褪黑素对糖尿病视网膜病变的保护作用及机制研究

发布时间：2018-03-28 23:29

本文选题：褪黑素　切入点：糖尿病视网膜病变　出处：《复旦大学》2014年博士论文

【摘要】：随着生活节奏和方式的转变,我国糖尿病的发病率逐年上升,现已高达9.7%[1],说明该疾病已成为我国主要的公共卫生问题之一。糖尿病可导致如白内障、青光眼、视神经病变等各种眼部并发症,而其中尤以糖尿病性视网膜病变(Diabetic Retinopathy, DR)最为严重,已成为获得性致盲的主要原因之一。研究表明,DR的发生发展涉及多条细胞信号转导通路,包括多元醇途径,蛋白激酶C激活,糖基化终末产物的积聚,氨基己糖途径等[2-3]。但深入研究阐明,氧化损伤是各条通路导致DR发生的根本机制[4],同时慢性炎症也是参与DR病程发展的重要因素[5]。因此,抗氧化和抗炎治疗将可能有效遏制疾病的进展,为患者提供一条新的治疗途径。褪黑素是一种主要由松果体分泌的激素,视网膜也有少量分泌[6]。据报道,它是目前发现的体内抗氧化应激能力最强的分子,可通过多种途径抗氧化[7-9]。同时也可以通过调节各种促炎症因子,发挥强大的抗炎效应[10]。因此褪黑素对于糖尿病视网膜病变具有潜在的治疗作用。本研究运用生物化学,分子生物学等方法,通过原代培养视网膜Muller细胞,建立糖尿病大鼠模型,从细胞水平和整体水平探讨褪黑素对糖尿病视网膜病变的保护作用,并进一步深入研究其作用机制,将为以后进行糖尿病视网膜保护新药的研发及药物的临床试验提供理论依据,为糖尿病视网膜病变的治疗提供新的途径。第一部分褪黑素对高糖所致视网膜Muller细胞损伤的保护作用及其机制研究目的：探讨褪黑素是否对高糖所致视网膜Muller细胞损伤具有保护效应,并进一步研究其作用通路。方法：酶消化法分离培养大鼠视网膜Muller细胞,免疫荧光法鉴定细胞性质和纯化度。细胞免疫荧光法检测Muller细胞褪黑素膜受体的分布。细胞分为低糖对照组(5.5mmol/l)、甘露醇对照组(5.5mmol/l+24.5mmol/l甘露醇)、高糖组(30mmol/l)、褪黑素+高糖组,褪黑素+高糖+P13k抑制剂(LY294002)组,褪黑素+高糖+褪黑素膜受体拮抗剂(Luzindole)组,分别用不同浓度的褪黑素干预(0.1umol/L,lumol/L,10umol/L,100umol/L),在干预不同时间(24h,48h,72h)后用Western Blot技术检测细胞中磷酸化的Akt水平,免疫荧光检测Nrf2的核转位,用Real time RT-PCR及Western Blot技术检测HO-1,GCLc,GCLm,IL-1 β,TNF-α,NF-κB的表达,ELISA法检测VEGF水平,综合分析褪黑素的保护作用。结果：酶消化法体外分离培养大鼠视网膜Muller细胞纯化度高。视网膜Muller细胞存在褪黑素膜受体MT1,MT2分布,且在高糖条件下受体表达升高。褪黑素可以诱导Akt的磷酸化,提高Nrf2及抗氧化酶H0-1,GSH,GCLc,GCLm的表达水平,促使Nrf2发生核转位,同时抑制高糖状态下显著提高的VEGF水平。而这些效应可被褪黑素膜受体拮抗剂Luzindole及P13K抑制剂LY294002部分阻断。高糖条件下,细胞内炎症因子IL-1β,TNF-α,NF-κB水平显著升高,褪黑素可降低其表达。结论：褪黑素对高糖所致视网膜Muller细胞损伤具有抗氧化及抗炎保护作用,主要体现在可以提高抗氧化酶水平以及抑制炎症因子表达。PI3K/Akt-Nrf2通路及褪黑素膜受体参与其抗氧化效应的实现,NF-K B在其抗炎效应中发挥重要作用。同时褪黑素可以抑制VEGF的表达。第二部分褪黑素对糖尿病大鼠视网膜组织损伤的保护作用及其机制研究目的：通过建立糖尿病大鼠模型,探讨褪黑素是否对糖尿病所致视网膜损伤具有保护作用,并进一步研究其作用机制。方法：成年雄性SD大鼠随机分为三组：正常对照组,糖尿病组,糖尿病+褪黑素组。采用STZ (60 mg/kg)一次性腹腔注射法建立糖尿病大鼠模型。三天后,尾静脉采血测血糖,血糖大于16.7 mol/L者为造模成功。糖尿病+褪黑素组在糖尿病造模成功后,给予褪黑素(10 mg/kg)每日腹腔注射。实验过程中定期检测实验动物的血糖和体重。血糖不符合者及时剔除。正常对照组给予相应溶剂腹腔注射。分别在造模4周,8周,12周后将动物处死,取出视网膜组织。免疫荧光法检测视网膜冰冻切片上褪黑素膜受体MT1, MT2的分布。Real time RT-PCR 及 Western Blot检测VEGF, HO-1, iNOS, IL1β, TNF-α及Nrf2水平,Western Blot检测磷酸化IK B,磷酸化Akt及细胞核内NF-κB水平。同时检测造模12周后各组大鼠ERG水平。结果：STZ腹腔注射法建立糖尿病大鼠模型方法可靠,成功率高,造模组动物血糖在整个实验中维持高水平,保持稳定。糖尿病大鼠体重与正常大鼠相比进行性降低。褪黑素干预对糖尿病大鼠体重和血糖无明显影响。免疫荧光检测显示MT1和MT2主要分布于视网膜神经节细胞层,内丛状层,且在糖尿病状态下表达升高。糖尿病大鼠视网膜VEGF水平较正常组显著提高,而褪黑素处理组可降低其表达。TNF-α,IL-1β等炎症因子,磷酸化IK B及细胞核NF-κB水平在糖尿病状态明显提高,褪黑素可以抑制其表达。褪黑素可以使磷酸化Akt水平提高,并提高抗氧化相关因子Nrf2, HO-1水平。造模12周后,糖尿病大鼠ERG的a波和b波振幅较正常组显著降低,而褪黑素干预组ERG与正常组相比无统计学差异。结论：褪黑素对糖尿病视网膜病变具有保护作用,主要通过抑制NF-κB及其下游炎症因子表达,及提高PI3K/Akt-Nrf2介导的抗氧化因子水平实现。同时褪黑素还可以抑制VEGF的表达,改善视网膜功能。因此,褪黑素对于糖尿病视网膜病变的治疗具有潜在价值。
[Abstract]:With the change of the rhythm of life and the way of our country, the incidence of diabetes has increased year by year, up to 9.7%[1], indicating that the disease has become one of the major public health problem in China. Diabetes can cause such as cataracts, glaucoma, optic neuropathy and other ocular complications, particularly in diabetic retinopathy (Diabetic Retinopathy DR) is the most serious, has become one of the main reasons for the blindness. Studies show that the occurrence and development of DR is involved in many cellular signaling pathways, including polyol pathway, activation of protein kinase C, advanced glycation end products [2-3]. accumulation, hexosamine pathway etc. but the research stated that oxidative damage is the the fundamental mechanism of DR pathway leading to [4], [5]. and an important factor of chronic inflammation is involved in the course of DR development so that the progress of antioxidant and anti-inflammatory treatment could effectively prevent disease, To provide a new therapeutic approach for patients. Melatonin is a hormone secreted by the pineal gland, retina also have a small amount of secretion of [6]. according to the report, it is the ability of anti oxidative stress found in the strongest antioxidant molecules, through a variety of ways [7-9]. also can be regulated by various proinflammatory cytokines, play an anti-inflammatory effect of [10]. so powerful melatonin for diabetic retinopathy has a potential therapeutic role. On the basis of Biochemistry, molecular biology methods, retinal Muller cells cultured by primary, establish the model of diabetic rats, to investigate the protective effect of melatonin on diabetic retinopathy at the cellular level and the overall level, and further study the mechanism of action, will to provide a theoretical basis for future clinical trials and research and development of new drugs for diabetic retinal protection, diabetes To provide a new way for the treatment of retinopathy. The melatonin protective effect and mechanism of injury of high glucose induced retinal Muller cells Objective: To investigate whether melatonin damage on retinal Muller cells induced by high glucose has a protective effect, and further study its pathway. Methods: cultured rat retinal Muller cells by enzyme digestion, immune cell identification fluorescence properties and purification of Muller cells was detected. The distribution of melatonin membrane receptor cell immunofluorescence. The cells were divided into low glucose control group (5.5mmol/l), mannitol group (5.5mmol/l+24.5mmol/l mannitol), high glucose group (30mmol/l), melatonin melatonin + + high glucose group, high glucose +P13k inhibitor (LY294002) group, high glucose + + melatonin melatonin membrane receptor antagonist (Luzindole) group, respectively with different concentrations of melatonin intervention (0.1umol/L, lumol/L, 10umol/L, 100umol/L), In the intervention of different time (24h, 48h, 72h) after Western cells were detected with phosphoric acid in the Blot technology of Akt, immunofluorescence detection of Nrf2 nuclear translocation was detected by HO-1 Real, time RT-PCR and Western Blot GCLc, GCLm, IL-1 beta, TNF- alpha, the expression of NF- kappa B, VEGF level detection ELISA by comprehensive analysis of the protective effect of melatonin. Results: the cultured rat retinal Muller cells of high purification enzyme digestion method in vitro. Retinal Muller cells exist melatonin receptors MT1, MT2 distribution, and the elevated expression in high glucose conditions. Melatonin can induce the phosphorylation of Akt, Nrf2 increased and antioxidant enzymes H0-1, GSH. GCLc, the expression level of GCLm, promote Nrf2 nuclear translocation, and inhibit the high glucose condition significantly increased levels of VEGF. These effects can be melatonin membrane receptor antagonist Luzindole and P13K inhibitor LY294002 partially blocked. Under high glucose condition, Intracellular inflammatory factor IL-1 beta, TNF- alpha, NF- kappa B had significantly higher levels of melatonin can reduce the expression. Conclusion: melatonin has antioxidant and anti-inflammatory protective effect on retinal Muller cells induced by high glucose injury, mainly reflected in can improve the levels of antioxidant enzymes and can inhibit the inflammatory cytokines expression of.PI3K/Akt-Nrf2 pathway and melatonin membrane receptor involved in the antioxidant the effect of NF-K, B play an important role in the anti-inflammatory effect. At the same time the expression of melatonin can inhibit the VEGF. The second part objective of melatonin on the protective effect and mechanism of retinal tissue injury in diabetic rats: by establishing the model of diabetic rats, to investigate whether melatonin on diabetic retinal injury has a protective effect, and further study its role the mechanism. Methods: adult male SD rats were randomly divided into three groups: normal control group, diabetes group, sugar The urine sickness + melatonin group. Using STZ (60 mg/kg) to establish the model of diabetic rats by intraperitoneal injection. After three days, blood glucose of tail vein was greater than 16.7 mol/L were successful. Diabetes + melatonin group rats in diabetes mellitus after administration of melatonin (10 mg/kg) intraperitoneal injection and blood glucose. The experimental animal weight detection regularly during the experiment. Blood sugar does not conform to timely removed. The normal control group were given intraperitoneal injection of the corresponding solvent. In 4 weeks, 8 weeks, 12 weeks after the animal is killed, remove the retinal tissue. Detection of retinal immunofluorescence on frozen sections of melatonin membrane receptor MT1,.Real time RT-PCR distribution VEGF MT2 and Western Blot detection, HO-1, iNOS, IL1 beta, TNF- alpha and Nrf2 level Western Blot IK detection of phosphorylated B, phosphorylation of Akt and nuclear NF- kappa B level. At the same time detected 12 weeks after modeling, the rats in ERG water Ping. Results: STZ intraperitoneal injection method to establish the diabetic rat model is reliable, high success rate, make blood sugar throughout the experiment animal model to maintain a high level, remained stable. The body weight of diabetic rats compared with normal rats was decreased. Melatonin intervention had no significant effect on body weight and blood glucose of diabetic rats was detected by immunofluorescence. MT1 and MT2 mainly distributed in retinal ganglion cell layer, inner plexiform layer, and in the condition of diabetes. Elevated expression of retinal VEGF levels in diabetic rats was higher than that in normal group and treatment group, melatonin can reduce the expression of.TNF- alpha, IL-1 beta and other inflammatory factors, the phosphorylation of IK B and nuclear NF- kappa B level significantly increased in the diabetic state, melatonin can inhibit its expression. Melatonin can improve the phosphorylation level of Akt, and improve the antioxidant related factor Nrf2, the level of HO-1. After 12 weeks, diabetic rats ER G A and b wave amplitude is significantly lower than the normal group, melatonin intervention group ERG no statistically significant difference compared with the normal group. Conclusion: melatonin has a protective effect on diabetic retinopathy, mainly inhibit the expression of NF- kappa B and its downstream inflammatory factors, and improve the level of antioxidant factor PI3K/Akt-Nrf2 mediated expression of melatonin and implementation. Also can inhibit VEGF and improve the function of retina. Therefore, melatonin has a potential value for the treatment of diabetic retinopathy.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R774.1

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