石斛多糖对糖尿病大鼠视网膜保护性作用的研究

发布时间：2018-02-03 06:00

本文关键词： 糖尿病视网膜病变石斛多糖炎症因子紧密连接蛋白血-视网膜屏障　出处：《蚌埠医学院》2016年硕士论文　论文类型：学位论文

【摘要】：研究目的近年来,越来越多的研究认为炎症作用机制在糖尿病视网膜病变(diabetic retinopathy DR)的早期发展中发挥着不可或缺的作用。由此推测,抗炎治疗有可能成为预防DR的发展及治疗提供一个新的思路。石斛作为我国传统名贵中草药之一,被认为具有滋阴补肾、清肝明目的功能。多糖是石斛的主要的药用活性成分之一,其化学成分和药理活性作用也日益引起人们的关注,研究表明,石斛多糖具有抗炎、抗氧化、抗肿瘤等作用。本研究以糖尿病大鼠为研究对象,观察石斛多糖与糖尿病大鼠血-视网膜屏障功能的关系及其具体的作用机制。实验方法动物实验:老鼠随机分为5组,进行实验:(1)正常组,(2)糖尿病组,(3)糖尿病+石斛多糖低剂量组[100mg/(kg?天)],(4)糖尿病+石斛多糖中剂量组[200mg/(kg?天)],(5)糖尿病+石斛多糖高剂量组[300mg/(kg?天)]。老鼠按照55mg/kg使用链脲佐菌素(streptozotocin,STZ)腹腔注射,正常组组大鼠腹腔注射同等剂量柠檬酸钠溶液,以后每周监测血糖一次。48小时血糖高于16.7mmol/L认为造模成功。建模成功6周后,石斛多糖各处理组进行为期8周灌胃治疗。8周后,2%戊巴比妥钠(40mg/kg)腹腔麻醉大鼠,取所需标本。采用蛋白印迹法(Westen Blot)检测大鼠视网膜内白介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumer factor-alpha,TNF-α)、血管内皮生长因子(vascular endothelial growth factor,VEGF)及视网膜内紧密连接蛋白家族(ZO-1、Occludin、Claudin-5)的蛋白表达水平,酶联免疫吸附试验(Enzyme Linked Immunosorbent Assay,ELISA)检测大鼠血清、视网膜内IL-6、TNF-α的含量,免疫组化观察大鼠视网膜内核转录因子κB(NF-κB)的表达水平,伊凡氏蓝(Evans blue)染色分析糖尿病大鼠血视网膜屏障功能。实验结果与正常组比较,糖尿病组大鼠视网膜内IL-6、TNF-α、VEGF的蛋白表达增加,紧密连接蛋白(ZO-1、Occludin、Claudin-5)表达水平降低,血清IL-6、TNF-α表达增加,大鼠视网膜渗透量增高,差异均具有统计学意义(P0.05)。与糖尿病组比较,石斛多糖低、中、高各剂量组大鼠视网膜内IL-6、TNF-α的蛋白表达明显降低,紧密连接蛋白(ZO-1、Occludin、Claudin-5)表达水平增高,血清IL-6、TNF-α的表达降低,石斛多糖各剂量组大鼠视网膜渗透量明显降低,差异均具有统计学意义(P0.05)。石斛多糖各剂量组之间各指标两两比较差异均无统计学意义(P0.05)。免疫组化切片观察,正常大鼠视网膜未见明显NF-κB阳性染色,糖尿病组大鼠视网膜内NF-κB表达高于正常组,与糖尿病组比较,石斛多糖各剂量组NF-κB表达降低。结论石斛多糖可以降低糖尿病视网膜病变大鼠体内炎症因子(IL-6、TNF-α)的表达,进而抑制VEGF表达上调并改善糖尿病大鼠血-视网膜屏障(blood-retinal barrier,BRB)的通透性。其作用机制可能与NF-κB通路有关。
[Abstract]:Research purposes in recent years. More and more studies suggest that the mechanism of inflammation in diabetic retinopathy / diabetic retinopathy DRR. Play an indispensable role in the early development of. Anti-inflammatory therapy may provide a new idea for the development and treatment of prevention Dr. As one of the traditional and precious Chinese herbal medicines, Dendrobium is considered to have nourishing yin and tonifying the kidney. Polysaccharide is one of the main medicinal active components of Dendrobium, and its chemical composition and pharmacological activity have attracted more and more attention. Studies show that Dendrobium polysaccharide has anti-inflammatory and antioxidant properties. In this study, diabetic rats were selected as the research objects. To observe the relationship between polysaccharide of Dendrobium and blood-retinal barrier function of diabetic rats and its specific mechanism. (2) Diabetic group (Dendrobium Dendrobium L.) low dosage of Dendrobium Dendrobium polysaccharide. [100 mg / kg? Dendrobium Dendrobiae (Dendrobium Dendrobium Dendrobium). [200 mg / kg? High dose group of Dendrobium Dendrobium polysaccharides. [300 mg / kg? Rats were injected intraperitoneally with streptozotocinia STZ (55 mg / kg), and rats in normal group were given the same dose of sodium citrate solution. Monitoring blood glucose once a week. 48 hours blood glucose was higher than 16.7 mmol / L, the model was successful. 6 weeks after the model was successfully established, Dendrobium polysaccharide treatment groups were treated for 8 weeks by gastric perfusion for 8 weeks. 2% pentobarbital sodium (40 mg / kg) was used to anesthetize rats intraperitoneally. Western blot was used to detect interleukin-6 interleukin-6 (IL-6) in rat retina. Tumor necrosis factor- 伪 tumer factor-alpha (TNF- 伪). Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor (endothelial growth factor-VEGF-1) and tight junction protein family (ZO-1) in the retina. The protein expression level of Occludinia Claudin-5 was detected by enzyme-linked immunosorbent assay (Elisa). The content of IL-6 TNF- 伪 in serum and retina of rats was detected by ELISA.The expression of NF- 魏 B in rat retina was observed by immunohistochemistry. The function of blood-retina barrier in diabetic rats was analyzed by Evans blueblue staining. The results were compared with those in the normal group, and the results showed that IL-6 TNF- 伪 was found in the retina of diabetic rats. The protein expression of VEGF was increased, the expression level of ZO-1 Occludin-Occludin-5) was decreased, and the expression of IL-6 TNF- 伪 was increased. Compared with the diabetic group, Dendrobium polysaccharide was low, medium and high doses of IL-6 in the retina of rats. The protein expression of TNF- 伪 was significantly decreased, the expression of tight junction protein ZO-1 Occludinia Claudin-5 was increased, and the serum IL-6 was increased. The expression of TNF- 伪 was decreased, and the retinal osmotic capacity of Dendrobium polysaccharide group was significantly decreased. The difference was statistically significant (P 0.05). There was no significant difference in each index between different dose groups of Dendrobium officinale. The expression of NF- 魏 B in retina of diabetic group was higher than that of normal group, which was higher than that of diabetic group. Conclusion Dendrobium polysaccharide can reduce the expression of IL-6 TNF- 伪 in diabetic retinopathy rats. And then inhibit the up-regulation of VEGF expression and improve the blood-retinal barrier blood-retinal barrier in diabetic rats. The mechanism of BRBs permeability may be related to NF- 魏 B pathway.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R276.7

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