AIM:To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms .METHODS: CFs were cultured using myocardial tissue with dry method .Hypoxic environment was established for CFs by continuous nitrogen supplement .Type I and type III collagens in supernatants were detected by ELISA.Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents .The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining .RESULTS:Un-der hypoxic condition , TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-de-pendent manner in the CFs .At the concentration of 5μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01).RXR agonist 9-cis-retinoic acid (9-cis-RA;10 -9 ~10 -6 mol/L) decreased TGF-β1 (5μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic con-dition.The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01).Smad2 inhibitor ( 20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF -β1 under hypoxic condition.Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly in-creased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05).CONCLU-SION:Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition .

[ABSTRACT]AIM:ToexploretheeffectofretinoidXreceptor(RXR)agonistbexarotene(Bex)andvitaminD receptor (VDR) agonist calcitriol (Cal) on the expression of nuclear factor-kappa B (NF-κB) and the development of atherosclerosis in streptozotocin-induced diabetic apolipoprotein E knockout ( STZ-ApoE-/-) mice.METHODS: Male mice were treated for 12 weeks as follows:(1) C57+vehicle;(2) ApoE-/-+vehicle;(3) STZ-ApoE-/-+vehicle;(4) STZ-ApoE-/-+Bex (10 mg· kg-1· d-1);(5) STZ-ApoE-/-+Cal (10 μg/kg, twice a week);(6) STZ-ApoE-/-+Bex (10 mg· kg-1· d-1) +Cal (10 μg/kg, twice a week).Intraperitoneal injection of STZ was performed to establish the diabetic animal model .Western blotting and immunohistochemical method was used to detect NF-κB level in the thorac-ic aorta.Plaque area in the thoracic aorta was measured using HE staining .RESULTS:Compared with the C57 mice, the fasting blood glucose in the ApoE-/-mice was not remarkably changed .The levels of total cholesterol ( TC) and low-densi-ty lipoprotein ( LDL) were greatly increased .The fasting blood glucose and lipid levels in STZ-ApoE-/-group were much higher than those in ApoE-/-group.Compared with STZ-ApoE-/-group, the fasting blood glucose and lipid levels in Bex group and Cal group were not significantly changed .Compared with the C57 mice, the protein expression of NF-κB in the ApoE-/-mice and the STZ-ApoE-/-mice was remarkably increased .Compared with STZ-ApoE-/-group, the levels of NF-κB in Bex group, Cal group and combination group were greatly decreased .Compared with STZ-ApoE-/-group, the thoracic artery plaque areas in Bex group and Cal group were inhibited (both P<0.05).Compared with Bex group, the plaque area of the thoracic artery in combination group was significantly decreased (P<0.05).CONCLUSION:Bexaro-tene or calcitriol decreases the development of atherosclerosis in streptozotocin -induced diabetic ApoE-/-mice.Bexarotene combined with calcitriol affords greater protection than monotherapy .The mechanism may be involved in down-regulating the expression of NF-κB.

Objective To examine the clinical effects of intravenous lyophilize recombinant human brain natriuretic peptide (rhBNP) in patients with refractory heart failure caused by coronary artery disease.Methods Seven patients with refractory heart failure caused by coronary artery disease were treated with rhBNP.The rhBNP nea grade,symptoms and signs,24 hours urine output,heart rate,blood pressure and central venous pressure were evaluated at 0,15,30 min and 1,2,4,8,12,24,and 48 h.Serum potassium,sodium,creatinine and plasma BNP before and after treatment were measured.Results After rhBNP therapy,dyspnea grade were improved in 5 patients.Symptoms and signs got better in 6 patients.Systolic blood pressure at 15 min of treatment distolic blood pressure was decreased slightly from (112.00±10.42) mm Hg to (105.14±7.76) mm Hg (P＜0.05) and became (108.71±6.63)mm Hg at 30 rain which was the same with that before treatment.There was no statistical significance in heart beat[ ( 88.57±16.92 ) vs.( 86.00±16.21 ) ] beat/min,serum sodium [ ( 133.57±5,38 ) mmol/Lvs.( 133.57±8.16) mmol/L ],serum potassium [ (3.83±0.37) mmol/L vs.(4.19±0.58 ) mmol/L ],ereatinine [ (93.11±27.90) μmol/L vs ( 123.01±93.01 ) μmol/L ] before and after treatment,and BNP[ ( 1218.43±847.83) vs.(1433.71±676.08)ng/L] before treatment and at24 h treatment,as well as urine output [(2329±1573 ) vs.(2126±1074) ml ] ( P ＞ 0.05 ).Urine output was increased during the treatment,but the usage of diuretic was remarkably decreased.Central venous pressure was gradually decreased from 30 rain to 48 h( P ＜ 0.05 ).Condusion rhBNP can decrease central venous pressure and increase urine output with exerts little side effects on electrolytes and renal function.Therefore rhBNP has positive clinical effects on refractory heart failure which is caused by coronary artery disease.

Objective To investigate the effect of 9-cis retinoid acid(c-RA),a retinoid X receptor(RXR)agonist,on hydrogen peroxide(H2O2)induced apoptosis in cultured rat neonatal cardiomyoeytes,and to explore the mechanism.Method Cultured cardiomyocpes were randomly divided into three groups:normal group treated with vehicle(N group),H2O2 group treated with 100 μmol/L H2O2(H group),and c-RA group pretreated with 100nmol/L c-RA(H+R group).Cell viability was detected by MTT.Morphological changes of apoptotic cardiomyocytes were observed by Hoechst 33258 staining under fluorescence microscope.The apoptotic rate was determined by flow cytometry.Mitochondrial membrane potential(△(ψ)m)was measured by JC-1 dye.Cellular reactive oxygen species(ROS)production was detected by CM-H2DCFDA fluorescent probe.All measurement data wIe expressed as(x±s),and statistically analyzed using one-way ANOVA analysis and Dunnett test.Differences were considered significant when P was＜0.05.Results Treatment with c-RA significantly enhanced cell viability,reduced apoptosis ratio,stabled mitoehondrial membrane potential and reduced level of cellular reactive oxygen species.Conclusions RXR agonist c-RA inhibits H2O2-induced myocyte apoptosis in cultured rat neonatal cardiomyocytes,which may be related to alleviate oxidative stress injury.

AIM:To explore the effects of atorvastatin (Atorv) on atherosclerosis in streptozotocin (STZ)-in-duced diabetic apolipoprotein E knockout ( ApoE-/-) mice with fat-rich diet and the possible mechanism .METHODS:C57 mice served as control.ApoE-/-mice (n=34) fed with high-fat diet were randomly divided into ApoE-/-group, STZ-ApoE-/-group and STZ-ApoE-/-+Atorv group.Intraperitoneal injection of streptozotocin was performed to create di-abetic animal model .Blood glucose was determined by glucose oxidase method .Blood lipid levels were detected by enzymic method or selective homogeneous method .The plaque area in the thoracic aorta was measured by HE staining .The protein level of nicotinamide-adenine dinucleotide phosphate ( NADPH) oxidase subunit gp91phox in the thoracic aorta was deter-mined by Western blotting .The levels of reactive oxygen species ( ROS) in blood and thoracic aorta homogenates were de-tected by Fenton reaction and Griess reagent .Human umbilical vein endothelial cells ( HUVECs ) were isolated from healthy umbilical cords by collagenase I and cultured .ROS production was detected by flow cytometry .NADPH oxidase ac-tivity was measured using lucigenin assay .Effects of retinoid X receptor α( RXRα) on inhibition of oxidative stress by ator-vastatin were evaluated by RNA interference and plasmid transfection .RESULTS: (1) Compared with C57 group, the plaque areas of the thoracic aorta in ApoE-/-group were increased .No difference of the fasting glucose between the 2 groups was observed.The levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in ApoE-/-group than those in C57 group.(2) Compared with ApoE-/-group, the plaque areas of the thoracic aorta in STZ-ApoE-/-group were further enlarged [(314.13 ±35.72) μm2 vs (215.88 ±34.19) μm2, P<0.05].The levels of blood glucose, TG, TC and LDL-C, thoracic aorta gp91phox protein and ROS in blood and thoracic aorta homogenates were higher in STZ-ApoE-/-group than those in ApoE-/-group (P<0.05).(3) Compared with STZ-ApoE-/-group, the plaque areas of the thoracic aorta in STZ-ApoE-/-+Atorv group were reduced [(217.47 ±24.56) μm2 vs (314.13 ±35.72) μm2, P<0.05].The levels of blood glucose , LDL-C, TC, HDL-C and TG showed no significant difference between the 2 groups.Thoracic aorta gp91phox protein level and ROS production in blood and thoracic aorta homogenates were lower in STZ -ApoE-/-+Atorv group than those in STZ-ApoE-/-group (P<0.05).(4) High glucose-induced increases in NADPH oxidase activity and gp91phox expression were significantly inhibited by atorvastatin (10-6 mol/L) in HUVECs.The inhibitory effects of atorvasta-tin on high glucose-induced ROS production and NADPH oxidase activation were largely impaired when the cells were trans -fected with RXRαsiRNA.However , the effect of atorvastatin was significantly strengthened when RXRαwas over-expressed in the HUVECs transfected with RXRαplasmid.CONCLUSION: Atorvastatin inhibits atherogenesis by depressing high glucose-induced oxidative stress in diabetic ApoE-/-mice with fat-rich diet.The anti-oxidative stress effect of atorvastatin is mediated by RXRα.

OBJECTIVETo explore the effect of retinoid X receptor (RXR) agonist bexarotene on atherosclerosis and the potential mechanism in streptozotocin (STZ) induced diabetic apolipoprotein E knockout (apoE(-/-)) mice.

METHODSEight C57BL/6 mice served as control, 46 apoE(-/-) mice were randomized into 4 groups: apoE(-/-) group (n = 10), STZ+apoE(-/-) group (n = 12), STZ+apoE(-/-)+Bex 10 (10 mg×kg⁻¹×d⁻¹)group (n = 12), STZ+ apoE(-/-)+Bex 30 (30 mg×kg⁻¹×d⁻¹) group (n = 12). Diabetic apoE(-/-) animal model was established by intraperitoneal injection of STZ. Blood glucose was determined by glucose oxidase method. Patch area in thoracic aorta was measured by HE staining. Western blotting was used to determine the RXR and gp91(phox) protein level in thoracic aorta. Reactive oxygen species (ROS) level in blood and thoracic aorta homogenates was detected by Fenton and Griess method.

RESULTS(1) Patch areas of thoracic aorta were larger in apoE(-/-) group than in C57BL/6 group [(38.40 ± 8.95)µm² vs. (0.10 ± 0.01) µm², P < 0.01], further increased in STZ+apoE(-/-) group [(94.06 ± 8.04)µm², P < 0.05 vs. apoE(-/-) group] and significantly reduced in STZ+apoE(-/-)+Bex 10 group [(78.72 ± 4.62)µm², P < 0.05 vs. STZ+apoE(-/-) group] and further educed in STZ+apoE(-/-)+Bex 30 group [(46.13 ± 7.56)µm², P < 0.05 vs. STZ+apoE(-/-)+Bex 10 group]. (2) Blood glucose level, TG, TC, LDL-C, thoracic aorta gp91(phox) protein level and ROS level in blood and thoracic aorta homogenates were significantly higher in STZ+apoE(-/-) group than in apoE(-/-) group (all P < 0.05). Blood glucose level and TG, TC, LDL-C levels were similar between STZ+apoE(-/-)+Bex10 and STZ+apoE(-/-) group. Thoracic aorta gp91(phox) protein level and ROS level in blood and thoracic aorta homogenates were lower in STZ+apoE(-/-)+Bex 10 group than in STZ+apoE(-/-) group (P < 0.05). Blood glucose level, TG, TC, LDL-C levels, gp91(phox) expression in thoracic aorta, ROS level in blood and thoracic in STZ+apoE(-/-)+Bex 30 group were lower than in STZ+apoE(-/-) group (all P < 0.05).

CONCLUSIONBexarotene treatment could attenuate arteriosclerosis progression in STZ induced diabetic apoE(-/-) mice, the underlying mechanism might be related to suppressing oxidative stress and decreasing blood glucose level and improving lipids metabolism.

Animals ; Apolipoproteins E ; genetics ; Atherosclerosis ; etiology ; metabolism ; prevention & control ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; complications ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Oxidative Stress ; drug effects ; Reactive Oxygen Species ; metabolism ; Retinoid X Receptors ; agonists ; metabolism ; Tetrahydronaphthalenes ; pharmacology