Inflammation Stress Exacerbate The Disturbance Of Lipid Metabolism Via LDL Receptor Pathway In APOE/SRA/CD36 Knock Out Mice And The Molecular Mechanism

PARTⅠ: THE ESTABLISHMENT OF INFLAMMATORY MODELObjectives: To establish the low grade, chronic and systemic inflammatory model using casein to induce apolipoprotein E, scavenger receptors class A and CD36 triple knockout (ApoE/SRA/CD36 KO) mice that reproduce the state of human.Methods: 16 male ApoE/SRA/CD36 KO mice were randomly assigned to 2 groups: control group and inflammation group. All mice were fed with Western diet (TD88137, containing 21%fat and 15% cholesterol). The control group was subcutaneously injected with 0.5ml PBS while the inflammation group was injected with 0.5ml casein. After 14 weeks, all mice were sacrificed and collected the serum, liver, kidney and aorta. Then we tested the level of SAA and IL-6 in serum.Results: Contrasted with control group, the level of SAA and IL-6 in inflammatory group was obviously increased. The results were 26.60± 3.24ng/ml vs 14.35±1.73ng/m(lP<0.01),36.37±2.20pg/ml vs 18.02±4.87 pg/ml(P<0.01)respectively.Conclusion: After the injection of casein for 14 weeks, it produced the low grade, chronic and systemic inflammation in ApoE/SRA/CD36 KO mice which reached the require of the study. The inflammatory model has established successfully.PARTⅡ: INFLAMMATION EXACERBATES THE DISTURBANCE OF LIPID METABOLISM AND RELATIVE INDEX CHANGESObjects: To investigate the disturbance of lipid metabolism under low grade, chronic and systemic inflammation by detecting the level of lipid in serum, liver, kidney and aorta of ApoE/SRA/CD36 KO mice; to survey the effect of inflammation on the progress of fibrosis in liver and kidney.Methods: The level of lipid in serum was detected by kits. Parts of the liver, kidney and aorta were embedded in OCT and cut to make frozen sections. Parts were made to paraffin sections through ordinal steps. The lipid accumulation in liver, kidney and aorta was tested by oil red O staining; the lipid content in these tissues was quantified by enzymic method; test and analyze the ratio of the total atheromatous plaque area in aorta root to the perimeter of aorta. The mRNA and protein expression of collagenⅠ(Col-Ⅰ), collagenⅣ(Col-Ⅳ) and fibronectin (Fn) were analyzed by Real-Time Polymerase Chain Reaction (RT-PCR) and immunohistochemistry staining.Results:①Blood level of total cholesterol (TC), high density lipoprotein (HDL), and low density lipoprotein (LDL) in inflammatory group was statistically lower than the control group(P<0.05). While the oil red O staining showed that the lipid accumulation in liver, kidney and aorta was abnormally increased in inflammatory group compared to the control group. The intracellular cholesterol content in liver and kidney of inflammatory group was higher than that of control group, which was consistent with the oil red staining. The ratio of the total atheromatous plaque area in aorta root to the perimeter of aorta showed that the plaque area and the lipid content of aorta were statistically higher in inflammatory group than in control group(P<0.05).②The HE staining showed that there were pathological changes in liver and kidney. Although the protein expression of Col-Ⅰ,Col-Ⅳand Fn was not statistically increased of inflammatory group using immunohistochemistry staining, the mRNA expression of these genes were statistically raised.Conclusion: The low grade, chronic and systemic inflammation exacerbated the disturbance of lipid metabolism in ApoE/SRA/CD36 KO mice. Compared to control group, the level of lipid in serum was lower while the lipid accumulation was increased in liver, kidney and aorta in inflammatory group. Inflammation stress causes the redistribution of cholesterol from serum to tissues. In addition, inflammation potentially induced the progress of fibrosis in liver.PARTⅢ: INFLAMMATION AFFECTS THE MRNA AND PROTEIN EXPRESSION OF LDLR, SCAP, SREBP-2 AND THE MOLECULAR MECHANISMObjects: To investigate if inflammatory stress exacerbates the disturbance of lipid metabolism via low density lipoprotein receptor (LDLr) pathway in ApoE/SRA/CD36 KO mice by detecting the mRNA and protein expression of LDLr, sterol regulatory element-binding protein-2 (SREBP-2), SREBPs cleavage activating protein (SCAP).Methods: The mRNA and protein expression of SREBP-2, SCAP and LDLr were analyzed by Real-Time Polymerase Chain Reaction (RT-PCR) and Western Blot, immunohistochemistry staining.Results: The Real Time PCR showed inflammation increased the mRNA expression of LDLr (4.56 fold), SCAP (3.14 fold) and SREBP-2 (14.72 fold) in liver compared to controls. Immunohistochemical staining also indicated increased proteins expression in the liver, which was consistent with mRNA data.Conclusions: Inflammation up-regulated the mRNA and protein expression of L DLr,SREBP2 and SCAP which were the main elements in LDLr pathway. It certified our hypothesis that inflammation causes the disturbance of lipid metabolism in tissues via LDLr pathway. In addition, inflammation may cause the earlier fibrosis in liver and kidney.