川芎嗪通过PBEF-NF-κB途径拮抗LPS诱导的A549细胞炎性反应

发布时间：2018-05-29 16:09

本文选题：急性呼吸窘迫综合征 + 川芎嗪　；参考：《重庆医科大学》2017年硕士论文

【摘要】：背景:ARDS是严重感染、创伤、休克等多种病因下引起肺泡上皮细胞和毛细血管内皮细胞损伤,从而导致急性呼吸衰竭综合征。近年来针对ARDS的发病机制和诊断标准进行了深入了研究和不断更新,其发病率较前下降,但临床调查显示其病死率较高,仍然缺乏有效的治疗手段。因此深入探讨ARDS的发病机制并寻求干预是目前研究的重点。ARDS发病机制复杂,炎性反应是其本质。核因子κB(Nuclear factorκB,NF-κB)是细胞内最重要的核转录因子,在多种炎性刺激介导的细胞信号转录调控中起着至关重要的作用,NF-κB通道的激活与ARDS的发生发展密切相关。前B细胞克隆增强因子(pre-B cell colony-enhancing factor,PBEF)是一种具有多重生理功能的细胞因子,参与了细胞的代谢、炎性反应和免疫调节功能。现研究发现PBEF可通过促进肺泡上皮细胞损伤、肺微血管内皮损伤、肺泡通透性增加参与ARDS的病理生理过程,而抑制PBEF的活性可减轻ARDS炎性反应。研究表明PBEF和NF-κB可作为研究ARDS发病机制的靶点,为明确二者之间的联系,有学者在脐静脉内皮细胞和肺微血管内皮细胞炎性反应中做了探索性研究,目前尚不清楚PBEF和NF-κB二者在肺泡上皮细胞炎性反应中的具体联系。川芎嗪(Tetramethylpyrazine,TMP)是从中药川芎根中提取的活性成分,具有钙离子通道拮抗剂作用,临床上广泛应用于心脑血管疾病。有研究发现TMP能通过抑制肺泡巨噬细胞NF-κB活化减轻家兔失血性休克合并内毒素诱发的急性肺损伤;该课题前期实验发现在油酸诱导的大鼠ARDS模型中,TMP能下调PBEF等炎性因子的表达,发挥肺保护作用。但目前TMP在ARDS炎性反应中的具体作用机制并不清晰。目的:(1)体外培养人II型肺泡上皮细胞(A549细胞,来源于人肺腺癌),观察LPS和TMP对A549细胞增殖活性的影响,筛选下一步实验选用的LPS和TMP浓度。(2)体外探讨LPS作用于A549细胞后PBEF和其他炎症因子的变化。(3)探讨PBEF与其他炎症因子的联系以及和NF-κB的关联。(4)探讨TMP对LPS诱导的A549炎性反应的保护作用及机制。方法:(1)体外培养人II型肺泡上皮细胞(A549细胞,来源于人肺腺癌),MTT法检测LPS和TMP药物对A549细胞的增殖作用的影响,并选择LPS造模的最佳浓度和TMP作用于A549细胞的安全浓度范围。(2)设正常对照组(Control)、LPS组、LPS+TMP组和LPS+FK866组。LPS(终浓度5mg/L)刺激A549细胞6h、12h和24h后建立炎症模型,分别加入TMP(终浓度10mg/L)和PBEF抑制剂FK866(终浓度10nmol/L)进行干预,q-PCR和Western Blot分别检测炎症因子肿瘤坏死因子α(tumor necrosis factor,TNF-a)、白细胞介素-1β(interleukin-1,IL-1β)、白细胞介素-8(interleukin-8,IL-8)、PBEF的m RNA和蛋白表达水平。(3)通过Western Blot检测3个时间段的细胞核和细胞质内磷酸化P65蛋白水平动态变化来反映NF-κB的激活情况。结果:(1)LPS对A549细胞增殖影响在同一时间点,6h的5mg/L和10mg/L、20mg/L和50mg/L实验组间细胞抑制率(Inhibition rate,IR)比较差异无统计学意义(P0.05),其余实验组在同一时间段间两两比较差异有统计学意义(P0.05);1mg/L LPS刺激6h、12h和24h后IR差异无统计学意义(P0.05),其余实验组在任何时间点组间两两比较差异有统计学意义(P0.05)。(2)TMP对A549细胞增殖影响在同一时间点,10mg/L和50mg/L的TMP对A549细胞(survival rate,SR)比较差异不具有统计意义(P0.05),6h的200mg/L和300mg/L实验组比较差异不具有统计学意义(P0.05),其余实验组在同一时间组间亮亮比较差异具有统计学意义(P0.05);在不同时间点,10mg/L和50mg/L实验组差异不具有统计学意义(P0.05),200mg/L和300mg/L实验组在12h和24h组间比较差异不具有统计学意义(P0.05),其余实验组在任何时间点组间比较差异有统计学意义(P0.05)。(3)药物干预对TNF-a、IL-1β、IL-8和PBEFm RNA表达的影响LPS刺激A549细胞后TNF-a、IL-1β、IL-8和PBEF炎症因子m RNA的表达均较对照组明显增高(P0.001),并随着作用时间延长LPS组间比较明显升高(P0.001)。FK866干预后TNF-a、IL-1β和IL-8m RNA的表达较同一时间段内LPS组降低(P0.05)。TMP干预后TNF-a、IL-1β、IL-8和PBEFm RNA的表达较同一时间段内LPS组降低(P0.05)。(4)药物干预对TNF-a、IL-1β、IL-8和PBEF蛋白表达的影响LPS作用于A549细胞后TNF-a、IL-1β、IL-8和PBEF蛋白较对照组表达升高(P0.05),随着作用时间延长,上述炎症因子蛋白表达升高,LPS组在12h和24h与6h比较差异有统计学意义(P0.05)。在加入FK866干预后TNF-a、IL-1β和IL-8蛋白表达较同一时间段内LPS组下降(P0.05),TMP干预后TNF-a、IL-1β、IL-8和PBEF蛋白的表达较同一时间段内LPS组降低(P0.05)。(5)药物干预对NF-κB活性的影响LPS刺激A549细胞后后细胞核磷酸化P65蛋白表达较对照组显著增高(3个时间段均P0.001),并随着作用时间延长逐渐升高,LPS组间两两比较差异具有统计学意义(P0.05)。在FK866干预后细胞核磷酸化P65蛋白较LPS组明显下降(3个时间段均P0.001),上述指标在TMP干预后也较LPS组降低(P0.01)。LPS刺激A549细胞后后细胞质磷酸化P65蛋白表达较对照组显著增高(3个时间段均P0.001),随着作用时间延长磷酸化P65蛋白逐渐开始下降,LPS组间两两比较差异具有统计学意义(P0.05)。在FK866干预后细胞质磷酸化P65蛋白较LPS组下降(P0.01),不同时间段FK866干预组间两两比较差异具有统计学意义(P0.05)。TMP干预后同一时间段细胞质磷酸化P65蛋白也较LPS组降低(P0.05),在6h和24h时间段TMP组间比较差异具有统计学意义(P0.05)。结论:(1)LPS对A549细胞增殖抑制呈剂量-时间依赖效应。(2)低浓度时TMP对A549细胞增殖无影响,100~300 mg/L时则抑制生长。(3)PBEF可通过NF-κB参与调控LPS诱导的A549细胞炎性因子TNF-a、IL-1β和IL-8的表达和释放。(4)TMP能降低LPS诱导A549细胞的PBEF的表达和NF-κB的活性,并降低炎症因子TNF-a、IL-1β和IL-8的表达和释放。因此,TMP可能通过降低PBEF的表达,抑制NF-κB的活性,从而减轻II型肺泡上皮细胞炎性反应,发挥ARDS保护作用。
[Abstract]:Background: ARDS is a serious infection, trauma, shock and other causes that cause damage to alveolar epithelial cells and capillary endothelial cells, resulting in acute respiratory failure syndrome. In recent years, the pathogenesis and diagnostic criteria of ARDS have been deeply studied and updated, the incidence of which is lower than before, but the clinical investigation shows its disease. The death rate is high and still lacks effective treatment. Therefore, it is very complex to explore the pathogenesis of ARDS and to seek intervention. The inflammatory response is its essence. Nuclear factor kappa B (Nuclear factor kappa B, NF- kappa B) is the most important nuclear transcription factor in the cell, and it is mediated by a variety of inflammatory stimuli. Regulation plays a vital role. The activation of NF- kappa B channel is closely related to the development of ARDS. The pre B cell clone enhancement factor (pre-B cell colony-enhancing factor, PBEF) is a cytokine with multiple physiological functions and participates in cell metabolism, inflammatory response and immunoregulation. The present study found PBEF can be passed. Promoting alveolar epithelial cell injury, pulmonary microvascular endothelial damage and increased alveolar permeability are involved in the pathophysiological process of ARDS, and the inhibition of PBEF activity can reduce the ARDS inflammatory response. The study shows that PBEF and NF- kappa B can be used as a target for the study of the pathogenesis of ARDS, and to clarify the association between the two and some scholars in the umbilical vein endothelial cells and pulmonary Microblood. PBEF and NF- kappa B two are not clearly understood in the inflammatory response of endothelium cells. Ligustrazine (Tetramethylpyrazine, TMP) is an active ingredient extracted from Ligusticum chuanxiong root, which has calcium ion channel antagonist, and is widely used in cardiovascular and cerebrovascular diseases. Some studies have found that TMP can reduce the activation of NF- kappa B in the alveolar macrophages to alleviate acute lung injury induced by hemorrhagic shock and endotoxin in rabbits. In the earlier experiment, it was found that in the ARDS model of oleic acid induced rat, TMP could reduce the expression of PBEF and other inflammatory factors and play the role of lung protection. But at present, TMP is in the inflammatory response of ARDS. The specific mechanism is not clear. Objective: (1) in vitro culture of human type II alveolar epithelial cells (A549 cells, derived from human lung adenocarcinoma), observe the effects of LPS and TMP on the proliferation of A549 cells, select the LPS and TMP concentrations in the next experiment. (2) to explore the changes in PBEF and other inflammatory factors after the action of LPS in A549 cells in vitro. (3) to explore PBEF Association with other inflammatory factors and the association with NF- kappa B. (4) to explore the protective effect and mechanism of TMP on LPS induced A549 inflammatory response. Methods: (1) human II alveolar epithelial cells (A549 cells, derived from human lung adenocarcinoma) were cultured in vitro. MTT assay was used to detect the effect of LPS and TMP drugs on the proliferation of A549 cells, and selected the best LPS model. The concentration and TMP effect on the safe concentration range of A549 cells. (2) the normal control group (Control), the LPS group, the LPS+TMP group and the LPS+FK866 group.LPS (final concentration 5mg/L) stimulated A549 cell 6h, 12h and 24h to establish the inflammatory model. Detection of inflammatory factor tumor necrosis factor (TNF-a), interleukin -1 beta (interleukin-1, IL-1 beta), interleukin -8 (interleukin-8, IL-8), PBEF m and protein expression levels. (3) the dynamic changes of phosphorylated protein levels in the nucleus and cytoplasm of the 3 time periods were detected to reflect the nuclear kappa Results: (1) the effects of LPS on the proliferation of A549 cells at the same time point, 5mg/L and 10mg/L of 6h, and the cell inhibition rate (Inhibition rate, IR) between the 20mg/L and 50mg/L experimental groups were not statistically significant (P0.05), and the other experimental groups were statistically significant in the same time period. There was no statistically significant difference in IR after 4H (P0.05), and in the rest of the experimental groups, there was a significant difference between 22 groups in any time point group (P0.05). (2) the effect of TMP on A549 cell proliferation at the same time point, 10mg/L and 50mg/L TMP to A549 cells (survival rate) was not statistically significant. The difference was not statistically significant (P0.05), and the difference between the other experimental groups was statistically significant (P0.05) at the same time. The difference between 10mg/L and 50mg/L was not statistically significant (P0.05) at different time points (P0.05), and there was no statistical difference between the 200mg/L and 300mg/L groups in the 12h and 24h groups (P0.05), but the rest were not statistically significant (P0.05). There were significant differences between the experimental groups at any time point (P0.05). (3) the effects of drug intervention on the expression of TNF-a, IL-1 beta, IL-8 and PBEFm RNA were significantly higher than those of the control group after LPS stimulation of A549 cells, and the expression of IL-1 beta, IL-8 and PBEF inflammatory factors increased significantly. After.FK866, the expression of IL-1 beta and IL-8m RNA was lower than that of LPS group in the same time period (P0.05).TMP intervention. The expression of TNF-a, IL-1 beta, IL-8 and PBEFm decreased in the same time period. (4) the effect of drug intervention on the expression of beta, beta, and egg white in the same time period. The expression of white was higher than that in the control group (P0.05). The expression of the above inflammatory factor protein increased with the prolongation of the action time. There was a significant difference between the 12h and 24h in the LPS group and 6h (P0.05). The expression of IL-1 beta and IL-8 protein was lower than that in the LPS group (P0.05). The expression of LPS group was lower than that of the same time period (P0.05). (5) the effect of drug intervention on the activity of NF- kappa B was significantly higher than that of the control group (P0.001) after LPS stimulated A549 cells (all 3 time periods were P0.001), and with the prolongation of action time, the difference between 22 of LPS groups was statistically significant (P0.05). After intervention, the nuclear phosphorylation P65 protein was significantly lower than that in the LPS group (3 time periods P0.001), and the above indexes were also lower than those in the LPS group after TMP intervention (P0.01).LPS stimulated A549 cells after A549 cells, and the expression of P65 protein in the cytoplasm was significantly higher than that in the control group (3 periods of P0.001). As the time extended the phosphorylation P65 protein gradually began to begin. The difference between the 22 groups in the LPS group was statistically significant (P0.05). After FK866 intervention, the cytoplasmic phosphorylation P65 protein was lower than that in the LPS group (P0.01), and the difference between the FK866 intervention groups at different time periods was statistically significant (P0.05), and the cytoplasmic phosphorylation P65 protein was also lower than the LPS group (P0.05) at the same time after the intervention of.TMP (P0.05). The difference between the TMP groups in the time period was statistically significant (P0.05). Conclusion: (1) LPS has a dose time dependence effect on the proliferation inhibition of A549 cells. (2) TMP has no effect on the proliferation of A549 cells at low concentration, while 100~300 mg/L inhibits the growth. (3) PBEF can be involved in regulating LPS induced inflammatory cytokines through NF- kappa B. (4) (4) TMP can reduce the expression of PBEF and the activity of NF- kappa B in A549 cells, and reduce the expression and release of inflammatory factors TNF-a, IL-1 beta and IL-8. Therefore, TMP may reduce the activity of NF- kappa by reducing PBEF expression, thus alleviated the inflammatory response of alveolar epithelial cells and played a protective role.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

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