Preventive Effect And Its Mechanism Of Mixed Antigens On Atherosclerosis In ApoE^-/- Mice

BackgroundAtherosclerosis(AS)is a large number of atherosclerotic plaques formed at arterial wall site after extensive inflammatory infiltration,which is characterized by long-term,complex and high mortality.It is the main cause of cardiovascular disease.A large number of evidence suggests that the immune system is involved in the pathogenesis of atherosclerosis,active immunization and passive immunization as an immune regulation strategy for the formation of atherosclerosis has a protective effect,which has been confirmed by a large number of researchers,these advances in clinical studies also provide the possibility of immunoregulatory therapy,so atherosclerosis vaccination has also received a lot of attention.Purpose1.Screening the polypeptide vaccine for atherosclerosis prevention by database analysis;2.ApoE-/-mice were used as the research object to observe whether the antigens have the effect of preventing the formation of atherosclerosis and the immune effect of mixed antigens;3.Establish macrophage foam cell inflammation model to explore the molecular mechanism of antigen prevention of atherosclerosis.Methods1.Antigen sequence selection: The protein sequence of hHSP60 and Perilipin was obtained by NCBI,and predicting the polypeptide sequence that may function by online software BLAST,Immunomedicine Group etc.,the hHSP60 peptide sequence is303CGFGDNRKNQL313,the Perilipin peptide sequence is 1CSMNKGPTVLD11.2.Model building: Animal model: the ApoE-/-mice were randomly divided into groups and immunized by subcutaneous injection,the model of atherosclerosis was established after high fat diet;Cell model: The macrophages(RAW264.7),each experimental group to join the corresponding drug treatment,drug dose and mouse injection is equivalent to 20 μg for 24 h,the same amount of saline and same time for control group.3.Experimental test: The expression level of serum lipid and related factors were detected by ELISA,oil red O staining to observe the aortic plaque formation.The following experiments of macrophage were carried out: the formation of foam cells was detected by oil red O staining,the migration ability of macrophages was observed by scratch test,and the expressions of TLR4 and MyD88 in macrophages were detected by Western blotting and immunofluorescence.Results1.Animal experiments:(1)The higher titer of specific IgG can be detected in the serum of 2 weeks and 14 weeks in immunized mice,and lower titer of specific IgG in KLH immunization group;(2)The levels of TC,TG and LDL in the vaccine group were significantly lower,the level of HDL was significantly higher than those in the control group,and the difference was statistically significant(P < 0.05);(3)Similarly,the levels of IL10 and TGF-β in the immunized group were higher,TNF-α and IFN-γ were lower,compared with the control group,the difference was notable and statistically significant(P< 0.05);(4)oil red O staining and quantitative analysis results showed that: the plaque area of the vaccine group was significantly decreased,compared with the control group,the difference was statistically significant(P < 0.05);(5)Moreover,the mixed antigen immune group is better than the single antigen immune group.2.Cell experiments:(1)Macrophage migration ability was measured by scratch healing test.The results showed that the sclerosis was observed in the control group andthe KLH group at 0 ~ 48 h,and the scratches in the OXLDL group and the Mix group were almost unchanged,the hHSP60 group and the hHSP60 group had a certain degree of healing;(2)The formation of macrophage foam-like cells was detected by oil red O staining,results showed that foam cells formed the most after stimulated by oxidized low-density lipoprotein.hHSP60 group,Perilipin group,Mix group can also see a lot of foam cells formation,while the control group and KLH group had nothing;(3)The expression of TLR4,MyD88,IL10,TNF-α,LOX1 and NFkB-p65 protein in treated macrophages were detected by immunofluorescence and Western blotting,it was found that TLR4,MyD88,TNF-α,LOX1 and NFkB-p65 had stronger fluorescence,higher protein expression and lower IL10 protein expression after treatment with OXLDL,hHSP60,Perilipin and their mixtures,grayscale analysis found that compared with control group,OXLDL,hHSP60,Perilipin and its mixture treatment group was statistically significant(P < 0.05).Conclusions1.The hHSP60 and Perilipin peptides selected in our laboratory are effective sequences for the prevention of atherosclerosis;2.To a certain extent,compared with hHSP60 and Perilipin alone immunization,mixed immunization has a better preventive effect;3.hHSP60 and Perilipin can prevent the formation of atherosclerosis,and will affect the migration of macrophages and the ability to differentiate into foam cells,which may be through the TLR4/MyD88 signaling pathway.