白藜芦醇对糖尿病视网膜病变的保护作用及机制研究

发布时间：2018-04-09 04:22

本文选题：白藜芦醇　切入点：糖尿病视网膜病变　出处：《武汉大学》2016年博士论文

【摘要】：第一部分白藜芦醇对STZ诱导的糖尿病大鼠视网膜病变的影响目的：观察白藜芦醇(Resveratrol,Res)对链脲佐菌素(Streptozotocin,STZ)诱导的大鼠糖尿病视网膜病变的防治作用,探讨其可能的作用机制。方法随机将35只SD大鼠中的10只分为正常对照组(Control),剩余的25只腹腔注射STZ建立糖尿病模型,将造模成功的20只大鼠随机均分为糖尿病模型组(DM)和白藜芦醇治疗组(DM+Res)。确定造模成功次日即开始灌胃给药,分别于灌胃后的第0、4、8、12周时测量大鼠的体重和血糖,12周时处死大鼠取眼球,然后测定各组大鼠视网膜丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性；采用苏木素-伊红(HE)染色观察大鼠视网膜组织的形态学改变并测量视网膜厚度；脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测大鼠视网膜神经细胞的凋亡：透射电镜下观察大鼠视网膜神经节细胞和毛细血管管壁超微结构的变化；免疫组织化学染色法检测大鼠视网膜Bax和Bcl-2蛋白的表达。结果：DM+Res组大鼠4、8、12周时体重均较DM组重,但低于Control组；其血糖在上述3个时间点均低于DM组。与DM组相比,DM+Res组大鼠视网膜MDA含量明显降低(P0.01),SOD活性明显升高(P0.01),视网膜总厚度及视网膜内、外核层厚度均显著增加(p0.01),神经节细胞层和内核层凋亡指数均降低(P0.01)；电镜下,DM组可见神经节细胞核染色质凝聚,线粒体肿胀,胞质空泡化；DM+Res组神经节细胞未见明显的核染色质凝聚、边集,线粒体肿胀、嵴丢失及胞质空泡化；免疫组织化学法显示与DM组比较,DM+Res组Bcl-2表达增强,Bax表达强度减弱,Bcl-2/Bax比值显著升高。结论： Res可改善糖尿病大鼠体重,降低其血糖,增强其视网膜的抗氧化能力,从而减轻视网膜的氧化损伤,改善糖尿病所致的视网膜的病理改变,并可通过上调Bcl-2和下调Bax的表达,抑制视网膜神经节细胞凋亡,发挥对糖尿病视网膜病变的防治作用。第二部分白藜芦醇对高糖状态ARPE-19细胞的影响目的：研究高糖条件下不同时间及不同浓度Res对ARPE-19细胞活力的影响,以探寻最适宜ARPE-19细胞高糖损伤模型,筛选适宜干预的Res浓度,并探讨Res对高糖状态ARPE-19细胞的影响方法：将 ARPE-19细胞株分别置于正常糖状态(5.5mmol/L葡萄糖)、高糖状态(30mmol/L葡萄糖)和高渗状态(5.5mmol/L葡萄糖+24.5mmol/L甘露醇)培养24h、48h、72h,CCK-8法检测各个时间点细胞活性并进行对比,选择最佳作用时间点；再用不同浓度白藜芦醇(Oμmol/L、5μmol/L、10μmol/L、25μmol/L、 50μmol/L)处理高糖状态(30mmol/L葡萄糖)ARPE-19细胞株48h,通过CCK-8法检测各组细胞活性,选择最佳浓度；将ARPE-19细胞株置于含30mmol/L葡萄糖和10 μmol/L Res的DMEM培养基培养48h(同时设置正常、高糖对照和高渗对照)后用倒置显微镜观察各组细胞生长情况和形态变化。结果：CCK-8法检测显示,与正常对照组相比,高糖组ARPE-19细胞,24h时细胞活性降低但无统计学差异,48h时出现统计学差异,72h时细胞活性降低但与48h时相比无统计学差异。故后续实验取48h为最佳作用时间。而高渗对照组在24h、48h、72h细胞活性与正常对照组无明显差异。高糖状态(30mmol/L葡萄糖)白藜芦醇浓度为10 μmol/L时,培养48h后细胞活性最高；高糖能引起ARPE-19细胞形态的异常改变,Res可缓解高糖所致的细胞形态的异常改变。结论：30mM的高糖作用体外培养的ARPE-19细胞48h可抑制其活性；白藜芦醇作用于高糖状态ARPE-19细胞48h时能增加其活性,浓度为10μM时效果最佳;Res可缓解高糖对ARPE-19细胞的损伤。第三部分白藜芦醇对高糖状态下ARPE-19田胞的保护作用及机制研究目的：探讨白藜芦醇对高糖状态下ARPE-19细胞的保护作用及其机制方法：ARPE-19细胞株分为3组：正常对照组(Control):5.5mmol/L葡萄糖；高糖组(HG):30mmol/L葡萄糖；Res干预组(HG+Res):30mmol/L葡萄糖+10μmol/LRes,三组细胞条件培养48h,流式细胞仪检测细胞凋亡率,免疫荧光法和Western-Blot检测各组中bax、bcl-2的表达,流式细胞仪和免疫荧光法检测ROS的产生,RT-PCR法检测IL-1βmRNA、TNF-αmRNA的表达结果：与HG组相比,HG+Res干预组细胞的凋亡率降低,表达bcl-2增加、bax减少,bcl-2/bax值升高；且ROS的产生减少、IL-1βmRNA、TNF-αmRNA的表达降低。结论：Res能降低细胞的凋亡率,可能是通过增加bcl-2、减少bax、升高bc1-2/bax值,减少ROS的产生和减少炎症相关细胞因子的释放实现的
[Abstract]:Part 1 Effects of resveratrol on STZ induced diabetic retinopathy in rats Objective: To observe the effect of resveratrol (Resveratrol, Res) on streptozotocin (Streptozotocin, STZ) prevention and treatment induced diabetic retinopathy in rats, and to explore its possible mechanism. Methods 10 only 35 SD the rats were divided into normal control group (Control), 25 with intraperitoneal injection of STZ remaining to establish diabetes model. Then 20 rats were randomly divided into model for diabetic model group (DM) and resveratrol treatment group (DM+Res). The next day to determine the successful model intragastrically. Measurement of body weight and blood glucose of rats after intragastric administration respectively at 0,4,8,12 weeks, 12 weeks, the rats were sacrificed, the eye, and then determined the Rats Retinal malondialdehyde (MDA) content and superoxide dismutase (SOD) activity; using hematoxylin eosin (HE) staining of rat optic Omental tissue morphological changes and measurement of retinal thickness; terminal deoxynucleotidyl transfer enzyme mediated nick end labeling (TUNEL) detection of apoptosis of retinal nerve cells in rats: Observation of rat retinal ganglion cells and capillary wall micro structure changes under electron microscope; immunohistochemical staining was used to detect the expression of Bax in rat retina and the Bcl-2 protein. Results: DM+Res rats, 4,8,12 weeks of weight when compared with the DM group, but lower than that of Control group; the blood glucose levels were lower than that of group DM at the 3 time points. Compared with the DM group, retinal MDA in DM+Res rats were significantly decreased (P0.01), the activity of SOD was significantly increased (P0.01) the total retinal thickness and retinal, and the thickness of the outer nuclear layer were significantly increased (P0.01), ganglion cell layer and inner nuclear layer decreased the apoptosis index (P0.01); under electron microscope, DM group showed ganglion cell nuclear chromatin Condensation, mitochondrial swelling, cytoplasm vacuolation of ganglion cells; DM+Res group had no obvious nuclear chromatin condensation, margination, mitochondrial swelling, cristae loss and cytoplasmic vacuolization; immunohistochemical method showed that compared with DM group, DM+Res group enhanced the expression of Bcl-2, reduce the strength weak expression of Bax, Bcl-2/Bax ratio was significantly increased. Conclusion: Res can improve the body weight of diabetic rats, lower blood sugar, enhance the antioxidative ability of retina, thereby reducing oxidative damage in the retina, improve the pathological changes in diabetic retina, and through the upregulation of the expression of Bcl-2 and Bax under the regulation, inhibit the apoptosis of retinal ganglion cells, play a role in the prevention and treatment of diabetic retinopathy. The second part of resveratrol on high glucose ARPE-19 cells Objective: different time and different concentration of Res on high glucose conditions on ARPE-19 cell activity influence, to explore The most suitable ARPE-19 cells in high glucose injury model, screening the appropriate concentration of Res intervention, and to explore the effect of Res on high glucose ARPE-19 cells methods: ARPE-19 cells were cultured in normal glucose (5.5mmol/L glucose), high glucose (30mmol/L glucose) and hyperosmolar state (5.5mmol/L glucose +24.5mmol/L mannitol) cultured in 24h, 48h, 72h. Comparison of CCK-8 assay at various time points and cell activity, choose the best time point; with different concentrations of resveratrol (O mol/L, 5 mol/L, 10 mol/L, 25 mol/L, 50 mol/L) and high glucose (30mmol/L glucose) ARPE-19 cell line 48h cells were activated through CCK-8 method detection, choose the best concentration; the ARPE-19 cell line was placed in the containing 30mmol/L glucose and 10 mol/L Res DMEM 48h (medium and normal, high glucose control and hypertonic control) with inverted microscope The growth and morphological changes of cells in each group. Results: CCK-8 assay showed that compared with normal control group, high glucose group ARPE-19 cells, 24h cell activity decreased but the difference was not statistically significant, statistically significant differences between the 48h, 72h cell activity decreased when compared with 48h but no statistically significant difference. So the follow-up experiment 48h is the best the role of time. While hypertonic group in 24h, 48h, 72h cell activity had no significant difference with normal control group. High glucose (30mmol/L glucose) resveratrol concentration is 10 mol/L, the highest 48h activity of cultured cells; high glucose can cause abnormal changes of the morphology of ARPE-19 cells, Res can alleviate the abnormal changes induced by high glucose cell morphology. Conclusion: ARPE-19 48h cells were cultured with high glucose in vitro effect of 30mM can inhibit the activity of resveratrol in high glucose; ARPE-19 cells can increase the activity of 48h, the concentration of 10 M aging The best fruit; Res can alleviate the damage of high glucose on ARPE-19 cells. The third part of resveratrol on the objective to study the protective effect and mechanism of ARPE-19 field cells under high glucose condition: To investigate the protective effect and mechanism of resveratrol on ARPE-19 cells under high glucose condition: ARPE-19 cells were divided into 3 groups: normal control group (Control) 5.5mmol/L: glucose; high glucose group (HG): 30mmol/L glucose; Res intervention group (HG+Res): 30mmol/L glucose +10 mol/LRes, 48h three groups of cell culture conditions, cell apoptosis was detected by flow cytometry, immunofluorescence and Western-Blot were detected in Bax, the expression of Bcl-2, flow cytometry and immunofluorescence method the detection of ROS, detection of IL-1 beta mRNA RT-PCR method, the expression of TNF- alpha mRNA: compared with HG group, the apoptosis of HG+Res cells decreased the rate of the intervention group, the expression of Bcl-2 increased, Bax decreased, bcl-2/bax increased and ROS; The expression of IL-1 beta mRNA and TNF- alpha mRNA decreased. Conclusion: Res can reduce the apoptosis rate of cells, which may be achieved by increasing Bcl-2, decreasing Bax, increasing bc1-2/bax value, reducing ROS production and reducing inflammatory related cytokines release.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R774.1

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