Effects Of Serum Amyloid P Component On Atherosclerosis In Apoe^-/- Mice

BackgroundAtherosclerosis(AS)is a chronic pathological process triggered by abnormally deposition of lipids.During the development of atherosclerosis,abundant lipids deposit in the intima of involved artery and form fatty streaks,then fibrous plaque and finally atheromatous plaque.The rupture of plaque can induce thrombosis and incidentally ischemia of relevant tissue.With the development of resident living standards,the morbidity and mortality of atherosclerotic cardiovascular disease(ASCVD)is increasing remarkably and threatening people's health all around the world.Since AS is critical pathophysiologic basis of ASCVD,researchers devote to explore the pathogenesis and therapies of AS.Currently,the most recognized theories of AS include lipid,inflammatory and infection theory.While the pathology of AS is still not well elucidated and more intensive researches are needed.As yet,researchers have looked for variable treatments to AS,however,none of which is quite effective.More efficiently therapeutic targets should be explored.It's known to all that inflammation and immune reaction exist through the development of AS.Abundant inflammatory cells and cytokines have been discovered to infiltrate in the atherosclerotic plaque.Macrophage phagocytosis ox-LDL and promote the genesis of AS.The phagocytic macrophage can secrete IL-6,MCP-1 and other inflammatory cytokines which can accelerate instability of plaque.Thus,anti-inflammatory therapies turn to be essential for anti-atherosclerosis.Serum amyloid P component(SAP)is a kind of glycoprotein with a characteristic pentameric organization and highly preserved in evolution.As a soluble pattern recognition molecule,SAP is an important part of innate immunity.Since SAP mainly combines with amyloid deposits,it's discovered in amyloidosis diseases at the beginning.Although both SAP and CRP belong to short pentameric protein superfamily,they play different role in inflammation and innate immunity.In recent years,it was found that,besides Alzheimer,SAP is closely related to unstable angina(UA)and myocardial infarction(MI).On the other hand,SAP is also related to Type 2 Diabetes Mellitus.Epidemiological evidence show that serum SAP is remarkably higher in CHD patients than normal.Mass of SAP was detected within human atherosclerotic plaque.Abundant evidence shows that SAP plays an important role in atherosclerosis,while the specific function and mechanism of SAP in AS is still unclear.ObjectivesOur study aims to observe the effect of SAP on atherosclerosis in ApoE-/-mice through intraperitoneal injection of exogenous SAP.Meanwhile we aim to explore the effect of SAP on serum lipid,inflammatory cytokines and cholesterol export rate of macrophage and further to explore the possible mechanism.Methods1.Groups and interventionsTwelve 8 weeks old male C57BL6 mice were fed with chow diet as the normal group.Twenty-four 8 weeks old ApoE-/-mice were fed with high-fat diet.At the 12th week of experiment,twelve ApoE-/-mice were randomly divided into two groups:PBS group and SAP group.The mice in SAP group were given intraperitoneal injections of SAP(6mg/g)every other day and totally for 2 weeks.The mice in normal and PBS group were injected with PBS of the same volume every other day and totally for 2 weeks.The normal group was continually fed with chow diet while the PBS and SAP group with high-fat diet until the sixteenth week.2.Specimen CollectionAt the 16th week of experiment,mice were anaesthetized with diethyl ether after 12 hours of empty stomach.Blood samples were obtained and centrifuged for 10min at 3000r/min.The blood vessels were washed with PBS and fixed with 4%paraformaldehyde.Then the aorta was separated with general microscope from the start of the ascending aorta to common iliac artery aortic bifurcation.Some aortic root was immediately stored at-20? to produce frozen sections for Oil Red O staining.The other was fixed in 4%paraformaldehyde to make paraffin sections for Hematoxylin-eosin(HE)staining and immunohistochemical detection.3.Detection of Serum LipidsTake out the cryopreserved mice serum specimens and detect the levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)using enzymatic kit according to manufacturer's instruction.4.Assessment of Serum PON1 Activity and Macrophage Reverse Cholesterol Export CapacitySerum PON1 activity was detected with Phenyl acetate as a substrate;Macrophage Reverse Cholesterol Export Capacity was measured using[3H]cholesterol in liquid flash counter.5.Detection of Serum Inflammatory Cytokines IL-6,IL-10,MCP-1 and SAPSerum IL-6,IL-10,MCP-1 and SAP were detected with enzyme-linked immunosorbent assay(ELISA)kits according to manufacturer's instruction.6.Quantification of atherosclerotic plaquesHE staining was used to observe the formation of atherosclerotic plaques.Oil red O staining was performed to observe lipid accumulation within atherosclerotic plaques.7.Immunohistochemistry of CD68 Positive Macrophages and SAPImmunohistochemistry was performed to detect the CD68 positive macrophage and SAP expression in atherosclerotic plaques.8.Statistical AnalysisAll measurement dates are presented as mean ±standard deviation(X±SD)and analyzed using SPSS 20.0 software.Independent sample t-test was used to compare means between two groups.One-way ANOVA was used to analyze means among three groups.Welch calibration test was used for variance heterogeneity.Post-hoc comparisons-were made using least significant difference(LSD)test.Dunnett's T3 test was used when the variances are heterogeneities.P<0.05 represented a statistically significant difference.Results1.Comparison of serum TC,TG,LDL-C and HDL-CCompared with normal group,TC,TG and LDL-C levels were significantly increased in the PBS and SAP group,while HDL-C level significantly decreased(P<0.05).But no statistical significance was observed between PBS and SAP group in TG,TC,LDL or HDL-C level.2.Comparison of serum PON1 activity and cholesterol efflux rateCompared with normal group(146.3±28.3 ku/L),serum PON1 activity markedly decreased in both PBS(62.1±21.7 ku/L,P=0.000)and SAP group(95.8±29.7 ku/L,P=0.005).Compared with the PBS group,SAP can increase PON1 activity slightly(P=0.046).Compared with normal group(13.7±2.5%),macrophage cholesterol efflux rate significantly decreased in PBS(7.2±1.8%,P=0.000)and SAP group(9.4±1.7%,P=0.000).Compared with the PBS group,SAP can increase macrophage cholesterol efflux rate significantly(P=0.017).3.Comparison of serum IL-6,IL-10,MCP-1 and SAPCompared with normal group(14.4±3.4 pg/ml),serum levels of IL-6 elevated significantly in PBS group(21.3 ±1.8 pg/ml,P=0.000)and SAP group(23.9±1.9 pg/ml,P=0.000).Compared with the PBS group,serum IL-6 obviously increased in SAP group(P = 0.025).Serum levels of IL-10 were decreased significantly in PBS group(106.7±20.9 pg/ml,P=0.000)while no significant difference in SAP group(153.4±57.0 pg/ml)compared with normal group(199.3±58.2 pg/ml).Serum IL-10 obviously increased in SAP group compared with the PBS group(P=0.012).Serum levels of MCP-1 were significantly higher in PBS(689.8±274.8 pg./ml,P=0.000)and SAP group(465.4±63.4 pg./ml,P=0.003)compared with normal group(125.0±45.5 pg/ml).Compared with the PBS group,serum MCP-1 was obviously less in SAP group(P=0.032).Serum expression of SAP was detected using ELISA kits,and the normal group of mice(15.8±8.1ug/ml)showed the lowest level of SAP,followed by the PBS group(83.2±39.5ug/ml).However,there was no significant difference between PBS and SAP group(95.6±39.1 ug/ml).4.Quantification of atherosclerotic plaquesHE staining showed that plaque area in SAP group(14.5±5.3%)was significantly less than PBS group(23.2±7.2%,P=0.039).Compared with the PBS group(7.2±2.1×105 um2),SAP can remarkably alleviate Oil red O positive area in aortic sinus(5.5±1.4×105 um2,P=0.028).Oil red O staining of whole aorta showed that SAP mitigated plaque development compared with PBS(4.1 ±1.6%vs 7.6±2.8%,P=0.023).5.Detection of CD68 positive macrophages and SAP in atherosclerotic lesionsImmunohistochemistry showed that SAP obviously alleviated CD68 positive macrophages infiltration within atherosclerotic plaques(18.0±3.7%vs 28.4±7.1%,P=0.01).Immunohistochemistry revealed higher percentage of SAP to total plaque area in normal SAP group(2.14±0.45%)of mice than that of PBS group(1.62±0.14%)(P=0.034).Conclusion1.SAP improved HDL function,increased serum PON1 activity,and promote macrophage cholesterol efflux rate,which can delay the development of AS.2.SAP played an important role in inflammatory reaction.SAP increased IL-10 and inhibited MCP-1 to exert anti-inflammatory function.Meanwhile,SAP also upregulated pro-inflammatory factor IL-6 expression.3.SAP alleviated CD68 positive macrophage infiltration within plaque,reduced the formation of foam cells from macrophage.4.SAP can effectively mitigate the development of atherosclerosis.5.SAP can improve HDL function,upregulate IL-10,down-regulate MCP-1 and alleviate CD68 positive macrophage infiltration,thus exerts an anti-atherosclerosis property.