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Background: Familial hypercholesterolemia (FH) is commonly associated with mutations in-LDL receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Aim: To identify genetic variants associated with FH in a population of children and adolescents with hypercholesterolemia or a family history of-demonstrated early CVD. Material and Methods: Clinical and biochemical parameters were evaluated, and nine genes related to FH were sequenced namely LDLR, APOB, PCSK9, LDLRAP1, LIPA, APOE, ABCG5, ABCG8 and STAP1, in 55 children and adolescents aged 1 to 18 years old, from non-consanguineous families. Results: Mutations associated with FH were found in 17 children and adolescents, corresponding to p.Asp47Asn, duplication of exons 13-15 and p.Ser326Cys of the LDLR gene; p.Glu204* and Ile268Met of the APOE gene. Thirteen patients were heterozygous, two homozygous, two compound heterozygous, and one double heterozygous. Conclusions: Children and adolescents carrying mutations associated with FH were found by selective screening, which constitutes the first stage in the identification of genetic variants in our country.
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Humans , Infant , Child, Preschool , Child , Adolescent , Proprotein Convertase 9/genetics , Hyperlipoproteinemia Type II/diagnosis , Hyperlipoproteinemia Type II/genetics , Hyperlipoproteinemia Type II/epidemiology , Chile , MutationABSTRACT
Objective To explore the potential mechanisms of low density lipoprotein receptor (LDLr) in high glucose peritoneal dialysis solution (PDS)-induced peritoneal fibrosis.Methods Human peritoneal mesothelial cells (PMCs) were applied.In pre-experiment,human PMCs were cultured with 1.5％ PDS,2.5％ PDS and 4.25％ PDS for 6 h,12 h and 24 h.4.25％ mannitol was used as high osmotic pressure control.In formal experiment,PMCs were divided into the control group (treated with phosphate buffer saline) and the high glucose PDS group (treated with 4.25％ PDS for 24 h).Morphological change of PMCs was observed by inverted microscope.The mRNA and protein expressions of extracellular matrix proteins such as α-smooth muscle actin (α-SMA),fibroblast specific protein-1 (FSP-1) and collagen Ⅰ in PMCs were respectively measured by real-time PCR and Western blotting.The lipid accumulation was observed by oil red O staining and filipin staining,and the content of intracellular cholesterol ester was detected by high-performance liquid chromatography.The co-expression of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) with golgin was observed with immunofluorescent staining.The mRNA and protein expressions of LDLr,SREBP-2 and SCAP were respectively detected by real-time PCR and Western blotting.The mRNA and protein expressions of mammalian target of rapamycin (mTOR),eukaryotic initiation factor 4E-binding protein 1 (4EBP1),and p70 S6 kinase (S6K1) were respectively detected by real-time PCR and Western blotting.Results (1) Compared with the 1.50％ PDS stimulation,4.25％ PDS for 24 h intervention significantly increased the expression of LDLr in PMCs (P ＜ 0.05),and high osmotic pressure control at 6 h,12 h and 24 h had no statistical difference (P ＞ 0.05).(2) Compared with those in the control group,in high glucose PDS group PMCs showed notable elongation consistent with the morphology of myofibroblasts,the expressions of α-SMA,FSP-1 and collagen Ⅰ were increased (all P ＜ 0.05),and the intracellular cholesterol were enhanced (P ＜ 0.05).Meanwhile,the co-expression of SCAP with golgin was enhanced,and the mRNA and protein expressions of LDLr,SREBP-2 and SCAP were up-regulated in high glucose PDS group (all P ＜ 0.05).Further,the mRNA and protein phosphorylation of mTOR,4EBP1 and S6K1 were increased (all P ＜ 0.05).Conclusions The disruption of LDLr feedback regulation is involved in high glucose PDS-mediated cholesterol accumulation in PMCs by mammalian target of rapamycin complex 1 (mTORC1) pathway,which promotes the accumulation of extracellular matrix and peritoneal fibrosis.
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Objective To investigate the difference between histopathological changes of brain white matter in low-density lipoprotein receptor (LDLR) homozygous mutation rats with hypercholesterolemia and wild-type rats.Methods Thirty LDLR-/-rats and 28 wild-type rats were selected.Plasma cholesterol levels were measured by enzyme-linked immunosorbent assay at 15,18 and 26 weeks old respectively.The axonal structure of the corpus callosum area was observed by transmission electron microscopy.The myelin basic protein (MBP) of the corpus callosum area was quantitatively analyzed by Western blotting.In addition,at 26 weeks old,the myelin sheaths were stained by fast blue staining.The expression level of MBP in white matter was further detected by immunofluorescence staining,and the morphological changes of glial cells were observed.Results Compared with the wild-type rats,the plasma cholesterol concentration in LDLR-/-rats increased significantly,and it could be as high as 3.3 times at 26 weeks.The results of electron microscopy showed that the LDLR-/-rats had axonal injury at 15 weeks and aggravated gradually over time.At 26 weeks,Western blot analysis of the LDLR-/-rats showed that the MBP expression level of the corpus callosum area decreased significantly.Fast blue staining showed loosening of nerve fibers,diffuse vacuole formation,and myelinated nerve fiber loss in the corpus callosum area.In addition,it was also found that the number of oligodendrocytes in LDLR-/-rats was significantly reduced,and large numbers of astrocytes and microglia were activated.Conclusions LDLR-/-rats will have spontaneous hypercholesterolemia.Axonal injury,demyelination,decreased oligodendrocytes,as well as the abnormal activation of astrocytes and microglia are present in the early adult brain white matter area.
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Objective To investigate the protective mechanism of MEK1/2 inhibitor PD98059 on ox-LDL induced injury of human umbilical vein endothelial cells (HUVEC),and its influence on the expression of LOX-1.Methods HUVEC damage models were established by using ox-LDL and were treated with PD98059 later,divided into the negative control group,the ox-LDL group,the positive control group and the PD98059+ox-LDL group.The effect of inhibition of MEK1/2 on ox-LDL induced HUVEC damage was measured.Results Compared with the negative control group,the levels in the ox-LDL group of LOX-1,pMEK1/2,RhoA,ROCK1,ROCK2,TNF-α and IL-6 were increased significantly,the proliferations of HUVEC and the productions of NO were decreased (P＜0.05).Compared with the ox-LDL group,the levels in the positive control group and the PD98059+ox-LDL group of pMEK1/2,RhoA,ROCK1,ROCK2,TNF-α and IL-6 were decreased,the proliferation of HUVEC and the production of NO were increased (P＜0.05).Conclusion PD98059 inhibit the MEK1/2 signaling pathway to suppress the ox-LDL induced damage of HUVEC by decreasing the expression of LOX-1.
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Humans , Atherosclerosis/therapy , LDL-Receptor Related Proteins/metabolism , Proprotein Convertases/metabolism , Proprotein Convertases/therapeutic use , Serine Endopeptidases/metabolism , Serine Endopeptidases/therapeutic use , Cholesterol, LDL/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Polymorphism, Genetic , Proprotein Convertases/pharmacology , Risk Factors , Serine Endopeptidases/pharmacologyABSTRACT
Objective To investigate the effects of low density lipoprotein receptor (LDLr) pathway on podocyte injury in diabetic nephropathy (DN) under inflammatory stress.Methods Male db/db mice and db/m mice were randomly divided into four groups (8 mice in each group):db/m group (control),casein injected db/m group (db/m + casein),db/db group (db/db),and casein injected db/db group (db/db + casein).An inflamed model of DN was established according to our previous study.24-hour urinary protein was measured every week.The plasma lipid profile was detected by clinical biochemistry assay.Podocyte changes were evaluated by electron microscope and immunofluorescent staining.Lipid accumulation in the kidney was evaluated by oil red O staining and intracellular cholesterol quantitative assay.The protein expression of Wilm's tumor-1 (WT-1),nephrin,α-smooth muscle actin (t-SMA),and molecules correlated with LDLr pathway were examined by immunohistochemical staining or Western blotting.The colocalized protein expression of LDLr with WT-1 was examined by immunofluorescent staining and laser confocal microscopy.Results There were no differences in plasma levels of LDL and HDL among four groups.Compared with db/db group,the db/db+ casein group showed markedly increased 24-hour urinary protein,more significant podocyte foot process effacement and podocyte damage,increased lipid droplet accumulation in kidneys,increased protein expressions of LDLr,SCAP and SREBP-2 in kidneys (all P ＜ 0.05).Interestingly,increased LDLr protein expression in kidneys of db/db mice was negatively correlated with decreased nephrin protein expression (r =-0.855,P ＜ 0.01) and positively correlated with increased α-SMA protein expression (r=0.768,P ＜ 0.01).Conclusions The disruption of LDLr pathway induced by inflammation contributes to podocyte injuries in diabetic nephropathy.
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Objective To explore the changes of serum lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and the level of lipids in patients with obstructive sleep apnea hypopnea syndrome (OSAHS).Methods A total of 94 candidates with sleep disorders between January-July 2011 at outpatient department were monitored with polysomnography.According to apnea-hypopnea index (AHI),they were divided into mild-to-moderate OSAHS (5 ≤ AHI ≤ 30) (n =27),severe OSAHS (AHI ＞ 30) (n =37) and normal control groups (AHI ＜ 5) (n =30).After polysomnography,their blood samples were obtained to measure the levels of serum LOX-1,triglycerides (TG),cholesterol (TC),high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C).Results The serum level of LOX-1 in severe OSAHS group was significantly higher than that in the mild-to-moderate and control groups (P ＜ 0.01).The serum level of LOX-1 in OSAHS patients was positively correlated with AHI and longest apnea time (LAT)(r =0.645 & 0.501 respectively,both P ＜ 0.01) and was negatively correlated with SaO2 (r =-0.647,P ＜0.01).No significant difference existed in serum lipids in all groups (P ＞ 0.05).Conclusions Intermittent hypoxia caused by OSAHS increases the level of LOX-1 to further promote the formation and development of atherosclerotic in patients with OSAHS.The levels of lipid can not effectively predict the severity of lipid metabolism disorder in patients with OSAHS.
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Objective To investigate the effects of simvastatin on the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) induced by oxidized low-density lipoprotein (ox-LDL) or Glucose in U937 macrophages, and explore the role of NF-κB in modulating of LOX-1 expression. Methods U937 macrophages were treated with PMA to induce differentiation, which were co-cultured with 50 mg/L ox-LDL or/and 25 mmol/L glucose. Pyrrolidine dithiocarbamate (PDTC) and simvastatin (1 μmol/L or 10 μmol/L) were used to treat cells. The expression of LOX-1 protein and NF-κB ac- tivity were detected by enzyme-linked immunosorbent assay technology. The expression of LOX-1 mRNA was measured by RT-PCR. Results The expression of LOX-1 was up regulated by ox-LDL, glucose and combination of both. The inhibitor of NF-κB PDTC suppressed this up-regulation. Simvastatin suppressed the expression of LOX-1 induced by ox-LDL, and showed a significant effect in the higher concentration. There was no significant effect of simvastatin on the expression of LOX-1 induced by glucose. The variation of NF-κB activity was similar to that of LOX-1 expression. Conclusion Simvas- tatin suppressed the expression of LOX-1 induced by ox-LDL, while no significant effect on the expression of LOX-1 in- duced by glucose. The expression and regulation of LOX-1 were related with NF-κB pathway.
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Objective To investigate whether low density lipoprotein receptor (LDLr) pathway involves in the progression of vascular calcification (VC) in hemodialysis patients under microinflammation.Methods Twenty-eight hemodialysis patients were divided into control and inflammation group according to plasma C-reactive protein level.Surgically removed tissues from radial artery of patients receiving arteriovenostomy were used in experiments.Foam cell formation and calcification deposition were observed by hematoxylin-eosin (HE) and alizarin red S staining respectively.VC-related protein expression,such as bone morphogenetic proteins-2 (BMP-2),collagen Ⅰ,alkaline phosphatase (ALP),and LDLr and its related nuclear factor of transcriptional regulation,such as sterol regulatory element binding protein-2 (SREBP-2) and SREBP cleavage-activating protein (SCAP),were detected by immunohistochemistry and immunofluorescence staining.Results HE and alizarin red S staining showed that there were parallel increased foam cell formation and calcium deposit in continuous cross-sections of radial arteries in inflammation group compared to control group,which were closely correlated with increased protein expressions of LDLr,SREBP-2,BMP-2,and collagen Ⅰ as shown by immunohistochemical and immunofluorescent staining.Confocal microscopy confirmed that inflammation enhanced the translocation of SCAP/SREBP-2 complex from endoplasmic reticulum to Golgi,thereby activating LDLr gene transcription.Inflammation increased protein expression of ALP and reduced protein expression of alpha-smooth muscle actin,contributing to the phenotype conversion of vascular smooth muscle cells in calcified vessels from the fibroblastic to the osteogenic,which were the main cell components in VC.Further analysis showed that the disruption of LDLr pathway induced by inflammation was positively correlated with the enhanced expression of BMP-2 and collagen Ⅰ (r=0.782,P＜0.01; r=0.644,P＜0.05).Conclusion Inflammation accelerates the progression of VC in hemodialysis patients through the disruption of LDLr feedback regulation.
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Objective To observe how ox-LDL impacts the mRNA expressions of MMP-9 and LOX-1 of human umbilical endothelial cells (HUEVC) and how LOX-1 acts as in inflamation course.Methods HUVEC were incubated in vitro.mRNA Expressions of MMP-9 and LOX-1 were determined by reverse transcription polymerase chain reaction (RT-PCR).Results Compared to the control group(0.252±0.032;0.279 ±0.041 ),ox-LDL significantly increased the mRNA expressions of MMP-9 and LOX-1 (25 ng/group 0.486 ± 0.012,0.586 ± 0.02;50 ng/L group 0.668 ± 0.011,0.739 ± 0.014; 100 ng/group 0.817 ±0.030,0.872 ±0.003,P ＜0.01 ).Those expressions were increased by ox-LDL( 1.020 ±0.039)in a concentration-and time-dependent manner.MMP-9 mRNA(0.872 ±0.046) was reduced when LOX-1 was inhibited by polyinosinic acid ( P ＜ 0.01 ).Conclusions The mRNA expressions of MMP-9 and LOX-1 were induced by ox-LDL in HUVEC.Inhibition of LOX-1 may decrease the expression of MMP-9.Those data demonstrate that LOX-1 is involved in the process of ox-LDL-induced MMP-9 expression.
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Low density lipoprotein receptor (LDLR) plays an important role in the cholesterol homeostasis. We examined the possible circadian regulation of LDLR and mechanism(s) underlying it. In mice, blood glucose and plasma triglyceride, total and high density lipoprotein cholesterol varied distinctively throughout a day. In addition, LDLR mRNA oscillated in the liver in a functional clock-dependent manner. Accordingly, analysis of human LDLR promoter sequence revealed three putative E-boxes, raising the possible regulation of LDLR expression by E-box-binding transcription factors. To test this possibility, human LDLR promoter reporter constructs were transfected into HepG2 cells and the effects of CLOCK/BMAL1, Hes1, and Hes6 expression were analyzed. It was found that positive circadian transcription factor complex CLOCK/BMAL1 upregulated human LDLR promoter activity in a serum-independent manner, while Hes family members Hes1 and Hes6 downregulated it only under serum-depleted conditions. Both effects were mapped to proximal promoter region of human LDLR, where mutation or deletion of well-known sterol regulatory element (SRE) abolished only the repressive effect of Hes1. Interestingly, hes6 and hes1 mRNA oscillated in an anti-phasic manner in the wild-type but not in the per1-/-per2-/- mouse. Comparative analysis of mouse, rat and human hes6 genes revealed that three E-boxes are conserved among three species. Transfection and site-directed mutagenesis studies with hes6 reporter constructs confirmed that the third E-box in the exon IV is functionally induced by CLOCK/BMAL1. Taken together, these results suggest that LDLR expression is under circadian control involving CLOCK/BMAL1 and Hes family members Hes1 and Hes6.
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Animals , Humans , Male , Mice , ARNTL Transcription Factors/physiology , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/genetics , CLOCK Proteins/physiology , Cholesterol/blood , Circadian Rhythm , E-Box Elements , Exons , Gene Expression Regulation , Hep G2 Cells , Homeodomain Proteins/genetics , Homeostasis , Liver/metabolism , Mice, Inbred C57BL , Promoter Regions, Genetic , Receptors, LDL/genetics , Repressor Proteins/genetics , Transcription, GeneticABSTRACT
ObjectiveTo investigate the effect of Simvastatin on LOX-1 and ROS in NRK52E induced by ox-LDL.MethodsNRK-52E cells were divided into three groups: Control group,ox-LDL group (50 μg/ml ox-LDL) and Simvastatin group (10 μmol/L Simvastatin +50 μg/ml ox-LDL).After incubation for 24 h,the expression of LOX-I was analyzed by Western blotting,and production of reactive oxygen species (ROS) was analyzed with confocal laser scanning microscopy.ResultsNRK-52E expressed LOX-1 at low level,50 μg/ml ox-LDL increased the expression of LOX-1 by 6.80 times.Pre - treatment with Simvastatin decreased LOX-1 expression by 65％.There was little ROS generation in NRK52E cells,50μg/ml ox-LDL promoted the expression of LOX-1 by 4.86.times.Pre - treatment with Simvastatin decreased ROS generation by 60％.ConclusionsSimvastatin upregulate LOX-1 expression and ROS generation induced by Ox-LDL in NRK52E cells.
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Objective To investigate the low density lipoprotein receptor (LDLR)gene and apolipoprotein (Apo) B gene mutation in a Chinese family with familial hypercholesterolemia(FH) and give the kindrids clinical check-ups. Methods After physical examination, the kindreds underwent ECG and ultrasound checks. Blood samples were tested for lipid profiles. The promoter and all eighteen exons of LDLR gene were investigated by using PCR and agarose gel electrophoresis in combination with DNA sequence analysis. The results were compared with the normal sequences in GenBank and FH database ( www. ucl. ac. uk/fh ) to find mutations. In addition, the apolipoprotein B100 gene for known mutations (R3500Q,R3531C,R3501W and R3480W)that cause familial defective ApoB100 (FDB)was also tested using the same method. Results A novel homozygous G ＞ A mutation at the 1581 bp of exon 10 was detected in the proband and his siblings. It caused a substitution of amimo acid Glu to Gly at codon 496. A novel heterozygous G ＞A mutation at the 1581 bp of exon 10 was detected in his parents. No mutations of R3500Q,R3531C,R3501W and R3480W of ApoB100 were observed. ECGs were normal. Atherosclerosis were found in all family members by ultrasound checks. Conclusions The homozygous G ＞ A mutation at the 1581 bp of exon 10 was first determined in our country. The change of amino acid Glu to Gly is responsible for FH of the family. The type of the gene mutation was not found in the FH database( www. ucl.ac. uk/ih). It's a new type of LDLR mutation.
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A nanoemulsão lipídica (LDE) se concentra nas células neoplásicas e pode ser utilizada como transportador de derivado lipofílico da daunorrubicina, como o Noleil- daunorrubicina (oDNR). Neste estudo, a LDE-oDNR foi preparada por homogeneização em alta pressão e sua toxicidade e atividade anti-tumoral testadas. A associação LDE-oDNR teve rendimento elevado e permaneceu estável por longo período. Em camundongosC57BL/6J, a dose máxima tolerada (DMT) foi 65 vezes maior e a DL50 48 vezes maior no tratamento LDE-oDNR comparado ao tratamento DNR comercial, resultando em alta redução da toxicidade. Em camundongos implantados com células de melanoma B16, a preparação LDE-oDNR (7,5 mol/kg) levou a redução de 59 ± 2% do crescimento do tumor comparado a redução de 23 ± 2% para o tratamento DNR comercial na mesma dose (p<0,001). A probabilidade de sobrevida teve aumento pronunciado nos animais tratados com LDE-oDNR comparado à DNR comercial (p <0,01). Além disso, apenas 30% dos animais portadores de melanoma submetidos ao tratamento com LDE-oDNR apresentaram metástases, comparado a 82% quando tratados com DNR comercial. Uma forte redução de toxicidade também foi observada pela redução da anemia e leucopenia nos animais tratados com LDE-oDNR, em comparação com DNR comercial. A preparacao LDE-oDNR foi eficaz também no quadro de trombocitose induzida por tumor. Os testes com fragmentos extraídos de tumores dos animais tratados mostraram que a LDE-oDNR foi mais eficaz na destruição das células neoplásicas comparado ao tratamento DNR comercial (9% de células viáveis com tratamento LDE-oDNR, 27% sob tratamento DNR). O estudo mostrou que o tratamento proposto com o derivado ODNR associado à nanoemulsão (LDE-oDNR) é efetivo no combate às células tumorais, seletivo, menos tóxico e melhor tolerado. Os estudos de farmacocinética e biodistribuição somam a este protocolo informações importantes relacionadas às propriedades de absorção, distribuição, metabolismo e excreção...
A lipidic nanoemulsion (LDE) that concentrates in neoplastic cells can be used as vehicle to daunorubicin lipophylic derivatives, such as N-oleyl-daunorubicin (oDNR). Here, LDE-oDNR was prepared by high pressure homogenization to test toxicity and anti-tumor activity. LDE-oDNR association yield was high and stable for long period. In mice, maximum tolerated dose was 65 and LD50 was 48-fold greater in LDE-oDNR than in commercial DNR treatment, showing very strong toxicity reduction. In melanoma B16-tumor bearing mice, LDE-oDNR (7.5 mol/Kg) reduced tumorgrowth by of 59±2%, and DNR by only 23±2% at same dose level (p<0.001). Survival was pronouncedly increased in LDE-oDNR compared to DNR treatment (p<0.01). Furthermore, the number of melanoma-bearing mice with metastasis was 30% under LDE-oDNR, compared to 82% under DNR treatment. Strong reduction of toxicity was also observed by reduction of anemia and leucopenia under LDE-oDNR, compared to commercial DNR tumor-induced thrombocytosis was more effective with LDE-oDNR than with DNR. Tests with fragments extracted from tumors of treated animals showed that LDE-oDNR was more effective in killing neoplastic cells than DNR (9% of viable cells under LDE-oDNR; 27% under DNR). The pharmacokinetics and biodistribution studies add important information to this protocol related to the properties of absorption, distribution, metabolism and excretion of the formulation under study compared to free DNR. The remarkable toxicity reduction and increase in pharmacological action supports novel LDE-oDNR as a promising weapon in cancer treatment.
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Drug Screening Assays, Antitumor , Daunorubicin/analogs & derivatives , Nanotechnology , Receptors, LDLABSTRACT
Objective To analyse the mutation of low density lipeprotein receptor (LDLR) gene associated with familial hypercholesterolemia (FH) and make a discussion on the relationship between genotype and phenotype. Methods The blood fat, electrocardiogram, heart and great vessels color Doppler were examined in propositus and family member. The promoter and all eighteen exons of LDLR gene were investigated by polymerase chain reaction (PCR),degenerate high performance liquid chromatogram (DHPLC) and DNA sequence analysis. The results were compared with the normal sequences in GenBank and FH database (www.ucl.uk/fh) to find the mutation. In addition,the apolipoprotein B100(ApoB100) gene for the known mutations(Q3500R) that cause familial defective ApoB100(FDB) was detected by directed screening.Results Two novel heterozygous c.1864 G→A (Asp622Asn) and c.1959 C→T(Val 653Val) mutations in the exon 13 of LDLR in promoter were detected. And Asp622Asn mutation segregated with the disease. No mutation Q3500R of ApeB100 was observed. Conclusions The heterozygous c.1864 G→A (Asp622Asn)mutation of LDLR gene is firstly determined in China. The heterozygous c.1864 G→A (Asp622Asn)mutation of LDLR gene is probably responsible for FH. Perhaps it is a particular pathogenesis for Chinese people. PCB-DHPLC could be used for detecting the mutation.
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Objective To investigate the effect of lectin like oxidized low density lipoprotein receptor 1 ( LOX-1 ) in NRK52E intaking lipid induced by oxidized low density lipoprotein ( ox-LDL). Methods NRK-52E was incubated with ox-LDL (0,25,50, and 100 g/ml ) for 24 hours or pre-treated with the chemical blocker of LOX-1 receptor- polyI or carrageenan, and then exposed to 50 μg/ml of ox-LDL. LOX-Ⅰ mRNA was examined by real-time PGR. LOX-1 protein was assessed by Western blot analysis. Lipid deposit was examined by oil red O. Results LOX-1 mRNA expression in 25,50,100 μg/ml ox-LDL group was 2. 13, 10. 14, 20. 81 times of that in 0 g/ml ox-LDL group ( P <0. 05 ,respectively). LOX-1 protein expression in 25,50,100 μg/ml ox-LDL group was 2. 53,12. 18,21.45 times of that in 0 μg/ml ox-LDL group( P <0. 05 ,respectively). Following the increased LOX-1, lipid intake increased. Pre-treatment with Poly Ⅰ or carrageenan, LOX-1 mRNA expression deceased by 48% or 47%, LOX-1 protein deceased by 72% or 65%, lipid intake induced by 41% or 49% ( P <0.05 ,respectively). Lipid had a close relationship with LOX-1 ( r = 0. 87, P < 0. 05). Conclusion Ox-LDL induced NRK52E to express LOX-1 and promoted NRK52E to intake lipid, and this effect could be partly blocked by LOX-1 blocker.
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Hypercholesterolemia and coronary heart disease caused by it have become the most important factors threatening human health. The lipid metabolism-related studies are increasingly receiving attention. Recent studies have demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9), a family member of precursor protein-converting enzyme, plays an important role in the regulation of lipid metabolism. The expression and mutation of PCSK9 gene are closely correlated with the content of low-density lipoprotein receptor (LDLR). The excessive expression of PCSK9 promotes the degradation of LDLR, thereby increasing the levels of plasma LDL; whereas the inhibition of PCSK9 gene expression causes the decreased levels of plasma LDL. Therefore, it is promising to develop novel medications for treating hypercholesterolemia, controlling hyperlipermia and preventing coronary heart disease by studying the mechanism of PCSK9.
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AIM To explore effects of policosanol on depressing cholesterol in hyperlipidemia rats and the correlated biochemistry mechanism. METHODS The rats were randomly divided into normal control, policosanol 4 mg·kg~(-1) prevention, hyperlipidemia model, policosanol 4, 6 and 8 mg·kg~(-1) and lovastatin positive control groups. The later 5 group rats were fed with high-cholesterol diets for 4 weeks in order to make hyperlipidemia model and beginning from the 5th week, in addition to the normal control and model groups, other groups were ig given policosanol or lovastatin once a day for 6 weeks, respectively, and policosanol protection group rats were ig given with policosanol 4 mg·kg~(-1) once a day for 10 weeks, together with high-cholesterol diets everyday. Total cholesterol(TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) concentrations in the serum and fecal bile acid (FBA) in the exrement were determined by auto-biochemistry analyzer. The activity of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase in hepatocellular microsomes was detected by ultraviolet spectrophotometric analysis and activity of low density lipoprotein receptor (LDL-R) in peripheral blood lymphocyte was detected by fluorescence labelled integrator method. RESULTS Compared with hyperlipemia model group, the levels of TC decreased (39.1%-46.4%), LDL-C decreased (66.6%-80.7%), and FBA increased (9.7%-19.0%), the activity of HMG-CoA reductase decreased （13.8%-23.6%）, and activity of LDL-R increased (27.5%-129.6%) in policosanol prevention, policosanol 4, 6 and 8 mg·kg~(-1) and lovastatin groups, respectively; HDL-C increased （12.2%-16.7%） in policosanol prevention and policosanol 8 mg·kg~(-1) groups; TG decreased in lovastatin group. CONCLUSION Policosanol has significant effects on decreasing cholesterol. The decreasing cholesterol mechanism should include: ① increasing FBA excretion; ② decreasing the activity of HMG-CoA reductase; ③ increasing activity of LDL-R.
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Objective To investigate low density lipoprotein receptor (LDLR)gene mutation in familial hypercholesterolemia (FH) patients. Methods The proband was given clinical diagnosis of homozygous FH based on marked features and blood lipid tests results. After apoB100R3500Q mutation was excluded, the promoter region and all of the 18 exons of LDLR gene were amplified by touch-downpolymerase chain reaction (PCR). The PCR products were analyzed by single-strand conformationalpolymorphism (SSCP). The PCR products with abnormal single strands were sequenced directly. Thesecondary structures of the mutational and wild type proteins were analyzed and compared byANTHEPROT5.0, and then the tertiary structures of the mutant and wild type LDLR were predicted atSWISS MODEL homepage online. Results A homozygous mutation A606T at exon 13 of the patients wasfound by SSCP and confirmed by DNA sequencing. GOR Ⅰ method in ANTHEPROT5.0 indicates that therandom coils and turns would replace some helixes at the mutation site. The online prediction from theSWISS MODEL homepage indicates the backbone structure of the mutant LDLR has no difference from thewild type one. Conclusion The results suggest the A606T mutation of LDLR gene is the cause of the FH inthis pedigree.
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Objective To observe the impact of IL-1β on the expression of lectin-like oxidized LDL receptor 1 (LOX-1) and ATP-binding cassette transporter A1 (ABCA1) in human mesangial cell line (HMCL), and its association with cholesterol homeostasis of HMCL. Methods Levels of LOX-1 and ABCA1 of HMCL induced by IL-1β were examined by using real-time PCR and Western blot. Results IL-1β up-regulated LOX-1 mRNA and protein expression. Treated with 5 μg/L IL-1β, the levels of LOX-1 mRNA and protein reached the peak after 6 h and 24 h of stimulation and were 6.87 folds and 1.88 folds of control rspectively. The expression of ABCA1 mRNA and protein of lipid-loaded HMCL was down-regulated by IL-1β Stimulated with 5 μg/L IL-1β the expression of ABCA1 mRNA and protein decreased to the lowest level, 19.0% and 50.62% of the baseline respectively. Conclusions The expression of LOX-1 can be up-regulated while the expression of ABCA1 can be decreased by the stimulation of IL-1β. IL-1β can enhance dyslipidemia and influence the balance of cholesterol homeostasis of HMCL.