Research On The Effect Of Chronic Inflammation On The Expression Of Endothelial Lipase In Aorta Of Mice

1.IntroductionCoronary heart disease(CHD) and cerebrovascular disease have become one of the biggest threaten of public health.There are many factors contribute to CHD.among them.Atherosclerosis is one of the most important one.It is widely accepted that dyslipidemia and inflammation are two causal risk factors of the development of atherosclerosisAlthough they are two individual risk factors of atherosclerosis,many studies have suggested that there is an interactive relationship between them,which has direct consequences for the development of atherosclerosis.Cholestrol,fatty acids and some modified lipids can directly activate the inflammatory cells and some inflammatory pathways.A long time cholestrol diet can increase the levels of circulating C-reactive protein(CRP),serum amyloid A(SAA) and some cytokins,which is so called "metabolic inflammation".Although the exact mechanisms of this effect are still under investigation,it may be partly caused by the increased translocation of lipopolysaccharide(LPS) from the gut into the circulation.Researches of hyperlipidemic mouse models showed that high circulating levels of chylomicrons,(V)LDL and their metabolic products lead to hepatic inflammation.In addition,circulating modified lipoproteins can enhance the vacular inflammation by activating some inflammatory signal pathways,such as NF-κB pathway and JNK pathway.On the other hand,observations in patients have showed that acute inflammation caused by bacterial or viral infection is accompanied by temporary hyperlipidemia,which is considered to play a vital role in the defense of the body to the infection.However,these changes during the chronic inflammation are always been prolonged and likely contribute to the development of atherosclerosis.Therefor,the interaction of dyslipidemia and inflammation can accelerate the progress of atherosclerosis.Endothelial lipase (EL) is a member of the lipoprotein lipase(LPL) gene family.Similar to hepatic lipase(HL) and lipoprotein lipase(LPL),the other two members of this family,EL also play an important role in lipid metabolism.Endothelial lipase has an activity of phospholipase and is a key enzyme that modulates HDL-C metabolism.EL is mainly synthesized by endothelial cells,besides that it is also expressed to a less extent in hepatic cells,smooth muscle cells and macrophages.HDL-C is an protective factor to a body during the process of atherosclerosis.In that case,EL inactivation may inhibit the progress of atherosclerosis.EL expression is regulated by a lot of factors,in which inflammation is one of the most important one.Therefor,further investigation of the mechanisms by which inflammation regulates EL expression can help us to build a new bridge between dyslipidemia and inflammation,so as to provide us a new sight of researches and interventions of atherosclerosis.lipopolysaccharide(LPS) is an important inflammatory stimulus which can act selectively on Toll-like receptor 4(TLR4) on the cell surface to activate intracellular inflammatory signal pathways,such as NF-κB pathway.Previously studies have suggested that LPS can promote the progress of atherosclerosis of human and animals.NF-κB and p38MAPK signal pathways are two important pathways in inflammatory diseases and may be involved in the progress of atherosclerosis caused by some risk factors.In this study,wild type C57BL/6 mice and ApoE-/-mice were given an intraperitoneal injection of LPS in order to investigate the effect of LPS to the expression of EL,NF-κB , p38MAPK and phosphorylated p38MAPK in aorta under circumstance of chronic inflammation.Atorvastatin was give by oral gavage so as to inhibit this effectln addition,PDTC and SB203580,the inhibitors of NF-κB and p38MAPK pathway were given by an intraperitoneal injection accompanied with LPS injection to further confirm that LPS regulates the expression of EL in aorta partly through NF-κB and p38MAPK pathway.2.Methods2.1 Animal modelsFourty 8-week-old male C57BL/6 mice and eight 8-week-old male ApoE-/-mice were obtained from the Vitalriver company in Beijing and all mice were given a high-fat diet(0.25% cholesterol and 15% cocoa butter).Mice were randomized to 6 groups(LPS(C57BL/6)group,LPS(ApoE-/-) group,atorvastatin group,normal control group,PDTC group and SB203580 group)with 8 mice of each group.Except normal control group,mice in other 5 groups were all given an intraperitoneal injection of LPS(2mg/kg)three times a week for 12 weeks.Besides,mice in PDTC and SB203580 groups were also given an intraperitoneal injection of PDTC(20mg/kg/d) and SB203580(5ug/kg/d).Daily administration of atorvastatin by oral gavage were given to mice in atorvastatin group for 12 weeks.All mice were sacrificed at the end of the study.2.2 Serum lipidsMice in the first four groups were fasted overnight and blood samples were collected before sacrificed.Serum levels of total cholesterol(TG),triglyceride(TC),low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol were detected.2.3 Serum levels of TNF-α,IL-1βand ANGPTL3ELISA kits were used to detect serum levels of TNF-α, IL-1β and ANGPTL3.The absorbance at 450nm was determined using a microplate reader.2.4 H&E stainingSections with the length of 1-1.5 cm from the arch to the descending aorta were removed,dehydrated and embedded in paraffin,and then were cut into 5um sections.Sections were stained with hematoxylin and eosin(H&E).2.5 Immunohistochemical AnalysesImmunohistochemistry staining was used to analyze the expression of EL in atherosclerotic plaques.The anti-EL antibody was diluted 1:200. Image pro-plus software was applied to analyze the data.2.6 Western BlotsTotal proteins were extracted from the aorta by lysis buffer(PMSF was added in it). The expression of EL(diluted 1:800),NF-κB p65(diluted 1:1000),p38MAPK(diluted 1:1000),and phosphorylated p38MAPK(diluted 1:1000) proteins were detected by Western Blots.Relative protein levels were measured by Image J software.2.7 StatisticsData were presented as means±SD.Group differences were analyzed by Student’s t test and one-way analysis of variance(ANOVA).All statistical analysis were performed by use of the Graph Prism5 program.p<0.05 was considered statistical significant.3.Results3.1 Serum lipidsCompared with normal control group,LPS can elevate serum levels of total cholesterol(TG) and lower serum levels of high density lipoprotein cholesterol(HDL-C) of mice in LPS(C57BL/6) group and LPS(ApoE-/-) group.(p<0.05)Administration of atorvastatin can inhibit this effect.3.2 Serum levels of TNF-a and IL-1βCompared with normal control group, LPS elevated the serum levels of TNF-a and IL-1β of mice in LPS(C57BL/6) group and LPS(ApoE-/-) group.(p<0.05)Administration of atorvastatin can inhibit this effect.3.3 Serum levels of ANGPTL3ANGPTL3 has been recognized as an inner inhibitor of EL.Results showed that serum levels of ANGPTL3 of mice in LPS(C57BL/6) group and LPS(ApoE-/-) group were decreased with the stimuli of LPS compared with normal control group.(p<0.05)Administration of atorvastatin can inhibit this effect.3.4 Expressions of EL proteins in aortaLPS increased the expressions of EL proteins in aorta of mice in LPS(C57BL/6) group and LPS(ApoE-/-) group,when compared with normal control group.The relative quantity of EL proteins of LPS(C57BL/6) group,LPS(ApoE-/-) group and NC group were 143.2±9.2,222.9±9.1,45.3±17.2, p<0.05.Administration of atorvastatin can inhibit this effect.3.5 Expressions of NF-κB p65,p38MAPK and p-p38MAPK proteins in aortaLPS increased NF-κB p65,p38MAPK and p-p38MAPK proteins in aorta of mice in LPS(C57BL/6) group and LPS(ApoE-/-) group,when compared with normal control group.(p<0.05)Administration of atorvastatin can inhibit the activation of p38MAPK pathway but merely have no effect on NF-κ B pathway.3.6 PDTC stimulation reduced the expression levels of ELPDTC inhibited the increased expression of EL in aorta induced by LPS.The expressions of EL proteins in aorta of mice in PDTC group were reduced compared with LPS(C57BL/6) group.(p<0.05).3.7 SB203580 stimulation reduced the expression levels of ELSB203580 inhibited the increased expression of EL in aorta induced by LPS.The expressions of EL proteins in aorta of mice in SB203580 group were reduced compared with LPS(C57BL/6) group.(p<0.05).3.8 HE staining and immunohistochemistryHE staining of sections at the level of arch revealed that the aorta intima was smooth and endothelial cells were continuous with small intervals and presented normal configuration without edema in mice of normal control group. ApoE-/-mice developed severe atherosclerosis predominantly both in the intimal area and in the media. Disorders and disruption of elastic fiber were observed in the media layer. The intimal layer became thick and upheaved, and some endothelium cells were detached with widened intervals in ApoE-/- mice.Compared with normal control group, ApoE-/- mice demonstrated a marked increase of EL protein expression localized predominantly in the intimal area adjacent to the media, especially the atherosclerotic lesions, represented by brown color.4.Conclusions(1) LPS can elevate the serum levels of TNF-a and IL-1β of mice and the chronic inflammation induced by LPS increases the expression of EL in aorta of C57BL/6 mice and ApoE-/- mice.(2) The effect that LPS increases the expression of EL in aorta may be caused partly by the activation of NF-κB and p38MAPK pathways.(3) LPS may increase the expression of EL in aorta of mice by inhibiting the expression of ANGPTL3.(4) Atorvastatin can inhibit the increased expression of EL in aorta of mice induced by LPS partly by inhibiting the activation of p38MAPK pathway.(5)Endothelial lipase may accelerate the development of atherosclerosis in aorta of ApoE-/-mice by the activation of NF-κB and p38MAPK pathways.