Evaluation Of The Role Of Epitopes Of Perilipin On The Formation Of Atherosclerotic Lesion In ApoE^-/- Mice

BackgroundAtherosclerosis,a chronic inflammatory disease of the dysfunction of the immune system is a main cause that result to a worse consequence of ischemic cardiovascular or cerebrovascular disease.Many more studies have confirmed that vaccination of animal with atherosclerosis-related antigen or derivatives could reduced the extent of the lesions in atherosclerosis-prone mice.And the in vivo and in vitro studies have identified that perilipin take the role of "molecular switch" in the fat metabolism.What's more,the extent of lesions in perilipin gene knockout mice have also reduced.We hypothesized that vaccination with KLH-conjugated peptides from the major outer membrane protein of perilipin could inhibit the progress of atherosclerosis in atherosclerosis-prone mice.ObjectiveIn this study,we investigate the antigenic effect of the peptide of preilipin on atherosclerotic lesion formation and the involved mechanism in ApoE^-/-mice infected with perilipin.Methods1.Searching for the perilipin polypeptide sequence in The National Center for Biotechnology Information?NCBI?and screening out the possible epitopes.Synthesis of perilipin polypeptide sequence,keyhole limpet hemocyanin?KLH?and KLH coupled perilipin polypeptide?KLH-perilipin peptide?.2.The male ApoE^-/-mice of 6 weeks old were divided into normal group?group NC n=10?,atherosclerosis model group?group AS,n=10?and vaccine immunization group?VA group,n=10?.The immune antigen in normal group and atherosclerosis model group is KLH,240 ul antigen soluble emulsion?20 ?g KLH+120 ?l Freund's adjuvant + 120?l physiological saline solution?were given to ApoE^-/-mice in abdomen and back of 12 sites by the subcutaneous injection.The vaccine group were injected with KLH-perilipin in the same way.Mice were given injection 4 times at the day 3,6,9,and 12 after the initial immunization.Complete adjuvant was adopted in the first and second immunization,and then incomplete adjuvant were used.3.Blood from eye socket was used to detect the specific antibody titers after two weeks of all immunizations.High fat diets were given to AS and VA groups,but not for the negative control group.Monitoring the changes of mice body weight until week 14.4.14 weeks after the first immunization,The levels of total cholesterol,triglyceride,high-density lipoprotein cholesterol?HDL?,and low density lipoprotein,?LDL?in serum samples were detected by ELISA kits.Compared the AS group of mice with NC group of mice,we randomly kill 3 AS group of mice to check whether the blood lipid result was significant.If there are a large number of aorta lipid plaque deposition,indicating that the success of the modeling.Finally all the mice were killed by removing eyeball and then harvest their plasma,heart and aorta.5.The IFN-?,TGF-?,TNF-?,IL-6,ox-LDL,IL-10 levels in the plasma samples were measured by ELISA following the manufacturer's instructions.6.The Bifurcation of common iliac artery to aortic root were dissected and expressed as the percentage of AS plaque occupied areas against the entire areas of aortic lumen via the oil red O staining.Aortic root of mice were entrapped in OCT gel as frozen sections.HE staining was used to observe the changes of aortic root morphology;Oil red O staining was performed to analyze the percentage of the aortic root plaque in all lumen area;EVG staining was used to check the ratio of elastic fibers in aortic root;Immunofluorescence was carried out to detect the relative content of inflammatory cytokines in AS plaque.Results1.Degree of specificity of antibody detection: 2 weeks after immunization and 14 weeks at the end of experiment,perilipin peptide-induced specific antibody in serum were increased significantly in VA group compared with either NC group or the AS group?P<0.05?.1.Serum lipid profile test: after the success of the modeling,TC,TG,and LDL were significantly increased,and HDL were significantly decreased?P<0.05?in serum of AS group compared to those in NC group.TC and LDL levels were significantly lower and HDL was significantly higher?P<0.05?in serum of VA group compared with those in AS group,in spite of that no significant difference was found on TG levels?P > 0.05?.2.Cytokines in serum detection: compared with NC group,AS group TGF-? content in serum no statistical difference?P > 0.05?,while TNF-?,ox-LDL,IL-6,IL-10,IFN-?has obvious increase,significant difference statistically significant?P<0.05?;Three inflammatory factors including TNF-?,oxLDL,IFN-? were significantly decreased,in contrast,two protective cytokines TGF-?,IL-10 were significantly increased in serum of VA group when compared with those in AS group?P<0.05-0.001?,however,there is no significant difference for IL-6 levels observed?P > 0.05?.3.Pathology examination: compared with NC group of ApoE^-/-mice,the high-fat feed the AS group and VA group fed with high-fat diet for 12 weeks,plaques occupied areas in VA group were significantly reduced in the descending aorta and aortic root showing 48.42% and 40.86% respectively?P<0.05?when compared with those in AS group.However,no significant difference was found on the ratio of elastic fiber?P > 0.05?.4.Immunofluorescence staining results showed that VA patchs CD4,CD11 c,CD68,TLR-4,MyD88 were found significantly lower percentage in contrast,foxp3 levels were significantly increased when compared to those in AS group?P<0.05?.Conclusion1.The amino terminal end of perilipin?1MSMNKGPTLLDGDLPEQ17?is an effective antigen epitope.2.The KLH-perilipin peptide vaccination can effectively reduce the lipid deposition in the aorta of ApoE^-/-mice which caused by feeding with high-fat diet.3.The mechanism of reducing atherosclerotic lesion may be due to the downregulation of TLR-4/MyD88 signalling pathway.