| Background:Methionine sulfoxide reductase A (MsrA), a specific intracellular oxidoreductase which exclusively reduces MetSO,serves as an essential factor in protecting cells against oxidative damage by methionine redox cycle. Atherosclerosis-induced cardiovascular disease is the most common cause of death and disability worldwide. Atherosclerosis is thought to be a chornic inflammatory disease associated with oxdative stress and dyslipidemia which making anti-oxidation, anti-inflammation and lipid lowering becomes the main strategies preventing from atherosclerosis. Although MsrA plays important anti-oxidation role intracellular, there were rare researchs about the relationship between MsrA and atherosclerosis. As an intracellular reducatase, MsrA recycle involves in thioredoxin system and additionally, proteins and nucleic acids are poorly transferred into cells for the permeability of cell membrane. So therapeutic use of exogenous MsrA in oxidative-induced diseases is limited. Nowadays, PEP-1 peptide become a novel, efficient and safe tool. PEP-1 peptide is a synthetic cell penetrating peptide (CPP) which already mediated several proteins, like superoxide dismutase (SOD), catalase (CAT) and paraoxonase-1 (PON1), transduced into cells and exerted beneficial effects. The aim of this study is to investigate whether the fusion of hMsrA with PEP-1, can carry out the protective effects in macrophages as well as atherosclerotic development in apolipoprotein E deficient (apoE-/-) mice.Methods:1. hMsrA and PEP-1-hMsrA fusion proteins were reconstructed in pET28a expression system. Transformed BL21DE3 E.coli were induced by IPTG and purified using Ni-NTA affinity chromatography. The second structures of proteins were tested by circular dichroism (CD) and activities were measured by DMSO.2. Different concentrations of proteins (0-18?M) were incubated with HeLa cells for 72 h for testing cell viability. Transduction of proteins into macrophages and HepG2 cells were examined by Western blot and immunofluorescence staining. After 1?M hMsrA or PEP-1-hMsrA incubated macrophages for 1h,1 mM H2O2 stimulated the cells for measuring intracellular reactive oxygen species (ROS) and cell death levels by flow cytometry:ROS was measured by the specific probe DCFH-DA and cell death level were measured by staining of Annexin V/PI.25 ng/ml LPS with 1?M hMsrA or PEP-1-hMsrA proteins incubated macrophages for analysis the inflammatory factors IL-1?, TNFa and IL-10 mRNA levels by RT-PCR.3. In vivo study,5.5nmol hMsrA or PEP-1-hMsrA proteins were intraperitoneally injected into 21-week old male apoE-/- mice every 36 h for 12 weeks. The control group was injected the same volume of 100 mM PBS. Mice were fed with Western diet for acceletating atherogensis. The last injection was 2 h before mice euthanasia. Blood samples were collected from mice after overnight fasting. Atherosclerotic lesions were analyzed by Oil Red 0 staining both in aortic roots, aortic archs and aortas. Total cholesterol (TC) and triglyceride (TG) levels in fresh plasma were measured by enzymatic colorimetric method. The macrophage contents and cell apoptosis ratios in aortic root lesion were confirmed by immunohistochemical and TUNEL staining. Plasma monocyte chemotactic protein-1 (MCP-1) level was assessed by ELISA and liver inflammatory factors TNFa and IL-6 mRNA level were measured by RT-PCR. Plasma PON1, SOD and myeloperoxidase (MPO) activities were assessed. Expression of PON 1 in liver were determined by RT-PCR and Western blot.Results:1. pET28a/hMsrA and pET28a/PEP-1-hMsrA plasmid vectors were successfully reconstructed. The DNA sequences were right and had no mutation. SDS-PAGE showed hMsrA and PEP-1-hMsrA proteins got up to 95% purity using affinity chromatography and the yield of these pure proteins were nearly 20 mg/L bacteria liquid (and about 5g bacterias). CD spectra showed that a-helix ratio of hMsrA and PEP-1-hMsrA proteins were analogous (18.1%vs.17.1%). Prokaryotic expressed hMsrA and PEP-1-hMsrA proteins exerted reduction activity and the activities of these two proteins had no differences.2. Even incubated with high concentrations (up to 18 ?M) proteins for 72 h, there was no influence on HeLa cells viability. PEP-1-hMsrA protein could efficiently transduce into macrophages and HepG2 cells with dose dependent manner verified by Western Blot and confocal and could maintain at least for 12 h intracellular. Transduction of PEP-1-MsrA significantly reduced the intracellular ROS level, cellular apoptosis and necrosis in H2O2-treated macrophages, but hMsrA protein could not. IL-1?, TNFa and IL-10 mRNA levels were increased in 25 ng/ml LPS-treated macrophages, PEP-1-hMsrA protein could decrease pro-inflammatory IL-1? and TNFa mRNA levels but elevate anti-inflammatory IL-10 mRNA level.3. In vivo study, compared with the control group, injection of hMsrA and PEP-1-hMsrA proteins had no effects on mice body weights, spleen weights, plasma TC, plasma TG and plasma IgG levels. But plasma PON1 and SOD activities were significant increased and MCP-1 level was decreased in PEP-1-hMsrA group. PEP-1-MsrA injection significantly reduced the aortic atherogenesis in aortic roots, aortic archs and aortas which accompanied with the reduction of macrophages and cells apoptosis ratios in lesions. Moreover, PEP-1-MsrA protein indirectly increased hepatic PON1 expression level. TNF-a, IL-6 mRNA level in liver were also decreased in PEP-1-hMsrA protein treated mice.Conclusion:Successfully reconstructed pET28a/hMsrA and pET28a/PEP-1-hMsrA plasmid vectors and the pure PEP-1-hMsrA protein could remarkablely increased the anti-oxidative stress and anti-inflammtion effects in macrophages. In vivo, PEP-1-hMsrA protein also prominently attenuated atherogenesis in Western diet fed apoE-/- mice. The mechnism may rely on improvement of anti-oxidative stress and anti-inflammtion environment by PEP-1-hMsrA protein into vascular and liver. Our study provides the evidence that PEP-1-hMsrA may be a potential therapeutic protein for reducing atherosclerosis-related cardiovascular diseases. |
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