吳茱萸堿對脂多糖誘導的ARDS大鼠的保護作用及機制研究
發(fā)布時間：2018-11-04 21:30
【摘要】：目的:探討吳茱萸堿(Evodiamine,EVO)對脂多糖(lipopolysaccharide,LPS)誘導的急性呼吸窘迫綜合征(ARDS)大鼠的保護作用及分子機制。方法:1.按照隨機數字表法將72只成年雄性SD大鼠隨機分為正常對照組(氣管滴注生理鹽水)、EVO單獨處理組(氣管滴注1mg/kg EVO)、LPS模型組(氣管滴注3mg/kg LPS)和EVO治療組(氣管滴注1mg/kg EVO和3mg/kg LPS),每組18只,通過氣管注射LPS(3mg/kg)建立ARDS模型,于建模6h后處死大鼠并收集各組動脈血、肺組織和肺泡灌洗液,通過動脈血氣分析、肺濕/干重比值、蘇木精-伊紅染色(HE染色)觀察各組大鼠肺組織的損傷情況;利用酶聯(lián)免疫吸附試驗(ELISA)和實時定量反轉錄-聚合酶鏈反應(q PCR)的方法檢測肺組織和肺泡灌洗液(BALF)中炎癥介質(TNF-α、IL-1b、HMGB1、IL-6)分泌水平;使用Western Blot方法測量各組肺組織中NF-kB信號通路相關蛋白(p50、p65)的表達。2.體外培養(yǎng)巨噬細胞系RAW264.7細胞,細胞經LPS(10μg/ml)單獨刺激或LPS(10μg/ml)與EVO(50μM)聯(lián)合作用后,于不同時間點收集各組細胞的培養(yǎng)上清及細胞團塊。通過ELISA法檢測細胞上清中炎癥介質(TNF-α、IL-1β、HMGB1、IL-6)的含量及細胞核中NF-k B p65 DNA的結合活性;利用Western Blot檢測各組細胞核內NF-κB家族蛋白(p50、p65)的表達。結果:1.LPS刺激6h后,大鼠精神不振、蜷縮少動、呼吸急促,進食減少,但EVO治療組中大鼠的精神和活動狀態(tài)較LPS模型組有明顯好轉。與LPS模型組相比較,EVO治療后可明顯減輕LPS所導致的肺水腫和組織病理學損傷,改善氧合功能,同時降低肺組織和BALF中炎癥介質(TNF-α、IL-1β、HMGB1、IL-6)的水平,Western Blot結果顯示EVO對LPS作用下肺組織胞核中NF-kBp65和p50的高表達具有明顯的下調作用。2.體外實驗中,LPS活化的RAW264.7細胞經EVO干預后,上清液中炎癥介質(TNF-α、IL-1β、HMGB1、IL-6)含量顯著降低,細胞核內NF-kB p50和p65表達顯著減少,同時NF-κB p65的DNA結合活性明顯受抑制。結論:1.EVO可有效減輕LPS誘導的ARDS大鼠肺組織損傷。2.EVO可顯著減少LPS刺激下炎性巨噬細胞中炎癥介質的分泌,該作用可能是通過抑制NF-κB p65和p50的入核及其DNA結合活性來實現的。
[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- 偽 (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- 偽, 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- 偽, 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- 偽, 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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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- 偽 (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- 偽, 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- 偽, 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- 偽, 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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R563.8
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