Metformin inhibits nuclear factor kappaB activation and decreases serum high-sensitivity C-reactive protein level in experimental atherogenesis of rabbits.

Previous studies demonstrated that metformin has obvious antiatherogenic properties, but the exact mechanism remains unclear. Therefore, we established an atherosclerotic rabbit model in order to investigate the potential effects of metformin on transcription factor nuclear factor kappaB (NF-kappaB) and serum high-sensitivity C-reactive protein (hs-CRP) level, which had been regarded as proatherogenic factors. New Zealand rabbits were randomly divided into three groups: a control group (n = 8), an atherosclerotic group (AS group, n = 8), and a metformin treatment group (Met group, n = 8). The experimental atherosclerotic rabbit model was successfully established at the end of the 8th week. From the 9th week, rabbits in the Met group were administered with 150 mg/kg metformin daily by gavage. Blood samples were collected at days 0 and 8, and at 16 weeks to detect the level of blood lipid and serum glucose. At the end of the experiment, blood samples were withdrawn for determining serum hs-CRP. Aortic samples were harvested for histomorphometric analysis. Immunohistochemistry and Western blotting were used to detect the expression of NF-kappaB subunit p65 in nuclear extracts and phosphorylation of inhibitor of nuclear factor kappaB (IkappaB) in cytoplasmic extracts. An experimental atherosclerotic rabbit model was successfully established. The expression of nuclear NF-kappaB subunit p65 and cytoplasmic phosphorylation of IkappaB protein in the vessel wall was enhanced (P < 0.01, respectively) in the AS group, and serum hs-CRP level was significantly increased in the AS group compared with the control group (3.90 +/- 0.25 mg/l versus 1.36 +/- 0.14 mg/l, P < 0.01). Treatment with metformin significantly attenuated the progression of aortic atherosclerosis. In the Met group, there was a marked reduction in nuclear NF-kappaB subunit p65 and cytoplasmic phosphorylation of IkappaB protein expression (P < 0.01). Serum hs-CRP concentration was also significantly decreased (3.20 +/- 0.20 mg/l versus 3.90 +/- 0.25 mg/l, P < 0.05). Metformin inhibits the phosphorylation of IkappaB and the activation of NF-kappaB in the vessel wall of experimental atherogenesis of rabbits, as well as decreasing the serum level of hs-CRP, thus suggesting that metformin has vascular anti-inflammatory properties, which may be one of its antiatherogenic mechanisms.

Antiapoptosis and mitochondrial effect of pioglitazone preconditioning in the ischemic/reperfused heart of rat.

PURPOSE: Pioglitazone, used clinically in the treatment of type 2 diabetes mellitus, has been implicated as a regulator of cellular inflammatory and ischemic responses. The present study examined whether pioglitazone could inhibit cardiomyocyte apoptosis and reduce mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of the rat. Furthermore, we investigated whether the protective effect of pioglitazone was related to opening of the mitochondrialATP-sensitive potassium channels. METHODS: Adult male Sprague-Dawley rats were subjected to 30 min of ischemia followed by 4 h of reperfusion. At 24 h before ischemia, rats were randomized to receive 0.9% saline, 5-hydroxydecanoate (5-HD, 10 mg kg(-1), i.v.) plus pioglitazone (3 mg kg(-1), i.v.) or pioglitazone (3 mg kg(-1), i.v.). One group served as sham control. We investigated mitochondrial structure, apoptosis rate and Bcl-2, Bax and Caspase-3 proteins by immunohistochemistry staining. RT-PCR was used to determine the expression of P38MAPKmRNA and JNKmRNA. Western blotting was used to measure the expression of P38MAPK, JNK and NFkappaB P65. A second group of rats were randomly divided into sham-operated, ischemia/reperfusion (I/R), pioglitazone treatment, 5-HD + pioglitazone and 5-HD groups and the size of myocardial infarction was determined. Primary cultured cardiomyocytes of neonatal Sprague-Dawley rats were divided into control, hypoxia reoxygenation, different concentrations of pioglitazone and 5-HD + pioglitazone groups. JC-1 staining flowcytometry was used to examine mitochondrial membrane potential (DeltaPsim). RESULTS: Pioglitazone decreased mitochondrial ultrastructural damage compared to I/R, and reduced infarct size from 34.93 +/- 5.55% (I/R) to 20.24 +/- 3.93% (P < 0.05). Compared with the I/R group, the apoptosis rate and positive cell index (PCI) of Bax and Caspase-3 proteins in the pioglitazone group were significantly decreased (P < 0.05), while the PCI of Bcl-2 protein was increased (P < 0.05). There was no significant difference between the I/R and 5-HD + pioglitazone groups. Compared with the sham-operated group, the expression of P38MAPK mRNA, JNK mRNA and protein of P38MAPK, JNK and NFkappaB P65 in I/R was increased (P < 0.05). Pioglitazone did inhibit the increase in expressions vs I/R (P < 0.05). The rate of loss DeltaPsim cells in the pioglitazone group was significantly lower than in the hypoxia reoxygenation group, while the addition of 5-HD inhibited the effect of pioglitazone. CONCLUSION: Pioglitazone inhibited cardiomyocyte apoptosis and reduced mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of rat. These protective effects of pioglitazone may be related to opening mitochondrial(ATP)-sensitive potassium channels.

Folic acid reduces chemokine MCP-1 release and expression in rats with hyperhomocystinemia.

OBJECTIVE: This study aimed to investigate the effects of folate on the monocyte chemoattractant protein-1 (MCP-1) expression and release in rats with hyperhomocystinemia induced by ingestion of excess methionine. METHODS AND RESULTS: Thirty male Sprague-Dawley rats (200+/-20 g) were randomly divided into three groups (n=10 for each group): control group (Control), high-homocystinemia (Hhcy) group, and folate treatment (FA) group. They were fed with a normal regular diet, enriched by 1.7% methionine plus 1.7% methionine and 0.006% folate for 45 days. Our study showed the following: (a) A high methionine diet for 45 days is sufficient to induce hyperhomocystinemia; folate supplementation to the rats fed the high-methionine diet prevented an elevation homocysteine (Hcy) levels in the blood (P<.01). (b) Compared with the Control group, the Hhcy group had elevated plasma levels of MCP-1, and Hcy was significantly correlated with MCP-1 (P<.05). (c) The protein and mRNA expression of MCP-1 in the aorta was higher in rats from the Hhcy group than in rats from the Control group. (d) Most important, after folic acid supplementation, the lowering of Hcy levels was accompanied by a marked reduction of MCP-1 expressed in aortae and released from plasma and peripheral blood mononuclear cells (PBMCs) stimulated by oxidized low-density lipoprotein (P<.05, P<.01). CONCLUSION: Folic acid supplementation not only can blunt the rise in Hcy and reduce MCP-1 released from both plasma and PBMCs of rats with hyperhomocystinemia but also can downgrade MCP-1 expression in the aorta of rats with hyperhomocystinemia.

Role of norepinephrine in development of short-term myocardial hibernation.

AIM: To investigate the role of norepinephrine in the development of short-term myocardial hibernation. METHODS: Hearts were removed from rats and set up as isometrically beating or short-term hibernation models. The hearts were perfused with modified Krebs-Henseleit buffer under a controlled perfusion pressure. The myocardial ultrastructure was examined, and the content of ATP, phosphocreatine, and glycogen in myocardium, the extent of myocyte apoptosis, and the amount of Bcl-2 and Bax products were determined after 120-min ischemia assessed by TUNEL and immunocytochemistry. RESULTS: There was no significant difference between the reserpinized hearts and the NS control group with respect to heart function, myocardial ultrastructure, ATP, phosphocreatine, or glycogen content, myocyte apoptosis, or amount of Bax or Bcl-2 products. However, relative to the normal saline group, in the norepinephrine-treated hearts, heart function, and myocardial ultrastructure deteriorated significantly, apoptosis and amount of Bax product increased significantly, and the ATP, phosphocreatine, and glycogen content decreased significantly, as did the amount of Bcl-2 product. CONCLUSION: Myocardial norepinephrine does not contribute to the development of short-term hibernation, but that exogenous NE can induce progressive decreases in coronary flow and cardiac performance, which might result from the increases in apoptosis and necrosis. Norepinephrine may be an important factor in the deterioration of myocardial structure and function during hibernation, and that anti-adrenergic treatment may be helpful for the development and sustainment of short-term myocardial hibernation.

Folic acid reduces adhesion molecules VCAM-1 expession in aortic of rats with hyperhomocysteinemia.

To investigate effects of supplementation of folic acid on the expression of adhesion molecules VCAM-1 in the aortas of rats with hyperhomocysteinemia. Thirty male SD rats (200 +/- 20 g) were invided into 3 groups (n = 10 for each group): control group(Control), high Met group(Met) and Met plus Folate group(Met + Folate), fed. for 45 days. Plasma Hcy levels were higher with the high-methionine diet (140.68 +/- 36.87 micromol/L vs 6.47 +/- 1.10 micromol/L in control rats) an effect which was reduced by folate. Respectively, the aortic expression of adhesion molecules VCAM-1 at protein and mRNA levels were higher in the Met groups than those in the control groups or the Met + Folate groups. A high methionine diet for 45 days was sufficient to induce hyperhomocysteinemia. Folate supplementation prevented elevation of Hcy levels in the blood, and reduced expression of the adhesion molecule VCAM-1. Hyperhomocysteinemia is now regarded as one of the important risk factors for cardiovascular and cerebralvascular disorders.[Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998; 38(15):1042-50.] Several plausible mechanisms for Hcy-induecd atherosclerosis have been proposed. These include endothelial dysfunction, enhancement of oxidative stress, reduction in NO bioavailability, and augmentation of thrombus formation.[Holven KB, Holm T, Aukrust P, et al. Effect of folic acid treatment on endothelium-dependent vasodilation and nitric oxide-derived end products in hyperhomocysteinemic subjects . Am J Med 2001;110(7):536-42; Guba SC, Fonseca V, Fink LM. Hyperhomocysteinemia and thrombosis. Semin Thromb Hemost 1999;25(3):291-309.] However, the precise molecular mechanism is still unclear. Recent reports have suggested a role for inflammatory processes in the pathogenesis of atherosclerosis.[Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol 2001;2(2):108-15.] Dysfunction of endothelial cells is the key process promoting inflammatory reactions. On injury, endothlial cells are capable of producing various cytokines that participate in inflammatory reactions in the arterial wall. Although results from in vitro studies suggest that Hcy, at pathophysiological concentrations, stimulates chemokine expression in vascular cells, it is unknown whether hyperhomocysteinemia can initiate similar changes, leading to enhanced momocyte adhesion/binding to the vascular endothelium in vivo.[Zeng X, Dai J, Remick DG, Wang X. Homocysteine mediated expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human monocytes. Circ Res 2003;93(4):311-20.] On the basis of the potential pathogenic role of chemokines in atherogenesis, the objective of the present study was to investigate that homocsteine may exert its effect in part though adhesion molecules VCAM-1 and that folic acid supplementation may downregulate these inflammatory responses. Male Sprague-Dawley rats (bred from animal centers of Tongji Medical College, Huazhong Science and Technology University) aged 8 weeks were divided into 3 groups(n=10 for each group) and maintained for 45 days on the following diets before the experiments: (1) regular diet; (2) high-metheionine diet, consisting of regular diet plus 1.7% methionine; and (3) high-methionine plus folate -rich diet, consisting of regular diet plus 1.7% methionine and 0.006% folate.[Boisvert WA, Curtiss LK, Terkeltaub RA. Interleukin-8 and its receptor CXCR2 in atherosclerosis. Immunol Res 2000;21(2-3):129-d37.] Plasma and serum samples wee colleced and stored at -80 degrees C after 45 days until analysis. The plasma homocysteine concentration of rats in three groups were determined by high-pressue liquid chromatography. To detect the endothelial expression of adhesion molecules VCAM-1, the thoracic aorta was isolated and dived into segments. These segments were immersion-fixed in 10% neutral-buffered formalin overlight and then embedded in paraffin. Sequential 5 mum paraffin-embedded cross sections were prepared. Immunohistochemical analyisis was performed to detect vascular cell adhesion molecule(VCAM)-1, The fixed cryosections were immediately blcked in 10% horse serum and phosphate baffered saline(PBS) at room temperature for 30 min. Goat polyclonal andibodies against rat VCAM-1(Santa Cruz Biotechnology) were diluted 1:100 in PBS and incubated with the cryosections for 1 h of room temperature. After three washes, the sections were incubated with biotin-conjugated rabbit anti-goat immunoglobulins(Dako) at 1:250 dilution in PBS. After three washes, the samples were mounted in 90% glycerol-PBS. Photographs were taken by use of a light microscope at a mignification of x200.

[Effects of atorvastatin on the function of dendritic cells in patients with unstable angina pectoris].

OBJECTIVE: To investigate the function of dendritic cells (DC) in patients with unstable angina pectoris (UAP) and the effects of atorvastatin on it. METHODS: 27 patients with UAP were divided into two groups treated respectively with regular pharmacotherapy and regular pharmacotherapy plus atorvastatin. PBMC from the UAP patients (before and 2 weeks after the treatment) and 11 healthy subjects were incubated and induced to mature DC in a completed medium containing GM-CSF and IL-4. Flow cytometric analysis was used to detect the expression of co-stimulating factor CD86 (B7-2) on DC. The stimulating capacity of DC was determined in allogenic mixed lymphocyte reaction (MLR). ELISA was used to analyze the level of cytokines (IL-1beta, IL-6, IL-10 and TNF-alpha) in the medium of MLR. Relationship of expression of CD86 to risk factors and blood CRP level was also analyzed. RESULTS: When compared with normal group, CD86 on DC was much more expressed in UAP patients; the stimulating capacity of DC in MLR was higher; T lymphocytes in MLR secreted higher levels of pro-inflammation cytokines (IL-1beta, IL-6 and TNF-alpha) and lower level of anti-inflammation cytokine (IL-10). Blood LDL-C before treatment was positively related to the expression of CD86. Atorvastatin inhibited the function of DC and lowered blood level of CRP and CD86, the levels of which were significantly positively correlated. CONCLUSIONS: DC in UAP are activated, which may play an important role in initiating immune reaction in the plaque. LDL-C may be one of the activators of DC; inhibitory effect of atorvastatin on inflammation in UAP may be due to its inhibition on DC.

[The relationship between gap junctional remodeling and atrial fibrillation in patients with rheumatic heart disease].

OBJECTIVE: To investigate the relationship between the alternations in the expression and distribution of connexin 40 (Cx40) and connexin 43 (Cx43) and atrial fibrillation (AF) in patients with rheumatic heart disease. METHODS: Biopsy was conducted to take specimens of right atrial appendage (RAA) from 32 patients with rheumatic heart disease, 21 with AF and 11 with sinus rhythm (SR) during valve replacement. The protein levels of Cx40 and Cx43 were examined by immunoconfocal microscopy and Western blotting. The former was also used to observe the distribution of Cx40 and Cx43 double-labeled with antibodies against Cx40 and Cx43. RESULTS: Immunoconfocal microscopy showed that the relative expression level of Cx40 protein in the specimens of RAA in the chronic AF group was (0.67 microm(2)/ microm(3) +/- 0.09 microm(2)/ microm(3)), significantly lower than that in the SR group (1.45 microm(2)/ microm(3) +/- 0.16 microm(2)/ microm(3)) (P < 0.01). There was no significant difference in the expression of Cx43 protein between the two groups. Distributed inhomogeneously, Cx40 and Cx43 were mainly located at the side to side interconnections, instead of being confined to the intercalated discs, as observed in the patients with SR. The results of Western blotting were similar to those of immunoconfocal microscopy. CONCLUSION: The reduction and redistribution of Cx40 and Cx43 contribute to initiation and persistence of AF in patients with rheumatic heart disease.

[Adenosine protects cardiomyocytes from hypoxia/reoxygenation injury].

The aim of this study was to investigate the protective effect of adenosine (ADO) on cardiomyocytes following hypoxia/reoxygenation (H/R) and its molecular mechanism. Primary cultured cardiomyocytes of neonatal rats were divided into two groups, namely H/R (control) and ADO (1.0 micromol/L) groups. The morphologic changes in cardiomyocytes were observed under an inverted phase-contrast microscope. The following parameters of the two groups were determined: lactate dehydrogenase (LDH) activity, intracellular calcium concentration and malondialdehyde (MDA) content. Tumor necrotic factor (TNF-alpha) assay was performed using an ELISA kit and NF-kappaB in the nucleus was analyzed by electrophoretic mobility shift assay (EMSA). The results are as follows: (1) after H/R injury, cardiomyocytes contracted, tending to get round in shape and its pseudopods decreased, while marked morphological changes were not observed in ADO group; (2) LDH leakage maintained at a lower level in ADO group than that in the control group during H/R (both P<0.01); (3) ADO significantly reduced the concentration of calcium in cells and prevented calcium overload during H/R (both P<0.01); (4) ADO markedly reduced the content of MDA during H/R (both P<0.01); (5) ADO inhibited the production of TNF-alpha during H/R (both P<0.01); and (6) ADO down-regulated NF-kappaB binding activity of cardiomyocytes during H/R (both P<0.01) The results suggest that (1) exogenous ADO attenuates H/R injury of cultured cardiomyocytes; (2) exogenous ADO inhibits the production of TNF-alpha after H/R injury; (3) exogenous ADO prevents the activation of NF-kappaB, which may be the molecular mechanism of down-regulation of TNF-alpha expression.