槲皮素对镉诱导雄性大鼠睾丸组织损伤的保护机制研究

发布时间：2018-05-14 08:22

本文选题：氯化镉 + 槲皮素　；参考：《沈阳农业大学》2017年硕士论文

【摘要】：目的:本试验探究抗氧化剂槲皮素通过调控Nrf2-Keap1途径拮抗镉对大鼠生殖机能造成的应激损伤。通过体内试验对其机制进行深入研究,为研究镉的生殖毒性及槲皮素通过调控Nrf2-Keap1路径保护机体免受氧化损伤的研究奠定了基础。方法:通过分组饲养,定时灌胃处理,建立试验模型,4周之后取出睾丸组织,通过检验睾丸组织的谷胱甘肽过氧化酶(Gsh-Px),过氧化氢酶(CAT),超岐物过氧化物酶(SOD)等抗氧化酶水平,睾酮分泌量以及Nrf2-Keap1抗氧化通路相关因子2(Nrf2)、谷胱甘肽过氧化物酶(Gsh-Px)、血红素加氧酶1(HO-1)、Y-谷氨酰半胱氨酸合成酶(γ-GCS)、醌氧化还原酶1(NQO1)mRNA及蛋白的表达量,进一步判定槲皮素作为抗氧化剂对染镉的干预作用。结果:1.通过对大鼠灌胃处理后,5天作为一个周期,对大鼠体重进行测定,发现染镉组大鼠体重明显低于空白组及槲皮素组,槲皮素组的体重和空白组的体重相比差异显著。槲皮素治疗组的大鼠体重测定值明显高于染镉组。2.通过对睾丸组织内必要的抗氧化酶检测,对试验数据进行分析发现,与空白组相比,染镉组丙二醛(MDA)活性明显升高(p0.01),差异极显著,槲皮素组丙二醛活性低于空白组(p0.01),槲皮素治疗组与染镉组比较,丙二醛活性低于染镉组(p0.01)。多岐物过氧化物酶(SOD)活性,过氧化氢酶(CAT),谷胱甘肽过氧化物酶(Gsh-Px),染镉组与实验空白组相比,活性明显降低(p0.01),槲皮素治疗组与染镉组相比,抗氧化酶的活性明显升高,差异极显著(p0.01)。数据表明槲皮素对镉引起的机体损伤具有很好的缓和作用,促使机体的抗氧化反应能力增强。3.通过Elisa酶联免疫吸附试验测定发现,与空白组作比较,染镉组睾酮含量明显降低(p0.01),槲皮素组的睾酮含量与空白组相比明显增加(p0.01),槲皮素治疗组与染镉组相比,睾酮含量明显上升,差异显著(p0.05)。证明槲皮素能够拮抗镉引起机体睾酮分泌量的减少,减弱对机体的损伤。4.通过对睾丸组织的HE染色镜检分析,发现与空白组相比,染镉组睾丸组织存在生精上皮生殖细胞大量脱落,各级生殖细胞数量减少,曲细精管中央伸长型精子细胞数量减少的现象,槲皮素治疗组部分生精上皮细胞损伤脱落,内皮细胞损伤情况不显著,这表示槲皮素对镉引起的大鼠睾丸组织损伤能够起到较好的缓解作用。5.通过q-PCR技术和Western Blot蛋白技术检测组织Nrf2抗氧化通路中Nrf2,HO-1,NQO1,Gsh-Px,γ-GCS,Keap1的mRNA和蛋白水平,发现与空白组相比,染镉组Keap1的mRNA和蛋白水平高于空白组(p0.01),然而Nrf2,HO-1,NQO1,Gsh-Px,y-GCS的mRNA和蛋白水平明显低于空白组(p0.01,p0.05)。对于槲皮素治疗组,Keap1的表达水平明显低于染镉组(p0.01),Nrf2,HO-1,NQO1,Gsh-Px,y-GCS水平高于染镉组(p0.01,p0.05)。结论:镉会损害大鼠睾丸组织的生精功能,降低睾丸组织对抗氧化反应的能力,造成生殖系统明显损伤,而槲皮素具有拮抗镉诱导损害作用的能力。槲皮素可以激活Nrf2-Keap1抗氧化通路,调节抗氧化酶的表达水平,干预镉损伤,证明槲皮素对镉造成的内源性氧化反应起到防御功能。
[Abstract]:Objective: To explore the antagonistic effect of quercetin on the stress damage caused by cadmium on the reproductive function of rats by regulating the Nrf2-Keap1 pathway. Through in vivo experiments, the mechanism of quercetin was studied in order to study the reproductive toxicity of cadmium and the study of quercetin to protect the body from oxidative damage by regulating the Nrf2-Keap1 path. Methods: the experimental model was established by grouping and feeding the stomach at a time. After 4 weeks, the testicular tissue was removed, and the levels of antioxidant enzymes, such as glutathione peroxidase (Gsh-Px), catalase (CAT), superoxide peroxidase (SOD), and Nrf2-Keap1 antioxidant pathway related factor 2 (Nrf2), were examined, Gu Guanggan Peptide peroxidase (Gsh-Px), heme oxygenase 1 (HO-1), Y- glutamyl cysteine synthetase (gamma -GCS), quinone oxidoreductase 1 (NQO1) mRNA and protein expression, further determined that quercetin was used as an antioxidant to interfere with cadmium. The results were as follows: 1. of rats were treated by gavage for 5 days as a cycle, and the weight of rats was measured. It was found that the weight of rats in the cadmium dyed group was significantly lower than that in the blank group and the quercetin group. The weight of the quercetin group was significantly different from that in the blank group. The weight determination of the rats in the quercetin group was significantly higher than that in the cadmium dyed group.2. through the test of the necessary antioxidant enzymes in the testicular tissue, and the test data were analyzed and compared with the blank group. The activity of malondialdehyde (MDA) in cadmium dyed group was significantly higher (P0.01). The activity of malondialdehyde in quercetin group was lower than that in blank group (P0.01). The activity of malondialdehyde in quercetin group and cadmium dyed group was lower than that of cadmium dyed group (P0.01). The activity of peroxidase (SOD), catalase (CAT), glutathione peroxidase (Gsh-Px), cadmium dyed group and solid of cadmium group were compared with that of cadmium dyed group. Compared with the blank group, the activity of the quercetin was significantly lower (P0.01). The activity of antioxidant enzymes in the quercetin group was significantly higher than that in the cadmium dyed group, and the difference was very significant (P0.01). The data showed that quercetin had a good mitigating effect on the damage caused by cadmium and enhanced the anti oxygenation ability of the organism by the Elisa enzyme-linked immunosorbent assay. Compared with the blank group, the testosterone content in the cadmium dyed group decreased significantly (P0.01), and the testosterone content in the quercetin group increased significantly compared with the blank group (P0.01). The content of testosterone in the quercetin group was significantly higher than that in the cadmium dyed group (P0.05). It was proved that quercetin could antagonize the decrease of the excretion of testosterone in the organism and the mechanism of reducing the mechanism of cadmium. The damage of.4. was analyzed by the HE staining microscopy of the testicular tissue. It was found that compared with the blank group, the testicular tissue in the cadmium dyed group had a large number of germ cells falling off, the number of germ cells at all levels decreased, the number of central elongated spermatozoa in the fine tubule decreased, and the injury of some spermatogenic epithelial cells in the quercetin group was lost. The damage of skin cells was not significant, which indicated that quercetin could play a better role in alleviating the damage of testicular tissue in rats induced by cadmium..5., HO-1, NQO1, Gsh-Px, gamma -GCS, Keap1 mRNA and protein levels were detected by q-PCR technology and Western Blot protein technology. The level of mRNA and protein was higher than that in the blank group (P0.01), but the level of mRNA and protein in Nrf2, HO-1, NQO1, Gsh-Px, y-GCS was significantly lower than that in the blank group (P0.01, P0.05). Quercetin can activate Nrf2-Keap1 antioxidant pathway, regulate the expression level of antioxidant enzymes, interfere with the cadmium damage, and demonstrate the endogenous oxidation caused by quercetin to cadmium. The reaction serves as a defensive function.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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