Intervention Study Of Methionine Sulfoxide Reductase A In Macrophages On Atherosclerosis

Backgroud:Oxidative stress plays a privotal role in the ragulation of numerous signal pathways. Atherosclerosis(As) was confirmed to associate with oxidative stress. Keeping the balance of redox is important to understand the potential mechanisms of As and prevent the body from atherosclerostic diseases. Methionine is one of the most easily oxidized amino acids, whose oxidative modification was participated into some degenerative diseases and the reduction of oxidized methionine contributed to repair the physiological function of protein. Methionine sulfoxide reductases A (MsrA) which is a kind of conservative and ubiquitous enzyme in intracellular redox could reduce methionine-S-sulfoxide specificly.Methods:The hMsrA DNA sequence was inserted into eukaryotic plasmid pWPI, the blank pWPI plasmids containing green fluorescent protein(GFP) were considered control. pWPI plamids were cotransfected into293t cells with package plasmid Δ8.91and envelope plasmid pD2G respectively. Mature lentiviral particles were collected, concentrated and determined the titers. In vitro, the lentivirus containing hMsrA (LV-hMsrA) were assigned to infecte primary macrophages and Raw264.7cell line with proper multiplicity of infection (MOI). Biological activity of macrophages with hMsrA overexpression was investigated responding to oxLDL. The cellular lipid accumulation of foam macrophages were measured by oil red O. The production of intracellular ROS was detected by fluorescence probe DCF. The content of inflammatory TNF-a, IL-6in medium was estimated by Elisa kits. Finally, the change of lipid transport associated ABCA1, ABCG1, SRBI and LXR was investigated by RT-PCR and Western Blotting. In vivo, the hematopoietic progenitor cells (HPCs) infected by LV-hMsrA and LV-GFP were transplanted into apoE-/-mice by bone marrow transplantation. After12weeks of western diet, the apoE-/-mice were executed by euthanasia. hMsrA overexpression in the spleen and HPCs would be identified by Western Blotting. And the lipid level, antioxidant and antiinflammatory activity in plasma would also be detected by enzymetic methods. The degree of atherosclerosis in aortic wall was evaluated via slicing and staining aortic sinus with oil red O, and analyzing lension of thoraco-abdominal aorta by en face.Results:The lentiviral vector containing hMsrA gene was constructed successfully, the inserted protein hMsrA is verified by sequencing analysis and Western Blotting that no mutation were occurred in the process of amplification and digestion. In the condition of oxidative stress, hMsrA overexpresion in macrophages could decrease the cellular lipid accumulation and the production of ROS, and it also inhibited the secretion of proinflammatory IL-6and TNF-a (P<0.05). To contrast with LV-GFP group, the expression of ABCG1, LXR, ABCA1and SRBI were upregulated in LV-hMsrA group (P<0.05). Then if no oxidative stimulus was administrated, differences weren’t observed among groups (P>0.05). In vivo, hMsrA were stably and highly expressed in the spleen and HPCs for14weeks after bone marrow transplantation. Macrophages with hMsrA overexpression in apoE-/-mice was attenuated the lesion of aorta, increased the antioxidant activity of PON1and SOD, and decrease the content of inflammatory MCP-1(P<0.05). Then the differences of the lesion in aortic sinus, the level of IL-6and TNF-a, the content of serum lipid were not observed between LV-GFP mice group and LV-hMsrA mice group (P>0.05).Conclution:High expression of MsrA in macrophages could decrease the cellular inflammatory level and prevent from the formation of macrophage foam cell via accelerating the lipid efflux from macrophages. Furthermore, in apoE-/-mice high macrophage-derived hMsrA expression could attenuate the development of atheroscleosis through improve the anti-inflammatory and anti-oxidative ability in local even systemic aortic wall.