑块、细胞内神经纤维缠结、神经元大量丧失,以及金属离子的过量堆积等临床病理特征。近年来的研究表明:金属离子(如Cu2+)引起的氧化应激和异常聚集的Aβ可能是导致AD的主要原因。具有氧化还原活性的Cu2+能催化产生自由基,破坏线粒体的正常生理功能,诱导细胞膜、组织和酶氧化损伤,最终导致神经变性疾病;Aβ聚集形成的聚集物(寡聚体、纤维等)可以破坏细胞膜,引起炎症反应,导致神经元凋亡。本文以米糠为原料,提取其中的粗黄酮,研究米糠粗黄酮对金属离子引起的氧化应激和异常聚集的Aβ导致的神经毒性的影响。利用CCA荧光法、原子力显微镜、噻哇蓝、MTT实验、Hoechst 33258染色实验、DCFH-DA测内源性RO实验、western blot实验探讨了米糠粗黄酮(Crude flavonoids from Rice bran,FRB)抑制羟基自由基和 Aβ诱导的 SH-SY5Y细胞凋亡的作用及机理的初步研究。通过本论文的研究发现:1.利用酶解辅助超声的方法提取米糠粗黄酮并对其提取工艺进行优化,米糠粗黄酮的提取率达到3.26%。HPLC初步检测米糠粗黄酮中含有柚皮素成分,含量占4.47%。2.米糠粗黄酮能减少Cu2+催化H2O2、Cu2+诱导抗坏血酸(AA)产生的羟基自由基。CCA荧光实验表明米糠粗黄酮能分别使Cu2+催化H202、Cu2+诱导AA产生的羟基自由基的量降低61.82%、57.46%。通过MTT实验发现60 μg/mL的米糠粗黄酮使羟基自由基损伤的SH-SY5Y细胞的存活率由(25.97±2.06)%增加至(76.96±4.39)%。因此,米糠粗黄酮可以通过抑制羟基自由基的生成,从而抑制SH-SY5Y细胞的凋亡。3.米糠粗黄酮能抑制Aβ(1-42)毒性聚集物的产生。利用AFM检测发现米糠粗黄酮能减少Aβ(1-42)毒性聚集物(寡聚体、纤维前体、纤维)的产生,改变Aβ(1-42)的聚集路径。在一定浓度范围内,米糠粗黄酮浓度越高,抑制Aβ(1-42)聚集物生成的效果更明显。4.米糠粗黄酮能抑制Aβ(1-42)毒性聚集物诱导的SH-SY5Y细胞的凋亡。通过MTT和Hoechst 33258实验发现80μM Aβ(1-42)处理的细胞存活率为(52.25±1.34)%,且细胞呈极亮的蓝色。120μM米糠粗黄酮使SH-SY5Y细胞的存活率由(52.25±1.34)%增加至(91.47±1.45)%,细胞极亮的蓝光减少,细胞核形态呈均一饱满状。说明米糠粗黄酮能明显抑制Aβ(1-42)诱导的SH-SY5Y细胞的凋亡。5.米糠粗黄酮能减少细胞内源性ROS和下调caspase-3,从而抑制Aβ(l-42)诱导SH-SYSY细胞的凋亡。通过DCFH-DA测细胞内源性ROS发现,米糠粗黄酮使细胞内源性ROS由1.72±0.02降低到1.12±0.04,从而保护SH-SY5Y细胞免受Aβ(1-42)诱导的损伤。western blot实验结果表明Aβ(1-42)能极显著性(p0.01)的上调细胞caspase-3的表达,而120 μM的米糠粗黄酮使caspase-3的蛋白表达相对量由1.73± 0.07降低到1.07±0.02。因此米糠粗黄酮可以通过下调caspase-3,从而抑制Aβ(1-42)诱导的SH-SY5Y细胞凋亡。综上,本文对米糠粗黄酮的提取工艺进行优化,并利用HPLC初步测定其成分;研究了米糠粗黄酮对羟基自由基和Aβ诱导SH-SY5Y凋亡的抑制作用,并对其机理进行了初步探究;为米糠的综合利用提供了新的研究方向,为AD的治疗提供了新思路,为开发新的治疗药物提供了理论依据,具有重大的现实意义。
[Abstract]:Alzheimer's disease is a common neurodegenerative disease, patients with brain atrophy, extracellular amyloid plaques appear, intracellular neurofibrillary tangles, neuronal loss, excessive accumulation of metal ions and the clinical pathological features. Recent studies show that metal ions (such as Cu2+) induced by oxidative stress and abnormal aggregation A beta may be the main cause of AD. Cu2+ has redox activity to catalyze the production of free radicals, the normal physiological function of mitochondria induced damage, cell membrane, tissue and enzymatic oxidative damage, eventually lead to neurodegenerative diseases; the aggregates formed by the aggregation of beta A (oligomers, fibers) can destroy the cell membrane, causing inflammation leads to neuronal apoptosis. In this paper, rice bran as raw material, the extraction of crude flavonoids from oxidative stress and the differentiation of rice bran flavonoids due to metal ions used to gather A caused by God The toxicity effect. By using CCA fluorescence spectrometry, atomic force microscopy, thiophene wow blue, MTT assay, Hoechst 33258 staining experiments, measurement of endogenous RO DCFH-DA Western blot experiment, experimental study of rice bran flavonoids (Crude flavonoids from Rice bran, FRB) a preliminary study on the inhibitory effect and mechanism of apoptosis of SH-SY5Y cells and A beta hydroxyl radicals the induction. Through the research of this paper found: 1. using enzyme assisted ultrasonic method for the extraction of rice bran crude flavonoids and the extraction process was optimized, the extraction rate of rice bran crude flavonoids to contain naringin component 3.26%.HPLC preliminary detection of yellow ketone content in rice bran, rice bran flavonoids can reduce 4.47%.2. catalyzed by Cu2+ H2O2. Cu2+ induced by ascorbic acid (AA) produced by hydroxyl radical.CCA fluorescence experiments showed that rice bran crude flavonoids respectively catalyzed by Cu2+ H202, Cu2+ AA induced by hydroxyl radicals generated was reduced by 61.82%, 57.46%. Through the MTT experiment found that rice bran flavonoids of 60 g/mL hydroxyl free radical damage to the survival rate of SH-SY5Y cells by (25.97 + 2.06)% increased to (76.96 + 4.39)%. Therefore, the rice bran flavonoids by inhibiting the production of hydroxyl radicals, the apoptosis of.3. rice bran flavonoids inhibit SH-SY5Y cell can inhibit A beta (1-42) toxic aggregates. Found that rice bran crude flavonoids can reduce A beta detected by AFM (1-42) toxic aggregates (oligomers, fiber precursor fiber) generation, change A beta (1-42) aggregation path. In a certain range of concentration, the higher the concentration of Flavonoids from rice bran. Inhibition of A beta (1-42) aggregates generated more obvious effect of.4. rice bran crude flavonoids can inhibit the apoptosis of A beta (1-42) toxic aggregates of SH-SY5Y cells induced by MTT and Hoechst. 33258 experiments showed that 80 M A beta (1-42) the cell survival rate was (52.25 + 1.34)%, and the cells a very bright blue.1 20 M rice bran flavonoids increased the survival rate of SH-SY5Y cells by (52.25 + 1.34)% increased to (91.47 + 1.45)%, bright blue cells decreased, nuclear morphology showed uniform full shape. The rice bran crude flavonoids can inhibit the apoptosis of.5. A beta (1-42) rice bran flavonoids induced SH-SY5Y cell to reduce the endogenous ROS and down-regulation of Caspase-3, thereby inhibiting A beta (L-42) induced apoptosis in SH-SYSY cells. DCFH-DA cells by measuring endogenous ROS found that rice bran flavonoids increased endogenous ROS from 1.72 + 0.02 reduced to 1.12 + 0.04, which protects SH-SY5Y cells against A beta (1-42) injury induced by.Western blot experiments show that A beta (1-42) can significantly (P0.01) expression of Caspase-3 cells, and rice bran crude flavonoid 120 M caspase-3 protein relative amount from 1.73 + 0.07 to 1.07 + 0.02. therefore reduce rice bran crude flavonoids by down regulating the expression of caspase-3, Thus inhibition of A beta (1-42) induced apoptosis in SH-SY5Y cells. In conclusion, this paper optimize the extraction process of rice bran crude flavonoids, and preliminary determination of its composition by HPLC; study the inhibitory effect of rice bran flavonoids on hydroxyl radical and A beta induced SH-SY5Y apoptosis, and the mechanism provides a preliminary inquiry; a new research direction for comprehensive utilization of rice bran, provides a new idea for the treatment of AD, and provides a theoretical basis for the development of new therapeutic agents, is of great practical significance.

【学位授予单位】：中南林业科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151.2
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本文编号：1455203