Gao Dengfeng

ASSA13-06-6 Prevention of Cardiac Remodelling by Gene Silencing of Toll-Like Receptor-4 in Mice with Diabetic Cardiomyopathy

Objective Left ventricular (LV) remodelling contributes to the development of cardiac dysfunction and is considered as a key determinant of morbidity, mortality and prognosis in patients who developed diabetic cardiomyopathy (DCM). Stimulation of toll-like receptor 4 (TLR4) can lead to impairment of myocyte contractility and activation of several intracellular mediators such as kinases, which are known as critical regulators of myocardial remodelling. Up-regulation of TLR4 expression has been shown in myocardial tissue of diabetic mice. This work is aimed to investigate whether TLR4 has influences on cardiac function and remodelling in a mouse model of DCM. Methods Diabetes was induced in C57/B16 mice by the intraperitoneal injection of streptozotocin. Diabetic mice were treated with TLR4 siRNA or scrambled siRNA as control once a week. After treatment for 6 weeks, echocardiographic parameters and invasive cardiac function detection were performed. LV tissues were collected for the assessment of collagen density and cardiomyocyte hypertrophy. Meanwhile, the expression of myeloid differentiation factor 88 (MyD88) and c-Jun N-terminal kinases (JNK) were also detected by real- time PCR and western blot. Results Compared with diabetic mice and scrambled siRNA treated diabetic mice, TLR4 siRNA treated diabetic mice showed improved systolic and diastolic LV functions and decreased LV remodelling as evidenced by reduced collagen density and levels of plasma atrial natriuretic peptide. Interstitial fibrosis and myocardial hypertrophy were also reduced by TLR4 siRNA treatment. These were associated with down- regulations of intracellular TLR4 adapter MyD88 and significantly increased expression of the anti-hypertrophic JNK, suggesting the JNK pathway may be also involved in TLR4 triggered LV remodelling in DCM. Conclusions In summary, we present direct evidence that TLR4 plays a critical role in cardiac remodelling in DCM. TLR4 may be a novel therapeutic target in the treatment of DCM.

GW24-e1159 Curcumin inhibits LPS-induced inflammation in rat vascular smooth muscle cells in vitro via ROS-relative TLR4-MAPK/NF-κB pathways

Objectives To investigate whether curcumin (Cur) suppressed lipopolysaccharide (LPS)-induced inflammation in vascular smooth muscle cells (VSMCs) of rats, and to determine its molecular mechanisms. Methods Primary rat VSMCs were treated with LPS (1 mg/L) and Cur (5, 10 or 30 mmol/L) for 24 h. The levels of MCP-1, TNF-α and iNOS were measured using ELISA and real-time RT-PCR. NO level was analysed with the Griess reaction. Western-blotting was used to detect the activation of TLR4, MAPKs, IκBα, NF-κB p65 and the p47phox subunit of NADPH oxidase in the cells. Results Treatment of VSMCs with LPS dramatically increased expression of inflammatory cytokines MCP-1 and TNF-α, expression of TLR4 and iNOS, and NO production. LPS also significantly increased phosphorylation of IκBα, nuclear translocation of NF-κB (p65) and phosphorylation of MAPKs in VSMCs. Furthermore, LPS significantly increased production of intracellular ROS, and decreased expression of p47phox subunit of NADPH oxidase. Pretreatment with Cur concentration-dependently attenuated all the aberrant changes in LPS-treated VSMCs. The LPS-induced overexpression of MCP-1 and TNF-α, and NO production were attenuated by pretreatment with the ERK inhibitor PD98059, the p38 MAPK inhibitor SB203580, the NF-κB inhibitor PDTC or anti-TLR4 antibody, but not with the JNK inhibitor SP600125. Conclusions Cur suppresses LPS-induced overexpression of inflammatory mediators in VSMCs in vitro via inhibiting the TLR4-MAPK/NF-κB pathways, partly due to block of NADPH-mediated intracellular ROS production.

GW24-e1872 Oxidised low-density lipoprotein induces inflammatory responses in cultured human mast cells via Toll-like receptor 4

Objectives Oxidized low-density lipoprotein (ox-LDL) is a powerful atherogenic factor during atherosclerosis. Toll-like receptor 4 (TLR4) has a pathophysiological role in regulating the inflammatory responses and atherosclerosis. Mast cells can infiltrate in the atheromatous plaque and secrete various pro-inflammatory cytokines, which significantly amplify the atherogenic processes and promote plaque vulnerability. We evaluated whether ox-LDL-induced inflammation depended in part on the activation of TLR4-dependent signalling pathway in cultured human mast cell line (HMC-1). Methods HMC-1 cells were cultured, and treated with ox-LDL or inhibitors of TLR4, phosphorylation of the mitogen-activated protein kinase (MAPKs), or nuclear factor-κB (NF-κB). The expressions of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) were measured after these treatment. Results Ox-LDL increased the expression of TLR4 and secretion of MCP-1, TNF-α and IL-6. Moreover, ox-LDL stimulated the transport of NF-κB, a critical mediator of inflammation, from the cytoplasm into nucleus. As well, phosphorylation of the MAPKs pathway was greatly increased. These ox-LDL-induced alterations were significantly attenuated by pretreatment with TLR4-siRNA. Conclusions Ox-LDL may induce inflammatory responses in cultured HMC-1 cells to cause NF-κB nuclear translocation and phosphorylation of the MAPKs pathway, a process mediated in part by TLR4.

GW24-e1158 Aliskiren/amlodipine versus aliskiren/hydrochlorothiazide in hypertension: Indirect-comparison meta-analysis of randomised controlled trials

Objectives Aliskiren, a direct renin inhibitor, is effective for reducing blood pressure (BP) in patients with hypertension when combined with a calcium channel blocker such as amlodipine or a diuretic such as hydrochlorothiazide (HCTZ). However, the efficacy and tolerability between the 2 combinations are unclear. We performed a systematic review of randomised controlled trials of aliskiren/amlodpine and aliskiren/HCTZ for hypertension. Methods The Cochrane Central Register of Controlled Trials, MEDLINE, Embase and the Novartis clinical trial database were searched through December 2012 for reports of RCTs of aliskiren/amlodpine and aliskiren/HCTZ versus monotherapy in patients with hypertension. The main outcome measures were reduction in systolic BP (SBP) and diastolic BP from baseline and rates of therapeutic response and BP control. Tolerance of aliskiren/amlodipine and aliskiren/HCTZ was also analysed. Outcomes were initially pooled by standard random-effects methods, producing a weighted mean difference (WMD) or risk ratio (RR) and 95% confidence intervals (95% CIs). The pooled estimates were then used for adjusted indirect comparisons. Results We selected 19 reports of trials involving 13,614 participants. Aliskiren/amlodpine and aliskiren/HCTZ were more effective than monotherapy in controlling BP. Aliskiren/amlodipine was significantly more effective than aliskiren/HCTZ in reducing SBP (WMD -3.36 mmHg, 95% CI -4.64–2.07 mmHg) and DBP (-3.49 mmHg, -4.34–2.63 mmHg). As compared with aliskiren/HCTZ, alikiren/amlodipine was associated with higher rate of therapeutic response (RR 1.23, 95% CI 1.14–1.33) and BP control (RR 1.24, 1.11–1.39). Number of adverse events and withdrawals due to adverse events were similar with aliskiren/amlodipine and aliskiren/HCTZ. Conclusions BP control is better with aliskiren combined with amlodipine or HCTZ than with monotherapy, aliskiren/amlodipien being more effective than aliskiren/HCTZ.

ASSA13-06-3 Selenium Prevents Cardiomyocyte Apoptosis During Hyperglycemia in Diabetic Rats Via TLR4 Pathway

Objective Previous studies have showed that TLR4 (Toll-like receptor 4) may trigger apoptosis of cardiomyocytes under conditions of cardiac inflammation and oxidative stress. Studies also presented that oxidative stress played an important role in the development of diabetic cadiomyopathy. Selenium (Se) is an essential trace element which is known as an antioxidant agent. This work aimed to investigate the cadioprotective effect and the possible mechanism of sodium selenite treatment in experimental diabetic rats. Methods Diabetes was induced in SD rats using intraperitoneal injections of streptozotocin (STZ). Rats were divided into four groups: nondiabetic rat group, nondiabetic rats received sodium selenite (Se) intraperitoneally for 3 weeks, diabetic rats received Se (DMSe group) intraperitoneally for 3 weeks and the diabetic rats were injected saline intraperitoneally for 3 weeks. Myocardial apoptosis was detected using TUNEL assay, TLR4 and caspase-3 expression was measured by RT-PCR and western-blot, left ventricular functions was examined by cardiac catheterization, NADPH activity and intracellular reactive oxygen species (ROS) were also assessed. Results After treatment with STZ, the rats developed diabetes evidenced by hyperglycemia. Compared with nondiabetic rats, myocardial apoptosis was detected in rat with hyperglycemia, and then left ventricular data demonstrated a significant depression in the left ventricular developed pressure (LVDP) measurement. Mechanistically, significantly increased expression of TLR4 and caspase-3 in myocardial tissue was observed, meanwhile, increased NADPH oxidase activity and ROS production was also detected in rats with hyperglycemia. In contrast, treatment of Se significantly reduced hyperglycemia up-regulated TLR4 and caspase-3 expression, minimised apoptosis in myocardial tissues, and inhibited NADPH oxidase activity and ROS production. Furthermore, the diabetic rats with Se treatment eventually showed greater improvement of LVDP compared to the untreated diabetic rats. Conclusions We concluded that hyperglycemia induces TLR4 expression in cardiac tissue; Se may act effectively against hyperglycemia-induced cardiac apoptosis as an antioxidant by modulating TLR4 signalling.

ASSA13-03-38 Selenium Ameliorate Myocardial Fibrosis in Diabetic Rats Through Inhibiting GPx- ROS-Smad- CTGF Signalling Pathway

Objective It has been mentioned that myocardial fibrosis which contributes to cardiac dysfunctions is one of the hall markers of diabetic cardiomyopathy. Up-regulation of reactive oxidative species (ROS)/TGF- beta 1/Smad signalling was described to enhance collagen synthesis and fibroblasts proliferation through activating connective tissue growth factor (CTGF). As an essential trace element, selenium (Se) has cardioprotective effects because of its antioxidant property. However, its role in diabetic cardiomyopathy against myocardial fibrosis has not been elucidated. In this study, we investigated effects of Se on myocardial fibrosis and its possible mechanisms. Methods I.p. injection of streptozotocin (STZ) at 50mg/Kg was administrated to induce type 1 diabetes in rats. Supplementation of Se was performed by i.p. injection of sodium selenite. Animals were divided into 4 groups: control group (Ctrl), diabetic group (D), normal animal with Se treatment (N+Se) and diabetic animal with Se treatment (D+Se). Intracellular generation of ROS was detected by 2’7- dichlorofluorescein staining. Activity of cellular glutathione peroxidase (GPx) was measured by spectrophotometrical method. Sirius red and Masson staining, gene expression examination of collagen type I (Coll-I) and fibronectin (FN) were employed to detect myocaridal fibrosis. Gene expression examination of TGF-beta 1, phosphorylated- Smad2, Smad2, phosphorylated- Smad3, Smad3 and CTGF was aimed to demonstrate the expression of Smad- CTGF signalling pathway. Results Significant myocardial fibrosis characterised by Mason staining, Sirius red staining, up-regulation of Coll-I and FN gene expression was found in diabetic rats (D), which was then attenuated by Se treatment (D+Se). Mechanically, accompanied by myocardial fibrosis, intracellular GPx activity decreased significantly in D compared with Ctrl and N+Se, resulting in increased formation of intracellular ROS. Meanwhile, activation of Smad-CTGF signalling pathway marked by up- regulation of TGF-beta 1, phosphorylated- Smad2/Smad2, phosphorylated-Smad3/Smad3 and CTGF was detected in D. However, after treatment of Se, down- regulation of Smad-CTGF signalling pathway in D+Se compared with D resulted in alleviation of myocardial fibrosis. Conclusions These results demonstrate that Se supplementation ameliorates myocardial fibrosis in diabetic cardiomyopathy through modulating GPx/ROS/Smad/CTGF signalling pathway, whose activation may participate in myocardial fibrosis in diabetes.

ASSA13-03-20 Selenium Attenuates High Glucose- Induced Cardiomyocyte Apoptosis by Modulating TLR-4/Myd88 Signalling Pathway

Objective Diabetic cardiomyopathy is one of the most serious complication of diabetes mellitus. Cell injury and apoptosis of myocytes are involved in diabetic cardiomyopathy. As activation of toll- like receptor 4 (TLR-4)/myeloid differentiation factor 88 (Myd88) signalling pathway which triggers cell apoptosis is correlated with generation of reactive oxygen species (ROS), it is logical to speculate the activation of TLR-4/Myd88 signalling pathway in myocytes play a role in diabetic cardiomyopathy. It is mentioned production of ROS contributes in onset, development and progression of diabetic cardiomyopathy. Selenium (Se) is an important trace element which is known to protect against ROS related cell injury by its antioxidant property. In this study, we investigated protective and anti- apoptosis effects of selenium on cardiomyocytes via ROS and TLR-4/Myd88 signalling pathway. Methods Myocytes isolated from neonate rats were cultured whether in standard medium or high glucose medium (concentration of glucose = 25.5mM) to mimic sustained hyperglycemia in diabetic cardiomyopathy. Sodium selenite solution was applied to myocytes after incubation of high glucose medium. ROS was monitored by cell fluorescent staining of 2.7- dichlorofluorescein (DCFH-DA); activity of cellular glutathione peroxidase (GPx) was measured spectrophotometrically; apoptosis of myocytes was examined by flow cytometry; expression of TLR-4, Myd88, apoptosis-inducing factor (AIF) and Caspase-3 were assessed by real- time PCR and western blotting. Results We found increased high glucose- induced cell apoptosis and up-regulated molecular expression of TLR-4/Myd88/AIF/Caspase-3 cascade, accompanied by increase of ROS generation. We also showed that supplement of selenium attenuated cell apoptosis of high glucose incubated myocytes, and mechanically, the protective effect was found dependent on attenuating oxidative status by decreasing level ROS, increasing activity of GPx, as well as inhibition of molecular expression of TLR-4/Myd88/AIF/Caspase-3 cascade in myocytes. Conclusions These results suggest activation of TLR-4/Myd88 signalling pathway plays an important role in high glucose-induced cardiomyocyte apoptosis. Additionally, by modulating TLR-4/Myd88 signalling pathway which is linked with ROS formation, selenium exerts its antioxidative and anti-apoptosis effects in high glucose incubated cardiomyocytes.

ASSA13-03-37 Adriamycin Induced Cardiac Dysfunction is Attenuated by Selenium Via Restoring Oxidative Stress Mediated Expression of ATP Sensitive Potassium Channels in Rats

Objective Cardiac toxicity is an important complication of chemotherapy. Selenium (Se) acts as antioxidant agent to protect myocytes from oxidative damage. Lack of Se would lead to enhancement of oxidative stress. It has been mentioned that impairment of ATP sensitive potassium channels (KATP) which are expressed in myocytes are correlated with oxidative mediated cardiac dysfunction. The possible mechanism of adriamycin (ADR) and/or Se deficiency induced cardiac dysfunction, and cardioprotective effects of Se against ADR induced cardiac toxicity were investigated in this study. Methods Animal model of cardiac dysfunction was induced by adriamycin inraperitoneal injection or/and Se deficit diet. Then by intraperitoneal injections, Se supplementation was applied to rats. We assessed cardiac function by serum brain natriuretic peptide (BNP) level, echocardiographic and hemodynamic parameters. Cardiac glutathione peroxidase (GPx) activity which mirrors oxidative stress indirectly was measured spectrophotometrically. Expression levels of KATP channels were evaluated by real-time polymerase chain reaction (PCR) and western blotting. Results The results showed that cardiac function and cardiac GPx activity decreased remarkably after administration of ADR or Se deficiency; more dramatic impairment of cardiac function and cardiac GPx activity were observed after co-administration of ADR and Se deficiency. Mechanically, down-regulation of KATP subunits gene expression in cardiac tissue was found after administration of ADR or Se deficiency; more significant inhibition of KATP subunits gene expression was identified after co-administration of ADR and Se deficiency. However, cardiac toxicity of ADR was alleviated by Se supplementation, accompanied by restoring of cardiac GPx activity and KATP subunits gene expression. Conclusions These results indicate that decreased oxidative mediated expression of KATP is involved in adriamycin and/or Se deficiency induced cardiac dysfunction; Se deficiency exacerbates adriamycin induced cardiac dysfunction by future inhibition of oxidative mediated KATP expression; Se supplementation seems to protect against adriamycin induced cardiac dysfunction by alleviating oxidative stress and then restoring KATP expression, showing potential clinical application in cancer chemotherapy.

ATP-binding cassette transporter G1 protects against endothelial dysfunction induced by high glucose

Introduction It is known that hyperglycaemia can contribute to defects in endothelial function, which is the early characteristic of atherosclerosis. ATP-binding cassette transporter G1 (ABCG1), a regulator of reversing cholesterol efflux, is highly expressed in endothelial cells. To gain further insight into the mechanism of endothelial dysfunction induced by high glucose, we strive to examine the effect of ABCG1 on endothelial function. Methods Human umbilical vein endothelial cells (HUVECs) were incubated with D-glucose (5.6 and 30 mmol/l) for one to seven days in vitro. Expression of ABCG1 in HUVECs was measured by real-time PCR and Western blot. The rate of cholesterol efflux to HDL was measured by scintillation counting, and intracellular lipid content was measured by enzymatic fluorometric method. The level of IL-6 and TNFα were measured by ELISA, and eNOS expression and activity of NO were examined. Results Compared with 5.6 mmol/l glucose, high glucose (30 mmol/l) decreased the mRNA and protein expressions of ABCG1 in HUVECs in a time-dependent manner. Consistent with downregulation of ABCG1, free cholesterol efflux to HDL was reduced. However, intracellular cholesterol levels were not changed. In addition, secretions of IL-6 and TNFα in HUVECs cultured in high glucose for seven days were increased three and four times respectively compared to normal glucose. In contrast, eNOS expression was downregulated for 20%, and activity of NO was inhibited. In addition, upregulation of ABCG1 gene expression by LXR agonist 22(R)-hydroxycholesterol reversed the levels of IL-6, TNFα in high glucose and upregulated the eNOS expression and NO activity. Conclusions These results suggest that high glucose concentration decrease ABCG1 expression in ECs and that this effect is associated with endothelial dysfunction induced by high glucose. Upregulation of ABCG1 has an effective effect on protection of endothelial function.