The aim of this study was to investigate the expression of circulating microRNAs (miRNAs) in apolipoprotein E (apoE) knockout mice (apoE-/-) and to validate the role of these miRNAs in human coronary artery disease (CAD). Pooled plasma from 10 apoE-/- mice and 10 healthy C57BL/6 (B6) mice was used to perform the microarray analysis. The results showed that miR-34a, miR-21, miR-23a, miR-30a and miR-106b were differentially expressed in apoE-/- mice, and these expression changes were confirmed by real-time quantitative reverse-transcription PCR. Then, miR-34a, miR-21, miR-23a, miR-30a and miR-106b were detected in the plasma of 32 patients with CAD and of 20 healthy controls. Only miR-34a, miR-21 and miR-23a were significantly differentially expressed in the plasma of CAD patients (all P<0.01). In conclusion, miR-34a, miR-21 and miR-23a were elevated in CAD patients, which means that these miRNAs might serve as biomarkers of CAD development and progression.
Aged ; Animals ; Apolipoproteins E/deficiency ; Biomarkers ; Case-Control Studies ; Coronary Artery Disease/*genetics ; Disease Models, Animal ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Male ; Mice ; Mice, Knockout ; MicroRNAs/*genetics ; Middle Aged ; Pilot Projects ; Reproducibility of Results ; Risk Factors

OBJECTIVETo study the effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice.

METHODEight male C57BL/6J mice were selected in the normal group (NF), 40 male ApoE -/- mice were fed for 16 weeks, divided into the model group (HF), the rosiglitazone group ( LGLT), the Jinlida low-dose group (JLDL), the Jinlida medium-dose group (JLDM), the Jinlida high-dose group (JLDH) and then orally given drugs for 8 weeks. The organization free fatty acids, BCA protein concentration determination methods were used to determine the skeletal muscle FFA content. The Real-time fluorescent quantitative reverse transcription PCR ( RT-PCR) and Western blot method were adopted to determine mRNA and protein expressions of mice fatty acids transposition enzyme (FAT/CD36), carnitine palm acyltransferase 1 (CPT1), peroxide proliferators-activated receptor α( PPAR α).

RESULTJinlida could decrease fasting blood glucose (FBG), cholesterol (TC), triglyceride (TG), free fatty acid (FFA) and fasting insulin (FIns) and raise insulin sensitive index (ISI) in mice to varying degrees. It could also up-regulate mRNA and protein expressions of CPT1 and PPARα, and down-regulate mRNA and protein levels of FAT/CD36.

CONCLUSIONJinlida can improve fat-induced insulin resistance ApoE -/- in mice by adjusting the changes in expression of skeletal muscle lipid transport enzymes.

Animals ; Apolipoproteins E ; deficiency ; genetics ; Blood Glucose ; metabolism ; CD36 Antigens ; genetics ; metabolism ; Carnitine O-Palmitoyltransferase ; genetics ; metabolism ; Dietary Fats ; adverse effects ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Hypoglycemic Agents ; administration & dosage ; Insulin ; metabolism ; Insulin Resistance ; Lipid Metabolism ; drug effects ; Male ; Metabolic Diseases ; drug therapy ; enzymology ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Muscle, Skeletal ; drug effects ; metabolism

OBJECTIVETo investigate the mechanism of liraglutide-mediated protection against nonalcoholic fatty liver disease (NAFLD) using aApoE knockout (KO) mouse with high-fat diet (HFD) and Acrp30 knockdown.

METHODSFifty-six male ApoE KO mice were divided into the following six modeling and experimental groups:regular chow fed (ApoE KO, n=10), HFD fed (HF, n=10), HFD+Adenovirus (Ad)-small hairpin (sh) Acrp30 (Ad-shAcrp30, n=10), HFD+Ad-shGreen Fluorescent Protein (GFP) (Ad-shGFP, n=6), HFD+Ad-shAcrp30+liraglutide (liraglutide, n=10), and HFD+Ad-shAcrp30+saline (saline, n=10). Weight-matched C57BL/6 mice on the regular chow diet were used as the control group (WT control, n=10).All mice were fed their assigned diet for 16 weeks.The Ad-shGFP or Ad-shAcrp30 was injected by tail vein at the end of 14 and 15 weeks.Mice in the liraglutide group received 1 mg/kg of the drug, twice daily, intraperitoneally for a total of 8 weeks (from the 9th to 16th week).Fasting blood samples were collected for testing levels of fasting plasma glucose (FPG), triglycerides (TGs), total cholesterol (TC), free fatty acid (FFA), alanine aminotransferase (ALT), Acrp30 and insulin.Liver tissue was procured for histological examination.Expression of mRNA was detected by real-time RT-PC and of protein was detected by western blot analysis.

RESULTSThe Ad-shAcrp30 treated mice had reduced expression of Acrp30 at both the mRNA and protein levels in adipose tissues and plasma, as compared with the AdshGFP treated mice (all P < 0.01).Compared to the WT and ApoE KO groups, the HF group showed higher levels of FPG, FFA, TGs and TC (all P < 0.01); furthermore, the Ad-shAcrp30 treatment compounded these changes.The Ad-shAcrp30 treated group had markedly higher hepatic TC and TGs than the HF group (P < 0.01 and P < 0.05).Oil Red O staining showed that there was more lipid droplets in the liver tissue of the Ad-shAcrp30 treated group than in that of the HF group (P < 0.01), and hematoxylin-eosin staining confirmed these results.Liraglutide treatment prevented the increase in body weight, FPG, FFA, TGs, TC and ALT levels, as compared to the saline controls (all P < 0.01), but the plasma Acrp30 levels and the Acrp30 mRNA and protein expression in adipose tissues were elevated (all P < 0.01).Oil-Red O staining indicated that the liraglutide group had a significantly lower hepatic lipid content than the saline group, and total hepatic TG and TC were reduced in the former group (P < 0.01 and P < 0.05).The liraglutide treatment significantly attenuated the mRNA expression of ACC and FAS (both P < 0.01) but increased AMPK phosphorylation (P < 0.01).

CONCLUSIONAdministration of liraglutide prevented the development of HFD-and hypoadiponectinemia-induced metabolic disturbance and accumulation of hepatic lipids in this mouse model system of NAFLD.

AMP-Activated Protein Kinases ; metabolism ; Adiponectin ; deficiency ; metabolism ; Adipose Tissue ; Alanine Transaminase ; Animals ; Apolipoproteins E ; deficiency ; metabolism ; Cholesterol ; Diet, High-Fat ; Disease Models, Animal ; Glucagon-Like Peptide 1 ; analogs & derivatives ; Insulin ; Liraglutide ; Male ; Metabolism, Inborn Errors ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Non-alcoholic Fatty Liver Disease ; metabolism ; Protective Agents ; RNA, Messenger ; Triglycerides

The microRNA (miRNA) regulation mechanisms associated with atherosclerosis are largely undocumented. Specific selection and efficient validation of miRNA regulation pathways involved in atherosclerosis development may be better assessed by contemporary microarray platforms applying cross-verification methodology. A screening platform was established using both miRNA and genomic microarrays. Microarray analysis was then simultaneously performed on pooled atherosclerotic aortic tissues from 10 Apolipoprotein E (apoE) knockout mice (apoE-/-) and 10 healthy C57BL/6 (B6) mice. Differentiated miRNAs were screened and cross-verified against an mRNA screen database to explore integrative mRNA-miRNA regulation. Gene set enrichment analysis was conducted to describe the potential pathways regulated by these mRNA-miRNA interactions. High-throughput data analysis of miRNA and genomic microarrays of knockout and healthy control mice revealed 75 differentially expressed miRNAs in apoE-/- mice at a threshold value of 2. The six miRNAs with the greatest differentiation expression were confirmed by real-time quantitative reverse-transcription PCR (qRT-PCR) in atherosclerotic tissues. Significantly enriched pathways, such as the type 2 diabetes mellitus pathway, were observed by a gene-set enrichment analysis. The enriched molecular pathways were confirmed through qRT-PCR evaluation by observing the presence of suppressor of cytokine signaling 3 (SOCS3) and SOCS3-related miRNAs, miR-30a, miR-30e and miR-19b. Cross-verified high-throughput microarrays are optimally accurate and effective screening methods for miRNA regulation profiles associated with atherosclerosis. The identified SOCS3 pathway is a potentially valuable target for future development of targeted miRNA therapies to control atherosclerosis development and progression.
Animals ; Aorta/metabolism/pathology ; Apolipoproteins E/*deficiency/metabolism ; Atherosclerosis/genetics/pathology ; Down-Regulation/genetics ; Gene Expression Profiling ; *Gene Expression Regulation ; Gene Regulatory Networks/genetics ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/*genetics/metabolism ; Models, Biological ; *Oligonucleotide Array Sequence Analysis ; RNA, Messenger/genetics/metabolism ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction/*genetics ; Suppressor of Cytokine Signaling Proteins/genetics/metabolism ; Up-Regulation/genetics

BACKGROUNDAtherosclerosis is the primary cause of cardiovascular disease, carotid artery disease, and peripheral vascular disease. However, it is hard to obtain human arterial tissue at different stages of atherosclerosis for a systematic study. The ApoE-deficient (ApoE(-/-)) mice predictably develop spontaneous atherosclerotic plaques with numerous features similar to the human lesions and contain nearly the entire spectrum of lesions observed during atherogenesis in humans. MicroRNA expression profiles at different stages of atherosclerosis in ApoE-deficient mice were screened to find out the differentially expressed microRNAs.

METHODSApoE-deficient mice were euthanized at 4, 8, and 20 weeks of age and divided into three groups according to the three time points, including groups A4 (fed a Western-type diet for 0 week), A8 (fed a Western-type diet for 4 weeks), and A20 (fed a Western-type diet for 16 weeks). Atherosclerotic lesions were analyzed. Fifteen aortas were collected and combined into three pools (five aortas in one pool) in each group. MicroRNA microarray analysis was replicated thrice in each group. The threshold of fold change ≥ 2.0 was used to screen up or down-regulated microRNAs. Differentially expressed microRNAs were subsequently verified with quantitative real-time polymerase chain reaction. Those increasingly up or down-regulated microRNAs during the progression of atherosclerosis were selected.

RESULTSAtherosclerotic lesions first appeared in the aortic arch in group A8. Severe atherosclerotic lesions were observed in group A20. In group A8, seven MicroRNAs were up-regulated while two were down-regulated. In group A20, 15 microRNAs were up-regulated while two were down-regulated. miR-34a-5p and miR-497-5p were increasingly up-regulated, while miR-434-3p was progressively down-regulated when atherosclerosis progressed.

CONCLUSIONSIn this study, we described that microRNAs are differentially expressed at different stages of atherosclerosis in ApoE-deficient mice. Those increasingly up or down-regulated microRNAs during the progression of atherosclerosis may play an important role in the pathogenesis of atherosclerosis and provide us opportunities for investigating atherosclerosis from early to advanced stages.

Animals ; Apolipoproteins E ; deficiency ; genetics ; Atherosclerosis ; genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs ; genetics ; Real-Time Polymerase Chain Reaction

BACKGROUNDPreeclampsia, especially early onset of preeclampsia (PE), is a common and serious disorder with high maternal and perinatal morbidity and mortality. Dietary factor is one of the most important factors which may affect the occurrence and development of the disease. The aim of this study is to investigate the effects of dietary factors on pathological changes of liver and placenta in preeclampsia-like mouse model by establishing the model at multiple stages of gestation.

METHODSWild-type (WT) mice were injected subcutaneously with nitric oxide synthase (NOS) inhibitor L-arginine methyl ester (L-NAME, 50 mg×kg(-1)×d(-1)) to establish PE-like model (L-NAME group) at early-, mid-, and late-pregnant periods respectively; simultaneously, the control mice were injected with normal saline (NS group). All the groups were divided into subgroups, standard chow group (SC), and high-fat diet group (HF). ApoE(-/-) pregnant mice served as a control group. Systolic blood pressure (SBP), urine protein, and histopathologic changes of placenta and liver in all groups were observed and statistically analyzed.

RESULTSIn WT and apoE(-/-) L-NAME subgroups, blood pressure and urine protein were significantly higher than those in all the gestational age matched NS groups (P < 0.05). Compared to other groups, remarkable liver fatty infiltration and lipid storage in placenta were found in early- and mid-L-NAME subgroups in apoE(-/-) mice (P < 0.05), especially in the early- and mid-HF+L-NAME subgroups in apoE(-/-) mice (P < 0.05). More lipid storage droplets both in liver and placenta were found in ApoE(-/-) mice than that of WT groups (P < 0.05). Morphology histopathologic examination of placentas showed varying degrees of fibrinoid necrosis and villous interstitial edema in early- and mid-L-NAME both in HF and SC of apoE(-/-) and WT subgroups compared to NS controls (P < 0.05). But there was no significant difference between HF and SC subgroups (P > 0.05), and no difference between apoE(-/-) and WT groups (P > 0.05).

CONCLUSIONSPreeclampsia-like conditions could be induced by L-NAME in mice at different gestational stages. Both WT and apoE(-/-) genotype mice with preeclampsia-like symptoms in early and mid stages of pregnancy presented lipid deposition in the placenta and hepatic fatty infiltration. To alter the environmental condition by feeding high-fat diet was harmful to the mother and the fetus. High-fat diet aggravated the impact of liver fatty infiltration at early and mid gestational stages especially in the apoE(-/-) mouse model. These results further revealed the association between early-onset preeclampsia and the dysoxidation of fatty acids.

Animals ; Apolipoproteins E ; deficiency ; genetics ; Diet, High-Fat ; Female ; Genotype ; Liver ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Mutant Strains ; NG-Nitroarginine Methyl Ester ; pharmacology ; Placenta ; drug effects ; metabolism ; Pregnancy

OBJECTIVETo observe the effect of Huxin Formula on expressions of the chief reverse cholesterol transport (RCT) associated genes, caveolin-1 and scavenger receptor-BI (SR-BI) in ApoE-gene knockout [ApoE (-/-)] mice.

METHODSThirty ApoE (-/-) mice of 4-6 weeks old were randomly divided into three groups (A-C). After being fed with high-fat diet for 16 weeks, they were treated with HXF (1 mL/100 g), pravachol (0.3 mg/100 g), and saline in equal volume respectively for 16 weeks successively; in addition, a blank group was set up with 10 C57BL/6J mice of 6-week old received 16-week high-fat feeding and saline treatment. Animals were sacrificed at the termination of the experiment, their paraffin sections of aortic tissue were used to measure the size of plaque, expressions of cavolin-1 and SR-BI were detected by immunological histochemical method.

RESULTSAs compared with the blank group, levels of caveolin-1 and SR-BI were increased in Groups A and B (P<0.01); but the increase in Group A was more significant than that in Group B (P<0.05). The plaque/aorta area ratio decreased significantly in Groups A and B, but showed insignificant difference between the two groups.

CONCLUSIONHXF could obviously increase the expressions of RCT associated genes, caveolin-1 and SR-BI, promote the RCT process, so as to reduce the formation of aorta atherosclerotic plaque in ApoE (-/-) mice.

Animals ; Aorta ; drug effects ; pathology ; Apolipoproteins E ; deficiency ; genetics ; Atherosclerosis ; pathology ; Biological Transport ; drug effects ; Caveolin 1 ; metabolism ; Cholesterol ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Plaque, Atherosclerotic ; pathology ; Receptors, Scavenger ; metabolism

OBJECTIVETo study possible influences of 1,25(OH)(2)D(3) on endothelial cell proliferation, apoptosis and endothelial nitric oxide synthase (eNOS) expression of aorta in apolipoprotein E-deficient (apoE(-/-)) mice and to explore the relationship between vitamin D and atherosclerosis.

METHODEndothelial cell of aorta in apoE(-/-) mice were isolated and cultured, and the influence of 1,25(OH)(2)D(3) on endothelial cell proliferation were observed by MTT, apoptosis of cells were quantitated by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling, Bcl-2 mRNA, fas mRNA and eNOS mRNA was detected by reverse transcription-polymerase chain reaction.

RESULTEndothelial cell proliferation rate of aorta did not significantly change in the two control groups (0.162 ± 0.031 vs. 0.158 ± 0.006, P > 0.05). Compared with control groups, 1,25(OH)(2)D(3) stimulated endothelial cell proliferation of aorta (P < 0.05), but endothelial cell proliferation rate did not significantly change in different 1,25(OH)(2)D(3) concentration groups [1,25(OH)(2)D(3) concentration: 10(-4)mol/L, 10(-5) mol/L, 10(-6) mol/L, 10(-7) mol/L, 10(-8) mol/L, endothelial cell proliferation rate: 0.189 ± 0.013 vs. 0.285 ± 0.011 vs. 0.296 ± 0.026 vs. 0.284 ± 0.017 vs. 0.233 ± 0.010, P > 0.05]. 1,25(OH)(2)D(3) research concentration as chosen as 10(-6) mol/L. In 1,25(OH)(2)D(3) 10(-6) mol/L group, the expression of Bcl-2, eNOS mRNA was significantly increased (0.78 ± 0.16 vs. 0.46 ± 0.21 vs. 0.42 ± 0.17, 0.56 ± 0.16 vs. 0.39 ± 0.13 vs. 0.35 ± 0.11, 0.46 ± 0.2 vs. 10.42 ± 0.17 vs. 0.78 ± 0.16, 0.79 ± 0.21 vs. 0.81 ± 0.20 vs. 0.43 ± 0.12), apoptotic index, Fas mRNA was significantly decreased (15.14 ± 3.19 vs. 18.94 ± 4.22 vs. 19.27 ± 4.58, 0.43 ± 0.12 vs.0.79 ± 0.21 vs. 0.81 ± 0.20)(P < 0.05). The quantity of eNOS gene expression was inversely associated with apoptosis index and Fas mRNA, was positively associated with Bcl-2 mRNA (r = -0.676, -0.758, 0.762, P < 0.01).

CONCLUSION1,25(OH)(2)D(3) stimulated endothelial cell proliferation, inhibited apoptosis and increased eNOS expression of aorta in apoE(-/-) mice. These results may deepen understanding of the pathogenesis of atherosclerosis.

Animals ; Aorta ; metabolism ; Apolipoproteins E ; deficiency ; Apoptosis ; drug effects ; Calcitriol ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelial Cells ; metabolism ; Female ; Male ; Mice ; Nitric Oxide Synthase Type III ; metabolism ; RNA, Messenger ; genetics

OBJECTIVETo explore the correlation between Fc γ RIII A (CD16A) and aortic atherosclerotic plaque destabilization in apoE knockout (apoE KO) mice and the intervention effects of effective components of chuanxiong rhizome and red peony root.

METHODSEight 8-week-old male C57BL/6J mice were selected as the control group. Forty 8-week-old male apoE KO mice were randomly divided into the model group, apoE KO + intraperitoneal injection immunoglobulin group (IVIG), apoE KO + simvastatin group (Sm), apoE KO + high dosage of xiongshao capsule (XSC) group (XSCH), and apoE KO + low dosage of XSC group (XSCL), 8 mice in each group. Mice in the control group were put on a normal diet, and others were fed with a high-fat diet. After 10-week different interventions, monocyte CD16 expression was detected by flow cytometry, aortic matrix metalloproteinase-9 (MMP-9) mRNA expression was detected using reverse transcription polymerase chain reaction, and serum tumor necrosis factor (TNF)-α level was detected using enzyme-linked immunosorbent assay.

RESULTSCompared with the control group, monocyte CD16 expression, aortic MMP-9 mRNA expression, and serum TNF-α level in the model group increased obviously (P<0.01). Injections of apoE KO mice with intraperitoneal immunoglobulin during a 5-day period significantly reduced the monocyte CD16 expression, aortic MMP-9 mRNA expression, and serum TNF-α level (P<0.01 or 0.05) over a 10-week period of high-fat diet. Indices above in the Sm group, XSCH group, and XSCL group decreased in a different degree. Of them, the aortic MMP-9 mRNA expression in XSCH group was lower than that in Sm group (P<0.05) and the monocyte CD16 expression and serum TNF-α level showed no significant difference between XSCH group and Sm group (P>0.05). Correlation analyses suggested positive correlation between monocyte CD16 expression and aortic MMP-9 mRNA expression or serum TNF-α level in IVIG group, XSCH group, and XSCL group.

CONCLUSIONSFcγR III A mediates systemic inflammation in the progression of coronary heart disease with blood stasis syndrome. XSC could stabilize atherosclerotic plaque by suppressing inflammation and its target was relative with FcγRIII A.

Animals ; Aorta ; drug effects ; enzymology ; pathology ; Apolipoproteins E ; deficiency ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Flow Cytometry ; Gene Expression Regulation, Enzymologic ; drug effects ; Lipopolysaccharide Receptors ; metabolism ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Monocytes ; drug effects ; metabolism ; Paeonia ; chemistry ; Phytotherapy ; Plant Roots ; chemistry ; Plaque, Atherosclerotic ; blood ; drug therapy ; enzymology ; pathology ; RNA, Messenger ; genetics ; metabolism ; Receptors, IgG ; metabolism ; Tumor Necrosis Factor-alpha ; blood

Animals ; Apolipoproteins E ; deficiency ; Female ; Hyperlipoproteinemia Type III ; blood ; metabolism ; Lipid Metabolism, Inborn Errors ; blood ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Receptors, Calcitriol ; metabolism ; Vitamin D ; blood