| BackgroundWith the development of society and changes in lifestyle, a large amount of fat intake cause the increasing incidence of obesity and its related metabolic disorders, especially type2diabetes mellitus year by year,which are seriously endangering people's physical and mental health.Obesity and diabetes may cause a series of changes in physiological functions,of which its macrovascular complications such as atherosclerosis is a direct threat to life, thereby increasing the risk of all-cause death.Traditional treatments including dietary control, physical exercises,medicine and surgery,are more or less in limit due to maintain difficulty,poor compliance,indefinition on surgical complications and long-term effect. Therefore, search of new therapies is absolutely necessary.Lots of evidence has shown that adipose tissue is not only a energy store but also an important endocrine organ,which releases large amounts of bioactive substances into circulation.These substances then coordinate energy metabolism, insulin sensitivity, inflammation and vascular reaction through local and systemic action. Adipocyte fatty-acid-binding protein(A-FABP,FABP4,ap2),belonging to FABP family,is one of these substances,which expresses mainly in adipocytes and macrophages, and also can be secreted into circulation.It participates transport and synthesis of free fatty acid(FFA) and other kinds of lipid hormones,thus regulates systemic insulin sensitivity and energy metabolism, the aggregation of inflammatory cell factors and cholesterol esters in macrophage.Studies in zoopery and clinic have proved the level of FABP4is closely related to the development of hyperglycemia, hyperinsulinism, hyperlipoidemia,metabolic syndrome and atherosclerosis,which are pathologic status of obesity and metabolic disturbance. FABP4-deficient mice and mice taking small-molecule inhibitor of FABP4have shown almost complete resistance to dietary or genetic obesity-induced insulin resistance, Dyslipidemia, atherosclerosis,fatty liver disease,and so on.Therefore,researchers insist that inhibiting the function of this lipid chaperone can significantly improve several aspects of metabolic syndrome.FABP4may be a new target to the therapies of obesity,diabetes and atherosclerosis.However, gene knock-out technique is difficult and infeasible,and taking inhibitor is also of poor compliance.Objective Based on the theory and practice above,here we first constructed vaccines against FABP4with different carriers,and immunized wild-type and gene-deficient mice with high-fat diet respectively. Immune effects and security were observed in order to supply theoretical and experimental evidence in new prevention and treatments of high-fat-induced insulin resistance and its macroangiopathy complications.Methods1,First, biosynthesis method was used to obtain depurated mouse FABP4,as one of the vaccines.In order to enhance the immunogenicity,two types of carriers:Virus-like particles (VLPs) formation by modified Hepatitis B virus core (HBc) and keyhole limpet hemocyanin (KLH), acting as a kind of immune-enhancing vaccine carrier, were chosen and fused with FABP4.Thus we have constructed in all three types of vaccines,HBc-FABP4,KLH-FABP4,FABP4,respectively.2,These three vaccines were used to immunized wild-type mice as experimental groups,with their corresponding carriers and adjuvant as control groups.The security and tolerance were observed,and the best immunizing dose and interval were researched.ELIS A was used to test the antibody titer of each group.3,The best strategy of immunization was selected to immunize wild-type C57BL/6J mice with high-fat diet.Body weight,food-intake and fasting blood glucose were measured during the whole immune period.After16weeks of the first vaccination,IPGTT was performed on each mouse,and the levels of serum insulin and adiponectin were tested.Then the mice were executed and frozen sections of their livers were made.Oil red O staining and imag-pro plus were used to compare the areas of fat deposition.4,The best strategy of immunization was also selected to immunize Apo E-/-mice with high-fat diet. Body weight,food-intake and fasting blood glucose were measured during the whole immune period. After12weeks of the first vaccination,IPGTT was performed on each mouse,and index related to glucose and lipid metabolism such as the levels of serum insulin,adiponectin,TC,TG,FFA,HDL,LDL were tested.Then the mice were executed and frozen sections of their aortas were made. Oil red O staining and imag-pro plus were used to compare the areas of atherosclerosis lesions.Results:1,Depurant FABP4and immune recombinant HBc protein were successfully expressed by the biosynthesis method.FABP4vaccines attached to the VLPs carrier derived from HBc and to the KLH respectively were successfully achieved.In conclusion,we successfully constructed3types of vaccines against FABP4, which established a good foundation for further immunization in mice.2,After immunized by the3types of vaccinations respectively,mice in every immune group all response with high, peptide-specific IgG antibody titers,which were not observed in control groups injected with carriers or adjuvant.That proved the3types of vaccinations can break the immunologic tolerance to FABP4in the hosts. Among them,the titer induced by FABP4was higher than that induced by HBc-FABP4or KLH-FABP4(P<0.05),and was considered to be the best immune strategy.3-weeks interval was effective,and the antibody titer of each group was stable after the3rd booster.During the immune period,there are some mice both in immune groups and in control groups appeared depilation and subcutaneous induration among injection sites,and no other adverse events or death happened.3,FABP4-immunized wild-type C57BL/6J mice with high-fat diet (treating group) were compared with adjuvant-injected mice with high-fat diet(control group) and mice with normal diet(normal group).In female mice:After16weeks of the first immunization,the body weight in treating group was higher than that in normal group but lower than that in control group (P<0.05). food-intake was higher than that in normal group (P<0.05),and had no difference with that in control group (P>0.05).The levels of fasting blood glucose,fasting insulin, HOMA-IR index,glucose AUC of IPGTT were all lower than those in control group(P<0.05),while adiponectin level was higher than that in control group (P<0.05),and had no differences with those in normal group (P>0.05). In male mice:After16weeks of the first immunization,the body weight in treating group was higher than that in normal group (P<0.05), and had no difference with that in control group (P>0.05). food-intake had no difference with that in either control group or normal group (P>0.05).The levels of fasting blood glucose, glucose AUC of IPGTT were lower than those in control group (P<0.05),while adiponectin level was higher than that in control group (P<0.05),and had no differences with those in normal group (P>0.05).The levels of fasting insulin and HOMA-IR index were higher than those in normal group but lower than those in control group (P<0.05).In both male and female mice,frozen sections of liver showed that the area and IOD of fat disposition in treating group were higher than those in normal group but much lower than those in control group (P<0.05)4,FABP4-immunized male Apo E-/-mice with high-fat diet (treating group) were compared with adjuvant-injected male Apo E-/-mice with high-fat diet(control group). After12weeks of the first immunization,the body weight and food-intake in treating group had no difference with those in normal group (P>0.05).The levels of fasting blood glucose, fasting insulin,HOMA-IR index,glucose AUC of IPGTT,TC,HDL,LDL and TC/HDL were lower than those in control group (P<0.05),while adiponectin level was higher than that in control group (P<0.05) The levels of fasting TG,FFA,LDL/HDL had no difference with that in control group (P>0.05).Frozen sections of aortas showed that the percentage of plaque area to the whole lumens area in treating group was much lower than that in control group(P<0.05)ConclusionIn conclusion,we successfully constructed3types of vaccines against FABP4,which could all induce specific humoral immunity and produce high titer of specific antibody in mice.FABP4vaccination effectively improved the levels of blood glucose,insulin,blood lipid of mice with high-fat diet,and also reduced fat disposition in liver and foam cell aggregation in aortas. Our study further proved the feasibility of treatment targeting to FABP4in high-fat induced insulin resistance and atherosclerosis,and offers theoretical and experimental basis for the brand-new immunologic intervention.
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