Research On Iron Lipid Metabolic Disorders In Atherosclerosis

Section 1:Research on mechanisms of focal autocrine formation of hepcidin induced iron retention in atherosclerotic plaque macrophagesObjective:To explore the potential mechanisms of iron trapped by autocrine formation of hepcidin in plaque macrophages and its roles in atherogenesis.Methods:Forty female ApoE^–/–mice were randomly divided into two groups with the following diets:1)normal diet?ND?and 2)high fat diet?HFD?.Fifteen female age-matched wild-type C57BL/6J mice?WT?receiving a normal chow diet served as controls.Mice were fed for 16 weeks until sacrificed after an overnight fasting.The aortas were excised for Oil Red O and hematoxylin and eosin?H&E?staining.Using fluorescent double-staining to colocalize macrophages with oxidized low-density lipoprotein?ox-LDL?,Toll-like receptor 4?TLR4?,p-p65,hepcidin and ferritin light chain?ferritin-L?in human and mice atherosclerotic plaques.Quantitative reverse transcriptase?qRT?-PCR was used to quantify messenger RNA?mRNA?levels of TLR4,hepcidin,mannose receptor and ferritin-L.According to the experimental design,hepcidin silencing in cultured RAW264.7macrophages was achieved by an adenovirus interference approach.Macrophages were exposed to ox-LDL alone or together with lipopolysaccharide?LPS,agonist of TLR4?,TAK-242?an antagonist of TLR4?,exogenous hepcidin,ferric ammonium citrate?FAC?,deferoxamine?DFO,iron chelator?,27-hydroxycholesterol?27HC?and cyclosporin A.Macrophages that received different treatments were harvested and Western bloting were used to examine the expression of TLR4,p-p65,hepcidin,ferritin-L,ferritin heavy chain?ferritin-H?,CYP27A1 and cholesterol influx/efflux-related proteins.Intracellular labile iron pool,intracellular lipids and cholesterol concentrations were determined.Dil-labeled-ox-LDL uptake and cholesterol efflux assays were conducted.Results:1)Oil Red O and H&E staining displayed that the aortic plaque area was increased75%in HFD group than that of ND group.Double immunofluorescence staining indicated that compared to WT and ND group,colocalization of ox-LDL,TLR4,p-p65,hepcidin and ferritin-L with macrophages was more abundant in the aortic plaques of HFD group.Increased double-positive staining was present in the regions of foam cell accumulation.TLR4,hepcidin,MR and ferritin-L were significantly upregulated in aortic tissues from the HFD group compared to the control WT and ND groups?P<0.05?.The messenger RNA level of hepcidin was positively correlated with MR,TLR4and ferritin-L?P<0.05?.3)The expression level of TLR4,p-p65,hepcidin,ferritin-L and ferritin-H in RAW264.7 macrophages were increased about 55%,34%,48%,60%and 42%after ox-LDL?50?g/mL?exposure?P<0.05?,which were enhanced when cells were cotreated with LPS?P<0.05?.Moreover,ox-LDL-derived induction of TLR4,p-p65,hepcidin,ferritin-L,ferritin-H and lipid accumulation were attenuated in the presence of TAK-242?P<0.05?.4)In the hepcidin silenced macrophages,the expression of TLR4 and p-p65 were increased about 41%and 34%after ox-LDL exposure,which was further promoted by FAC?P<0.05?.Meanwile,the oil red O staining area and cellular cholesterol level were significantly increased.The HDL-and ApoA I-induced cholesterol efflux were decreased about 35%and 68%?P<0.05?.On the contrary,the iron chelation effectively suppressed the expression of TLR4 and p-p65 in the hepcidin pre-treated macrophages and inhibited cellular cholesterol accumulation?P<0.05?.5)The expression of CYP27A1 was decreased about 50%in macrophages after hepcidin incubation?P<0.05?.Hepcidin aggravates cellular cholesterol disequilibrium by upregulating the expression of CD36?increased about 31%than control group,P<0.05?and suppressing the expression of LXR?,ABCA1 and ABCG1?decreased about31%,29%and 37%than control group,respectively.P<0.05?.Conclusions:Iron deposition in plaque macrophages was trapped by ox-LDL-triggered autocrine formation of hepcidin through TLR4/NF-?B pathway activation.Excessive iron retention in macrophages could in turn activate the TLR4/NF-?B pathway,forming a vicious cycle between autocrine formation of hepcidin and iron retention through the TLR4/NF-?B pathway and the superposed influence on the CYP27A1/27HC axis via CD36-mediated cholesterol influx and PPAR?-LXR-ABCA1/G1 pathway-dependent cholesterol efflux.Section 2: Effects of dietary iron overload on systemic iron lipid metabolic disorders in Apo E–/– mice: i TRAQ-based proteomic analysisObjective: To test the iron hypothesis in Apo E–/– mice fed with high fat diet contained 2% carbonyl iron.The i TRAQ-based proteomic analysis was used to explore the potential mechanisms of systemic iron overload diminishing atherosclerosis in Apo E–/– mice.Methods: Sixty female Apo E–/– mice were randomly divided into three groups with the following diets: 1)normal diet?ND?,2)high fat diet?HFD?and 3)high fat diet containing 2% carbonyl iron?HFD+Fe?.Fifteen female age-matched wild-type C57BL/6J mice?WT?receiving a normal chow diet served as controls.Mice were fed for 16 weeks until sacrificed after an overnight fasting.The aortas and livers were excised for histopathological examination.Serum lipids and iron levels were determined as well as serum beta-hydroxybutyric acid,lactic acid,alanine aminotransferase?ALT?and aspartate aminotransferase?AST?levels.Moreover,Hepatic lipids,iron content,ATP,acetyl coenzyme A,hepatocytes apoptosis levels and hepatic redox status were meseared.Hepatic proteins of mice from ND,HFD and HFD+Fe groups were labeled by i TRAQ after digestion.Equal amount of labeled-samples were used for liquid chromatography-tandem mass spectrometry?LC-MS/MS?analysis.Quantitative reverse transcriptase?q RT?-PCR and Western blotting were used to verify the differential proteins screened by proteomic analysis.Primary mouse hepatocytes were isolated and incubated with free fatty acids and/or ferric ammonium citrate?FAC?.Intracellular triglyceride?TG?level was measeared.Western blotting and immunofluorescence staining were used to examine the expression levels of CD36 and liver-fatty acid binding protein?FABP1?.The free fatty acid uptake assay was performed and fluorescent double-staining used to colocalize fatty acids with endoplasmic reticulum.Results: 1)In comparison with HFD group,there was a significant 65% reduction in en face aortic atherosclerotic lesion area and serum total cholesterol?TC?,TG and low-density lipoprotein cholesterol?LDL-C?levels in HFD+Fe group were decreased 25%,30% and 70%,respectively?P<0.05?.Hepatic TG and TC levels were significantly lowered in HFD+Fe group than HFD group?P<0.05?.2)Serum total iron,hepatic iron content and ferritin expression level were increased about 42%,51% and 57% in HFD+Fe group than that of HFD group?P<0.01?.3)According to the screening criteria?fold change>1.5 or <0.67,P<0.05?,a total of 333 differential proteins were identified between HFD and ND group.These proteins were enriched in the fatty acid metabolism pathway,fatty acid degradation pathway,peroxisome and glutathione metabolism pathway.A total of 203 differential proteins were identified between HFD+Fe and HFD group.These proteins were enriched in the citrate cycle?TCA?pathway,pyruvate metabolism pathway,fatty acid degradation pathway and glutathione metabolism pathway.4)In comparison with ND group,fatty acid biosynthesis pathway was significantly suppressed while fatty acid degradation pathway was greatly activated in HFD group.There was a significant deduction in protein expression of fatty acid synthase?decreased about 55% than ND group?and phosphorylated-acetyl Co A carboxylase?decreased 65% than ND group?while protein expression level of acyl-Co A dehydrogenase,acetyl-Co A acetyltransferase and peroxisome proliferator activated receptor alpha were significantly upregulated about 64%,88% and 95% than ND group?P<0.05?.In comparison with HFD group,fatty acid biosynthesis pathway was significantly activated while fatty acid degradation pathway was parently blocked in HFD+Fe group?P<0.05?.5)Fatty acid uptake and trafficking-related proteins,including CD36,liver fatty acid binding protein?FABP1?and intestinal fatty acid binding protein?FABP2?in HFD+Fe group were significantly decreased about 75%,57% and 52% than that of HFD group,respectively.?P<0.05?.The serum free fatty acid was increased 35% in HFD+Fe group?P<0.05?.In vitro study indicated that FAC treatment significantly alleviated intracellular lipids accumulation and suppressed CD36,FABP1 and FABP2 protein expression in FFA-incubated primary mouse hepatocytes?P<0.05?.6)Several TCA-related critical proteins were inhibited in HFD+Fe group?P<0.05?,including pyruvate dehydrogenase E1 Beta subunit,dihydrolipoyl dehydrogenase,Aconitase-1,mitochondrial malate dehydrogenase and Fumarate hydratase.Hepatic acetyl-Co A and ATP levels were lowered in HFD+Fe group than HFD group,which were only about 54% and 65% of which in HFD group?P<0.05?.7)Proteins involved in glutathione metabolism pathway were apparently downregulated,such as leucine aminopeptidase 3,glutathione synthetase,glutathione peroxidases 1 and glutathione S-transferase in HFD+Fe group?P<0.05?.Hepatic proteins modified by 4-HNE in HFD+Fe group were much more than that of HFD group,which was in line with the increased hepatic MDA level?increased 34% than HFD group?while decreased SOD?decreased 64% than HFD group?and GSH levels?decreased 31% than HFD group,P < 0.05?.More serious degree of hepatocyte apoptosis was observed in HFD+Fe group,which was agree with the remarkably increased serum ALT and AST levels,which were increased about 35% and 30% than that of HFD group,respectively?P<0.05?.Conclusions: Dietary iron overload provoked systemic iron lipid metabolic disorders in Apo E–/– mice,which was characterized by hepatic fatty acid uptake and trafficking impairment and energy dysmetabolism triggered by the blockage of fatty acid degradation and TCA pathway.In this case,the diminished aortic atherosclerotic plaque burden,an "illusion of improvement",is actually a manifestation of liver fatty acid metabolism dysfunction-and energy deficiency-induced occult liver injury.