中药复方PRD对OLETF大鼠视网膜自杀相关因子和半胱天冬酶-8表达的影响

发布时间：2018-08-29 18:06

【摘要】：糖尿病(diabetes mellitus,DM)在眼部的主要并发症是糖尿病视网膜病变(diabetic retinopathy,DR),它作为一种不可逆盲,严重威胁患者的视功能。因DR发病机制复杂,目前尚未阐明。随着对DR研究的不断深入,人们认识到DR是一种神经血管性疾病,且神经组织的损伤可能早于微血管病理改变。研究发现细胞凋亡是参与DM视网膜神经组织损伤发病过程的重要事件。自杀相关因子(factors associated suicide,fas)和半胱天冬酶-8(cysteine aspartic acid specific protease-8,caspase-8)是细胞凋亡中死亡受体途径中重要的靶标因子,共同参与细胞凋亡。菩人丹(Purendan superfine powder,PRD)是以苦瓜为君药,丹参、人参为臣药,葛根、何首乌及水蛭为佐使药,六味药共同组成的中药复方。本研究旨在观察PRD对Otsuka Long-Evans Tokushima Fatty(OLETF)大鼠视网膜fas和caspase-8表达的影响,为临床可能应用中药复方制剂PRD治疗DM视网膜神经组织损伤提供理论依据。目的:观察中药复方PRD对OLETF大鼠视网膜中fas和caspase-8表达的影响,探讨PRD对DM大鼠视网膜神经组织可能的保护作用。方法:1以雄性自发性2型DM大鼠模型OLETF大鼠和非DM对照组LETO大鼠为实验对象,以血糖峰值16.7 mmol/L和负荷后120 min血糖11.1mmol/L作为成模标准。造模成功后,将24只成模的OLETF大鼠随机分为PRD治疗组和DM模型组,每组各12只;同周龄12只雄性LETO大鼠为正常对照组。PRD治疗组大鼠连续PRD(1.8 g超微粉/kg/d)灌胃2个月,正常对照组和DM模型组给予等量蒸馏水灌胃。2苏木素-伊红染色观察各组大鼠视网膜的形态结构改变。3 TdT介导的dUTP缺口末端标记法(TUNEL)检测各组大鼠视网膜神经细胞的凋亡情况。4sp免疫组织化学染色法及免疫印迹法检测各组大鼠视网膜fas和caspase-8表达的蛋白。5逆转录聚合酶链反应(rt-pcr)的方法检测各组大鼠视网膜中fas和caspase-8mrna的表达。结果:1各组大鼠视网膜组织的病理改变正常组:高倍光镜下可见视网膜各层结构完整、排列整齐,细胞大小均匀,内界膜完整。模型组:视网膜结构层次欠清楚,视网膜节细胞(retinalganglioncells,rgcs)排列紊乱,数量减少,有空泡样改变,并可见核固缩及部分核溶解,内界膜肿胀增厚、粗糙不平,可见部分内界膜破裂。prd组:视网膜的层次清楚,rgcs排列轻度紊乱,数量稍减少,内界膜表面肿胀增厚减轻,粗糙稍欠平。2tunel法检测视网膜细胞凋亡结果阳性产物表现为棕褐色、颗粒状,位于胞核中,在各组大鼠视网膜切片均可见。阳性细胞散在分布于内核层和rgcs层。与正常对照组相比,dm组大鼠视网膜神经细胞的ai显著升高(p0.01);与dm组比较,prd治疗组视网膜神经细胞的ai明显降低(p0.01)。3fas蛋白及mrna在各组大鼠视网膜的表达免疫组织化学显色结果显示,fas的阳性产物表现为棕黄色、细颗粒状,位于胞质中。免疫阳性细胞主要分布部位为内核层和rgcs层。与正常对照组相比,dm组大鼠视网膜fas蛋白及mrna表达显著升高(p0.01);与dm组比较,prd治疗组大鼠视网膜fas蛋白及mrna表达显著降低(p0.01)。4caspase-8蛋白及mrna在各组大鼠视网膜的表达免疫组织化学显色结果显示,caspase-8的阳性产物表现为棕黄色、细颗粒状,位于胞质中。免疫阳性细胞主要分布部位为内核层和rgcs层。与正常对照组相比,dm组大鼠的视网膜caspase-8蛋白及mrna显著升高(p0.01);与dm组相比,prd治疗组大鼠的视网膜caspase-8蛋白及mrna显著降低(p0.01)。结论:1prd可抑制fas、caspase-8蛋白及mrna在糖尿病视网膜组织中的表达。2 PRD对DR有保护作用,其机制可能是通过抑制fas及caspase-8蛋白及mRNA的表达,减少糖尿病视网膜神经细胞凋亡来实现的。
[Abstract]:The main complication of diabetes mellitus (DM) in the eye is diabetic retinopathy (DR). As an irreversible blindness, DR seriously threatens the visual function of the patients. The pathogenesis of DR is complex and has not been elucidated yet. With the deepening of the study of DR, people realize that DR is a neurovascular disease, and DR is an irreversible blindness. Apoptosis is an important event involved in the pathogenesis of retinal nerve injury in DM. Suicide-associated factors (fas) and cysteine aspartate acid specific protease-8 (caspase-8) are death receptors in apoptosis. Purendan superfine powder (PRD) is a Chinese herbal compound consisting of balsam pear, Salvia miltiorrhiza, ginseng, kudzu root, Polygonum multiflorum and leech as adjuvant and six kinds of herbs. The aim of this study was to observe the effect of PRD on Otsuka Long-Evans Tokushima Fatty (OLETF) rat retina. Objective: To observe the effect of PRD on the expression of Fas and caspase-8 in the retina of OLETF rats and explore the possible protective effect of PRD on the retinal nerve tissue of DM rats. Twenty-four OLETF rats were randomly divided into PRD treatment group and DM model group with 12 rats in each group and 12 male LETO rats at the same age as normal control group. Rats in the PRD treatment group were given PRD (1.8 g ultrafine powder/kg/d) for 2 months. Rats in the normal control group and DM model group were given the same amount of distilled water. 2 Hematoxylin-eosin staining was used to observe the morphological changes of retina in each group. 3 TdT-mediated dUTP nick end labeling (TUNEL) was used to detect the apoptosis of retinal neurons in each group. Immunohistochemical staining and Western blotting were used to detect the expression of Fas and caspase-8 in the retina of rats in each group. the expression of Fas and caspase-8 mRNA was detected by reverse transcription polymerase chain reaction (rt-pcr). In model group, the retinal structure was not clear, the retinal ganglion cells (rgcs) were disorderly arranged, the number of RGCs was reduced, vacuole-like changes were observed, nucleus shrinkage and partial nucleolysis were observed, the inner limiting membrane was swelling and thickening, rough and uneven, and part of the inner limiting membrane ruptured. Rd group: the retinal layers were clear, RGCs were slightly disordered, the number of RGCs was slightly reduced, the swelling and thickening of the inner limiting membrane was alleviated, rough and slightly uneven. 2 TUNEL assay showed that the positive products of retinal cell apoptosis were brown, granular, located in the nucleus of the retina. the positive cells were scattered in the inner nuclear layer and R section of the retina. Compared with the normal control group, AI of retinal nerve cells in DM group increased significantly (p0.01); compared with DM group, AI of retinal nerve cells in PRD group decreased significantly (p0.01). 3 Fas protein and mRNA expression in the retina of rats in each group showed that the positive products of Fas were brown yellow, fine granular. Compared with the normal control group, the expression of Fas protein and mRNA in the retina of DM group increased significantly (p0.01); compared with the DM group, the expression of Fas protein and mRNA in the retina of PRD group decreased significantly (p0.01). 4 caspase-8 protein and mRNA in the retina of rats in each group were significantly lower (p0.01). Immunohistochemical staining showed that the positive products of caspase-8 were brown-yellow, fine granular and located in the cytoplasm. CONCLUSION: 1prd can inhibit the expression of fas, caspase-8 protein and mRNA in diabetic retina. 2PRD can protect DR by inhibiting the expression of fas, caspase-8 protein and mRNA and reducing the apoptosis of diabetic retinal neurons.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R276.7

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