小窝蛋白-1脚手架区多肽通过增强血红素加氧酶-1活性发挥抗炎作用
发布时间：2018-11-20 04:12
【摘要】：血红素加氧酶-1 (heme oxygenase-1,HO-1)可以通过结合内源性的小窝蛋白-1(caveolin-1,Cav-1)使得其活性受到抑制。研究发现,Cav-1结合并抑制HO-1活性的关键氨基酸区域为小窝蛋白-1脚手架区(caveolin-1 scaffolding domain,CSD)第97-101位氨基酸。于是,我们猜想CSD多肽能够解除Cav-1对HO-1内源性抑制,从而增强HO-1活性。本课题以原代分离培养的大鼠肺泡巨噬细胞为体外研究对象,通过脂多糖(lipopolysaccharide,LPS)诱导建立细胞炎症损伤。前期利用HO-1激动剂血晶素以及HO-1活性抑制剂锌原卟啉,发现HO-1抗炎作用依赖于HO-1活性。为解决HO-1的内源性抑制问题,本课题通过生物信息学技术分析得出两种突变多肽可能与Cav-1竞争性结合HO-1。人工合成三种多肽分别为野生型WTCSD、截短型△101CSD以及第99位氨基酸突变型F99ACSD多肽。在LPS诱导的肺泡巨噬细胞炎症模型上发现,三种多肽预处理后可以显著降低细胞炎症因子的基因表达水平,促进巨噬细胞从M1型向M2型极化,使得HO-1和Cav-1蛋白表达发生改变,并且可以抑制细胞浆中IκB蛋白降解;与LPS诱导的炎症损伤相比较,野生型WTCSD和截短型A101 CSD多肽预处理细胞后,可以减少细胞内Cav-1与HO-1蛋白之间的结合、增加细胞内HO-1的活性且可以降低MAPK信号通路中相关蛋白的磷酸化水平。而突变型F99A CSD多肽预处理则降低HO-1的活性。从研究结果中可以看出野生型WTCSD多肽解除HO-1内源性抑制效果最为显著。体内实验则采用LPS气管滴定建立小鼠急性肺损伤模型,预处理野生型WT CSD多肽验证其在体内的作用效果。结果显示,WTCSD多肽预处理后可以使肺组织的肺水肿、炎性细胞浸润及肺泡壁增厚现象明显减轻,并且降低炎症因子的表达。同时,WTCSD多肽预处理后可以减少肺组织中Cav-1与HO-1之间的结合并增加HO-1的活性;HO-1活性抑制剂(锌原卟啉)可以解除WT CSD多肽对LPS诱导的小鼠急性肺损伤的抗炎作用说明CSD多肽的抗炎作用是依赖于HO-1活性的。总之,CSD多肽可以通过解除Cav-1对HO-1的内源性抑制作用,从而增强HO-1活性,发挥抗炎作用。
[Abstract]:Heme oxygenase-1 (heme oxygenase-1,HO-1) inhibits its activity by binding to endogenous nest protein-1 (caveolin-1,Cav-1). It was found that the key amino acid region of Cav-1 binding and inhibiting the activity of HO-1 was the 97-101 amino acid in the small nest protein-1 scaffold region (caveolin-1 scaffolding domain,CSD). Therefore, we hypothesized that CSD peptides could remove the endogenous inhibition of HO-1 by Cav-1, thus enhancing the activity of HO-1. In this study, primary cultured rat alveolar macrophages were isolated and cultured in vitro to establish inflammatory injury induced by lipopolysaccharide (lipopolysaccharide,LPS). By using HO-1 agonist hemagglutinin and zinc protoporphyrin, an inhibitor of HO-1 activity, it was found that the anti-inflammatory effect of HO-1 depended on the activity of HO-1. In order to solve the problem of endogenous inhibition of HO-1, through bioinformatics analysis, it is concluded that two kinds of mutated polypeptides may be competitively bound to HO-1. with Cav-1. The three synthetic peptides were wild-type WTCSD, truncated 101CSD and 99th amino acid mutant F99ACSD polypeptide. In the model of alveolar macrophage inflammation induced by LPS, it was found that pretreatment with three kinds of polypeptides could significantly reduce the gene expression level of cytokines and promote macrophage polarization from M1 to M2. The expression of HO-1 and Cav-1 protein was changed, and the degradation of I 魏 B protein in cytoplasm was inhibited. Compared with the inflammatory injury induced by LPS, wild type WTCSD and truncated A101 CSD peptide pretreated cells could reduce the binding between Cav-1 and HO-1 protein. The activity of HO-1 in cells was increased and the phosphorylation level of related proteins in MAPK signaling pathway was decreased. The pretreatment of mutant F99A CSD peptide decreased the activity of HO-1. From the results, it can be seen that the wild-type WTCSD polypeptide is the most effective in removing endogenous inhibition of HO-1. In vivo, LPS titration was used to establish acute lung injury model in mice. Wild type WT CSD peptide was pretreated to verify its effect in vivo. The results showed that pretreatment with WTCSD peptide could significantly reduce pulmonary edema, inflammatory cell infiltration and alveolar wall thickening, and decrease the expression of inflammatory factors. At the same time, pretreatment with WTCSD peptide could reduce the binding between Cav-1 and HO-1 and increase the activity of HO-1 in lung tissue. HO-1 activity inhibitor (zinc protoporphyrin) can relieve the anti-inflammatory effect of WT CSD polypeptide on acute lung injury induced by LPS in mice. The anti-inflammatory effect of CSD polypeptide is dependent on HO-1 activity. In a word, CSD peptides can enhance the activity of HO-1 and play an anti-inflammatory effect by removing the endogenous inhibitory effect of Cav-1 on HO-1.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
本文编号：2343678
[Abstract]:Heme oxygenase-1 (heme oxygenase-1,HO-1) inhibits its activity by binding to endogenous nest protein-1 (caveolin-1,Cav-1). It was found that the key amino acid region of Cav-1 binding and inhibiting the activity of HO-1 was the 97-101 amino acid in the small nest protein-1 scaffold region (caveolin-1 scaffolding domain,CSD). Therefore, we hypothesized that CSD peptides could remove the endogenous inhibition of HO-1 by Cav-1, thus enhancing the activity of HO-1. In this study, primary cultured rat alveolar macrophages were isolated and cultured in vitro to establish inflammatory injury induced by lipopolysaccharide (lipopolysaccharide,LPS). By using HO-1 agonist hemagglutinin and zinc protoporphyrin, an inhibitor of HO-1 activity, it was found that the anti-inflammatory effect of HO-1 depended on the activity of HO-1. In order to solve the problem of endogenous inhibition of HO-1, through bioinformatics analysis, it is concluded that two kinds of mutated polypeptides may be competitively bound to HO-1. with Cav-1. The three synthetic peptides were wild-type WTCSD, truncated 101CSD and 99th amino acid mutant F99ACSD polypeptide. In the model of alveolar macrophage inflammation induced by LPS, it was found that pretreatment with three kinds of polypeptides could significantly reduce the gene expression level of cytokines and promote macrophage polarization from M1 to M2. The expression of HO-1 and Cav-1 protein was changed, and the degradation of I 魏 B protein in cytoplasm was inhibited. Compared with the inflammatory injury induced by LPS, wild type WTCSD and truncated A101 CSD peptide pretreated cells could reduce the binding between Cav-1 and HO-1 protein. The activity of HO-1 in cells was increased and the phosphorylation level of related proteins in MAPK signaling pathway was decreased. The pretreatment of mutant F99A CSD peptide decreased the activity of HO-1. From the results, it can be seen that the wild-type WTCSD polypeptide is the most effective in removing endogenous inhibition of HO-1. In vivo, LPS titration was used to establish acute lung injury model in mice. Wild type WT CSD peptide was pretreated to verify its effect in vivo. The results showed that pretreatment with WTCSD peptide could significantly reduce pulmonary edema, inflammatory cell infiltration and alveolar wall thickening, and decrease the expression of inflammatory factors. At the same time, pretreatment with WTCSD peptide could reduce the binding between Cav-1 and HO-1 and increase the activity of HO-1 in lung tissue. HO-1 activity inhibitor (zinc protoporphyrin) can relieve the anti-inflammatory effect of WT CSD polypeptide on acute lung injury induced by LPS in mice. The anti-inflammatory effect of CSD polypeptide is dependent on HO-1 activity. In a word, CSD peptides can enhance the activity of HO-1 and play an anti-inflammatory effect by removing the endogenous inhibitory effect of Cav-1 on HO-1.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
【参考文献】
相关期刊论文 前2条
1 舒娟;刘晓菊;褚旭;邱敬满;曾晓丽;包海荣;;细颗粒物对慢性阻塞性肺疾病小鼠肺泡巨噬细胞吞噬功能的影响[J];中华医学杂志;2016年04期
2 何俊;徐文莉;曾思;曾因明;庞庆丰;;大鼠肺泡巨噬细胞原代培养技术的改良措施[J];徐州医学院学报;2010年05期
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