小窩蛋白-1腳手架區(qū)多肽通過增強血紅素加氧酶-1活性發(fā)揮抗炎作用
發(fā)布時間：2018-11-20 04:12
【摘要】：血紅素加氧酶-1 (heme oxygenase-1,HO-1)可以通過結合內源性的小窩蛋白-1(caveolin-1,Cav-1)使得其活性受到抑制。研究發(fā)現,Cav-1結合并抑制HO-1活性的關鍵氨基酸區(qū)域為小窩蛋白-1腳手架區(qū)(caveolin-1 scaffolding domain,CSD)第97-101位氨基酸。于是,我們猜想CSD多肽能夠解除Cav-1對HO-1內源性抑制,從而增強HO-1活性。本課題以原代分離培養(yǎng)的大鼠肺泡巨噬細胞為體外研究對象,通過脂多糖(lipopolysaccharide,LPS)誘導建立細胞炎癥損傷。前期利用HO-1激動劑血晶素以及HO-1活性抑制劑鋅原卟啉,發(fā)現HO-1抗炎作用依賴于HO-1活性。為解決HO-1的內源性抑制問題,本課題通過生物信息學技術分析得出兩種突變多肽可能與Cav-1競爭性結合HO-1。人工合成三種多肽分別為野生型WTCSD、截短型△101CSD以及第99位氨基酸突變型F99ACSD多肽。在LPS誘導的肺泡巨噬細胞炎癥模型上發(fā)現,三種多肽預處理后可以顯著降低細胞炎癥因子的基因表達水平,促進巨噬細胞從M1型向M2型極化,使得HO-1和Cav-1蛋白表達發(fā)生改變,并且可以抑制細胞漿中IκB蛋白降解;與LPS誘導的炎癥損傷相比較,野生型WTCSD和截短型A101 CSD多肽預處理細胞后,可以減少細胞內Cav-1與HO-1蛋白之間的結合、增加細胞內HO-1的活性且可以降低MAPK信號通路中相關蛋白的磷酸化水平。而突變型F99A CSD多肽預處理則降低HO-1的活性。從研究結果中可以看出野生型WTCSD多肽解除HO-1內源性抑制效果最為顯著。體內實驗則采用LPS氣管滴定建立小鼠急性肺損傷模型,預處理野生型WT CSD多肽驗證其在體內的作用效果。結果顯示,WTCSD多肽預處理后可以使肺組織的肺水腫、炎性細胞浸潤及肺泡壁增厚現象明顯減輕,并且降低炎癥因子的表達。同時,WTCSD多肽預處理后可以減少肺組織中Cav-1與HO-1之間的結合并增加HO-1的活性;HO-1活性抑制劑(鋅原卟啉)可以解除WT CSD多肽對LPS誘導的小鼠急性肺損傷的抗炎作用說明CSD多肽的抗炎作用是依賴于HO-1活性的�？傊�,CSD多肽可以通過解除Cav-1對HO-1的內源性抑制作用,從而增強HO-1活性,發(fā)揮抗炎作用。
[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.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96
本文編號：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.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96
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