| BackgroundAtherosclerosis (AS), which is a chronic inflammatory disease initiated by endothelial dysfunction and abnormal lipid metabolism, can lead to coronary artery disease (CAD), stroke and other diseases threatening human health seriously. High-density lipoprotein-cholesterol (HDL-C) is an independent protective factor for CAD. Clinical and epidemiological studies have shown that plasma HDL-C concentration is inversely correlated with the incidence of coronary CAD. As multifunctional protein complexes, HDL is thought to protect against AS by promoting reverse cholesterol transport (RCT), inhibiting the oxidative modification and vascular inflammation, inhibiting thrombosis, enhancing endothelial repair, regulating plasma glucose levels and suppressing the proliferation of hematopoietic stem cells. The most important atheroprotective function of HDL is the promotion of reverse cholesterol transport as well as anti-oxidant and anti-inflammatory.However, accumulating evidence has demonstrated that the level of HDL-C cannot represent the whole function of HDL accurately, and quantifying HDL-C concentration alone only provides limited information about HDL’s cardioprotective effects. Structural modification and compositional alteration of HDL particles as a result of chronic inflammation and acute phase responses may adversely affect or reverse their normal biological function. Structural modifications of HDL may reduce the efficiency of RCT, HDL’s anti-inflammatory and anti-oxidative effects, and even convert HDL into a pro-inflammatory, pro-oxidative and inhibiting RCT agent which may subsequently promote the development of atherosclerosis. Therefore, it is more important to pay close attention to the change of HDL properties than HDL-C levels to investigate the association between HDL and CAD risk. Khera etc in2011has reported that cholesterol efflux capacity from macrophages, a metric of HDL function, has a strong inverse association with CAD, independently of the HDL-C level. Visibly, the assessment of cholesterol efflux capacity to evaluate the change of HDL function and development of atherosclerosis is very important.Studies have shown that inflammation and immune response plays an important role in AS. HSP65, a major autoantigen, has strong immunogenicity and contributes to the initiation and development of autoimmunity and atherosclerosis. Numerous studies have demonstrated that immunization with mycobacterial HSP65administered subcutaneously increased levels of proinflammatory cytokines and accelerated atherosclerosis in mice, indicating that HSP65could promote the development of AS through inflammatory immune response. It has been reported that inflammation could induce the change of HDL function, however, it remains unclear whether HSP65could influence RCT, anti-oxidant and anti-inflammatory functions of HDL.In our study, we investigated whether subcutaneous immunization with different dose of HSP65would influence the properties of HDL in Apolipoprotein E knockout (Apoe-/-) mice. Furthermore, we explored the underlying relationship among HSP65-specific immune response, the function of HDL and atherosclerosis.ObjectivesWe set out to investigate whether the anti-inflammative and anti-oxidative functions of HDL and cholesterol efflux rate are impaired in atherosclerosis ApoE-/-mice by subcutaneous immunization with different dose of HSP65. What’s more, we explore the mechanism of the change of cholesterol efflux rate influenced by HSP65. Methods1. Experimental animalsEight8weeks old C57BL/6J mice were fed with regular diet as a normal group. Twenty four8weeks old ApoE-/-mice fed with high-fat Western-type diet were randomly divided into three groups:phosphate-buffered saline (PBS) group,5μgHSP65group and25μgHSP65group. The ApoE-/-mice were boosted twice with either PBS or HSP65(5μg and25μg) under the same protocol following three and six weeks. All mice were sacrificed at16weeks.2. Serum lipid analysisMice were anaesthetized by ethyl ether at16weeks end of the experiment. Blood samples were obtained by cardiac puncture. Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (HDL-C) concentrations were measured by using an automated biochemical analyzer.3. Detection of anti-HSP-65antibodies, IL-10and IFN-ySerum anti-HSP-65antibodies were measured using enzyme-linked immunosorbent assay (ELISA). Serum IL-10and IFN-y were quantified by ELISA kits (Assay, USA) according to the manufacturer’s instruction.4. Assessment of the anti-oxidant and anti-inflammatory function of HDL and cholesterol efflux rate from macrophagesAt16weeks, blood samples were obtained. Serum PON1activity was measured by using phenylacetate as a substrate. Serum MPO activity were tested by chromatometry kit according to the manufacturer’s instructions. High-density lipoprotein inflammatory index (HII) was determined by the CFA assay.[3H] labeled cholesterol efflux rate was evaluated by liquid scintillation spectrometry.5. Detection of SR-B1, ABCA1, ABCG1, PPAR-y and LXR-a mRNA in liver tissuesThe total RNA was isolated from mice liver by a Trizol reagent. Quantitative real-time PCR was performed to detected SR-B1, ABCA1, ABCG1, PPAR-y and LXR-a mRNA expression in liver.6. Detection of SR-B1, ABCA1, ABCG1, PPAR-y and LXR-a protein expression in liver tissues and peritoneal macrophagesThe liver tissues and peritoneal macrophages were lysised with RIPA lysis buffer. BCA protein assay kit was used to measure protein concentration. The expressions of SR-B1, ABCA1, ABCG1, PPAR-y and LXR-a were detected with Western blot.7. Assessment of atherosclerosisAt the end of16weeks, after sacrifice of mice, the entire aorta was moved, fixed in10%neutral buffered formaldehyde solution. Cross-sections of the artery were evaluated for atherosclerotic plaques after staining with hematoxylin and eosin (H&E). HE-stained sections were examined under microscope and atherosclerotic plaques were quantified by Image Pro Plus6.0.8. Statistical analysisDatas are presented as mean±SD. SPSS13.0software was used for statistical analysis. Independent sample t-test was used for analyzing the differences in variables between two groups at the same time point. One-way ANOVA was used for analyzing differences in variables between groups at the same time point. Post-hoc comparisons were made among the various groups using least significant difference (LSD) method. When variances are not homogeneity, Welch method was used for analyzing differences in variables between groups at the same time point, and Dunnett’s T3method was used for Post-hoc comparisons. P<0.05indicated a statistically significant difference. Results1. Serum lipid levels in experimental miceThe levels of serum TC, TG, and LDL-C in three treatment groups, i.e. PBS,5μgHSP65and25μgHSP65, were increased compared with normal group, while HDL-C level decreased. Serum HDL-C in25μgHSP65group, was decreased compared with PBS or5μgHSP65(P<0.05), but no significant differences statistically were observed between PBS and5μgHSP65groups. The three treatment groups did not differ in serum TC, TG, and LDL-C levels.2. Comparison of antibodies to HSP65and cytokine assayThe anti-HSP65antibodies level in the normal group was similar to PBS group (0.19±0.03vs0.22±0.05, P>0.05). Anti-HSP65antibodies level in the5μgHSP65group was significantly higher than levels in the PBS group(0.55±0.15vs0.22±0.05, P<0.05); In25μgHSP65group, anti-HSP65antibodies level was markedly elavated in comparison with5μgHSP65group(1.08±0.24vs0.55±0.15, P<0.05).There was no significant difference in IL-10and IFN-y levels between the PBS group and the normal group [IL-10:(338.54±45.16)ng/L vs (350.98±56.87)ng/L, P>0.05; IFN-y:(121.01±21.48)ng/L vs (116.91±18.06)ng/L, P>0.05];25μgHSP65group had less IL-10levels than PBS or5μgHSP65group [(253.76±45.06)ng/L vs (338.54±45.16)ng/L,(253.76±45.06)ng/L vs (303.55±42.47)ng/L, P<0.05], but no significant differences statistically were observed between5μgHSP65and PBS groups[(303.55±42.47)ng/L vs (338.54±45.16)ng/L, P=0.15].5μgHSP65and25μgHSP65group both had higher IFN-y levels than PBS group [(139.34±11.43)ng/L vs (121.01±21.48)ng/L, P=0.04;(185.06±13.80)ng/L vs (121.01±21.48)ng/L, P<0.05].3. Comparison of cholesterol efflux rate and HDL anti-oxidant and anti-inflammatory functionsCompared with normal or PBS-treatment group, HSP65(5μg or25μg) markedly decreased cholesterol efflux rate and increased HII in a dose-dependent manner (P<0.05). In HSP-65immunized mice, only the group immunized with the higher dose (25μg) developed significantly higher MPO activity and lower PON1activity compared with PBS-treatment group. Although5μgHSP65immunized mice produced a slight increase in the MPO activity level and reduction in the PON1activity relative to the PBS-treatment group, the effect were not significant (P>0.05).4. Cholesterol transport proteins including SR-B1, ABCA1, ABCG1, PPAR-y and LXR-a mRNA expressions in hepatocytesThe results showed that compared with normal group, the expressions of cholesterol transport proteins mRNA were significantly decreased in PBS group (P<0.05). Compared with PBS group,5μgHSP65group showed significantly lower levels of SR-B1, ABCG1and PPAR-y mRNA (P<0.05), while there were no significant differences in the expressions of ABCA1and LXR-a mRNA between PBS group and5μgHSP65group (P>0.05).25μgHSP65group had decreased cholesterol transport proteins mRNA levels when compared with PBS group (P<0.05). Compared with5μgHSP65group,25μgHSP65group showed significantly lower levels of ABCG1, PPAR-y and LXR-a mRNA (P<0.05).5. The protein expressions of SR-B1, ABCA1, ABCG1, PPAR-y and LXR-a in hepatocytesCompared with normal group, SR-B1, ABCA1, ABCG1, PPAR-y and LXR-a protein expressions were significantly decreased in PBS group (P<0.05); Compared with PBS group, SR-B1, ABCA1and ABCG1protein expressions were significantly reduced in5μgHSP65group (P<0.05), while there were no significant differences in the expressions of PPAR-y and LXR-a between the two groups (P>0.05). Compared with PBS group,25μgHSP65had markedly decreased cholesterol transport proteins levels (P<0.05). Compared with5μgHSP65group,25μgHSP65group showed significantly lower levels of SR-B1, ABCA1, PPAR-γ and LXR-α protein expressions (P<0.05). 6. The protein expressions of SR-B1, ABCAl, ABCGl, PPAR-y and LXR-a in peritoneal macrophagesCompared with normal group, PBS group showed significantly lower levels of SR-B1, ABCA1, PPAR-y and LXR-a protein expressions in peritoneal macrophages (P<0.05). Compared with PBS group, SR-B1, ABCA1, ABCG1and PPAR-y protein expressions were significantly decreased in5μgHSP65group (P<0.05), while there was no significant difference in the expression of LXR-a between the two groups (P>0.05). Compared with PBS group, cholesterol transport proteins levels were significantly decreased in25μgHSP65group (P<0.05).25μgHSP65group showed significantly lower levels of ABCA1, ABCG1and LXR-α protein expressions than5μgHSP65group (P<0.05).7. Comparison of atherosclerotic lesionsThe size of the lesions in the aortic root in mice immunized with HSP65at25μg (385,121±97,695μm2) or with HSP65at5μg (221,636±86,752μm2) was significantly larger than in the PBS-immunized groups (88,919±46,369μm2; P<0.01). In the HSP65immunized mice, the group injected with the higher dose (25μg) developed significantly larger lesions compared with5ug HSP65immunized litter mates (P<0.01). Conclusion1. HSP65administered subcutaneously increased levels of proinflammatory cytokines and accelerated atherosclerosis in mice, indicating that HSP65could promote the development of AS through inflammatory immune response.2. Subcutaneous immunization with HSP65impaired HDL anti-inflammatory and antioxidant function by increasing MPO activity and inflammatory index, and by supressing serum PON1activity.3. HSP65decreased the expression of cholesterol transport proteins in macrophages and hepatocytes, which hindered the process of RCT and speed up the progression of atherosclerosis. |
PDF Full Text Request