| Objective Lipid metabolism disorders,chronic inflammation and excessive oxidative stress are the main factors in the development of metabolic diseases such as atherosclerosis and steatosis.Circulation of HDL play an important role in improving the body lipid disorders,promoting cholesterol efflux,anti-inflammatory and anti-oxidation.When the body is always in the lipid metabolism disorders,chronic inflammatory conditions,the structure and function of some improtant proteins in HDL are damaged,leading to the dysfunctional HDL.The dysfuntional HDL inhibits the reverse cholesterol transport,promotes systemic and vascular inflammation,exacerbates oxidative stress,and accelerates the process of arterial atherosclerosis.Methionine sulfoxide reductase A(MsrA)is an important intracellular antioxidant enzyme.Our previous study showed that MsrA overexpression in liver significantly decreased hepatic lipid deposition,decreased blood lipid level,improved oxidative inflammation and delay the process of atherosclerosis in apoE diffecient(apoE-/-)mice.In this study,we constructed an Epk-MsrA recombinant lentiviral vector with a secreted polypeptide-Epk which combined with HDL.Epk-MsrA was injected into the scavenger receptor type BI gene knockout(SR-BI-/-)mice in form of Letivirus,to investigate the effect of Epk-MsrA on the repair of HDL-related protein molecules in circulation and the effect of Epk-MsrA on lipid disorder,hyperinflation and hyperoxia in mice.Methods The recombinant plasmid pWPI-GFP-Epk and pWPI-GFP-Epk-MsrA was constructed using the plasmid PUC-signal peptide histag-EPK and pWPI-hMsrA.In the cell experiment,pWPI-GFP-Epk and pWPI-GFP-Epk-MsrA were transfected into 293T cells respectively,and the expression of Epk-MsrA protein in medium was confirmed by Western Blotting.Then,pWPI-GFP-Epk and pWPI-GFP-Epk-MsrA with the lentiviral packaging plasmids plp1,plp2 and VsVg were co-transfected into 293T cells by liposomes,respectively.The culture medium was collected and concentrated to obtain high titer lentivirus particles Lv-GFP,Lv-GFP-Epk and Lv-GFP-Epk-MsrA.SR-BI-/-mice were randomly divided into three groups:Lv-GFP,Lv-GFP-Epk and Lv-GFP-Epk-MsrA.Lentivirus particles were injected into SR-BI-/-mice by intravenously.After 2 weeks of injection,the mice were fed with Western diet to accelerate lipid metabolism disorders.The mice were monitored for weight and subject to retrobulbar venous blood sampling every 3 or 4 weeks.The mice were euthanized for analysis after 12 weeks of Western diet.Plasma cholesterol(TCH),free cholesterol(FCH),triglycerides(TG),high density lipoprotein cholesterol(HDL-C)were measured by enzymatic methods.Fast protein liquid chromatography(FPLC)was used to detect the distribution of plasma lipoprotein cholesterol.Plasma superoxide dismutase(SOD)and myeloperoxidase(MPO)were measured.The activity of plasma paraoxonase(PON1)was measured continuously by using paraoxon as substrate.The activity of plasma lecithin cholesterol esterase transferase(LCAT)was measured by the amount of formed nitrophenol by using p-nitrophenylbutyric acid as substrate.High expression of Epk-MsrA was confirmed by liver frozen section and plasma Western Blotting.Liver lipid deposition was detected by histochemical method.Liver lipid content was determined by liver lipid extraction method.Plasma SAA,apoAIV and A1AT were determined by Western Blotting to reflect plasma oxidative and inflammatory status.Real-time quantitative PCR(QPCR)and Western Blotting were used to detect expression of lipid metabolism and inflammation-related genes and proteins in mice liver.Results The recombinant plasmid pWPI-GFP-Epk-MsrA was successfully constructed,which was confirmed by DNA sequencing.In the cell experiment,the pWPI-Epk-MsrA-GFP plasmid was successfully transfected into 293T cells,and the recombinant protein of Epk-MsrA successfully secreted into the medium detected by Western Blotting.In animal experiments,high titer lentiviral particles were obtained by co-transfection into 293T cells and then infected them into mice.After 12 weeks of high fat diet,mice were euthanized and Epk-MsrA was confirmed to secrete into plasma and specific bind to HDL fraction.There were no significant difference in body weight and the ALT activity,but the spleen weight of Lv-GFP-Epk group and Lv-GFP-Epk-MsrA group were lower compared with Lv-GFP group.The contents of TCH in plasma of Lv-GFP-Epk group and Lv-Epk-MsrA-GFP group were unchanged,however plasma HDL-C level was significantly increased,and FCH and TG levels both decreased(p<0.05).PON1 activity and LCAT activity were significantly increased in Lv-GFP-Epk group and Lv-GFP-Epk-MsrA group(p<0.01),the activity of MPO and SAA protein levels were significantly decreased(p<0.05).ApoAI as the main component in HDL was significantly increased and A1AT and apoAIV protein levels were significantly decreased(p<0.05).The oil red staining and HE staining of liver showed that hepatic lipid deposition in Lv-GFP-Epk group and Lv-GFP-Epk-MsrA group were decreased.The data of hepatic lipid extraction were also showed that TC level had no difference in three group,however,the contents of hepatic FC and TG were significantly decreased(p<0.05).furthermore,the expression of genes related to lipid metabolism in liver were detected,the expression of FASN in Lv-GFP-Epk and Lv-Epk-MsrA-GFP group were decreased significantly(p<0.05),the mRNA level of ACAT,LXRa,ABCG8,and PON1 increased significantly(p<0.05),while the expression of inflammatory factors TNFa and IL-6 significantly decreased(p<0.05),and the protein levels of ACAT,LXRa,ABCAlwere also confirmed to increased significantly(p<0.05).Conclution We successfully construct pWPI-Epk-MsrA-GFP plasmid through a HDL-binding Epk peptide,and Epk-MsrA could highly screte into plasma and bind with HDL in SR-BI-/-mice.Epk-MsrA increased the antioxidant activity of the circulatory system,reduced the levels of inflammatory factors,improved the lipid metabolism and HDL function and reduced the hepatic lipid deposition in SR-BI-/-mice. |
PDF Full Text Request