重组PEP-1-hMsrA蛋白对动脉粥样硬化的影响

发布时间：2018-09-19 13:40

【摘要】：背景：甲硫氨酸亚砜还原酶A(MsrA)是细胞内特异还原S型甲硫氨酸亚砜(MetSO)的还原酶类,是细胞内重要的抗氧化屏障。动脉粥样硬化所致的心脑血管疾病目前是世界致死、致残率最高的一类疾病,动脉粥样硬化是公认的慢性炎症性疾病,氧化应激和血脂紊乱也是其主要的诱因。因此,抗氧化抗炎及降脂成为防治动脉粥样硬化的主要策略。细胞内抗氧化酶MsrA与动脉粥样硬化发生发展的相关性并不十分明确,由于该蛋白酶必须在细胞内依赖硫氧还蛋白系统恢复其活性,而作为生物大分子又难以直接穿过细胞膜进入细胞内发挥作用,目前利用外源性MsrA研究对动脉粥样硬化等氧化应激所致疾病的作用及相关机制尚无报道。近年有报道,细胞穿膜肽PEP-1作为一种新型、有效、安全的工具肽,介导超氧化物歧化酶(SOD)、过氧化物酶(CAT)、对氧磷酯酶1(PON1)等抗氧化蛋白成功穿入细胞并发挥有效的抗氧化功能。本文拟构建穿膜活性肽PEP-1与人源MsrA(hMsrA)的重组PEP-1-hMsrA融合蛋白,以巨噬细胞及载脂蛋白E基因敲除(apoE-/-)小鼠模型为研究对象,旨在探究PEP-1-hMsrA蛋白在动脉粥样硬化发生发展中的调节作用及可能的相关机制。方法：1.构建含有hMsrA或PEP-1-hMsrA的重组原核表达载体pET28a/hMsrA及 pET28a/PEP-1-hMsrA,分别转化至BL21DE3大肠杆菌、经IPTG诱导表达及Ni-NTA亲和层析获得纯品hMsrA及PEP-1-hMsrA蛋白。圆二色光谱法鉴定两种蛋白的二级结构,并以DMSO为底物测定MsrA蛋白的还原酶活性。2.细胞实验：以不同浓度(0-18μM)的hMsrA或PEP-1-hMsrA蛋白孵育Hela细胞72h,MTT法测细胞存活率；以适宜的浓度及时间孵育小鼠巨噬细胞及人HepG2细胞,Western Blot及免疫荧光方法检测PEP-1-hMsrA蛋白的穿膜效率；1 μ.M hMsrA或PEP-1-hMsrA蛋白分别孵育细胞1h后1mM过氧化氢(H202)刺激小鼠巨噬细胞,观察PEP-1-hMsrA对氧化应激下巨噬细胞内ROS水平及细胞坏死的影响：荧光探针DCFH-DA检测细胞内活性氧簇(ROS)水平；AnnexinV-FITC/PI双染细胞凋亡试剂盒检测细胞凋亡坏死率：25ng/ml脂多糖(LPS)与1μM hMsrA或PEP-1-hMsrA蛋白共孵育小鼠巨噬细胞,RT-PCR检测细胞内炎症相关基因IL-1β、TNFα、IL-10的转录水平。3.动物实验：选择21周龄雄性apoE-/-小鼠作为实验对象,经腹腔连续注射5.5nmol hMsrA或PEP-1-hMsrA蛋白,设空白组注射等体积磷酸盐缓冲液(100 mMPBS,pH7.4),每36h一次。实验中给予小鼠高脂饮食加速动脉粥样硬化进程,于第12周最后一次注射蛋白2h后小鼠行安乐死。采取禁食12h后小鼠全血分离血浆,采用小鼠主动脉窦冰冻切片、主动脉弓及主动脉、油红0染色进行斑块横切面及en face剖面分析主动脉粥样硬化斑块面积；酶法测定血清总胆固醇(TC)和甘油三酯(TG)浓度；利用免疫组化及TUNEL染色检测主动脉斑块中巨噬细胞数量及细胞凋亡比例；ELISA法测定血清炎症因子MCP-1蛋白水平,RT-PCR检测肝脏组织中炎症因子TNFα和IL-6的mRNA水平；检测血清PON1和SOD活性,RT-PCR及Western Blot同时检测肝脏组织中PONI的表达水平。结果：1.成功构建重组原核表达载体pET28a/hMsrA和pET28a/PEP-1-hMsrA,DNA测序证实碱基序列正确；S DS-PAGE鉴定经亲和层析获得的hMsrA和PEP-1-hMsrA蛋白纯度均高于95%,得率约为20 mg/L菌液(菌体重量约为5g)；圆二色光谱法鉴定,hMsrA和PEP-1-hMsrA蛋白的α螺旋结构无明显差异(18.1%vs.17.1%)；体外对比MsrA的还原酶活性,结果显示hMsrA和PEP-1-hMsrA的酶活性无明显差异。2.细胞实验中发现即使高浓度(18pM)的hMsrA或PEP-1-hMsrA蛋白长时间(72h)孵育Hela,对细胞活力没有影响：以不同浓度蛋白孵育巨噬细胞或人肝细胞,蛋白免疫印迹及激光共聚焦均证实PEP-1-hMsrA蛋白具备有效穿膜效应且表现一定的浓度依赖性,在细胞中可持续存在达12h。与hMsrA组相比,PEP-1-hMsrA蛋白可显著降低H202诱发的胞内ROS水平,并明显降低H202引起的细胞坏死率及凋亡率；LPS刺激炎症相关基因IL-1β、TNFα、IL-10 mRNA的表达,PEP-1-hMsrA蛋白可显著降低胞内致炎因子I]L-1β、TNFα的mRNA水平,但可以明显提高抗炎因子IL-10的mRNA水平。3.动物实验中,与注射PBS的对照组比较,hMsrA和PEP-1-hMsrA蛋白注射对小鼠体重、脾重、血浆TC、TG、IgG水平并无明显影响；但P EP-1-hMsrA组可显著提高血中抗氧化蛋白PON1、SOD的活性,并降低血清中炎症因子MCP-1的蛋白水平；与PBS对照组及hMsrA组比较,给予PEP-1-hMsrA蛋白可明显降低高脂喂养下小鼠主动脉窦部粥样斑块面积,同时显著降低主动脉弓和主动脉的粥样斑块面积比例,并使主动脉窦部斑块中巨噬细胞数量减少,并且细胞的凋亡比率也明显降低；同时发现,PEP-1-hMsrA蛋白间接地提高了小鼠肝脏中PON1的mRNA和蛋白表达水平；肝脏中炎症因子TNFa及IL-6的mRNA水平也下降。结论：成功构建并获得具有穿膜效应的PEP-1-hMs rA蛋白,证实其可明显提高巨噬细胞的抗氧化、抗炎能力,并显著减缓高脂饮食下apoE-/-小鼠动脉粥样硬化斑块的发展进程,其机制可能与PEP-1-hMsrA进入血管、肝组织细胞,改善局部及血循环系统的氧化、炎症状态有关。本研究为动脉粥样硬化的抗氧化防治提供了新的策略和依据。
[Abstract]:BACKGROUND: Methionine sulfoxide reductase A (MsrA) is a reductase that specifically reduces S-type methionine sulfoxide (MetSO) in cells and an important antioxidant barrier in cells. Oxidative stress and dyslipidemia are also major contributors to atherosclerosis. Therefore, antioxidant, anti-inflammatory and lipid-lowering are the main strategies for preventing and treating atherosclerosis. However, as a biological macromolecule, it is difficult to directly penetrate the cell membrane into the cell to play a role. Exogenous MsrA has not been reported to study the role of oxidative stress induced diseases such as atherosclerosis and related mechanisms. Antioxidant proteins such as dismutase (SOD), peroxidase (CAT) and paraoxonase-1 (PON1) successfully penetrate into cells and play an effective antioxidant role. In this study, the recombinant PEP-1-hMsrA fusion protein of PEP-1 and human MsrA (hMsrA) was constructed. The macrophages and apoE-knockout mice were used as the research objects. Methods: 1. Recombinant prokaryotic expression vectors containing hMsrA or PEP-1-hMsrA, pET28a/hMsrA and pET28a/PEP-1-hMsrA, were constructed and transformed into E. coli BL21DE3 by IPTG induction and Ni-NTA affinity chromatography. The secondary structures of the two proteins were identified by circular dichroism spectroscopy, and the reductase activity of MsrA protein was measured by DMSO as substrate. 2. Cell experiments: Hela cells were incubated with different concentrations of hMsrA or PEP-1-hMsrA protein for 72 hours, and the cell viability was measured by MTT assay. The penetration efficiency of PEP-1-hMsrA protein was detected by Western Blot and immunofluorescence assay in human HepG2 cells, and the effects of PEP-1-hMsrA protein on ROS level and cell necrosis in macrophages under oxidative stress were observed by DCFH-DA with fluorescent probe. Cellular reactive oxygen species (ROS) level was measured; Annexin V-FITC/PI double staining apoptosis kit was used to detect apoptosis and necrosis rate: 25ng/ml lipopolysaccharide (LPS) was co-incubated with 1 mu mhMsrA or PEP-1-hMsrA protein in mouse macrophages, and RT-PCR was used to detect the transcription level of inflammation-related genes IL-1beta, TNF-a and IL-10.3. Animal experiment: 21 weeks old. Male apoE-/-mice were injected with 5.5 nmol hMsrA or PEP-1-hMsrA protein through abdominal cavity. The blank group was injected with equal volume phosphate buffer (100 mMPBS, pH 7.4) every 36 hours. The mice were given a high fat diet to accelerate the atherosclerosis process. The mice were euthanized 2 hours after the last injection of protein at the 12th week. After fasting for 12 hours, the plasma was isolated from the whole blood of mice. The aortic sinus, aortic arch and aorta were stained with oil red 0, and the plaque area was analyzed by transverse section and en face section. The serum total cholesterol (TC) and triglyceride (TG) levels were measured by enzyme method, and the aorta was detected by immunohistochemistry and TUNEL staining. The number of macrophages and the proportion of apoptosis in the plaque were measured by ELISA. The levels of serum inflammatory factors MCP-1 protein were measured by ELISA. The mRNA levels of inflammatory factors TNFalpha and IL-6 in liver tissue were detected by RT-PCR. The activities of serum PON1 and SOD were detected. The expression of PONI in liver tissue was detected by RT-PCR and Western Blot. The prokaryotic expression vectors pET28a/hMsrA and pET28a/PEP-1-hMsrA were confirmed by DNA sequencing. The purity of hMsrA and PEP-1-hMsrA proteins obtained by affinity chromatography identified by S DS-PAGE was higher than 95%, and the yield was about 20 mg/L bacterial fluid (body weight was about 5 g); circular dichroism spectroscopy showed that the alpha helix structure of hMsrA and PEP-1-hMsrA proteins was not obvious. The activity of MsrA and PEP-1-hMsrA was not significantly different in vitro. 2. Cell experiments showed that even high concentration (18pM) of hMsrA or PEP-1-hMsrA protein incubated Hela for a long time (72h) had no effect on cell viability: macrophages or human hepatocytes were incubated with different concentrations of proteins. PEP-1-hMsrA protein could effectively penetrate the membrane and exhibit a certain concentration-dependent effect. It could persist for 12 hours. Compared with hMsrA group, PEP-1-hMsrA protein could significantly reduce the intracellular ROS level induced by H202 and the cell necrosis rate and apoptosis rate induced by H202. The expression of IL-1beta, TNFalpha, IL-10 mRNA and PEP-1-hMsrA protein could significantly decrease the mRNA levels of intracellular inflammatory factors I]L-1beta and TNFalpha, but could significantly increase the mRNA levels of anti-inflammatory factors IL-10. 3. In animal experiments, compared with the control group injected with PBS, hMsrA and PEP-1-hMsrA protein injected mice weight, spleen weight, plasma TC, TG, I. There was no significant effect on the level of gG, but PEP-1-hMsrA significantly increased the activity of antioxidant protein PON1 and SOD, and decreased the level of inflammatory factor MCP-1 in serum. Compared with PBS control group and hMsrA group, PEP-1-hMsrA protein significantly decreased the area of atherosclerotic plaque in aortic sinus of mice fed with high-fat diet, and also significantly decreased the area of atherosclerotic plaque. The proportion of atherosclerotic plaque area in aortic arch and aorta decreased the number of macrophages in aortic sinus plaque and the rate of apoptosis of the cells. It was also found that PEP-1-hMsrA protein indirectly increased the expression of PON1 mRNA and protein in mice liver, and the levels of inflammatory factors TNFa and IL-6 mRNA in liver were also increased. CONCLUSION: PEP-1-hMs rA protein with membrane-penetrating effect was successfully constructed and obtained, which proved that PEP-1-hMs rA protein could significantly improve the antioxidant and anti-inflammatory capacity of macrophages, and significantly slow down the development of atherosclerotic plaques in apoE-/-mice fed with high-fat diet. The mechanism may be related to the entry of PEP-1-hMs rA into blood vessels, hepatocytes, and the improvement of local and blood levels. This study provides a new strategy and basis for the antioxidant prevention and treatment of atherosclerosis.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R543.5

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