中药菩人丹对OLETF大鼠视网膜磷酸化c-Jun氨基末端激酶和半胱氨酸天冬氨酸激酶-3表达的干预作用

发布时间：2018-03-26 01:57

本文选题：菩人丹超微粉　切入点：OLETF大鼠　出处：《承德医学院》2016年硕士论文

【摘要】：随着人均寿命的延长和人们生活方式的改变,糖尿病视网膜病变(diabetic retinopathy,DR)的患病率、致盲率正逐年升高,成为人类不可逆性盲的重要原因。因此,DR的防御及治疗成为糖尿病及其并发症研究领域的一个重要课题。近年来研究表明,DR不仅是一种微血管病变更是一种视网膜神经组织损伤的退行性变,视网膜神经细胞凋亡是DR早期最主要的病理表现形式。JNK信号通路作为介导细胞凋亡的一条重要通路在DR的发生发展过程中发挥着重要作用,c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和半胱氨酸天冬氨酸激酶-3(cysteine aspartic acid specific protease-3,Caspase-3)作为JNK信号通路中的两个重要因子与DR关系密切。中药复方在防治DR方面具有多靶位、多途径的独特优势。菩人丹超微粉(Purendan superfine powder,PRD)是针对糖尿病(diabetes mellitus,DM)病机关键“热、虚、瘀”而设的中药组方,具有益气清热、活血通络之功效[1]。本研究采用自发性2型糖尿病大鼠模型OLETF大鼠为研究对象,探讨PRD对2型糖尿病大鼠模型视网膜病变的保护作用,为PRD治疗DR提供理论基础和研究依据。目的:探讨PRD对糖尿病大鼠视网膜p-JNK和Caspase-3表达的影响。方法:1以自发性雄性2型糖尿病大鼠模型OLETF大鼠和其同系非糖尿病对照鼠LETO雄性大鼠为实验对象,空腹血糖(fasting blood-glucose,FBG)峰值16.7 mmol/L和负荷后120 min血糖11.1 mmol/L作为造模成功标准,将24只雄性成模OLETF大鼠按随机数字表法分为DR模型组、PRD治疗组,每组12只,同时选取12只同周龄雄性leto大鼠为空白对照组(正常组)。成功建立模型后,prd治疗组大鼠给予prd(1.8g/kg/d)灌胃2个月。2采用快速血糖仪法监测大鼠血糖,观察大鼠生存状况。3he染色观察视网膜形态结构的变化。4采用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情况。5sp免疫组织化学染色法和westernblotting法检测视网膜磷酸化jnk(p-jnk)、caspase-3蛋白的表达。6采用逆转录聚合酶链法(reversereanscription-polymerasechainereaction,rt-pcr)检测大鼠视网膜jnkmrna、caspase-3mrna的表达。结果:1造模前各组大鼠fbg无明显差距,成模时除正常组外,dr模型组、prd治疗组fbg显著升高,差异有统计学意义(p0.01)。用药后,与dr模型组比较,prd治疗组fbg水平显著降低,有统计学意义(p0.01)。2各组大鼠视网膜病理学改变:正常组大鼠视网膜组织结构完整、内界膜光滑、分层清晰;节细胞圆形、椭圆形,染色浅,排列整齐;内网状层较厚、疏松;内核层染色稍深,由3~5层细胞构成;外网状层较内网状层明显变薄;外核层染色深,由8~10层细胞组成,排列较紧密;外界膜边界清楚、整齐。dr模型组可见视网膜内界膜明显肿胀、增厚,部分内界膜破裂,内界膜界线不清晰,部分细胞空泡样变、细胞核固缩、溶解。prd治疗组视网膜分层较为清晰,内界膜轻度肿胀,可见少量空泡样变细胞。3各组大鼠视网膜神经细胞的凋亡情况:正常组大鼠视网膜切片未见明显tunel染色阳性细胞;dr模型组及prd治疗组阳性细胞分布范围扩大,tunel染色阳性产物表现为褐色、颗粒状,位于胞核中,主要分布部位为视网膜内核层和神经节细胞(retinalganglioncells,rgcs)层。3组大鼠视网膜中ai的总体比较差异有统计学意义,与正常组相比,dm模型组大鼠视网膜神经细胞ai明显升高(p0.01);与dm模型组相比,prd治疗组大鼠视网膜神经细胞ai明显降低(p0.01)。4各组大鼠视网膜p-JNK的表达:免疫组织化学染色结果显示,p-JNK免疫阳性产物为棕黄色、细颗粒状物质,位于胞核和胞质中,阳性细胞主要分布部位为RGCs层及内核层。Western blotting法结果显示,p-JNK蛋白条带位于42 kDa处,β-actin条带位于43 kDa处。RT-PCR结果显示,JNK mRNA条带位于422 bp处,β-actin条带位于425 bp处。糖尿病模型组大鼠与正常组大鼠相比,糖尿病模型组大鼠视网膜组织p-JNK蛋白和mRNA表达明显升高(P0.01);PRD治疗组大鼠与糖尿病模型组大鼠相比,PRD治疗组大鼠视网膜组织p-JNK蛋白和mRNA表达明显降低(P0.01)。5大鼠视网膜Caspase-3的表达:免疫组织化学染色结果显示,Caspase-3免疫阳性产物为棕黄色、细颗粒状物质,位于胞核中,阳性细胞主要分布部位为内核层和RGCs层。Western blotting法结果显示,Caspase-3蛋白条带位于34 kDa处,β-actin条带位于43 kDa处。RT-PCR结果显示,Caspase-3 mRNA条带位于329 bp处:β-actin条带位于425 bp处。糖尿病模型组大鼠与正常组大鼠相比,糖尿病模型组大鼠视网膜组织Caspase-3蛋白和mRNA表达明显升高(P0.01);PRD治疗组大鼠与糖尿病模型组大鼠相比,PRD治疗组大鼠视网膜组织Caspase-3蛋白和mRNA表达明显降低(P0.01)。结论:PRD可以改善糖尿病视网膜病变中神经组织的损伤,表现为神经细胞凋亡的减少,这可能与下调p-JNK、Caspase-3的表达相关。
[Abstract]:With the extension of life expectancy and the change of people's lifestyle, diabetic retinopathy (diabetic, retinopathy, DR) the prevalence of blindness rate is increasing year by year, has become an important cause of human irreversible blindness. Therefore, the prevention and treatment of DR become an important research topic in the field of diabetes and complications in recent years. Research shows that DR is not only a change of microvascular disease is a degenerative retinal nerve tissue injury and apoptosis of retinal nerve cells is an important pathway in early DR, the main pathological manifestation of the.JNK signal pathway is mediated by apoptosis plays an important role in the development of DR, c-Jun terminal kinase (c-Jun N-terminal, kinase, JNK) and cysteine aspartate kinase -3 (cysteine aspartic acid specific protease-3, Caspase-3) as the two important in the JNK signaling pathway The factor is closely related with DR. Traditional Chinese medicine compound with multi targets in the prevention and treatment of DR, the unique advantages in many ways. Purendan Supermicropowder (Purendan superfine powder PRD (diabetes) for diabetes mellitus, DM) the key pathogenesis of heat, deficiency, blood stasis and prescription of traditional Chinese medicine ", has the effect of heat. Effect of Huoxue Tongluo [1]. the spontaneous type 2 diabetes OLETF rats as the research object, to investigate the protective effect of PRD on retinal lesions in type 2 diabetic rat model, and provides a theoretical basis and research basis for the treatment of PRD DR. Objective: to discuss the impact of PRD on the expression of p-JNK and Caspase-3 in retina of diabetic rats methods: 1 OLETF male spontaneous type 2 diabetic rat model and rat syngeneic non-diabetic control rats LETO rats as experimental subjects, fasting blood glucose (fasting, blood-glucose, FBG) 16.7 mmol/L and peak load of 120 min Blood glucose of 11.1 mmol/L as the successful model of the standard, 24 male model OLETF rats were randomly divided into DR model group, PRD treatment group, 12 rats in each group at the same time, a total of 12 week old male Leto rats into normal control group (normal group). After the model, PRD treatment rats were given PRD (1.8g/kg/d) by gavage for 2 months.2 glucose by fast blood glucose meter monitoring rats,.3he rats survival staining nick end labeling changes of.4 retinal morphology using terminal deoxynucleotidyl transferase mediated (TUNEL) detection of rat retinal cell apoptosis.5sp immunohistochemical staining of retinal phosphorylated JNK method and westernblotting method (p-JNK), the expression of.6 caspase-3 protein by reverse transcriptase polymerase chain reaction (reversereanscription-polymerasechainereaction, RT-PCR) detection of jnkmr in retina of rats Na, the expression of Caspase-3mRNA. Results: 1 of the rats in FBG no obvious difference between model except the normal group, Dr model group, PRD treatment group, FBG was significantly increased, the difference was statistically significant (P0.01). After treatment, compared with Dr model group, PRD treatment group and FBG level were significantly decreased. There was statistical significance (P0.01) pathological changes of retina of rats in.2 groups: normal rat retinal tissue structural integrity, membrane smooth, clear stratification; ganglion cell round, oval, stained and neatly arranged; the inner plexiform layer is thick and loose; the core layer was slightly darker, composed of 3~5 layers of cells; the outer plexiform layer than the inner reticular layer was thinner; the outer nuclear layer was deep, composed of 8~10 layers of cells, arranged more closely; outside the membrane boundary clear, neat.Dr model group showed retinal membrane swelling, thickening, part of the internal limiting membrane rupture, internal limiting membrane boundary is not clear, cell vacuolar degeneration fine. Pyknosis, dissolved in.Prd treatment group layered retina is clear, the internal limiting membrane swelling, apoptosis of a small amount of vacuolar degeneration of cells in.3 group rat retinal neural cells: the positive cells of normal rats retinal slices without obvious TUNEL staining; Dr model group and PRD treatment group distribution of positive cells to expand, tunel positive product is brown, granular, is located in the nucleus, the main distribution of retinal inner nuclear layer and ganglion cell layer (retinalganglioncells, RGCs) general.3 rats retina in AI had a significant difference, compared with the normal group, DM model group of rat retinal nerve cells significantly increased AI (P0.01); compared with the DM model group, PRD treatment significantly decreased the AI of retinal nerve cells in rats (P0.01) expression of p-JNK in retina of rats in.4 groups: immunohistochemical staining showed that p-JNK immune The positive product was brown yellow, fine granular material, is located in the nucleus and cytoplasm, the positive cells mainly distributed position display for the RGCs layer and core layer.Western blotting results, p-JNK protein bands at 42 kDa and beta -actin bands located at 43 kDa.RT-PCR results showed that the JNK mRNA band at 422 BP -actin, beta band at 425 BP. The diabetic model rats compared with normal rats, the expression of diabetic retinal tissue of rats in the model group of p-JNK protein and mRNA increased significantly (P0.01); PRD rats in the treatment group compared with the diabetic rats in the model group, PRD treatment group significantly decreased expression of rat retina p-JNK protein and mRNA (P0.01) expression in Caspase-3 rat retina.5: immunohistochemical staining showed that Caspase-3 immunoreactive product was brown yellow, fine granular material, is located in the nucleus. The positive cells mainly distributed to the kernel layer and parts RGCs.Western blotting assay showed that Caspase-3 protein band at 34 kDa, beta -actin bands located at 43 kDa.RT-PCR results showed that the Caspase-3 mRNA band at 329 BP beta -actin band at 425 BP. The diabetic model rats compared with normal rats, the expression of diabetes model the retinal tissue of rats Caspase-3 protein and mRNA increased significantly (P0.01); PRD rats in the treatment group compared with the diabetic rats in the model group, PRD treatment group significantly decreased expression of rat retinal tissue Caspase-3 protein and mRNA (P0.01). Conclusion: PRD can improve the nerve tissue of the diabetic retinopathy is to reduce nerve damage, cell apoptosis, which may be related to the down-regulation of p-JNK, Caspase-3 expression.

【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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