同型半胱氨酸对实验性结肠炎大鼠肠纤维化的影响及机制研究

发布时间：2019-04-18 17:36

【摘要】：背景：克罗恩病（Crohn’s disease, CD）是一种病因未明的慢性非特异性肠道肉芽肿性疾病，肠纤维性狭窄是CD常见并发症之一，目前尚无有效的治疗药物，手术是最终治疗手段，术后仍有约70%患者再次发生狭窄，，反复发生的肠狭窄严重影响患者的生活质量。同型半胱氨酸（homocysteine, Hcy）是一种含硫氨基酸，是甲硫氨酸去甲基化过程的中间产物，研究显示Hcy与心、肝、肾、肺等多种组织器官纤维化过程密切相关，多个研究发现在CD患者血浆和肠黏膜中Hcy水平明显增高，但对于Hcy是否参与CD肠纤维化的病理过程目前国内外尚无报道。因此，探讨Hcy对CD肠纤维化的影响及其可能机制对于CD肠纤维化狭窄的防治具有重要的理论和实际意义。 目的：探讨同型半胱氨酸（Hcy）对实验性结肠炎大鼠肠纤维化的影响及其可能机制。 方法：1.采用2,4,6-三硝酸苯磺酸(TNBS)灌肠制备大鼠CD模型。对照组以等体积的生理盐水（NS）灌肠；2.SD大鼠随机分为四组（n=8）：正常对照组（NS灌肠+皮下注射NS）、正常对照+Hcy注射组（NS灌肠+皮下注射Hcy）、TNBS模型对照组（TNBS/乙醇灌肠+皮下注射NS）、TNBS模型+Hcy注射组（TNBS/乙醇灌肠+皮下注射Hcy）；3.记录大鼠体重变化并进行疾病活动指数评分（DAI），实验结束后取部分结肠行大体损伤评分（CMDI），HE染色和Masson染色行结肠组织学损伤评分（HI）及纤维化评分；4.高效液相荧光检测法（HPLC-FD）检测大鼠血浆及结肠组织Hcy水平，结肠匀浆检测MDA含量及SOD、GSH-PX、MPO活性，酶联免疫法（ELISA）测定结肠匀浆IL-1β、IL-6、TNF-α、TGF-β1、CTGF、MMP-2、MMP-9、Ⅰ型和Ⅲ型胶原水平，逆转录聚合酶链反应（RT-PCR）检测TGF-β1、MMP-2、MMP-9和TIMP-1mRNA表达水平，免疫组化SP法检测结肠组织MMP-2和MMP-9蛋白表达水平。 结果：与正常对照组比较，TNBS结肠炎大鼠体重明显减轻，出现不同程度的肉眼血便，DAI评分增高（p0.05）；CMDI、HI及纤维化评分均显著增高。结肠组织MPO活性增高，SOD、GSH-PX活性降低，MDA、IL-1β、IL-6、TNF-α、TGF-β1、CTGF、MMP-2、MMP-9、Ⅰ型和Ⅲ型胶原水平增高，MMP-2和MMP-9蛋白表达增强，TGF-β1、MMP-2、MMP-9和TIMP-1mRNA表达增强（p0.05）。与TNBS模型对照组比较，皮下注射Hcy的结肠炎大鼠血浆及结肠组织Hcy水平明显增高，DAI、CMDI、HI及纤维化评分增高，结肠组织SOD、GSH-PX活性降低，MPO活性、MDA、IL-1β、IL-6、TNF-α、TGF-β1、CTGF、MMP-2、MMP-9、Ⅰ型和Ⅲ型胶原水平均明显增高（p0.05）。 结论：Hcy可加重实验性结肠炎大鼠肠纤维化，机制可能与增加结肠氧化及炎症损伤、促进TGF-β1、CTGF等促纤维化因子表达以及通过影响MMPs/TIMPs平衡导致细胞外基质代谢失衡有关。
[Abstract]:Background: Crohn's disease (Crohn's disease, CD) is a chronic nonspecific intestinal granulomatous disease with unknown etiology. Intestinal fibrous stenosis is one of the common complications of CD. About 70% of the patients suffered from recurrent stricture, and recurrent intestinal stenosis seriously affected the quality of life of the patients. Homocysteine (homocysteine, Hcy) is a sulfur-containing amino acid, which is the intermediate product of methionine demethylation. Studies have shown that Hcy is closely related to fibrosis of heart, liver, kidney, lung and other tissues and organs. Many studies have found that the level of Hcy in plasma and intestinal mucosa of patients with CD is significantly increased, but there is no report on whether Hcy is involved in the pathological process of CD intestinal fibrosis at home and abroad. Therefore, it is of great theoretical and practical significance to explore the effect of Hcy on intestinal fibrosis in CD and its possible mechanism for the prevention and treatment of intestinal fibrosis stenosis in CD. Objective: to investigate the effect of homocysteine (Hcy) on intestinal fibrosis in rats with experimental colitis and its possible mechanism. Methods: 1. The CD model of rats was established by (TNBS) enema with 2,4, 6-trinitrate benzenesulfonic acid. The control group was enema with the same volume of saline (NS). 2.SD rats were randomly divided into four groups (n = 8): normal control group (NS enema subcutaneously injected NS), normal control Hcy injection group; NS enema subcutaneously injected Hcy), TNBS model control group; TNBS/ ethanol enema subcutaneously injected NS),). TNBS model Hcy injection group (TNBS/ ethanol enema subcutaneously injected Hcy); 3. The body weight of the rats was recorded and the disease activity index score (DAI),) was carried out. After the end of the (DAI), experiment, partial colon was taken for gross injury score (CMDI), HE staining and Masson staining for colon histology injury score (HI) and fibrosis score; 4. High performance liquid phase fluorescence assay (HPLC-FD) was used to detect the level of Hcy in plasma and colon tissue of rats. The content of MDA and the activity of SOD,GSH-PX,MPO in colonic homogenate were measured. The levels of IL-1 尾 and IL-6,TNF- 伪 in colonic homogenate were determined by enzyme linked immunosorbent assay (ELISA). The levels of TGF- 尾 1, CTGF,MMP-2,MMP-9, type 鈪

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