Object To investigate the effect of PPARαactivation on PPARγ-induced fatty liver in the mouse. Methods Wild type mice ( C57BL/6) aged 4 to 5 weeks were used as animal models.All mice were divided into four groups.The mice in the first group were fed with chow diet.The mice in the second group were fed with a diet containing 0.125%Wy-14,643, an agonist of PPARa, for 8 days.The mice in the third group were injected with Ad/PPARγvia tail vein for 5 day.The mice in the fourth group were firstly fed with Wy-14,643 diet for 3 days and then injected with Ad/PPARγvia tail vein for another 5 day.Mouse livers were collected and photographed.The effect of PPARαactivation on PPARγ-induced fatty liver was observed by H&E and Oil red O staining.Results Compared with the controls, wild-type mice treated with Wy-14,643 for 8 days exhibited marked hypertrophy of hepatocytes with increased cytoplasmic eosinophil-ia and proliferation of peroxisomes.The liver size was significantly increased in the wild-type mice treated with Ad/PPARγfor 5 days, and over-expression of PPARγstrongly induced hepatic steatosis.Importantly, the wild-type mice pretreated with Wy-14,643 for 3 days and then given Ad/PPARγinjection exhibited dramatically the increase of liver size, which might be due to the dual function of PPARa and PPARγ.Compared with the Ad/PPARγgroup, the Wy-14,643 pretreat-ment group showed a reduced hepatic steatosis.Conclusions Activation of PPARαby Wy-14,643 effectively improves PPARγ-stimulated hepatic steatosis, which provides a novel target for prevention and therapy of fatty liver.

ABSTRACT:Objective To analyze the correlation between plasma nitric oxide (NO)level and atherosclerotic lesion in high cholesterol-fed (HCD ) rabbits.Methods Twenty male Japanese white rabbits were divided randomly into two groups,which were fed with normal diet (control group,n =10)or HCD (experimental group, n =10 )for 1 6 weeks.At the end of the experiment,plasma lipid and NO levels were measured.The gross atherosclerotic lesions in each group were detected by Sudan IV staining while intimal lesion area was measured by hematoxylin/eosin (HE)and elastica van gieson (EVG).Moreover,the macrophages (MΦ)and smooth muscle cells (SMC)were detected by immunohistochemical staining.The correlation analysis was made to reveal the relationship between atherosclerotic lesions and plasma NO level.Results Compared with those in control group, the total cholesterol (TC),triglyceride (TG),low-density lipoprotein cholesterol (LDL-C)and NO levels all increased significantly in experimental group.Atherosclerotic lesions appeared on the vascular wall in the latter group.The area of atherosclerotic lesions and MΦ in the plaque had a positive association with plasma NO level. Conclusion There is a relationship between plasma NO level and the size of HCD-induced atherosclerotic lesions in rabbits.Meanwhile,the MΦ positive area in the atherosclerotic plaque is also associated with plasma NO level in cholesterol-fed rabbits,suggesting that plasma NO level may be associated with the occurrence and progress of early atherosclerosis.

Objective To investigate the effect of allicin on the development of atherosclerosis in apoE-/-mice and explore its underlying mechanism from the perspective of protein S-nitrosylation.Methods Thirty male apoE-/- mice were randomly divided into 3 groups:control group (saline,ig),low-dose group (allicin,9 mg/kg·d, ig)and high-dose group (allicin,18 mg/kg·d,ig).They were fed with high cholesterol diet for 12 weeks.The levels of plasma lipids,oxidized-LDL (ox-LDL),malondialdehyde,tumor necrosis factor-alpha and nitric oxide (NO)were measured.The atherosclerotic lesions in aortic root were evaluated after hematoxylin and eosin staining and elastica van Gieson and immunohistochemical staining,respectively.Furthermore,in vitro experiments were performed using human umbilical vein endothelial cells (HUVECs).The HUVECs were treated with allicin (10μmol/L or 20 μmol/L)for 24 hours in the presence of ox-LDL (50 μg/mL).The level of NO in supernatant was measured by a nitrate/nitrite assay. The protein S-nitrosylation of the HUVECs was detected through immunofluorescence.Results The histological analysis revealed that allicin treatment not only significantly decreased the areas of the atherosclerotic lesion (all P <0.05)but also suppressed the macrophage accumulation and smooth muscle cell proliferation in the lesion.There was no significant difference in the levels of plasma lipids between control and treated groups.However,allicin exerted obvious anti-oxidative and anti-inflammatory effects. Interestingly,the allicin treatment led to marked increase of the plasma NO level (P <0.05)and aortic protein S-nitrosylation.The experiments in vitro further proved that the allicin up-regulated the levels of NO and protein S-nitrosylation in HUVECs treated with ox-LDL (P < 0.01 ).Conclusion Allicin can inhibit the development of atherosclerosis.The mechanism is associated with the up-regulation of protein S-nitrosylation in endothelial cells, which plays an important role in anti-oxidization and anti-inflammation.

Objective The aim of this study was to generate human cholesteryl ester transfer protein ( CETP) transgenic rabbits and analyze their biological properties.Methods We generated human CETP transgenic rabbits by DNA microinjection, and detected the expression of human CETP by real-time PCR and Western blot assay.The activity of CETP was measured using an activity assay kit.Results Human CETP transgenic rabbits were successfully generated by DNA microinjection.Compared with wide type rabbits, the expression of human CETP was dramatically increased in the liver of the human CETP transgenic rabbits.The plasma CETP activity was also much higher in the liver of human CETP transgenic rabbits than that of control rabbits.Conclusions The model of human CETP transgenic rabbits is successfully established by DNA microinjection.It will provide a useful tool for the studies of CETP biological function and its involvement in the mechanisms of cardiovascular diseases.

OBJECTIVE: To construct an effective lentiviral vector for RNA interference (RNAi) with human glucose transporter 3 (GLUT3)gene.
 METHODS: Four pairs of shRNA sequences against different parts of GLUT3-mRNA were separately cloned into the RNAi plasmid vector pLV-shRNA by recombinant DNA technology to construct shRNA expression vectors pLV-shRNA-GLUT3-1, pLV-shRNA-GLUT3-2, pLV-shRNA-GLUT3-3, and pLV-shRNA-GLUT3-4. The vectors were transfected into HeLa cells to detect the effectiveness of GLUT3 gene silencing. One of effective vectors was selected and co-transfected into 293T cells with lentivirus packaging plasmids to obtain packaged lentivirus particles LV-GLUT3. After viral titer determination, U251 glioblastoma cells were infected with LV-GLUT3 at a multiplicity of infection (MOI) of 10. Finally, the expression of GLUT3 protein was detected by Western blot. 
 RESULTS: DNA sequencing demonstrated that the shRNA sequences were successfully inserted into the pLV-shRNA vectors. In HeLa cells, the expression of GLUT3-mRNA was significantly down-regulated by the recombinant vectors compared with negative control. The recombinant lentivirus LV-GLUT3 harvested from 293T cells had a titer of 1.5×10(9) TU/mL. After infection with LV-GLUT3, the expression of GLUT3 protein in U251 glioblastoma cells was down-regulated. 
 CONCLUSION An effective lentiviral shRNA expression vector targeting the GLUT3 gene is successfully constructed and can be used for further study on the functions of GLUT3 gene.
Genetic Vectors ; Glucose Transporter Type 3 ; genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Lentivirus ; Plasmids ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the effects of PPARγ overexpression on steatosis in mouse primary hepatocytes.

METHODSPrimary hepatocytes isolated from C57BL/6J mice were infected with either Ad/LacZ or Ad/PPARγ for 48 h. Steatosis of the primary hepatocytes was checked by Oil Red O staining. The mRNA and protein expression of adipocyte-specific genes PPARγ, aP2 and CideA were analyzed by using RT Real-time PCR and Western Blot.

RESULTSPrimary hepatocytes were small and even. Hepatocyte nuclei were round with dispersed chromatin and prominent nucleoli. Accumulated lipid droplets were observed in Ad/PPARγ-infected hepatocytes, but in Ad/LacZ-infected hepatocytes. Moreover, compared with Ad/LacZ-infected hepatocytes, the mRNA expression of PPARγ, aP2, FGF21 and CideA in Ad/PPARγ-infected hepatocytes were significantly induced, the protein expression of PPARγ and its target aP2 strongly increased.

CONCLUSIONover expression of PPARγ induces adipogenic steatosis in mouse primary hepatocytes.

Adipocytes ; metabolism ; Adipogenesis ; Animals ; Cells, Cultured ; Fatty Liver ; metabolism ; pathology ; Genetic Vectors ; Hepatocytes ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; PPAR gamma ; metabolism ; Transfection

【Objective】 To study the effect of macrophage mediator 1 (MED1) deficiency on atherosclerosis in female mice. 【Methods】 ApoE knockout (ApoE^-/-), LDLR knockout (LDLR^-/-), MED1^fl/fl, and macrophage MED1 knockout (MED1^△Mac) mice were recruited in the study. Two types of mouse model were constructed:ApoE and macrophage MED1 double knockout (MED1^△Mac/ApoE^-/-) mice and their littermate controls (MED1^fl/fl/ApoE^-/-). ② LDLR knockout (LDLR^-/-) mice receiving bone marrow from MED1^△Mac (MED1^△Mac→LDLR^-/-) or MED1^fl/fl (MED1^fl/fl→LDLR^-/-) mice. Female mice from these two models were fed a Western diet (21% fat and 0.15% cholesterol) for 12 weeks to promote the development of atherosclerosis. Body weight, total cholesterol (TC), and total triglyceride (TG) content in plasma were measured dynamically. After Western diet feeding for 12 weeks, aortic tree and aortic root were collected and hematoxylin-eosin (H&E) and oil red O staining were performed. 【Results】 Plasma TC and TG did not significantly differ between MED1^fl/fl/ApoE^-/- control group and MED1^△Mac/ApoE^-/-experimental group. However, the plaque area in aortic tree and aortic root was significantly increased in MED1^△Mac/ApoE^-/-mice. Moreover, compared with that in MED1^fl/fl→LDLR^-/- control group, the plaque area of aortic tree and aortic root had an increasing trend in MED1^△Mac→LDLR^-/- mice group. 【Conclusion】 MED1 deficiency in macrophages promotes the development of atherosclerosis in female ApoE or LDLR knockout mice.