在大鼠视网膜急性缺血前后Sirtuins在视网膜和视神经的表达变化以及白藜芦醇激活SIRT1调节线粒体动态

发布时间：2019-06-07 10:31

【摘要】：目的:氧化应激引起线粒体功能障碍并最终导致视网膜神经节细胞的死亡。白藜芦醇具有抗炎抗氧化应激作用,并且通过激活SIRT1来发挥其保护作用。但是,白藜芦醇是否可以通过激活SIRT1来保护线粒体处于未知阶段。目前关于Sirtuins的研究越来越多,但是在眼科领域还处于未知阶段。因此本研究主要探讨1:Sirtuin家族在大鼠急性缺血损伤视网膜和视神经中的表达情况。2.在视网膜缺血损伤动物模型中,白藜芦醇对线粒体的保护作用并且是否通过SIRT1来介导的。方法:(1)选取成年大鼠,随机选取右眼做急性短暂性视网膜缺血模型,左眼作为对照组,分别于1d、3d、7d提取视网膜组织蛋白检测OPA1、Drp1、SIRT1、Sirtuins蛋白表达。(2)取正常成年大鼠视网膜,通过免疫荧光检测SIRT1和OPA1在视网膜表达以及检测Sirtuins在视网膜和视神经的表达。(3)大鼠急性视网膜缺血损伤后立即给予白藜芦醇处理后7d提取视网膜组织检测OPA1、Drp1、SIRT1蛋白表达。(4)定量PCR检测opa1、drp1、sirt1和Sirtuins基因的表达。(5)通过免疫沉淀检测SIRT1和OPA1的相关性。结果:(1)正常大鼠视网膜中,Sirtuins、OPA1、SIRT1和Sirtuins阳性表达主要在视网膜神经节细胞层、内核层、外核层,其中OPA1和SIRT1共同在视网膜神经节细胞细胞质表达阳性表达最明显。(2)与正常组0d相比,Sirtuin家族变化不同。与正常组0d相比,视网膜缺血损伤后1d,SIRT1的表达基本不变,视网膜缺血损伤后3d、7d后SIRT1的表达下降,7d明显下降(*p0.05);与正常组0d相比,视网膜缺血损伤后1d,OPA1的表达升高,视网膜缺血损伤后3d、7d后OPA1的表达又下降,7d明显下降(*p0.05);与正常组0d相比,视网膜缺血损伤后1d,Drp1的表达下降(*p0.05),视网膜缺血损伤后3d,Drp1表达相比1d又升高(*p0.05),7d后Drp1的表达相比0d明显下降(**p0.01)。(3)与正常组0d相比,视网膜缺血损伤后7d OPA1表达明显下降(*p0.05);给予RSV处理后,OPA1的表达又升高且升高明显(*p0.05);与正常组0d相比,视网膜缺血损伤后7d Drp1表达明显下降(*p0.05);给予RSV处理后,Drp1表达相对0d下降(*p0.05),并且相对7d Drp1继续下降,但表达无明显差异(P0.05);与正常组0d相比,视网膜缺血损伤后7d SIRT1表达明显下降(*p0.05);给予RSV处理后,SIRT1的表达又升高且升高明显(*p0.05)。(4)与正常组0d相比,Sirtuin家族基因表达变化不同。与正常组0d相比,视网膜缺血损伤后7d opa1基因表达明显下降(*p0.05);给予RSV处理后,opa1基因表达又升高且升高明显(*p0.05);与正常组0d相比,视网膜缺血损伤后7d drp1基因表达明显下降(*p0.05);给予RSV处理后,drp1基因表达相对7d又明显升高(*p0.05)。(5)SIRT1和OPA1没有直接起作用。结论:(1)Sirtuin家族在大鼠急性缺血损伤视网膜和视神经表达不同。(2)视网膜急性缺血损伤后,SIRT1呈下降趋势,OPA1先升高再下降、Drp1表达和OPA1相反。(3)白藜芦醇对SIRT1有保护作用,同时保护线粒体,促进线粒体的融合、抑制其裂解。
[Abstract]:Objective: oxidative stress causes mitochondrial dysfunction and eventually leads to the death of retinal ganglion cells. Resveratrol has anti-inflammatory and antioxidant stress and plays a protective role by activating SIRT1. However, whether resveratrol can protect mitochondria at an unknown stage by activating SIRT1. At present, there are more and more research on Sirtuins, but it is still in an unknown stage in the field of ophthalmology. Therefore, the purpose of this study was to investigate the expression of 1:Sirtuin family in the retina and optic nerve of rats with acute ischemic injury. 2. In the animal model of retinal ischemic injury, resveratrol protects mitochondria and is mediated by SIRT1. Methods: (1) Adult rats were randomly selected to make acute transient retinal ischemia model in the right eye, and the left eye was used as the control group. The retinal tissue protein was extracted on the 1st day, the 3rd day, and the OPA1,Drp1,SIRT1, was detected on the 7th day. Expression of Sirtuins protein. (2) the retinal tissue of normal adult rats was taken from the retinal tissue of normal adult rats. The expression of SIRT1 and OPA1 in the retina and the expression of Sirtuins in the retinal and optic nerve were detected by immunofluorescence. (3) the retinal tissue was extracted to detect OPA1,Drp1, 7 days after acute retinal ischemia and injury. (3) 7 days after resveratrol treatment, the retinal tissue was extracted to detect OPA1,Drp1,. (4) quantitative PCR was used to detect the expression of opa1,drp1,sirt1 and Sirtuins genes. (5) the correlation between SIRT1 and OPA1 was detected by immunoprecipitation. Results: (1) the positive expression of Sirtuins,OPA1,SIRT1 and Sirtuins was mainly in the retinal ganglion cell layer, inner nuclear layer and outer nuclear layer of normal rats. The positive expression of OPA1 and SIRT1 in the cytoplasm of retinal ganglion was the most obvious. (2) the change of Sirtuin family was different from that of normal group on day 0. Compared with the normal group, the expression of SIRT1 remained unchanged on the 1st day after retinal ischemic injury, and the expression of SIRT1 decreased on the 3rd day and 7th day after retinal ischemic injury, and decreased significantly on the 7th day after retinal ischemic injury (* p0.05). Compared with the normal group, the expression of OPA1 increased on the 1st day after retinal ischemic injury, and the expression of OPA1 decreased again on the 3rd day after retinal ischemic injury, and decreased significantly on the 7th day after retinal ischemic injury (* p0.05). Compared with the normal group, the expression of Drp1 decreased on the 1st day after retinal ischemic injury (* p0.05), and increased on the 3rd day after retinal ischemic injury (* p0.05). Compared with the normal group, the expression of Drp1 decreased significantly on the 7th day (* p0.01). (3), and the expression of OPA1 on the 7th day after retinal ischemia injury was significantly lower than that of the normal group (* p0.05). After treatment with RSV, the expression of OPA1 increased significantly (* p0.05), and the expression of Drp1 decreased significantly on the 7th day after retinal ischemia injury compared with the normal group (* p0.05). After treatment with RSV, the expression of Drp1 decreased relative to 0 days (* p0.05), and the expression of Drp1 continued to decrease compared with 7 days, but there was no significant difference in the expression of Drp1 (P 0.05). Compared with the normal group on the 0th day, the expression of SIRT1 decreased significantly on the 7th day after retinal ischemic injury (* p0.05). After treatment with RSV, the expression of SIRT1 increased and increased significantly (* p0.05). (4), and the expression of Sirtuin family gene was different from that of normal group on day 0. Compared with the normal group, the expression of opa1 gene decreased significantly on the 7th day after retinal ischemia injury (* p0.05), and the expression of opa1 gene increased significantly after RSV treatment (* p0.05). Compared with the normal group, the expression of drp1 gene decreased significantly on the 7th day after retinal ischemia injury (* p0.05), and the expression of drp1 gene increased significantly on the 7th day after RSV treatment (* p0.05). (5). SIRT1 and OPA1 did not play a direct role. Conclusion: (1) the expression of Sirtuin family in retinal and optic nerve of rats with acute ischemic injury is different. (2) after acute ischemic injury of retinal, SIRT1 tends to decrease, OPA1 increases at first and then decreases. The expression of Drp1 is opposite to that of OPA1. (3) resveratrol has protective effect on SIRT1, at the same time, it can protect mitochondria, promote the fusion of mitochondria and inhibit its fragmentation.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R775

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