OBJECTIVE: To study the role of apolipoprotein E (APOE) in regulating endometrial cancer metastasis and explore the signaling pathway in the regulatory mechanism. METHODS: Human endometrial cancer cell line HEC-1B was transfected with a control siRNA (siCtrl) or a specific siRNA targeting APOE (siAPOE) or with either pEGFP-N1 plasmid or an APOEoverexpressing plasmid. The changes in migration, proliferation, apoptosis and cell cycle of the transfected cells were examined using wound healing assay, Transwell migration assay, MTT assay, flow cytometry, and Hoechst staining. The activity of the ERK/MMP9 signaling pathway in the transfected cells was assessed using RT-qPCR and Western blotting. The expression level of APOE in clinical specimens of endometrial cancer tissues were detected using immunohistochemistry and its correlation with differentiation of endometrial cancer tissues was analyzed. RESULTS: Wound healing assay and Transwell migration assay showed that compared with those in siCtrl group, HEC-1B cells transfected with siAPOE showed significantly reduced migration ability (P < 0.05), whereas APOE overexpression significantly promoted the migration of the cells (P < 0.05). Neither APOE knockdown nor overexpression produced significant effects on HEC-1B cell proliferation as shown by MTT assay and flow cytometry. Hoechst staining revealed that transfection with siAPOE did not significantly affect apoptosis of HEC-1B cells. APOE knockdown obviously reduced and APOE overexpression enhanced ERK phosphorylation and MMP9 expression in HEC-1B cells (P < 0.05). Treatment with U0126 partially reversed the effects of APOE overexpression on ERK phosphorylation, migration and MMP9 expression in HEC-1B cells (P < 0.05). APOE is highly expressed in clinical samples of endometrial cancer tissues as compared with the adjacent tissues. CONCLUSION APOE is highly expressed in endometrial cancer tissues to promote cancer cell migration by enhancing ERK phosphorylation and MMP9 expression.
Female ; Humans ; Matrix Metalloproteinase 9/metabolism* ; Cell Line, Tumor ; Signal Transduction ; Endometrial Neoplasms/genetics* ; Cell Proliferation ; Apoptosis ; Cell Movement ; RNA, Small Interfering ; Apolipoproteins E ; Apolipoproteins/pharmacology*

OBJECTIVETo investigate the effect of apolipoprotein B (apoB) and E (apoE) genetic variations on lipid profile at baseline (before treatment), and also on the subsequent response to simvastatin therapy.

METHODSEighty-eight patients with hyperlipidemia were treated with simvastatin 5mg daily. The plasma levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and apo B were measured pre-treatment and at the end of the 4th, 8th and 12th post-treatment week. Polymorphisms of apoB at XbaI locus and apoE were determined by restriction fragment length polymorphism (RFLP).

RESULTSIn all patients, relative frequencies of X- allele and X+ allele were 0.943 and 0.057 for apoB gene respectively. For apoE gene the relative frequency of epsilon2 allele was determined as 0.182, epsilon3 as 0.580 and epsilon4 as 0.238. The reduction in TC level was more prominent in patients carrying X- allele than in those with X+ allele following treatment (-23. 9% vs. -13. 6%, P < 0. 05). Compared with patients carrying epsilon3 or epsilon4 allele, those with epsilon2 allele showed a significantly higher percentage in reduction of apoB level after treatment (P < 0.05).

CONCLUSIONThe relative frequency of apoB X+ allele is high in patients with hyperlipidemia, in whom the TC-lowering efficacy is decreased following treatment of simvastatin. The relative frequencies of epsilon2 and epsilon4 are also high in hyperlipidemic patients, and the epsilon2 allele is associated with reduction in apoB level during lipid-relating therapy.

Aged ; Alleles ; Apolipoproteins B ; genetics ; Apolipoproteins E ; genetics ; Cholesterol ; blood ; Cholesterol, LDL ; blood ; Female ; Gene Frequency ; Humans ; Hyperlipidemias ; blood ; drug therapy ; genetics ; Male ; Middle Aged ; Mutation ; Polymorphism, Restriction Fragment Length ; Simvastatin ; pharmacology ; therapeutic use ; Triglycerides ; blood

OBJECTIVETo observe monocyte (Mo) development in wild type C57BL/6 mice and apoE gene knockout (apoE(-/-)) mice, and to evaluate the immuno-regulatory effect of Huanglian Jiedu Decoction (HJD) on peripheral Mo development in apoE(-/-) mice.

METHODSFour, 8, 12, and 16 weeks old female C57BL/6 mice were set up as control groups of different ages, while 4, 8, 12, and 16 weeks old female apoE(-/-) mice were set up as hyperlipidemia groups of different ages. Four-week old female C57BL/6 mice were recruited as a blank group. Four-week old female apoE(-/-) mice were randomly divided into the control group, the Western medicine group, and the Chinese medicine group by paired comparison, 5 in each group. Equivalent clinical dose was administered to mice according to body weight. Mice in the Western medicine group were administered with Atrovastatin at the daily dose of 10 mg/kg by gastrogavage, while those in the Chinese medicine group were administered with HJD at the daily dose of 5 g/kg by gastrogavage. Body weight was detected each week. After 4 weeks blood lipids levels (such as TG, TC, LDL-C, and HDL-C), and the proportions of Mo and Ly6c(hi) were detected.

RESULTSCompared with 4-week-old homogenic mice, the proportion of Mo decreased in 16-week-old C57BL/6 mice (P < 0.05). Levels of TC and TG, and the proportion of Ly6c(hi) subtype increased, but the proportion of Mo de- creased in 8-week-old apoE(-/-) mice (P <0. 05). Levels of TC, TG, and LDL-C increased in 12-week-old apoE(-/-) mice (P < 0.05). Levels of TC, TG, LDL-C, and HDL-C increased in 16-week-old apoE(-/-) mice (P < 0.05, P < 0.01). Compared with 8-week-old homogenic mice, the proportion of Mo decreased in 16-week-old C57BL/6 mice (P < 0.05); levels of TC and LDL-C increased in 12-week-old apoE(-/-) mice (P < 0.05); levels of TC and HDL-C increased in 16-week-old apoE(-/-) mice (P < 0.05, P < 0.01). Compared with C57BL/6 mice of the same age, TC and TG increased, HDL-C decreased (P < 0.01) in 4-and 8-week-old apoE(-/-) mice (P < 0.01); levels of TC, TG, LDL-C increased, and HDL-C level decreased in 12- and 16-week-old apoE(-/-) mice (P < 0.05, P < 0.01); the proportion of Mo increased in 4-week-old apoE(-/-) mice (P < 0.05); proportions of Mo and Ly6c(hi) increased in 8-week-old apoE(-/-) mice (P < 0.05). Compared with the blank control group, levels of TC, TG, and LDL-C, proportions of Mo and Ly6c(hi) increased (P < 0.01, P < 0.05), but HDL-C level decreased (P <0. 01) in the control group after intervention. Compared with the control group, body weight gained less in the Western medicine group and the Chinese medicine group (P < 0.05); the proportion of Ly6c(hi) subtype decreased in the Chinese medicine group (P < 0.05).

CONCLUSIONSIn development process blood lipids levels in apoE(-/-) mice are not only associated with age. Blood lipids levels induced growth changes in natural immune system are also correlated with age. In early stage of lipids development HJD intervention could correct this special immune disorder in apoE(-/-) mice.

Animals ; Apolipoproteins E ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Gene Knockout Techniques ; Hyperlipidemias ; Lipids ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Monocytes ; physiology

OBJECTIVETo explore if PPARgamma agonist rosiglitazone could enhance the anti-atherosclerotic effects of mouse peroxisome proliferator-activated receptor gamma1 (PPARgamma1) gene transfer in apolipoprotein-knock out mice.

METHODSAdult ApoE-knock out mice were fed a Western-diet for 20-weeks and then injected with PBS, Ad. PPARgamma1 (5 x 10(8)pfu) or Ad. GFP (5 x 10(8)pfu) via jugular vein. Another group of mice were intervened with rosiglitazone (dissolved in 0.5% cellulose acetate, 4 mg.kg(-1).d(-1), per gavage) 1 week before Ad. PPARgamma1 injection (n = 10, each group). Two weeks later, the lipid core and plaque composition were characterized with oil red O staining and Movat method respectively. The expression of PPARgamma, SM-actin, MOMA-2, MMP-9/TIMP-1, CD40/CD40L and TF antigens in aortic roots and plaques among four groups were compared semi-quantitatively using immunohistochemical technology.

RESULTSAll parameters were similar between AdGFP and PBS groups (P > 0.05). The area of plaque were significantly decreased and oil red O staining area significantly increased in AdPPARgamma1 [(0.86 +/- 0.12) mm(2), (150 +/- 35) x 10(3) microm(2)] and AdPPARgamma1 + RO [(0.79 +/- 0.15) mm(2), (270 +/- 49) x 10(3) microm(2)] treated mice compared with AdGFP group [(0.98 +/- 0.17) mm(2), (80 +/- 21) x 10(3) microm(2)] all P < 0.05. Elastic fiber, collagen and proteoglycan in plaques were also significantly increased in AdPPARgamma1 and AdPPARgamma1 + RO groups. Upregulation of PPARgamma, SM-actin, TIMP-1 antigen activity and downregulation of MOMA-2, MMP-9, CD40/CD40L and TF antigen activity in AdPPARgamma1 and most significantly in AdPPARgamma1 + RO group were observed (P < 0.05).

CONCLUSIONAnti-atherosclerotic effects of PPARgamma1 gene transfer in ApoE-knock out mice could be enhanced by PPARgamma agonist rosiglitazone.

Animals ; Apolipoproteins E ; deficiency ; genetics ; Atherosclerosis ; genetics ; Gene Transfer Techniques ; Male ; Mice ; Mice, Knockout ; PPAR gamma ; agonists ; genetics ; metabolism ; Peroxisome Proliferator-Activated Receptors ; metabolism ; Thiazolidinediones ; pharmacology ; Transfection

OBJECTIVETo investigate the pathologies of aortic root atherosclerotic lesion in uremic apoE-/- mice and explore the effect of serum from patients with chronic renal insufficiency (CRI) and the uremic toxin, indoxyl sulfate (IS), on the expression of cholesterol transporting receptors and lipid accumulation in macrophages in vitro.

METHODSThe uremic apoE-/- mouse model was established by surgical operation. Frozen sections of the aortic root were collected from uremic apoE-/- mice, sham-operated apoE-/- mice and C57BL/6J mice and stained with oil red O to calculate the relative area of atherosclerotic plaque. Murine macrophage RAW264.7 cell line was treated for 12 h with different concentrations of IS or serum samples from CRI patients and healthy individuals, and the mRNA expressions of cholesterol transporting receptors (SR-A1, CD36, ABCA1, ABCG1 and SR-B1) were detected. After treatment for 24 h, the cells were induced into foam cells to determine lipid contents using oil red O staining.

RESULTSThe relative area of the atherosclerotic plaques in the aortic root increased significantly in uremic apoE-/- mice compared with that in sham-operated apoE-/- mice. CRI serum (5%) and IS (250 µmol/L) obviously increased the mRNA expression of CD36 and lipid accumulation in the macrophages, but did not affect the mRNA expression of other cholesterol transporting receptors.

CONCLUSIONCRI can accelerate the progression of atherosclerosis through the mechanism that IS in CRI serum promotes lipid accumulation in macrophages by enhancing the mRNA expression of CD36, which contributes to the formation of foam cells.

Animals ; Apolipoproteins E ; Cell Line ; Foam Cells ; chemistry ; Humans ; Indican ; pharmacology ; Lipids ; chemistry ; Macrophages ; chemistry ; Mice ; Mice, Inbred C57BL ; Plaque, Atherosclerotic ; pathology ; Renal Insufficiency, Chronic ; blood

This study aims to explore the effect of Buyang Huanwu Decoction glycosides on the inflammatory response of apolipoprotein E~(-/-)(ApoE~(-/-)) mice and RAW264.7 cells through nuclear factor kappa-B(NF-κB) signaling pathway. In the in vivo experiment, ApoE~(-/-) mice were fed with high-fat diets for 12 weeks to induce the animal model of atherosclerosis, and 75 μg·mL~(-1) oxidized low-density lipoprotein(Ox-LDL) incubated RAW264.7 cells for 24 h to establish the atherosclerosis cell model. Automatic biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay(ELISA), Western blot, and droplet digital polymerase chain reaction(PCR) were used to determine the blood lipid levels, aortic intimal thickness, inflammatory factor content, NF-κB pathway-related proteins, and mRNA expression levels, and evaluate arterial atherosclerotic lesions and anti-atherosclerotic mechanisms of the drug. The model of atherosclerosis was successfully established in ApoE~(-/-) mice after 12 weeks of feeding with high-fat diets. In the model group, the plasma levels of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-C) were increased(P<0.01), the intima of the blood vessels was thickened, the levels of inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) were increased, and the protein and mRNA expressions of NF-κB and inhibitor of NF-κB(IκBα) were significantly increased as compared with the control group. Compared with the model group, the high-dose Buyang Huanwu Decoction glycoside group decreased the plasma levels of TC, TG, and LDL-C, reduced the plaque area and thickness and the content of inflammatory factor TNF-α, and inhibited the protein and mRNA expressions of NF-κB and IκBα, with the effect same as Buyang Huanwu Decoction. In the in vivo experiment, 75 μg·mL~(-1) Ox-LDL stimulated RAW264.7 cells for 24 h to successfully establish a foam cell model. As compared with the control group, the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα in the model group increased. Compared with the model group, the middle-dose and high-dose Buyang Huanwu Decoction glycoside groups decreased the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα. The above results show that the glycosides are the main effective substances of Buyang Huanwu Decoction against atherosclerosis, which inhibit the NF-κB pathway and reduce the inflammatory response, thus playing the role against atherosclerotic inflammation same as Buyang Huanwu Decoction.
Mice ; Animals ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Glycosides/pharmacology* ; Cholesterol, LDL ; Atherosclerosis/genetics* ; Signal Transduction ; Inflammation/drug therapy* ; Interleukin-6 ; Apolipoproteins E/pharmacology* ; RNA, Messenger/metabolism*

OBJECTIVETo study the regulatory effect of lipi tiaozhi capsule (LTC) on the expression of peroxisome proliferator-activated receptor (PPAR) alpha and gamma mRNA in dyslipidemia rats and ApoE(-/-) mice, and to explore its mechanisms for regulating lipid metabolism. Methods 48 Wistar rats were randomly divided into the blank group, the model group, the treatment group (treated by LTC), and the control group (treated by Xuezhi-kang Capsule). After four-week modeling (except the blank group) 30 ApoE(-/-) mice were randomly divided into the blank group, the treatment group, and the control group. LTC was given by gastrogavage to rats and ApoE(-/-) mice in LTC groups while XZKC was given to XZKC groups. The medication was conducted once daily for eight weeks. The serum TC and TG contents of rats and mice were determined by enzymic method. The serum high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were detected by precipitation method. PPARalpha and gamma mRNA expressions were detected in the liver tissue of the rats and mice by fluorescent PCR.

RESULTSCompared with the model group and the blank group, the serum contents of TC, TG, and LDL-C of rats or mice in the treatment group decreased significantly (P < 0.01). The serum content of HDL-C increased significantly (P < 0.01). PPARalpha and gamma mRNA expressions of rats or mice increased significantly (P < 0.01). Compared with the control group, the serum contents of TC, TG, and LDL-C of mice and rats in the treatment group decreased (all P < 0.05), the serum content of HDL-C increased significantly (P < 0.05, P < 0.01). And PPARalpha and gamma mRNA expressions of rats or mice increased significantly (P < 0.05).

CONCLUSIONLTC could significantly increase PPARa and y mRNA expressions of experimental dyslipidemia rats and ApoE(-/-) mice, playing roles in regulating nuclear factors and further effecting lipid metabolism.

Animals ; Apolipoproteins E ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Dyslipidemias ; drug therapy ; metabolism ; Male ; Mice ; Mice, Knockout ; Peroxisome Proliferator-Activated Receptors ; genetics ; metabolism ; Phytotherapy ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

BACKGROUNDStudy of the relationship between mast cells and atherosclerosis is mostly dependent on pathological observation and cytology experiments. To investigate the effects of mast cells degranulation on plaque and their possible mechanisms we used apolipoprotein E knockout mice which had been placed perivascular common carotid collar with mast cells degranulator compound 48-80.

METHODSForty apolipoprotein E knockout mice were fed a western-type diet and operated on with placement of perivascular right common carotid collar. Four weeks after surgery, the mice were intraperitoneally injected with compound 48-80 (0.5 mg/kg) or D-Hanks every other day for 4 times. The serum lipids and activity of tryptase were measured. Tissue sections were stained with hematoxylin and eosin. Corresponding sections were stained with toluidine blue and immunohistochemically with antibodies against macrophage-specific antigen, alpha-smooth muscle actin, interleukin-1beta and von Willebrand factor. Simultaneously, basic fibroblast growth factor was detected by in situ hybridization and immunofluorescence.

RESULTSNo pathological change was observed in common carotid non-collar placement but atherogenesis in common carotid collar placement of both groups. There was a significant increase in plaque area ((5.85+/-0.75) x 10(4) vs (0.86+/-0.28) x 10(4) microm(2), P<0.05), the degree of lumen stenosis ((81+/-15)% vs (41+/-12)%, P<0.05), the activity of tryptase in serum ((0.57+/-0.13) U/L vs (0.36+/-0.10) U/L, P<0.05), and the percentage of degranulated mast cells ((80.6+/-17.8)% vs (13.5+/-4.1)%, P<0.05). The expressions of macrophage-specific antigen, alpha-smooth muscle actin, interleukin-1beta, basic fibroblast growth factor and the density of neovessel in plaque were more in the compound 48-80 group than in the control group.

CONCLUSIONSPerivascular common carotid collar placement can promote atherosclerotic plaque formation in apolipoprotein E knockout mice. Compound 48-80 increases plaque area and the degree of lumen stenosis by the mechanism that compound 48-80 promotes proliferation of smooth muscle cells and aggregation of macrophages. Compound 48-80 promotes angiogenesis in plaque. The mechanism is potentially that compound 48-80 increases the expressions of basic fibroblast growth factor mRNA and protein in plaque. Compound 48-80 enhances the expression of interleukin-1beta in plaque.

Animals ; Apolipoproteins E ; genetics ; Atherosclerosis ; chemically induced ; genetics ; metabolism ; pathology ; Carotid Arteries ; drug effects ; pathology ; Fluorescent Antibody Technique ; Immunohistochemistry ; In Situ Hybridization ; In Vitro Techniques ; Male ; Mast Cells ; drug effects ; metabolism ; Mice ; Mice, Knockout ; p-Methoxy-N-methylphenethylamine ; pharmacology

OBJECTIVETo observe the effect of Rhizoma chuanxiong (RC), Radix Paeoniae rubra (RP) and Xiongshao Capsule (XC, a compound of their active ingredients, Chuanxingols and Paeoniflorins) on stability of atherosclerotic plaque in ApoE-/- mice and to explore the probable mechanisms.

METHODSThe effect of RC, RP and XC in stabilizing atherosclerotic plaque, in terms of pathologic morphology, cell composition and inflammatory reaction, in the atherosclerosis model established on ApoE-/- mice was studied by using optical microscope, immunohistochemical method and computerized imaging analysis respectively.

RESULTSAfter the ApoE-/- mice being fed with high fat diet for 26 weeks, obvious atherosclerotic lesion with typical unstable characteristics was found in their aortic root. Both RC and RP had certain effects in lowering total cholesterol and increasing the thickness of fibre cap. RC could also lower the serum triglyceride (TC) level and the lipid-core/plaque area ratio as well as reduce the macrocytic infiltration. In addition to the same effects as above mentioned, XS could also raise the levels of high density lipoprotein-cholesterol (HDL-C), lower TC/HDL-C ratio, reduce inflammatory reaction and enlarge the collagen area in plaque.

CONCLUSIONThe acting links of RC and RP on atherosclerosis are different, the compound of their active ingredients, XS, shows a more evident effect in intervening unstable plaque. It demonstrates the effect-enhancing power of TCM compound and is worth further studying.

Animals ; Apolipoproteins E ; genetics ; Atherosclerosis ; blood ; genetics ; prevention & control ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; therapeutic use ; Female ; Lipids ; blood ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Paeonia ; chemistry ; Random Allocation

OBJECTIVETo observe the effect of six common Chinese medicinal herbs for promoting blood circulation, including Radix Paeoniae rubra (I), Radix Salviae miltiorrhizae (II), Rhizoma Chuanxiong (III), Radix Notoginseng (IV), Semen Persicae (V) and wine steamed Radix et Rhizoma Rhei (VI), on blood lipids and inflammatory reaction of atherosclerotic plaques in ApoE gene deficiency mice.

METHODSNinety mice, 6 - 8 weeks old, were divided into 8 groups, the model group, the control group (treated with simvastatin) and the six treated groups treated with the above-mentioned 6 Chinese medicinal herbs respectively. All the mice were fed with the diet of western kind for 13 weeks until the mature atherosclerotic plaques formed in them. Then they were treated with respective drugs for another 13 weeks except those in the model group. All the mice were sacrificed at the end of experiment, their blood was collected for lipids determination, heart and aorta were taken out for determining the level of CD68 in root of aorta, as well as the expressions of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-a (TNF-a) by immunohistochemistry staining.

RESULTSAll the 6 Chinese herbs showed regulatory action on blood lipids. The positive expression of CD68 in the model group displayed the highest activity. As compared with the model group, the CD68 positive expressed cells in the control group and the groups treated with Chinese herbs II, III, and IV were lesser (P < 0.05), and the expression of inflammatory factors (MCP-1 and TNF-alpha) in atherosclerotic plaques was significantly lower in the control group and the group treated with Chinese herb VI (P < 0.05).

CONCLUSIONChinese medicinal herbs tested in this study can interfere the maturing progress of atherosclerotic plaques and stabilize the plaques in ApoE deficiency mice, the mechanisms may relate to its actions in regulating lipids metabolism and inhibiting inflammatory reaction. Different Chinese medicinal herbs for activating blood circulation of conventional dosage might show difference in potency and acting links.

Animals ; Apolipoproteins E ; genetics ; Arteriosclerosis ; drug therapy ; metabolism ; pathology ; Chemokine CCL2 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Inflammation ; Lipids ; blood ; Male ; Mice ; Mice, Inbred C57BL ; Phytotherapy ; Tumor Necrosis Factor-alpha ; metabolism