吴茱萸碱对脂多糖诱导的ARDS大鼠的保护作用及机制研究
发布时间：2018-11-04 21:30
【摘要】：目的:探讨吴茱萸碱(Evodiamine,EVO)对脂多糖(lipopolysaccharide,LPS)诱导的急性呼吸窘迫综合征(ARDS)大鼠的保护作用及分子机制。方法:1.按照随机数字表法将72只成年雄性SD大鼠随机分为正常对照组(气管滴注生理盐水)、EVO单独处理组(气管滴注1mg/kg EVO)、LPS模型组(气管滴注3mg/kg LPS)和EVO治疗组(气管滴注1mg/kg EVO和3mg/kg LPS),每组18只,通过气管注射LPS(3mg/kg)建立ARDS模型,于建模6h后处死大鼠并收集各组动脉血、肺组织和肺泡灌洗液,通过动脉血气分析、肺湿/干重比值、苏木精-伊红染色(HE染色)观察各组大鼠肺组织的损伤情况;利用酶联免疫吸附试验(ELISA)和实时定量反转录-聚合酶链反应(q PCR)的方法检测肺组织和肺泡灌洗液(BALF)中炎症介质(TNF-α、IL-1b、HMGB1、IL-6)分泌水平;使用Western Blot方法测量各组肺组织中NF-kB信号通路相关蛋白(p50、p65)的表达。2.体外培养巨噬细胞系RAW264.7细胞,细胞经LPS(10μg/ml)单独刺激或LPS(10μg/ml)与EVO(50μM)联合作用后,于不同时间点收集各组细胞的培养上清及细胞团块。通过ELISA法检测细胞上清中炎症介质(TNF-α、IL-1β、HMGB1、IL-6)的含量及细胞核中NF-k B p65 DNA的结合活性;利用Western Blot检测各组细胞核内NF-κB家族蛋白(p50、p65)的表达。结果:1.LPS刺激6h后,大鼠精神不振、蜷缩少动、呼吸急促,进食减少,但EVO治疗组中大鼠的精神和活动状态较LPS模型组有明显好转。与LPS模型组相比较,EVO治疗后可明显减轻LPS所导致的肺水肿和组织病理学损伤,改善氧合功能,同时降低肺组织和BALF中炎症介质(TNF-α、IL-1β、HMGB1、IL-6)的水平,Western Blot结果显示EVO对LPS作用下肺组织胞核中NF-kBp65和p50的高表达具有明显的下调作用。2.体外实验中,LPS活化的RAW264.7细胞经EVO干预后,上清液中炎症介质(TNF-α、IL-1β、HMGB1、IL-6)含量显著降低,细胞核内NF-kB p50和p65表达显著减少,同时NF-κB p65的DNA结合活性明显受抑制。结论:1.EVO可有效减轻LPS诱导的ARDS大鼠肺组织损伤。2.EVO可显著减少LPS刺激下炎性巨噬细胞中炎症介质的分泌,该作用可能是通过抑制NF-κB p65和p50的入核及其DNA结合活性来实现的。
[Abstract]:Aim: to investigate the protective effect and molecular mechanism of rutaecarpine (Evodiamine,EVO) on lipopolysaccharide (lipopolysaccharide,LPS) -induced acute respiratory distress syndrome (ARDS) rats. Methods: 1. Seventy-two adult male SD rats were randomly divided into two groups according to random number table: normal control group (treated with normal saline), EVO alone) (1mg/kg EVO), by trachea drip). The ARDS model was established in LPS model group (3mg/kg LPS) and EVO treatment group (18 rats in each group) by LPS (3mg/kg) injection. The rats were killed and arterial blood was collected 6 hours after the model was established. Lung tissue and alveolar lavage fluid were analyzed by arterial blood gas analysis, lung wet / dry weight ratio and hematoxylin eosin staining (HE staining). Enzyme linked immunosorbent assay (ELISA) and real-time quantitative reverse transcription-polymerase chain reaction (q PCR) were used to detect the level of TNF- 伪 (IL-1b,HMGB1,IL-6) in lung tissue and alveolar lavage fluid (BALF). The expression of NF-kB signaling pathway related protein (p50 p65) in lung tissues was measured by Western Blot method. 2. Macrophage cell line RAW264.7 cells were cultured in vitro. After stimulated by LPS (10 渭 g/ml) alone or combined with LPS (10 渭 g/ml) and EVO (50 渭 M), the supernatant and cell mass were collected at different time points. The content of TNF- 伪, IL-1 尾, HMGB1,IL-6 in the supernatant and the binding activity of NF-k B p65 DNA in the cell nucleus were detected by ELISA assay. The expression of NF- 魏 B family protein (p50 p65) was detected by Western Blot. Results: after 6 hours of 1.LPS stimulation, the rats suffered from mental retardation, curling, shortness of breath and decreased food intake. However, the mental and activity status of the rats in the EVO treatment group was significantly better than that in the LPS model group. Compared with LPS model group, EVO could significantly reduce pulmonary edema and histopathological injury induced by LPS, improve oxygenation function, and decrease the level of TNF- 伪, IL-1 尾, HMGB1,IL-6 in lung tissue and BALF. Western Blot results showed that EVO significantly down-regulated the expression of NF-kBp65 and p50 in lung nuclei induced by LPS. 2. In vitro, the content of TNF- 伪, IL-1 尾, HMGB1,IL-6 in supernatant of RAW264.7 cells activated by LPS decreased significantly, and the expression of NF-kB p50 and p65 in nucleus decreased significantly after EVO intervention. At the same time, the DNA binding activity of NF- 魏 B p65 was significantly inhibited. Conclusion: 1.EVO can effectively attenuate the lung injury induced by LPS in ARDS rats, and 2.EVO can significantly reduce the secretion of inflammatory mediators in inflammatory macrophages stimulated by LPS. This effect may be achieved by inhibiting the nucleation and DNA binding activity of NF- 魏 B p65 and p50.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8
[Abstract]:Aim: to investigate the protective effect and molecular mechanism of rutaecarpine (Evodiamine,EVO) on lipopolysaccharide (lipopolysaccharide,LPS) -induced acute respiratory distress syndrome (ARDS) rats. Methods: 1. Seventy-two adult male SD rats were randomly divided into two groups according to random number table: normal control group (treated with normal saline), EVO alone) (1mg/kg EVO), by trachea drip). The ARDS model was established in LPS model group (3mg/kg LPS) and EVO treatment group (18 rats in each group) by LPS (3mg/kg) injection. The rats were killed and arterial blood was collected 6 hours after the model was established. Lung tissue and alveolar lavage fluid were analyzed by arterial blood gas analysis, lung wet / dry weight ratio and hematoxylin eosin staining (HE staining). Enzyme linked immunosorbent assay (ELISA) and real-time quantitative reverse transcription-polymerase chain reaction (q PCR) were used to detect the level of TNF- 伪 (IL-1b,HMGB1,IL-6) in lung tissue and alveolar lavage fluid (BALF). The expression of NF-kB signaling pathway related protein (p50 p65) in lung tissues was measured by Western Blot method. 2. Macrophage cell line RAW264.7 cells were cultured in vitro. After stimulated by LPS (10 渭 g/ml) alone or combined with LPS (10 渭 g/ml) and EVO (50 渭 M), the supernatant and cell mass were collected at different time points. The content of TNF- 伪, IL-1 尾, HMGB1,IL-6 in the supernatant and the binding activity of NF-k B p65 DNA in the cell nucleus were detected by ELISA assay. The expression of NF- 魏 B family protein (p50 p65) was detected by Western Blot. Results: after 6 hours of 1.LPS stimulation, the rats suffered from mental retardation, curling, shortness of breath and decreased food intake. However, the mental and activity status of the rats in the EVO treatment group was significantly better than that in the LPS model group. Compared with LPS model group, EVO could significantly reduce pulmonary edema and histopathological injury induced by LPS, improve oxygenation function, and decrease the level of TNF- 伪, IL-1 尾, HMGB1,IL-6 in lung tissue and BALF. Western Blot results showed that EVO significantly down-regulated the expression of NF-kBp65 and p50 in lung nuclei induced by LPS. 2. In vitro, the content of TNF- 伪, IL-1 尾, HMGB1,IL-6 in supernatant of RAW264.7 cells activated by LPS decreased significantly, and the expression of NF-kB p50 and p65 in nucleus decreased significantly after EVO intervention. At the same time, the DNA binding activity of NF- 魏 B p65 was significantly inhibited. Conclusion: 1.EVO can effectively attenuate the lung injury induced by LPS in ARDS rats, and 2.EVO can significantly reduce the secretion of inflammatory mediators in inflammatory macrophages stimulated by LPS. This effect may be achieved by inhibiting the nucleation and DNA binding activity of NF- 魏 B p65 and p50.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8
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