紫檀芪對(duì)百草枯誘導(dǎo)肺纖維化的保護(hù)作用及機(jī)制
發(fā)布時(shí)間：2019-06-10 19:03
【摘要】：研究背景百草枯(paraquat, PQ)中毒可導(dǎo)致肺部損傷,該過程可明確劃分為兩個(gè)階段：急性肺損傷階段與肺纖維化階段。PQ通過誘導(dǎo)氧化應(yīng)激、炎癥以及纖維反應(yīng)產(chǎn)生毒性。PQ中毒主要機(jī)制是氧化還原反應(yīng)和活性氧(reactive oxygen species, ROS)的產(chǎn)生。 Sirt1(sirtuin type1)是一種重要的細(xì)胞生存蛋白,與氧化應(yīng)激有關(guān)。ROS水平增加可抑制Sirtl活性,這是因?yàn)镽OS能夠氧化修飾Sirt1半胱氨酸殘基。Sirt1的過表達(dá)能夠誘導(dǎo)抗氧化基因表達(dá),進(jìn)而起到抗氧化應(yīng)激作用。 紫檀芪(pterostilbene, PTS)在葡萄、藍(lán)莓和紫檀木中被發(fā)現(xiàn),具有止痛、抗衰老、抗癌、抗炎、抗氧化和抗增殖的藥理作用。 本實(shí)驗(yàn)分為兩個(gè)部分：第一部分旨在研究Sirt1是否與PQ誘導(dǎo)的急性肺損傷與肺纖維化有關(guān)；第二部分旨在研究紫檀芪能否通過抑制ROS的產(chǎn)生和調(diào)節(jié)Sirt1的表達(dá)而對(duì)PQ誘導(dǎo)的肺部纖維化起到保護(hù)作用。 方法第一部分實(shí)驗(yàn)：腹腔注射PQ30mg/kg,構(gòu)建C57BL/6小鼠(5-8周齡)急性肺損傷模型,實(shí)驗(yàn)分為正常對(duì)照組和PQ組。單次注射PQ8h后麻醉動(dòng)物進(jìn)行支氣管肺泡灌洗,取支氣管肺泡灌洗液(BALF)檢測灌洗液中細(xì)胞總數(shù)、總蛋白濃度,采用ELISA試劑盒檢測BALF中IL-6與TNF-α水平；取肺組織,HE染色觀察組織形態(tài)學(xué)的變化,DHE染色檢測ROS水平,TUNEL染色與Caspase-3活性檢測細(xì)胞凋亡狀況,Western Blot檢測組織中Sirt1的蛋白表達(dá)。腹腔注射PQ20mg/kg,構(gòu)建C57BL/6小鼠(5-8周齡)肺纖維化模型,實(shí)驗(yàn)分為正常對(duì)照組和PQ組。單次注射21天后造模結(jié)束,處理方法及檢測指標(biāo)同上,并做Masson染色檢測膠原沉積。 第二部分實(shí)驗(yàn)：紫檀芪干預(yù)實(shí)驗(yàn)分為正常對(duì)照組、模型組、溶媒組、紫檀芪低劑量組(PQ+PTS20mg/kg)、紫檀芪高劑量組(PQ+PTS40mg/kg)和紫檀芪組(PTS40mg/kg)。單次腹腔注射PQ20mg/kg,隨后每日進(jìn)行紫檀芪灌胃,連續(xù)灌胃21天,麻醉動(dòng)物進(jìn)行支氣管肺泡灌洗并取肺組織,方法及檢測指標(biāo)與肺纖維化模型相同。 結(jié)果 第一部分實(shí)驗(yàn)結(jié)果： 1.與正常對(duì)照組相比,急性肺損傷小鼠肺組織中出現(xiàn)肺泡間隔增厚、肺泡斷裂和炎性細(xì)胞浸潤；肺纖維化小鼠肺組織中無功能的肺纖維組織逐漸代替正常的肺泡組織,肺泡出現(xiàn)萎陷、肺泡間隔增厚、炎性細(xì)胞浸潤。Masson染色結(jié)果顯示肺纖維化肺組織出現(xiàn)膠原沉積。 2.與正常對(duì)照組相比,急性肺損傷與肺纖維化小鼠BALF中細(xì)胞總數(shù)增多,總蛋白濃度增加,IL-6與TNF-α水平升高。 3.與正常對(duì)照組相比,急性肺損傷與肺纖維化小鼠肺組織Caspase-3活性顯著增強(qiáng),TUNEL染色陽性細(xì)胞數(shù)增多。 4.與正常對(duì)照組相比,急性肺損傷與肺纖維化小鼠肺組織ROS水平升高。 5.與正常對(duì)照組相比,急性肺損傷與肺纖維化小鼠肺組織Sirt1蛋白表達(dá)降低。 6.與正常對(duì)照組相比,急性肺損傷與肺纖維化小鼠BALF中IL-6與TNF-α水平升高。 第二部分實(shí)驗(yàn)結(jié)果： 1.與PQ組相比,高劑量紫檀芪能夠減輕PQ對(duì)肺泡結(jié)構(gòu)的損傷,同時(shí)也減少膠原沉積。 2.與PQ組相比,高劑量紫檀芪能顯著減少BALF中細(xì)胞總數(shù)和總蛋白濃度。 3.與PQ組相比,高劑量紫檀芪可抑制肺組織Caspase-3活性,同時(shí)可使肺組織TUNEL染色陽性細(xì)胞數(shù)減少。 4.與PQ組相比,高劑量紫檀芪可降低肺組織ROS水平。 5.與PQ組相比,高劑量紫檀芪能夠上調(diào)肺組織Sirt1蛋白表達(dá)。 6.與PQ組相比,高劑量紫檀芪能顯著降低BALF中IL-6與TNF-α水平。 結(jié)論P(yáng)Q所介導(dǎo)的Sirt1蛋白表達(dá)下調(diào)以及ROS產(chǎn)生可能是其誘導(dǎo)小鼠產(chǎn)生急性肺損傷與肺纖維化的重要原因。紫檀芪可通過上調(diào)Sirt1的表達(dá)以及抑制活性氧產(chǎn)生和炎癥反應(yīng),從而減輕PQ誘導(dǎo)肺纖維化。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R285.5
本文編號(hào)：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R285.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 何興軒;Edward H.Schuchman;;酸性鞘磷脂酶在環(huán)境健康中的潛在作用(英文)[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2012年02期
2 張健;馮睿;馮翔;孫穎浩;王林輝;趙志青;郭明金;楊波;李文獻(xiàn);景在平;;離體腎動(dòng)脈瘤修補(bǔ)、腎動(dòng)脈重建和自體腎移植治療復(fù)雜性孤腎腎動(dòng)脈瘤一例并文獻(xiàn)復(fù)習(xí)[J];中華外科雜志;2007年18期
，本文編號(hào)：2496666
本文鏈接：http://sikaile.net/yixuelunwen/mazuiyixuelunwen/2496666.html
最近更新
教材專著
