紫檀芪对百草枯诱导肺纤维化的保护作用及机制
发布时间：2019-06-10 19:03
【摘要】：研究背景百草枯(paraquat, PQ)中毒可导致肺部损伤,该过程可明确划分为两个阶段：急性肺损伤阶段与肺纤维化阶段。PQ通过诱导氧化应激、炎症以及纤维反应产生毒性。PQ中毒主要机制是氧化还原反应和活性氧(reactive oxygen species, ROS)的产生。 Sirt1(sirtuin type1)是一种重要的细胞生存蛋白,与氧化应激有关。ROS水平增加可抑制Sirtl活性,这是因为ROS能够氧化修饰Sirt1半胱氨酸残基。Sirt1的过表达能够诱导抗氧化基因表达,进而起到抗氧化应激作用。 紫檀芪(pterostilbene, PTS)在葡萄、蓝莓和紫檀木中被发现,具有止痛、抗衰老、抗癌、抗炎、抗氧化和抗增殖的药理作用。 本实验分为两个部分：第一部分旨在研究Sirt1是否与PQ诱导的急性肺损伤与肺纤维化有关；第二部分旨在研究紫檀芪能否通过抑制ROS的产生和调节Sirt1的表达而对PQ诱导的肺部纤维化起到保护作用。 方法第一部分实验：腹腔注射PQ30mg/kg,构建C57BL/6小鼠(5-8周龄)急性肺损伤模型,实验分为正常对照组和PQ组。单次注射PQ8h后麻醉动物进行支气管肺泡灌洗,取支气管肺泡灌洗液(BALF)检测灌洗液中细胞总数、总蛋白浓度,采用ELISA试剂盒检测BALF中IL-6与TNF-α水平；取肺组织,HE染色观察组织形态学的变化,DHE染色检测ROS水平,TUNEL染色与Caspase-3活性检测细胞凋亡状况,Western Blot检测组织中Sirt1的蛋白表达。腹腔注射PQ20mg/kg,构建C57BL/6小鼠(5-8周龄)肺纤维化模型,实验分为正常对照组和PQ组。单次注射21天后造模结束,处理方法及检测指标同上,并做Masson染色检测胶原沉积。 第二部分实验：紫檀芪干预实验分为正常对照组、模型组、溶媒组、紫檀芪低剂量组(PQ+PTS20mg/kg)、紫檀芪高剂量组(PQ+PTS40mg/kg)和紫檀芪组(PTS40mg/kg)。单次腹腔注射PQ20mg/kg,随后每日进行紫檀芪灌胃,连续灌胃21天,麻醉动物进行支气管肺泡灌洗并取肺组织,方法及检测指标与肺纤维化模型相同。 结果 第一部分实验结果： 1.与正常对照组相比,急性肺损伤小鼠肺组织中出现肺泡间隔增厚、肺泡断裂和炎性细胞浸润；肺纤维化小鼠肺组织中无功能的肺纤维组织逐渐代替正常的肺泡组织,肺泡出现萎陷、肺泡间隔增厚、炎性细胞浸润。Masson染色结果显示肺纤维化肺组织出现胶原沉积。 2.与正常对照组相比,急性肺损伤与肺纤维化小鼠BALF中细胞总数增多,总蛋白浓度增加,IL-6与TNF-α水平升高。 3.与正常对照组相比,急性肺损伤与肺纤维化小鼠肺组织Caspase-3活性显著增强,TUNEL染色阳性细胞数增多。 4.与正常对照组相比,急性肺损伤与肺纤维化小鼠肺组织ROS水平升高。 5.与正常对照组相比,急性肺损伤与肺纤维化小鼠肺组织Sirt1蛋白表达降低。 6.与正常对照组相比,急性肺损伤与肺纤维化小鼠BALF中IL-6与TNF-α水平升高。 第二部分实验结果： 1.与PQ组相比,高剂量紫檀芪能够减轻PQ对肺泡结构的损伤,同时也减少胶原沉积。 2.与PQ组相比,高剂量紫檀芪能显著减少BALF中细胞总数和总蛋白浓度。 3.与PQ组相比,高剂量紫檀芪可抑制肺组织Caspase-3活性,同时可使肺组织TUNEL染色阳性细胞数减少。 4.与PQ组相比,高剂量紫檀芪可降低肺组织ROS水平。 5.与PQ组相比,高剂量紫檀芪能够上调肺组织Sirt1蛋白表达。 6.与PQ组相比,高剂量紫檀芪能显著降低BALF中IL-6与TNF-α水平。 结论PQ所介导的Sirt1蛋白表达下调以及ROS产生可能是其诱导小鼠产生急性肺损伤与肺纤维化的重要原因。紫檀芪可通过上调Sirt1的表达以及抑制活性氧产生和炎症反应,从而减轻PQ诱导肺纤维化。
[Abstract]:The study of paraquat (PQ) poisoning can lead to lung injury, which can be clearly divided into two stages: the stage of acute lung injury and the stage of pulmonary fibrosis. PQ is toxic by inducing oxidative stress, inflammation, and fiber response. The main mechanism of PQ poisoning is the production of redox and reactive oxygen species (ROS). Sirt1 (sirtuin type1) is an important cell survival protein, which is associated with oxidative stress. the increase in the level of ROS can inhibit the activity of Sirtl, since ROS can be oxidized to modify the Sirt1 cysteine residue The overexpression of the base. Sirt1 can induce the expression of the anti-oxidation gene and further act as an anti-oxidative stress. It has effects in relieving pain, resisting aging, resisting cancer, resisting inflammation, resisting oxidation, and resisting proliferation. The first part is to study whether Sirt1 is related to the acute lung injury induced by PQ and the pulmonary fibrosis. The second part is to study whether the red sandalwood can inhibit the production of ROS and regulate the expression of Sirt1 to cause the pulmonary fibrosis induced by the PQ. The first part of the method was to construct the model of acute lung injury in C57BL/6 mice (5-8 weeks of age) by injecting PQ30mg/ kg in the abdominal cavity. In the control group and PQ group, the animals were anesthetized with bronchoalveolar lavage after a single injection of PQ8h, the total cell count and total protein concentration in the lavage fluid were detected by the bronchoalveolar lavage fluid (BALF), and the level of IL-6 and TNF-1 in BALF was detected by the ELISA kit; and the lung tissue and the HE staining observation group were taken. The changes of the weaving morphology, the detection of ROS level by DHE staining, the TUNEL staining and the activity of Caspase-3 in the detection of cell apoptosis, and the detection of Sir in the tissue by Western Blot The expression of the protein of t1 was given by intraperitoneal injection of PQ20mg/ kg, and the model of pulmonary fibrosis in C57BL/6 mice (5-8 weeks of age) was constructed. The control group and the PQ group, after a single injection for 21 days, the model was finished, the treatment method and the detection index were the same as above, and the Masson staining was performed. Color detection of collagen deposition. Part 2: The intervention of the red sandalwood was divided into the normal control group, the model group, the vehicle group, the lower dose group of the red sandalwood group (PQ + PTS20mg/ kg), the high dose group (PQ + PTS40mg/ kg) and the red sandalwood group (PT S40mg/ kg. After a single intraperitoneal injection of PQ20mg/ kg, a daily dose of Pterocarpus santalensis was given intragastric administration for 21 days, and the anesthetic animals were given bronchoalveolar lavage and the lung tissue, method and detection index were taken. with the lung The fibrosis model is the same. Results:1. The experimental results of the first part:1. In the lung tissue of the mice with acute lung injury, the alveolar space thickening, the alveolar rupture and the infiltration of the inflammatory cells in the lung tissue of the mice with acute lung injury were compared with the normal control group, and the lung tissue of the pulmonary fibrosis mice had no function in the lung. The fibrous tissue gradually replaces the normal alveolar tissue, and the alveoli are withering. Depression, alveolar septum thickening, inflammatory cell infiltration. Masson staining results The total number of cells in the BALF of the mice with acute lung injury and pulmonary fibrosis increased, the total protein concentration was increased, compared with the normal control group. Increased levels of IL-6 and TNF-3.3. The activity of Caspase-3 in lung tissue of mice with acute lung injury and pulmonary fibrosis compared with the normal control group. 4. Compared with the normal control group, the number of TUNEL-stained positive cells increased significantly. The level of ROS increased in the lung of mice with lung injury and pulmonary fibrosis. The expression of Sirt1 protein in lung tissue of mice with lung fibrosis and pulmonary fibrosis decreased.6. Acute lung injury was associated with normal control. Pulmonary fibrosis mouse BAL The level of IL-6 and TNF-1 in F increased. The second part of the experiment results:1. Compared with the PQ group, high The dose of red sandalwood can reduce the damage of PQ to the alveolar structure, and also reduce the deposition of collagen. Compared with the Q group, the high-dose rosewood could significantly reduce the total number of cells in BALF and the total protein concentration. The activity of Caspase-3 can be made by TUNEL staining of lung tissue. Decrease in number of sex cells.4. High dose of rosewood could reduce lung tissue RO as compared to the PQ group. S-level.5. The high-dose rosewood was able to upregulate the expression of Sirt1 protein in lung tissue compared with the PQ group. 6. Compared with the PQ group, the high-dose rosewood could significantly lower the level of IL-6 and TNF-1 in BALF. The downregulation of rt1 protein and the production of ROS may be an important cause of the induction of acute lung injury and pulmonary fibrosis in mice.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285.5
本文编号：2496666
[Abstract]:The study of paraquat (PQ) poisoning can lead to lung injury, which can be clearly divided into two stages: the stage of acute lung injury and the stage of pulmonary fibrosis. PQ is toxic by inducing oxidative stress, inflammation, and fiber response. The main mechanism of PQ poisoning is the production of redox and reactive oxygen species (ROS). Sirt1 (sirtuin type1) is an important cell survival protein, which is associated with oxidative stress. the increase in the level of ROS can inhibit the activity of Sirtl, since ROS can be oxidized to modify the Sirt1 cysteine residue The overexpression of the base. Sirt1 can induce the expression of the anti-oxidation gene and further act as an anti-oxidative stress. It has effects in relieving pain, resisting aging, resisting cancer, resisting inflammation, resisting oxidation, and resisting proliferation. The first part is to study whether Sirt1 is related to the acute lung injury induced by PQ and the pulmonary fibrosis. The second part is to study whether the red sandalwood can inhibit the production of ROS and regulate the expression of Sirt1 to cause the pulmonary fibrosis induced by the PQ. The first part of the method was to construct the model of acute lung injury in C57BL/6 mice (5-8 weeks of age) by injecting PQ30mg/ kg in the abdominal cavity. In the control group and PQ group, the animals were anesthetized with bronchoalveolar lavage after a single injection of PQ8h, the total cell count and total protein concentration in the lavage fluid were detected by the bronchoalveolar lavage fluid (BALF), and the level of IL-6 and TNF-1 in BALF was detected by the ELISA kit; and the lung tissue and the HE staining observation group were taken. The changes of the weaving morphology, the detection of ROS level by DHE staining, the TUNEL staining and the activity of Caspase-3 in the detection of cell apoptosis, and the detection of Sir in the tissue by Western Blot The expression of the protein of t1 was given by intraperitoneal injection of PQ20mg/ kg, and the model of pulmonary fibrosis in C57BL/6 mice (5-8 weeks of age) was constructed. The control group and the PQ group, after a single injection for 21 days, the model was finished, the treatment method and the detection index were the same as above, and the Masson staining was performed. Color detection of collagen deposition. Part 2: The intervention of the red sandalwood was divided into the normal control group, the model group, the vehicle group, the lower dose group of the red sandalwood group (PQ + PTS20mg/ kg), the high dose group (PQ + PTS40mg/ kg) and the red sandalwood group (PT S40mg/ kg. After a single intraperitoneal injection of PQ20mg/ kg, a daily dose of Pterocarpus santalensis was given intragastric administration for 21 days, and the anesthetic animals were given bronchoalveolar lavage and the lung tissue, method and detection index were taken. with the lung The fibrosis model is the same. Results:1. The experimental results of the first part:1. In the lung tissue of the mice with acute lung injury, the alveolar space thickening, the alveolar rupture and the infiltration of the inflammatory cells in the lung tissue of the mice with acute lung injury were compared with the normal control group, and the lung tissue of the pulmonary fibrosis mice had no function in the lung. The fibrous tissue gradually replaces the normal alveolar tissue, and the alveoli are withering. Depression, alveolar septum thickening, inflammatory cell infiltration. Masson staining results The total number of cells in the BALF of the mice with acute lung injury and pulmonary fibrosis increased, the total protein concentration was increased, compared with the normal control group. Increased levels of IL-6 and TNF-3.3. The activity of Caspase-3 in lung tissue of mice with acute lung injury and pulmonary fibrosis compared with the normal control group. 4. Compared with the normal control group, the number of TUNEL-stained positive cells increased significantly. The level of ROS increased in the lung of mice with lung injury and pulmonary fibrosis. The expression of Sirt1 protein in lung tissue of mice with lung fibrosis and pulmonary fibrosis decreased.6. Acute lung injury was associated with normal control. Pulmonary fibrosis mouse BAL The level of IL-6 and TNF-1 in F increased. The second part of the experiment results:1. Compared with the PQ group, high The dose of red sandalwood can reduce the damage of PQ to the alveolar structure, and also reduce the deposition of collagen. Compared with the Q group, the high-dose rosewood could significantly reduce the total number of cells in BALF and the total protein concentration. The activity of Caspase-3 can be made by TUNEL staining of lung tissue. Decrease in number of sex cells.4. High dose of rosewood could reduce lung tissue RO as compared to the PQ group. S-level.5. The high-dose rosewood was able to upregulate the expression of Sirt1 protein in lung tissue compared with the PQ group. 6. Compared with the PQ group, the high-dose rosewood could significantly lower the level of IL-6 and TNF-1 in BALF. The downregulation of rt1 protein and the production of ROS may be an important cause of the induction of acute lung injury and pulmonary fibrosis in mice.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285.5
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