小窩蛋白-1腳手架區(qū)多肽通過(guò)增強(qiáng)血紅素加氧酶-1活性發(fā)揮抗炎作用
發(fā)布時(shí)間：2018-11-20 04:12
【摘要】：血紅素加氧酶-1 (heme oxygenase-1,HO-1)可以通過(guò)結(jié)合內(nèi)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(xiàn),Cav-1結(jié)合并抑制HO-1活性的關(guān)鍵氨基酸區(qū)域?yàn)樾「C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(duì)HO-1內(nèi)源性抑制,從而增強(qiáng)HO-1活性。本課題以原代分離培養(yǎng)的大鼠肺泡巨噬細(xì)胞為體外研究對(duì)象,通過(guò)脂多糖(lipopolysaccharide,LPS)誘導(dǎo)建立細(xì)胞炎癥損傷。前期利用HO-1激動(dòng)劑血晶素以及HO-1活性抑制劑鋅原卟啉,發(fā)現(xiàn)HO-1抗炎作用依賴于HO-1活性。為解決HO-1的內(nèi)源性抑制問(wèn)題,本課題通過(guò)生物信息學(xué)技術(shù)分析得出兩種突變多肽可能與Cav-1競(jìng)爭(zhēng)性結(jié)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(dǎo)的肺泡巨噬細(xì)胞炎癥模型上發(fā)現(xiàn),三種多肽預(yù)處理后可以顯著降低細(xì)胞炎癥因子的基因表達(dá)水平,促進(jìn)巨噬細(xì)胞從M1型向M2型極化,使得HO-1和Cav-1蛋白表達(dá)發(fā)生改變,并且可以抑制細(xì)胞漿中IκB蛋白降解;與LPS誘導(dǎo)的炎癥損傷相比較,野生型WTCSD和截短型A101 CSD多肽預(yù)處理細(xì)胞后,可以減少細(xì)胞內(nèi)Cav-1與HO-1蛋白之間的結(jié)合、增加細(xì)胞內(nèi)HO-1的活性且可以降低MAPK信號(hào)通路中相關(guān)蛋白的磷酸化水平。而突變型F99A CSD多肽預(yù)處理則降低HO-1的活性。從研究結(jié)果中可以看出野生型WTCSD多肽解除HO-1內(nèi)源性抑制效果最為顯著。體內(nèi)實(shí)驗(yàn)則采用LPS氣管滴定建立小鼠急性肺損傷模型,預(yù)處理野生型WT CSD多肽驗(yàn)證其在體內(nèi)的作用效果。結(jié)果顯示,WTCSD多肽預(yù)處理后可以使肺組織的肺水腫、炎性細(xì)胞浸潤(rùn)及肺泡壁增厚現(xiàn)象明顯減輕,并且降低炎癥因子的表達(dá)。同時(shí),WTCSD多肽預(yù)處理后可以減少肺組織中Cav-1與HO-1之間的結(jié)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(duì)LPS誘導(dǎo)的小鼠急性肺損傷的抗炎作用說(shuō)明CSD多肽的抗炎作用是依賴于HO-1活性的。總之,CSD多肽可以通過(guò)解除Cav-1對(duì)HO-1的內(nèi)源性抑制作用,從而增強(qiáng)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(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R96
本文編號(hào)：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(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R96
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