OBJECTIVETo investigate the protective effects of irbesartan against cardiac inflammation associated with diabetes and obesity in the db/db mouse model of type 2 diabetes and explore the underlying mechanisms.

METHODSTwenty- four 10-week-old diabetic db/db mice were equally randomized into irbesartan treatment (50 mg/kg per day) group and model group, using 12 nondiabetic littermates (db/+) as the controls, The mice were treated with irbesartan or saline vehicle for 16 consecutive weeks, after which the heart pathology was observed and the heart weight, body weight, and serum levels of fasting blood glucose (FBG), total cholesterol(TC), and triglycerides(TG) were measured. The expression of nuclear factor-kappaB (NF-κB) p65 in the myocardium was assessed with immunohistochemistry, the protein levels of P-IκBα ,IκBα and β-actin were analyzed with Western blotting, and the pro-inflammatory cytokines IL-6 and TNF-α mRNA were detected using quantitative real-time PCR (qPCR).

RESULTSCompared with db/+ mice, the saline-treated db/db mice developed obesity, hyperglycemia and hyperlipidemia (P<0.01). Histopathological examination of the heart tissue revealed inflammatory cell infiltration, increased myocardial interstitium and disorders of myocardial fiber arrangement. The diabetic mice showed increased P-IαBα and decreased IκBα protein levels, enhanced activity and expression of NF-κB in the hearts, and increased mRNA expression of IL-6 and TNF-α in the myocardium. These abnormalities were all associated with increased inflammatory response. Treatment with irbesartan improved the heart architecture and attenuated high glucose-induced inflammation in the diabetic mice.

CONCLUSIONTreatment with irbesartan attenuates cardiac inflammation in type 2 diabetic db/db mice, and this effect was probably associated with the suppression of cardiac angiotensin II and NF-κB signaling pathway.

Actins ; metabolism ; Angiotensin II ; metabolism ; Animals ; Biphenyl Compounds ; pharmacology ; Cardiovascular Diseases ; drug therapy ; Diabetes Mellitus, Experimental ; complications ; Diabetes Mellitus, Type 2 ; complications ; Inflammation ; drug therapy ; Interleukin-6 ; metabolism ; Mice ; Obesity ; complications ; Random Allocation ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Tetrazoles ; pharmacology ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo observe the effect of Tripterygium glycosides (TG) on the expression of hypoxia-inducible factor-1α and endothelin-1 in the kidney of diabetic rats and explore the possible mechanism underlying the protective effect of TG against diabetic nephropathy.

METHODSSixty 8-week-old male SD rats were randomly divided into normal control group (n=10) and streptozotocin-induced diabetes mellitus (DM) model group (n=50). The diabetic model rats were then randomly divided into DM group, low-dose (8 mg/kg) and high-dose (16 mg/kg) TG treatment groups, and Irbesartan (50 mg/kg) treatment group. After 8 weeks, the levels of blood glucose (BG), 24-h urine protein (24 h Upro), serum creatinine (Scr) and blood urea nitrogen (BUN) were measured. The pathological changes in the renal tissues were examined by optical microscopy, and the mean glomerular area (MGA) and mean glomerular volume (MGV) were measured with pathological image analysis. Immunohistochemical and Western blotting were used to detect the expression of HIF-1α and ET-1 protein in the renal tissue, and their mRNA expressions were detected using real-time fluorescence quantitative PCR.

RESULTSHIF-1α and ET-1 expression increased in the kidney of diabetic rats. Compared with the diabetic model rats, the rats receiving TG and Irbesartan treatment showed decreased levels of Scr, BUN, 24h Upro, MGA and MGV, improved renal histopathology, and reduced expression of HIF-1α and ET-1 mRNA and protein in the renal tissue. These changes were more obvious in high-dose TG treatment group. Correlation analysis showed that the expression of HIF-1α was positively correlated with that of ET-1, and they were both positively correlated with kidney weight index (KW/BW), 24 h Upro, MGA, and MGV.

CONCLUSIONHIF-1α and ET-1 are overexpressed in the kidney of diabetic rats. TG can improve kidney damage in diabetic rats and delay the development of diabetic nephropathy by inhibiting the HIF-1α and ET-1 expression.

Animals ; Biphenyl Compounds ; pharmacology ; Blood Glucose ; Blood Urea Nitrogen ; Creatinine ; blood ; Diabetes Mellitus, Experimental ; metabolism ; Endothelin-1 ; metabolism ; Glycosides ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Kidney ; drug effects ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Tetrazoles ; pharmacology ; Tripterygium ; chemistry

Hepatic steatosis is common in obese individuals with hyperinsulinemia and is an important hepatic manifestation of metabolic syndrome. Sterol regulatory binding protein-1c (SREBP-1c) is a master regulator of lipogenic gene expression in the liver. Hyperinsulinemia induces transcription of SREBP-1c via activation of liver X receptor (LXR) and specificity protein 1 (Sp1). Cilostazol is an antiplatelet agent that prevents atherosclerosis and decreases serum triglyceride levels. However, little is known about the effects of cilostazol on hepatic lipogenesis. Here, we examined the role of cilostazol in the regulation of SREBP-1c transcription in the liver. The effects of cilostazol on the expression of SREBP-1c and its target genes in response to insulin or an LXR agonist (T0901317) were examined using real-time RT-PCR and western blot analysis on cultured hepatocytes. To investigate the effect of cilostazol on SREBP-1c at the transcriptional level, transient transfection reporter assays and electrophoretic mobility shift assays (EMSAs) were performed. Cilostazol inhibited insulin-induced and LXR-agonist-induced expression of SREBP-1c and its downstream targets, acetyl-CoA carboxylase and fatty acid synthase, in cultured hepatocytes. Cilostazol also inhibited activation of the SREBP-1c promoter by insulin, T0901317 and Sp1 in a luciferase reporter assay. EMSA analysis showed that cilostazol inhibits SREBP-1c expression by repressing the binding of LXR and Sp1 to the promoter region. These results indicate that cilostazol inhibits insulin-induced hepatic SREBP-1c expression via the inhibition of LXR and Sp1 activity and that cilostazol is a negative regulator of hepatic lipogenesis.
Animals ; Cells, Cultured ; Hep G2 Cells ; Hepatocytes/drug effects/*metabolism ; Humans ; Hydrocarbons, Fluorinated/pharmacology ; Insulin/pharmacology ; Lipogenesis ; Mice ; Mice, Inbred C57BL ; Orphan Nuclear Receptors/agonists/*metabolism ; Promoter Regions, Genetic ; Protein Binding ; Rats ; Sp1 Transcription Factor/*metabolism ; Sterol Regulatory Element Binding Protein 1/genetics/*metabolism ; Sulfonamides/pharmacology ; Tetrazoles/*pharmacology

OBJECTIVETo investigate the role of Toll-like receptor 4 (TLR4) signaling pathway in the activation of rennin- angiotensin system (RAS) in adipose cells.

METHODS3T3-L1 cells induced with isobutylmethylxanthine, insulin and dexamethasone to differentiate into adipocytes were stimulated by LPS with or without irbesartan pretreatment. The expression levels of TLR4, angiotensinogen (AGT) and angiotensin II receptor type 1 (ATlR) mRNA in 3T3-L1 cells was determined by RT-PCR, and their protein expressions were detected with Western-blotting. Immunofluorescence double staining was used to observe the translocation of NF-κB p65 subunit in the cells.

RESULTSStimulation with LPS dose- and time-dependently increased the mRNA and protein expressions of TLR4, AGT and AT1R. LPS exposure resulted in enhanced translocation of NF-κB p65 subunit in the adipose cells, which was attenuated by irbesartan pretreatment.

CONCLUSIONActivation of TLR4 signaling pathway may trigger the activation of local RAS in adipose cells.

3T3-L1 Cells ; Adipocytes ; metabolism ; Angiotensinogen ; metabolism ; Animals ; Biphenyl Compounds ; pharmacology ; Mice ; RNA, Messenger ; Receptor, Angiotensin, Type 1 ; metabolism ; Renin-Angiotensin System ; Signal Transduction ; Tetrazoles ; pharmacology ; Toll-Like Receptor 4 ; metabolism ; Transcription Factor RelA ; metabolism

The aim of present study was to explore the vasodilatation mechanism of angiotensin II (AngII) at the molecular level by investigating the effect of AngII on large-conductance Ca²⁺-activated potassium channels (BK(Ca)) in human mesenteric artery smooth muscle cells. The effect of AngII on BK(Ca) was observed by using patch clamp single channel recording technique and amphotericin-perforated whole-cell recording technique. AngII type 1 receptor (AT₁R) and AngII type 2 receptor (AT₂R) mRNA expression in human mesenteric artery was detected by RT-PCR. In cell-attached patch (Vm = +40 mV), AngII (100 nmol/L) had no significant effect on BK(Ca). After pretreatment with Valsartan (a specific inhibitor of AT₁R, 10 μmol/L), 25, 100 and 250 nmol/L AngII stimulated BK(Ca) activity significantly in a dose response manner. After pretreatment of Valsartan, AngII (100 nmol/L) enhanced BK(Ca) open probability (NP(O)) from 0.010 ± 0.003 to 0.039 ± 0.015, decreased the mean close time (T(C)) of BK(Ca) markedly from (2 729.5 ± 808.6) ms to (487.7 ± 182.5) ms (n = 11, P < 0.05) , but AngII had no significant influences on the amplitude (Amp) and the mean open time (T(O)) of BK(Ca). Further PD123,319 (a specific inhibitor of AT₂R) treatment prevented the stimulatory effect of AngII: PD123,319 decreased the NP(O) of BK(Ca) from 0.016 ± 0.003 to 0.004 ± 0.001 (n = 5, P < 0.05), but had no significant influences on Amp, T(O) and T(C) of BK(Ca). In addition, after pretreatment with Valsartan and PD123,319, AngII (100 nmol/L) had no significant effect on BK(Ca). In the amphotericin-perforated whole-cell patch-clamp configuration, after pretreatment with Valsartan, the current density of BK(Ca) at the voltage of -60 - +30 mV had no significant changes before and after adding 100 nmol/L AngII, but the current density of BK(Ca) at the voltage of +40 mV, +50 mV and +60 mV increased significantly after adding 100 nmol/L AngII, from (9.03 ± 2.23) pA/pF, (12.88 ± 2.55) pA/pF and (17.26 ± 2.84) pA/pF to (12.47 ± 2.22) pA/pF, (18.71 ± 2.51) pA/pF and (27.21 ± 3.12) pA/pF (n = 6, P < 0.05), respectively. Using RT-PCR, the AT₁R mRNA and AT₂R mRNA from isolated human mesenteric artery were detected. So we can draw a conclusion, AngII can stimulate BK(Ca) activity in human mesenteric artery smooth muscle cells after pretreatment with Valsartan, which is possibly mediated by AT₂R.
Angiotensin II ; pharmacology ; Humans ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Mesenteric Arteries ; cytology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Patch-Clamp Techniques ; Receptor, Angiotensin, Type 1 ; metabolism ; Receptor, Angiotensin, Type 2 ; metabolism ; Tetrazoles ; pharmacology ; Valine ; analogs & derivatives ; pharmacology ; Valsartan ; Vasodilation

OBJECTIVETo investigate the effect of low-dose carvedilol combined with candesartan in the prevention of acute and chronic cardiotoxicity of anthracycline drugs in adjuvant chemotherapy of breast cancer.

METHODSForty patients were randomly divided into two groups: the experimental group with chemotherapy plus low-dose carvedilol combined with candesartan (20 cases) and control group with chemotherapy alone (20 cases). The same chemotherapy was given to the two groups. All the 40 patients had no contraindication for carvedilol and candesartan. Patients of the experimental group received low-dose carvedilol from 2.5 mg orally twice a day at first cycle to 5 mg twice a day gradually if no side reactions, and candesartan 2.5 mg orally once a day. Electrocardiogram, ultrasonic cardiogram, arrhythmia, troponin and non-hematologic toxicity were recorded and compared after the second, forth and sixth cycle of chemotherapy. Each cycle included 21 days.

RESULTSLVEF was decreased along with the prolongation of chemotherapy in the experimental group and control group. LVEDD and LVESD showed no significant changes in the experimental group, but gradually increased in the control group. After four and six cycles of chemotherapy, LVEF were (57.00 ± 5.13)% and (45.95 ± 3.68)%, respectively, in the control group, significantly lower than that of (67.00 ± 5.13)% and (57.50 ± 2.57)%, respectively, in the experimental group (P < 0.05). After six cycles of chemotherapy, LVEDD and LVESD were (50.00 ± 10.48) mm and (35.01 ± 2.99) mm, respectively, in the control group, significantly higher than those before chemotherapy (P < 0.05) and experimental group (P < 0.001). The rate of ST segment and T wave abnormalities was 80.0% in the control group after six cycles of chemotherapy, significantly higher than that of 25.0% after four cycles of chemotherapy (P = 0.001) and 10.0% after two cycles of chemotherapy (P < 0.001). The reduction of QRS voltage, arrhythmia and abnormal troponin were 55.0%, 45.0% and 45.0%, respectively, in the control group, significantly higher than those in the experimental group (20.0%, P < 0.05), (10.0%, P = 0.010) and (10.0%, P < 0.05), respectively. The rate of abnormal expression of troponin was 45.0% in the control group, significantly higher than the 10.0% in the experimental group (P < 0.05).

CONCLUSIONSThe use of low-dose carvedilol combined with candesartan can reduce the acute and chronic cardiotoxicity of anthracycline drugs, and with tolerable toxicities. This may provide a new approach to prevent cardiotoxicity of anthracycline drugs in adjuvant chemotherapy of breast cancer.

Adrenergic beta-Antagonists ; administration & dosage ; pharmacology ; Adult ; Aged ; Angiotensin II Type 1 Receptor Blockers ; administration & dosage ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; therapeutic use ; Arrhythmias, Cardiac ; chemically induced ; Benzimidazoles ; administration & dosage ; pharmacology ; Breast Neoplasms ; drug therapy ; surgery ; Carbazoles ; administration & dosage ; pharmacology ; Chemotherapy, Adjuvant ; Cyclophosphamide ; adverse effects ; therapeutic use ; Electrocardiography ; drug effects ; Epirubicin ; adverse effects ; therapeutic use ; Female ; Fluorouracil ; adverse effects ; therapeutic use ; Humans ; Mastectomy, Radical ; Middle Aged ; Propanolamines ; administration & dosage ; pharmacology ; Stroke Volume ; drug effects ; Tetrazoles ; administration & dosage ; pharmacology ; Troponin ; metabolism

The present study was to determine whether candesartan, an angiotensin II type 1 receptor blocker (ARB), exerts anti-inflammatory effects through inhibiting the toll-like receptor 4 (TLR4) pathway in human renal tubular epithelial cells (HKCs). The experiments were carried on cultured HKCs. By means of flow cytometry, Western blot, RT-PCR and ELISA techniques, the TLR4 protein, angiotensin II type 1 receptor (AT1R) and phosphorylated nuclear factor-kappa B (NF-κB) p65 protein level, mRNA levels of macrophage chemoattractant protein-1 (MCP-1) and regulated upon expression normal T cell expressed and secreted (RANTES), as well as MCP-1 and RANTES protein concentrations in conditioned media were measured. The results showed that lipopolysaccharide (LPS) upregulated the TLR4 protein level in cultured HKCs. Application of LPS increased NF-κB activation and induced release of its downstream inflammatory factors including MCP-1 and RANTES. Candesartan reversed LPS-induced upregulation of TLR4 expression, inhibited NF-κB activation, and reduced MCP-1 and RANTES release. However, knockdown on AT1R by siRNA did not change those previous effects of candesartan. These results suggest that candesartan-induced anti-inflammatory effect may be through a novel pathway, independent of AT1R.
Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Benzimidazoles ; pharmacology ; Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; Gene Expression Regulation ; Humans ; Kidney Tubules ; cytology ; Lipopolysaccharides ; NF-kappa B ; metabolism ; RNA, Messenger ; Receptor, Angiotensin, Type 1 ; metabolism ; Signal Transduction ; Tetrazoles ; pharmacology ; Toll-Like Receptor 4 ; metabolism ; Up-Regulation

OBJECTIVETo explore the effects of Candesartan cilexetil on the rats exposed to silica.

METHODNinety-six wistar rats were randomly divided into model-group, intervention-group and control-group (32 rats a group). The intervention-group, model-group and control group were orally exposed to Candesartan cilexetil (10 mg/kg) and normal solution for a week, respectively. Then the model and intervention groups were exposed to silica by intratracheal infusion of silica dust suspension (50 mg/ml), the control group was exposed to 0.5 ml normal solution for 2 days. On the 3rd, 7th, 14th and 28th days after exposure to silica, 8 rats of each group were sacrificed, respectively. The samples of lung tissues were collected. The lung/body coefficients were detected. The pathological examinations were performed by HE and Masson staining. The levels of ACE in the lung tissues were observed by immunochemistry staining. The levels of TGF-β1 and Ang II in the BALF were examined by ELISA.

RESULTSOn the 3rd, 7th, 14th and 28th days after exposure, the levels of alveolitis and pulmonary fibrosis in the intervention group were significantly alleviated as compared with model group, and the lung/body coefficients in the intervention group, which were significantly lower than those in model group respectively (P < 0.01). As compared with control group, the levels of TGF-β1 and Ang II of the BALF in the model and intervention groups significantly enhanced (P < 0.01). As compared with model group, the levels of TGF-β1 and Ang II of the BALF in the intervention group significantly decreased (P < 0.01). As compared with control group, the levels of ACE of the lung tissues in the model and intervention groups significantly increased (P < 0.01). But the level of ACE of the lung tissues in the intervention group was significantly lower than that in the model group (P < 0.01).

CONCLUSIONThe early Candesartan cilexetil intervention could significantly decrease the levels of alveolitis and lung fibrosis, declined the levels of TGF-β(1) and Ang II of BALF and downregulated the expression level of ACE in lung tissues in rats exposed to silica.

Angiotensin II ; metabolism ; Animals ; Benzimidazoles ; pharmacology ; therapeutic use ; Bronchoalveolar Lavage Fluid ; Female ; Lung ; drug effects ; pathology ; Male ; Pulmonary Fibrosis ; chemically induced ; drug therapy ; Rats ; Rats, Wistar ; Silicon Dioxide ; toxicity ; Tetrazoles ; pharmacology ; therapeutic use ; Transforming Growth Factor beta1 ; metabolism

BACKGROUNDAngiogenesis occurs commonly in various physiological and pathological processes. Improving blood supply through promoting angiogenesis is a novel approach for treating ischemic diseases. Angiotensin II type 1 receptor blockers (ARBs) dominate the management of hypertension, but evidence of their role in angiogenesis is contradictory. Here we explored the angiogenic effects of ARBs through characterizing gene expression of the human umbilical vein endothelial cell line EA.hy926 exposed to irbesartan.

METHODSThe human umbilical vein endothelial cell line EA.hy926 was grown for 72 hours after treatment with different concentrations of irbesartan. The cell proliferative capacity was assessed by CCK8 assay at 24, 48 and 72 hours. Gene expression levels in EA.hy926 cells responding to irbesartan were measured under optimal proliferation conditions by microarray analysis using Affymetrix U133 plus 2.0. The differential expression of genes involved in angiogenesis was identified through cluster analysis of the resulting microarray data. Quantitative RT-PCR and Western blotting analyses were used to validate differential gene expression related to the angiogenesis process.

RESULTSIn the 10(-4), 10(-5), 10(-6) mol/L treatment groups, cell proliferation studies revealed significantly increased proliferation in EA.hy926 cells after 24 hours of irbesartan treatment. However, after 48 and 72 hours of treatment with different concentrations of irbesartan, there was no significant difference in cell proliferation observed in any treatment group. We selected the group stimulated with irbersartan at a concentration of 10(-6) mol/L for microarray experiments. Statistical analysis of the microarray data resulted in the identification of 56 gene transcripts whose expression patterns were significantly correlated, negatively or positively, with irbesartan treatment. Cluster analysis showed that these genes were involved in angiogenesis, extracellular stimulus, binding reactions and skeletal system morphogenesis. Of these 56 genes we identified seven genes (VEGF, KDR, PTGS2, PLXND1, ROBO4, LMO2, and COL5A1) involved in the angiogenesis process. qRT-PCR analysis of these genes confirmed the microarray results. Protein expression of three VEGF pathway genes (VEGF, KDR, and PTGS2) was further confirmed by Western blotting.

CONCLUSIONSOur study showed that irbesartan may induce angiogenic effects in vascular endothelial cells. It suggested that the mechanism of angiogenic effects of ARBs might be attributed to the signaling cascade from angiotensin receptors in the VEGF pathway. It also provided evidence indicating that ARBs could be used as a novel therapeutic approach to treat chronic ischemic heart disease as well as anti-hypertensive agents.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Antihypertensive Agents ; pharmacology ; Biphenyl Compounds ; pharmacology ; therapeutic use ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Gene Expression Profiling ; Humans ; Myocardial Ischemia ; drug therapy ; Neovascularization, Physiologic ; drug effects ; Tetrazoles ; pharmacology ; therapeutic use

KR-31543, (2S, 3R, 4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro-2-dimethyoxymethyl-3-hydroxy-2-methyl-2H-1-benz opyran is a new neuroprotective agent for ischemia-reperfusion damage. It has also been reported that KR-31543 has protective effects on lipid peroxidation and H2O2-induced reactive oxygen species production. In this study, we investigated the anti-inflammatory and anti-atherogenic properties of KR-31543. We observed that KR-31543 treatment reduced the production of MCP-1, IL-8, and VCAM-1 in HUVECs, and of MCP-1 and IL-6 in THP-1 human monocytes. We also examined the effect of KR-31543 on monocytes migration in vitro. KR-31543 treatment effectively reduced the migration of THP-1 human monocytes to the HUVEC monolayer in a dose-dependent manner. We next examined the effects of this compound on atherogenesis in LDL receptor deficient (Ldlr-/-) mice. After 10 weeks of western diet, the formation of atherosclerotic lesion in aorta was reduced in the KR-31543-treated group compared to the control group. The accumulation of macrophages in lesion was also reduced in KR-31543 treated group. However, the plasma levels of total cholesterol, HDL, LDL, and triglyceride were not affected by KR-31543 treatment. Taken together, these results show that KR-31543 has anti-inflammatory properties on human monocytes and endothelial cells, and inhibits fatty streak lesion formation in mouse model of atherosclerosis, suggesting the potential of KR-31543 for the treatment for atherosclerosis.
Animals ; Aorta/pathology ; Atherosclerosis/blood/*drug therapy/pathology ; Benzopyrans/*pharmacology/therapeutic use ; Cholesterol, HDL/blood ; Cholesterol, LDL/blood ; Diet ; Disease Models, Animal ; Human Umbilical Vein Endothelial Cells/drug effects/metabolism ; Inflammation Mediators/*metabolism ; Interleukin-6/metabolism ; Interleukin-8/metabolism ; Macrophages/metabolism ; Mice ; Mice, Transgenic ; Monocytes/drug effects/*metabolism ; Neuroprotective Agents/*pharmacology/therapeutic use ; Receptors, CCR2/metabolism ; Receptors, LDL/genetics ; Tetrazoles/*pharmacology/therapeutic use ; Transendothelial and Transepithelial Migration/drug effects ; Triglycerides/blood ; Vascular Cell Adhesion Molecule-1/metabolism