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Objective: To study the effects of cellular repressor of E1 A stimulated genes(CREG) on lysosome biogenesis and expression of cathepsins in mouse peritoneal cavity macrophages. Methods: Lysotracker Red staining and transmission electron microscope were applied to observe the effect of CREG on lysosome biogenesis in mouse peritoneal cavity macrophages using gain-of-function and loss-of-function approaches. The effect of CREG on expression of cathepsins in mouse peritoneal cavity macrophages were studied by immunofluorescence staining and Western blot. Results: It was confirmed that CREG can promote lysosome biogenesis in mouse peritoneal cavity macrophages using Lysotracker Red staining and transmission electron microscope. The findings of immunofluorescence staining and Western blot provided evidence that CREG promoted expression of cathepsin B and cathepsin S in mouse peritoneal cavity macrophages. Conclusion: CREG promotes lysosome biogenesis and expression of cathepsin B and cathepsin S in mouse peritoneal cavity macrophages. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects and possible mechanism of astragalus radix extract(ARE) on high fat diet(HFD)induced obesity in mice. Methods: A total of 30 C57 BL/6 mice were randomly divided into normal diet(ND) group(n=10), HFD group(n=10) and HFD+ARE group. Body weight and food intake were recorded. After 16 weeks, epididymal white adipose tissue was weighted and size of adipocytes was observed by HE staining; fatty degeneration of liver was assessed by HE staining. Level of leptin and adiponectin were assessed by ELISA. Expression level of peroxisome proliferator activated receptor γ(PPARγ) was determined by Western Blot. Results:(1) Compared to ND group, body weight and calorie intake increased in HFD group, which indicates that model of obesity was successfully established; ARE group had lowered body weight with similar calorie intake in contrast to HFD group.(2)Compared to ND group, heavier white adipose tissue, increased adipocyte size and significant fatty degeneration were detected in HFD group; while the above mentioned indexes were greatly improved in ARE group compared to those in HFD group.(3) Compared to ND group, increased leptin and decreased adiponectin were found in HFD group; whereas lower leptin and higher adiponectin were found in ARE group compared to HFD group.(4) Compared to ND group, expression level of PPARγ significantly increased in HFD group, while ARE treatment reversed this change. Conclusion: ARE alleviates obesity induced by HFD in mice possibly through inhibiting PPARγ. [ABSTRACT FROM AUTHOR]

To investigate the role and possible mechanism of cellular repressor of E1A-stimulated genes (CREG) in oxidized low density lipoprotein (ox-LDL) induced proliferation of human vascular smooth muscle cells (VSMC). Methods: VSMC from human thoracic aorta were treated with 60 μg/mL ox-LDL. At different time points (0 h, 12 h, 24 h and 48 h), cell numbers were counted to plot growth curve; BrdU staining was performed to assess percentage of proliferative cells at 48 h; Western Blot was carried out to detect CREG expression at different time points. Then VSMC over-expressing CREG (VSMCCREG) was established by transfecting VSMC with a plasmid vector carrying CREG gene. These cells were also treated with ox-LDL. Cell counting, BrdU staining, and detection of CREG and phospho-Erk1/2 were performed as mentioned above. Compared to untreated VSMC, VSMC treated with ox-LDL had more total cells and higher ratio of BrdU positive cells; moreover, expression of CREG decreased gradually after treatment. In addition, compared to VSMC treated with ox-LDL, VSMCCREG treated with ox-LDL showed significantly less number of total cells, decreased ratio of BrdU positive cells, increased CREG expression while down-regulated phosphorylated Erk1/2. Over-expression of CREG could inhibit ox-LDL induced proliferation of human VSMC possibly through down-regulating Erk1/2 phosphorylation. [ABSTRACT FROM AUTHOR]

Objective: To investigate the influence of different salt concentration on the renal fibrosis and macrophages infiltration in salt sensitive hypertension. Methods: Dahl salt sensitive rats were randomly divided into the normal salt (0.3 ％ nacl) group,4 ％ high salt, 8 ％ high salt groups at six weeks continuously feeding for 8 weeks, each group contained 15 rats. Tail-cuff method was used to value rat blood pressure at 8 weeks, Masson trichromatic method was used to detect renal fibrosis of the three groups at 8 week. Immunohistochemistry and Western blot method were used to depict the renal macrophage infiltration at 8 week. Results:1) The blood pressure of 4 ％ salt and 8％ high salt group rats were significantly higher than those of the normal salt group at 8week,meanwhile the blood pressure of 8 ％ high salt was further increased than that of 4 ％ high salt group at 8 week. 2) The relative kidney weight and renal fibrosis of 4 ％ salt and 8 ％ high salt group rats were obviously higher than that of normal salt group at 8 week, meanwhile the relative kidney weight and renal fibrosis of 8 ％ high salt were further increased than those of 4 ％ high salt group at 8 week. 3) The macrophage infiltration of 4 ％ salt and 8％ high salt group rats were higher than that of the normal salt group at 8week, meanwhile the macrophage infiltration of 8 ％ high salt was further increased than that of 4 ％ high salt group at 8 week. Conclusion: Different high salt concentrations had different effect on the renal fibrosis and macrophage infiltration in the salt sensitive hypertension, high salt concentration could exacerbate the renal fibrosis and macrophage infiltration. [ABSTRACT FROM AUTHOR]

Objective: To make genetic diagnosis and discuss the clinical characteristics in two Chinese Han Marfan syndrome (MFS) families. Methods: Family investigation and pedigree analysis was earned out in two MFS families. Variants detection for whole exons of FBN1 gene were performed by polymerase chain reaction and DNA direct sequencing. Functional significances for variants were assessed using by Swiss-model. Polyphen-2 and SIFT software. Results: The two families were characterized as an autosomal dominant inheritance. A de novo variant of FBN1 gene, an insertion A at 1691 base of exon 13 was identified in the patients of family 1 (1691 ins A), which resulted in the premature termination of translation at 571 amino acid, hi addition, a known point mutation at exon 27 (3463 G>A) was found in the patients of family 2. which resulted in amino acid change from Asp to Asn at 1155. The two variants were absent in healthy family members and 50 healthy controls. Results of functional significance prediction indicated that these two variants might influence the structure or function of fibrillin-1 protein. Conclusions: A novel insertion variant (1691 ins A) and a known point mutation (3463 G>A) are found in two MFS families. The results enlarge the spectrum of FBN1 gene variants and further support the hypothesis that FBX1 gene variants are pathogenic variants for MFS. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relationship between serum CC chemokine ligand 5 (CCL5) level and the severity of coronary atherosclerosis. Methods: The expression of CCL5 protein in carotid atherosclerotic plaques obtained from carotid eversion endarterectomy (CEE) of 5 cases of patients with highgrade stenosis (>70 %) (AS group) and 3 cases of normal postmortem arteries (control group) were detected by Immunohistochemistry and Western blotting. The CCL5 expression was compared between the two groups. 110 cases of consecutive patients who underwent coronary angiography were selected and divided into the control group (Gensini's score = 0, n=27), mild lesions group (020, n=51). The blood pressure, blood lipid, blood glucose and serum CCL5 of all groups were investigated. Their serum CCL5 levels were measured by enzyme linked immunosorbent assay (ELISA). The relationship between risk factors, serum CCL5 level and Gensini score were analyzed. Results: The expression of CCL5 protein in the carotid artery atherosclerosis was evidently higher than that in the normal carotid artery, and the serum CCL5 level of CAD patients was significantly higher than that of the control group. Sex, smoking history, systolic pressure, diastolic pressure, glucose and serumCCL5 were significantly different among three groups. Correlation analysis indicated that diastolic pressure, glucose and serum CCL5 were positively correlated with Gensini's score (r=0.276, 0.418, 0.519; P<0.05). In multi-variant linear regression model, serum CCL5 (B=8.775; P=0.000) were established as independent predictors of Gensini's score. Conclusion: Serum CCL5 was positively correlated with the severity of coronary artery atherosclerosis and was proved to be an independent predictor of Gensini's score. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relationship between serum CC chemokine ligand 5 (CCL5) level and the severity of coronary atherosclerosis. Methods: The expression of CCL5 protein in carotid atherosclerotic plaques obtained from carotid eversion endarterectomy (CEE) of 5 cases of patients with highgrade stenosis (>70 %) (AS group) and 3 cases of normal postmortem arteries (control group) were detected by Immunohistochemistry and Western blotting. The CCL5 expression was compared between the two groups. 110 cases of consecutive patients who underwent coronary angiography were selected and divided into the control group (Gensini's score = 0, n=27), mild lesions group (020, n=51). The blood pressure, blood lipid, blood glucose and serum CCL5 of all groups were investigated. Their serum CCL5 levels were measured by enzyme linked immunosorbent assay (ELISA). The relationship between risk factors, serum CCL5 level and Gensini score were analyzed. Results: The expression of CCL5 protein in the carotid artery atherosclerosis was evidently higher than that in the normal carotid artery, and the serum CCL5 level of CAD patients was significantly higher than that of the control group. Sex, smoking history, systolic pressure, diastolic pressure, glucose and serum CCL5 were significantly different among three groups. Correlation analysis indicated that diastolic pressure, glucose and serum CCL5 were positively correlated with Gensini's score (r=0.276, 0.418, 0.519; P<0.05). In multi-variant linear regression model, serum CCL5 (B=8.775; P=0.000) were established as independent predictors of Gensini's score. Conclusions: Serum CCL5 was positively correlated with the severity of coronary artery atherosclerosis and was proved to be an independent predictor of Gensini's score. [ABSTRACT FROM AUTHOR]

Objective: CX3CL1 played an important role in atherosclerosis recently. We investigated the association between the chemokine CX3CL1 with coronary artery disease in type 2 diabetes mellitus. Methods: CX3CL1 expression in carotid artery specimens was analysed by immunohistochemistry. The study was conducted in 400 coronary artery disease in type 2 diabetes mellitus and 400 control subjects in a case-control study. CX3CL1 gene rs170364 variant was detected by the directing sequencing technology. Results: CX3CL1 expressions were increased markedly in carotid artery plaque than in normal carotid artery. CX3CL1 gene rs170364 genotype (GG, GT and TT) frequency in coronary artery disease in type 2 diabetes mellitus is 42.7%, 40.0% and 17.2%, and 50.2%, 39.6% and 10.2% in the control group. CX3CL1 gene rs170364 variant is a genetic risk factor for coronary artery disease in type 2 diabetes mellitus (P <0.05). Logistic regression analysis with adjustments for other risk factors showed that the CX3CL1 gene rs170364 T allele significantly increases the risk of coronary artery disease in type 2 diabetes mellitus compared with the control subjects (P <0.05). Conclusion: Our study shows that the CX3CL1 expression increased in the carotid plaques and serum of the coronary artery disease in type 2 diabetes mellitus. CX3CL1 gene rs170364 variant may be considered a genetic risk factor for coronary artery disease in type 2diabetes mellitus. [ABSTRACT FROM AUTHOR]

Objective: To explore the clinical significance of serum CX3CL1 level for coronary artery disease. Methods: The study was conducted in 250 cases of coronary artery disease patients(75 patients with stable angina pectoria,75 patients with unstable angina pectoris,50 patients with ST-elevation acute myocardial infarction,50 non ST-elevation acute myocardial infarction patients) and 200 cases of control subjects(normal coronay artery) in a case-control study. The CX3CL1 level in different groups were analyzed by ELISA and CX3CR1 expression on CD4~+CD28~-T cells was analyzed by flow cytometry. Results: The serum CX3CL1 level increased markedly in coronary artery disease patients compared with the control subjects(P<0.05). The serum CX3CL1 level of patients with stable unstable angina was increased in the patients with ST-elevation acute myocardial infarction than those with the non ST-elevation acute myocardial infarction patients(P<0.05). An increased number of CD4~+CD28~-CX3CR1+T cells was found in the patients with coronary artery disease patients than the control group especially in the ST-elevation acute myocardial infarction patient(P<0.05). Conclusion: CX3CL1 might be involved in the formation of coronary atherosclerotic plaque and played an important role in the atherosclerotic procession,which might be useful to predict the stability of plaque in the early stage of coronary artery disease. [ABSTRACT FROM AUTHOR]

Objective: To explore the clinical significance of serum CX3CL1 level for coronary artery disease. Methods: The study was conducted in 250 cases of coronary artery disease patients (75 patients with stable angina pectoria, 75 patients with unstable angina pectoris, 50 patients with ST-elevation acute myocardial infarction, 50 non ST-elevation acute myocardial infarction patients) and 200 cases of control subjects (normal coronay artery) in a case-control study. The CX3CL1 level in different groups were analyzed by ELISA and CX3CR1 expression on CD4+CD28-T cells was analyzed by flow cytometry. Results: The serum CX3CL1 level increased markedly in coronary artery disease patients compared with the control subjects (P<0.05). The serum CX3CL1 level of patients with stable unstable angina was increased in the patients with ST-elevation acute myocardial infarction than those with the non ST-elevation acute myocardial infarction patients (P<0.05). An increased number of CD4+CD28-CX3CR1+T cells was found in the patients with coronary artery disease patients than the control group especially in the ST-elevation acute myocardial infarction patient (P<0.05). Conclusion: CX3CL1 might be involved in the formation of coronary atherosclerotic plaque and played an important role in the atherosclerotic procession, which might be useful to predict the stability of plaque in the early stage of coronary artery disease. [ABSTRACT FROM AUTHOR]

Objective: To establish cardiac-specific Creg gene knockout mice and analyze their phenotype. Methods: Ordered mice with Creg gene flanked by loxP (Cregflox/flox) were crossed with transgenic mice with Cre recombinase gene driven by muscle creatine kinase (Cknun-cre) promoter to obtain Cregflox/+VCkmm-cre mice. Then mutual mating was earned out in Cregflox/+/Ckinm-cre mice to establish cardiac specific Creg conditional knockout (Creg cKO) mice with a genotype of Cregflox/floxCkmm-cre. Genotyping was realized by PCR. Expression of Creg gene in heart tissue was determined by Real-time PCR and Western Blot. Size and morphology of hearts from knockout mice and then wild type littennates were assessed by HE staining. Electrocardiography (ECG) was recorded and left ventricular contractility was measured by small animal echocardiography. Results: (1) Creg cKO mice were successfully established and confirmed by genotyping. (2) Compared with wild type control. Creg cKO mice showed no significant change in size and morphology of heart. ECG and ejection fraction of left ventricle. Conclusion: We successfully established cardiac-specific Creg cKO mice, which will provide powerful tools for further investigations on the role and mechanism of CREG in heart diseases. [ABSTRACT FROM AUTHOR]

Objective: To explore and optimize the isolation, culture and identification of cardiac primary fibroblasts of adult SD rats in vitro. Methods: The heart of Adult SD rat is cuted into small tissue blocks then using the following four methods (A: 0.08% trypsin +0.1% collageuase II. digestion 15 min; B: 0.2% collagenase II. digestion 15 min: C: 0.2% collagenase n. digestion 60 min: D: 0.2% collagenase II. digestion 90 min) to isolate primary cardiac fibroblast. At last primary cultured fibroblasts are cultured through differential adherent method. The basic morphological characteristics of fibroblasts are observed by inverted microscope, and the primary cells are identified by Vimentin inmmnofluorescence staining. The survival rate of the cultured fibroblasts is identified by using the method of cell counting. Results: The primary cardiac fibroblast can be isolated through above four methods, but the method of single enzyme digestion 60 min can culture more cells in good condition, and can go down to next generation at 3 d. The positive rate of Vimentin immunofluorescence staining is nearly up to 97% at 72 h. The cell death rate is significantly decreased by Blue staining, and cell count reveals that the cell growth status is excellent. Conclusion: The most efficient, fast and stable method for the extraction of the primary fibroblasts of adult SD rats is single enzyme digestion 60 min which provides an ideal experimental cell model for the cardiac basic and clinical research. [ABSTRACT FROM AUTHOR]

Objective: To observe the effect of C-C motif ligand2 (CCL2) on the expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs cells were isolated and cultured in vitro. HUVECs were plated into 6-well plate, when cells were grown to 80-90 %, CCL2 overexpression plasmid [pcDNA3.1 (+)-CCL2] or CCL2 small interfering RNA (si-CCL2) was transfected into HUVECs. At different time points after transfection (12 h, 24 h and 48 h), HUVECs were collected for RNA and protein extraction. The mRNA expressions of CCL2 and ICAM-1 were examined using quantitative RT-PCR. The protein expressions of CCL2 and ICAM-1 were examined using western blotting. Results: (1) Compared with pcDNA3.1 (+) group, the expressions of CCL2 mRNA and protein were significantly higher in pcDNA3.1 (+)-CCL2 group; Compared with si-Control group, the expressions of CCL2 mRNA and protein were significantly decreased in si-CCL2 group. (2) Compared with pcDNA3.1 (+) group, pcDNA3.1 (+)-CCL2 significantly increased the mRNA and protein expressions of ICAM-1 in HUVECs, and si-CCL2 significantly inhibited the mRNA and protein expressions of ICAM-1 in HUVECs compared with si-Control group. Conclusions: CCL2 could affect ICAM-1 expression in HUVECs, which may provide a theoretical basis for clarifying the mechanisms of atherosclerosis. [ABSTRACT FROM AUTHOR]

Objective: To investigate the association between the ACE gene ID variant with coronary artery disease (CAD) in Northern Han population of China. Methods: In this study we used the gene directing sequencing analysis to detect the ACE gene I/D variant among the 56S CAD patients and 529 control subjects by a case-control study. Results: hi CAD patients and controls subjects, we found that ACE gene ID variant conformed to the Hardy-Weinberg equilibrium well. ACE gene ID variant (II. ID and DD(genotype frequency was 50.3 %. 30.2 % and 19.5 % in CAD patients and 41.7 %. 47.2 % and 11.1 % in controls subjects. ACE gene ID variant maybe a genetic risk factor for CAD in Chinese Han population (P< 0.05). Logistic regression analysis with adjustments for the other risk factors of CAD showed that ACE gene I/D variant significantly increased the risk of CAD compared with the non-carriers (P<0.05). Conclusions: hi Northern Han population of China. ACE gene ID variant maybe considered a genetic risk factor for CAD. [ABSTRACT FROM AUTHOR]

Objective: To investigate the association between the interleukin 23 Receptor (IL23R) rs6682925 variant with coronary artery disease (CAD) in Northern Han population of China. Methods: The study was conducted in 568 coronary artery disease patients and 529 control subjects in a case-control study. We used the gene directing sequencing technology to detect the IL23R rs6682925 and rs1884444 variant. Results: We found that the IL23R rs6682925 and rs1884444 variant frequency conformed well to the Hardy-Weinberg equilibrium in both case and control subject. IL23R rs6682925 (CC, TC and TT) genotype frequency was 22.9 %, 39.6 % and 37.5 % in CAD group and 41.7 %, 47.2 % and 11.1 % in control group. IL23 R rs6682925 C allele was a genetic risk factor for coronary artery disease in Northern Han population of China (P<0.05). There were no significant differences in the genotype and allele distribution of IL23R rs1884444 variant between CAD and control subjects (P>0.05). Logistic regression analysis with adjustments for the other risk factors showed that the C allele carriers of IL23R rs6682925 variant significantly increased the risk of coronary artery disease compared with the non-carriers (P<0.05). Conclusions: IL23R rs6682925 variant may be considered a genetic risk factor for CAD in Northern Han population of China. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects and possible mechanism of astragalus radix extract (ARE) on differentiation of vascular smooth muscle cells (VSMCs) from mouse embryonic stem cells (ESCs). Methods: VSMCs differentiation model was established by seeding ESCs directly on collagen IV coated plates. Cells were divided into 3 groups according to dose of ARE supplemented into culture medium: (1) control group (no ARE); (2) low dose group (60 g/mL); and (3) high dose group (120 g/mL). Cells were treated for 14 days. Immunofluorescence staining, real-time PCR and Western Blot were employed to detect expression of differentiation marker of VSMCs. The cholinergic receptor agonist (carbacol) was used to assess contractility of VSMCs from different groups. Real-time PCR was performed to detect changes of key factors regulating VSMCs differentiation. Results: Compared to the control group, ARE treatment significantly increased expression of VSMCs differentiation markers in a dose dependent manner. ARE treatment also improved contractility of VSMCs. In addition, ARE treatment promoted expression of transforming growth factor (TGF- ). Conclusion: ARE promotes VSMCs differentiation fromESCs possibly through activating TGF- pathway. [ABSTRACT FROM AUTHOR]