葡萄籽提取物对博莱霉素诱导的小鼠肺纤维化的影响

发布时间：2018-01-15 12:16

本文关键词：葡萄籽提取物对博莱霉素诱导的小鼠肺纤维化的影响　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:肺纤维化是一种间质性肺部疾病,常由各种呼吸系统致病因素导致,它包括特发性肺纤维化,慢性阻塞性肺部疾病导致的气道肺纤维化和哮喘气道重塑。肺纤维化患者在确诊后一般仅有3-5年的存活期,并且会伴有一系列并发症。但目前对于肺纤维化的治疗及预后缺乏特效药,寻找有效治疗肺纤维化的理想药物已成为医药领域的重要研究课题。近年越来越多的研究表明,葡萄籽提取物具有较高的营养与药效价值。尤为葡萄籽提取物所含原花青素(Procyanidins)、白藜芦醇(Resveratrol)、脂类等成分具有预防治疗癌症、清除多余的自由基,保护心血管系统等作用,不失为一种来源于天然植物具有开发前景的保健品或药物。在我国,葡萄是大面积种植、食用且营养价值极其丰富的高产水果,因而葡萄籽的开发利用具有广阔的应用市场及前景,以及重大的社会学、经济学意义。本研究侧重探索葡萄籽提取物对博莱霉素诱导的小鼠肺纤维化的改善作用,并初步探讨其防治肺纤维化的作用机制。方法:第一部分:ICR小鼠50只随机分为5组,正常对照组,模型组,GSE50mg/kg、GSE100 mg/kg组,阳性对照(DXM 0.5 mg/kg)组。实验时,小鼠腹腔注射3%戊巴比妥钠(10 mL/kg),麻醉后仰位固定于试验台上,切开颈部皮肤,暴露气管,用注射器穿刺气管滴入BLM(2 mg/kg),再缝合颈部皮肤。空白对照组用等容量的生理盐水在小鼠气管内滴注。造模第二天分别按组进行药物干预,受试品溶液按0.2 mL/20g容量灌胃给予,正常组和模型组均给予等量溶媒,连续21天,给药过程定期记录小鼠体重。实验结束后采用Buxco动物肺功能分析系统测定小鼠肺功能。实验结束处死小鼠,结扎左肺,灌洗右肺肺泡,收集肺泡灌洗液(Bronchoalveolar lavage fluid,BALF),取上清液计数其中的白细胞总数、检测TGF-β1与LDH;沉淀用于计数巨噬细胞数、淋巴细胞数、中性粒细胞分类计数。取小鼠左肺肺组织制作组织切片,行HE染色,光学显微镜下观测肺组织形态。第二部分:ICR小鼠50只随机分为5组,正常对照组,模型组,GSE 50 mg/kg、GSE 100 mg/kg组,阳性对照(DXM 0.5 mg/kg)组。小鼠腹腔内注射3%戊巴比妥钠(10 mL/kg),麻醉后仰位固定于试验台上,注射器穿刺气管滴入BLM(2 mg/kg),再缝合颈部皮肤。空白对照组以等容量的生理盐水进行气管内滴注。造模第二天分别按组进行干预,受试品溶液按0.2 mL/20g容量灌胃给予,正常组和模型组均给予等量溶媒,持续进行21天。结束后杀死小鼠,结扎左肺,灌洗右肺肺泡,收集肺泡灌洗液(Bronchoalveolar lavage fluid,BALF)上清液用于检测TGF-β1含量。取小鼠部分左肺肺组织制作组织切片,行Masson染色和免疫组化,在光学显微镜下进行观测。另一部分左肺用于检测羟脯氨酸(Hydroxyproline,HYP)含量,并制备组织匀浆用实时定量PCR(qRT-PCR)法检测EMT相关因子。结果:第一部分:与模型组相比,灌胃GSE可显著改善BLM导致的肺纤维化小鼠肺功能Penh值升高,显著减少BALF中白细胞总数,中性粒细胞和巨噬细胞的增加。下调肺组织中IL-6、IL-1β mRNA高表达,缓解BLM诱发的肺泡结构破坏与肺组织实质病变减少肺泡灌洗液中LDH含量。第二部分:灌胃GSE可显著下调肺组织中MMP-9与TIMP-1 mRNA和蛋白高表达,减少BLM诱导的肺组织中的胶原纤维沉积及羟脯氨酸的水平,减少肺泡灌洗液中TGF-β1含量。GSE可通过抑制p38通路的激活调节纤维化相关因子E-cadherin、FN1与COL1A1 mRNA和蛋白表达,上调E-cadherin表达,下调FN1与COL1A1表达,从而改善肺纤维化程度。结论:经研究发现口服GSE不仅显著降低BLM诱导的肺部炎症细胞聚集和浸润,改善肺功能,并且能够显著抑制纤维化相关因子,如羟脯氨酸,TGF-β1,MMP-9,COL1A1和FN1的表达。GSE作为一种天然抗氧化剂,能够通过p38信号通路抑制EMT和细胞外基质的沉积,显著改善由博莱霉素导致的小鼠肺功能下降与肺组织损伤。
[Abstract]:Objective: pulmonary fibrosis is a kind of interstitial lung disease, often by a variety of respiratory pathogenic factors, including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease caused by airway pulmonary fibrosis and airway remodeling. The survival of patients with pulmonary fibrosis only 3-5 years after diagnosis, and will be accompanied by a series of complications. But the treatment and prognosis of pulmonary fibrosis and the lack of medicine, looking for the ideal drugs are effective in the treatment of pulmonary fibrosis has become an important research topic in the field of medicine. In recent years, more and more studies show that the nutrition and efficacy value of grape seed extract has high. Especially the grape seed extract containing proanthocyanidins (Procyanidins), white resveratrol (Resveratrol), lipid can prevent cancer, remove excess free radicals, protects the cardiovascular system, it is a source of natural plant Health care products or drugs with potential development. In our country, the grape is planted in large area, high yield and nutritional value of edible fruit is extremely rich, so the development and utilization of grape seeds with market and broad prospects, and major in sociology, economics significance. This study tries to improve the effect of grape seed extract on bleomycin induced pulmonary fibrosis in mice, and to explore its mechanism of prevention and treatment of pulmonary fibrosis. Methods: the first part: 50 ICR mice were randomly divided into 5 groups, normal control group, model group, GSE50mg/kg, GSE100, mg/kg group, positive control group (DXM 0.5 mg/kg). During the experiment, intraperitoneal injection of pentobarbital sodium in mice 3% (10 mL/kg), after anesthesia supine fixed on the test bench, the neck skin incision, exposing the trachea, tracheal instillation of BLM puncture syringe (2 mg/kg), and then suture the skin of the neck. The control group treated with normal saline In mice by intratracheal instillation. Rats in second days respectively by group intervention, by the test solution with 0.2 mL/20g capacity gavage, the normal group and the model group were given the same amount of solvent, for 21 consecutive days, the administration process regularly recorded in body weight. After the experiment using Buxco animal lung function analysis system for determination of lung the function of mice. The end of the experiment, mice were sacrificed, ligation of the left lung, right pulmonary alveolar lavage, bronchoalveolar lavage fluid (Bronchoalveolar lavage, fluid, BALF), the total number of white blood cell count in the supernatant, detection of TGF- beta 1 and LDH; precipitation is used to count the number of macrophages, lymphocyte count, neutrophil count from lung tissue. Left mouse tissue sections were made for HE staining of lung tissue morphology observation under optical microscope. The second part: 50 ICR mice were randomly divided into 5 groups, normal control group, model group, GSE 50 mg/kg, GSE 100 mg/kg group, positive control group (D XM 0.5 mg/kg) group. Mice received intraperitoneal injection of 3% pentobarbital sodium (10 mL/kg), after anesthesia supine fixed on the test bench, syringe needle tracheal instillation of BLM (2 mg/kg), and then suture the skin of the neck. The saline group with the equal volume of blank control of intratracheal instillation of second days of modeling. According to the group intervention by test solution with 0.2 mL/20g capacity gavage, the normal group and the model group were given the same amount of solvent, lasting for 21 days. After killing the mice, ligation of the left lung, right pulmonary alveolar lavage, bronchoalveolar lavage fluid (Bronchoalveolar lavage, fluid, BALF) was used to detect TGF- beta 1 the content of the mice. Part of left lung tissue and tissue sections were made for Masson staining and immunohistochemistry, in optical microscope observation. The other part of the left lung for the detection of hydroxyproline (Hydroxyproline, HYP) and the content of tissue homogenate was prepared by real-time quantitative PCR (qRT- PCR) assay for detection of EMT associated factors. Results: the first part: compared with the model group, intragastric administration of GSE pulmonary function in mice with pulmonary fibrosis caused by Penh significantly improved BLM value increased, BALF significantly reduced the total number of white blood cells, increased neutrophils and macrophages. The down-regulation of IL-6 in lung tissue, high expression of IL-1 beta mRNA. To alleviate the damage of alveolar structure and lesions of lung tissue induced by BLM decreased LDH content in bronchoalveolar lavage fluid. The second part: the intragastric administration of GSE could significantly decrease the MMP-9 and TIMP-1 mRNA and protein expression in lung tissue, decreased collagen deposition and hydroxyproline induced by BLM in lung tissue level, reduce alveolar lavage fluid in TGF- beta 1 the content of.GSE can inhibit the activation of p38 pathway regulating fibrosis related factors E-cadherin, FN1 and COL1A1 mRNA and protein expression, upregulating the expression of E-cadherin and down-regulation of FN1 COL1A1 expression, so as to improve the degree of pulmonary fibrosis. Theory: the study found that oral administration of GSE not only significantly reduce the pulmonary inflammatory cells of BLM induced aggregation and infiltration, improve lung function, and can significantly inhibit the fibrosis related factors, such as hydroxyproline, TGF- beta 1, MMP-9,.GSE and FN1 COL1A1 expression as a natural antioxidant, can inhibit the deposition of extracellular matrix by EMT and p38 the signal pathway, significantly improved by bleomycin induced mice reduced lung function and lung tissue damage.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

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