The Role Of ROS In The Expression Of Inflammatory Cytokines And Its Relationship With Hypertensive Cardiac Remodeling: The Interventional Effects Of Simvastatin

Background and objective Hypertension is the most important risk factor for cardiovascular disease. Cardiac remodeling in patients with hypertension is an independent risk factor for cardiovascular events, and is the key pathological manifestations during the transition of heart function from compensate to decompensate. Therefore, the study on the pathogenesis and control of cardiac remodeling has become a worldwide hot topic. In the past decades, many strides have been made in the field, but the pathogenesis mechanism of cardiac remodeling still remains unclear. It is wellknown that abnormal activation of neurohumoral factors is an important mechanism for cardiac remodeling. Nowadays, blockages of a variety of neurohumoral factors have become routine medication, however, the tendency of heart failure in patients is still unable to prevent and the incidence of heart failure remains high. Proinflammatory cytokines, the important mediators of inflammatory response, have become another important neurohumoral factors associated with cardiovascular system. A growing body of studies has shown that inflammatory cytokines play an important role in cardiac remodeling. Inflammatory cytokines promote the progression of cardiac remodeling in various ways, including cardiomyocytes hypertrophy, cell apoptosis and extracellular matrix synthesis and degradation. Furthermore, just like inflammatory cells, many kinds of cells in myocardium may synthesis and secrete inflammatory cytokines, which affect the injury, repairing and structural change of heart in autocrine and paracrine manner. But most of the studies are in vitro research-based, and the studies in vivo were mainly confined in the cardiovascular diseases with obvious inflammation response such as myocardial infarction and myocarditis. Clinical data show the serum levels of proinflammatory factors in patients with hypertension increase in comparison with normotensive, nevertheless the expression of inflammatory cytokine in local myocardium is not yet clear, which is more important in the formation of cardiac remodeling. Whether hypertension can cause expression of inflammatory cytokines in myocardium and the expression mechanism, is not yet clear. Latest studies show that ROS is associated with the genesis and development of hypertension. Increasing evidence is emerging that ROS is an important signaling molecule that can activate oxygen sensitivity kinases and transcription factors and modulate a variety of genes expression. Documents have reported that ROS is involved in the expression of inflammatory cytokines in vascular endothelial cells and macrophages. And up-regulation of ROS was detected in myocardial tissue of both patients with hypertension and hypertensive animals. But whether hypertension can up-regulate the expression of inflammatory cytokines by increasing ROS level of myocardium and subsequently promote the development of cardiac remodeling has not yet been reported. Our previous studies demonsrated that HMG-CoA reductase inhibitors, named as statins, inhibited the hypertrophy of cardiomyocytes and the proliferation and collagen synthesis of cardiac fibroblasts. Statins attenuated the cardiac hypertrophy in SHR model. These studies suggest the inhibitory effects of statins on cardiac remodeling, but the mechanism remains unclear. This study was therefore designed to observe the temporal profile of the expression of inflammatory cytokines and analyze its relationship with hypertensive cardiac remodeling; to study the role of ROS in the expression of cytokines in myocardium and cardiomyocytes; to investing the effects and the mechanism of simvastatin (Sim) in the prevention and treatment of cardiac remodeling. The purpose of the study is to elucidate the cellular and molecular mechanisms of cardiac remodeling associated with hypertension and the interventional effects of Sim on it, and provide novel theoretical evidences and strategy for prevention and treatment of hypertensive cardiac remodeling.Methods In this study, pressure-overload rats and cultured cardiac myocytes of neonatal SD rats were used as experiment models. Histopathologic assay, echocardiography, hemodynamic measurement, colorimetric method, fluorescent probe technique, cytochrome C reduction assay, ELISA, RT-PCR and Western blot were applied to identify: (1) the dynamic changes of expression of TNF-α, IL-6 and IL-10 in pressure-overload rats and its relationship with structure and function of heart; (2) the dynamic changes of ROS level and NADPH oxidase activity in pressure-overload rats; (3) the effects of LPS on the ROS level, NADPH oxidase activity and p38 MAPK activation in cultured cardiomyocytes. (4) the effects of N-acetyl cysteine (NAC) on the expression of inflammatory cytokines in myocardium of pressure-overload rats and cultured cardiomyocytes. (5) the effects of Sim on the ROS level and the expression of cytokines in myocardium of pressure-overload rats and cultured cardiomyocytes.Results (1) Compared with sham operation (Sham) rats, blood pressure in abdominal aorta coarctation (AC) rats significantly increased at 1 week postoperation (P<0.01). Left ventricular weight index (LVWI), ventricular wall thickness, myocyte diameter and myocardial collagen content were elevated at 2 week (P<0.01). Left ventricle diastolic dysfunction was found at 4week (P<0.05). All the changes further progressed then. (2) In AC rats, the mRNA expression of TNF-α, IL-6 and IL-10 in myocardium increased at 2 week, and peaked at 4 week. Compared with Sham rats, the mRNA expression of TNF-α, IL-6 and IL-10 were increased by 126%, 183% and 83% respectively (P<0.05 or P<0.01). (3) In AC rats, the protein content of TNF-α, IL-6 and IL-10 in myocardium increased at 4 week. Compared with Sham rats, the protein content of TNF-α, IL-6 and IL-10 were increased by 179%, 114% and 38% respectively (P<0.05 or P<0.01).There was no significant difference of content of TNF-α, IL-6 and IL-10 in serum between AC and Sham groups (P>0.05). (4) Correlation analysis indicated the protein levels of TNF-αand IL-6 in myocardium were positively correlated with myocardial collagen content, left ventricular end-diastolic pressure (LVEDP) but not myocyte diameter. There was no significant correlation between protein levels of IL-10 in myocardium with myocardial collagen content, LVEDP and myocyte diameter. (5) In AC rats, ROS level and NADPH oxidase activity increased at 1 week, and peaked at 2week, preceding the increase of inflammatory cytokines expression. The ROS level and NADPH oxidase activity at 2 week increased by 140% and 114% (P<0.01). (6) In AC rats treated with NAC (0.2g/kg/d, in drinking water), LVWI, ventricular wall thickness, myocardial collagen content and left ventricular diastolic function were greatly improved (P<0.05 or P<0.01). The myocyte diameter had no change after NAC treatment. Compared with AC group, the mRNA and protein level of TNF-αand IL-6 in myocardium of AC rats treated with NAC markedly decreased (P<0.01). There was no significant change of IL-10 level (P>0.05). (7) In AC rats treated with Sim at both early and late stage, LVWI, ventricular wall thickness, myocyte diameter, myocardial collagen content and left ventricular diastolic function were all significantly improved (P<0.05 or P<0.01), but there was no change of blood pressure. The effects of Sim treatment at early stage was better than that at late stage (P<0.05). (8) In AC rats treated with Sim at both early and late stage, the mRNA and protein level of TNF-αand IL-6 in myocardium significantly decreased in comparison with AC rats (P<0.05 or P<0.01), and the effects of Sim treatment at early stage was better than that at late stage (P<0.05 or P<0.01). The level of IL-10 expression didn't change after Sim treatment. (9) After Sim treatment, the ROS level and NADPH oxidase activity in myocardium significantly decreased (P<0.05 or P<0.01), and the effects of Sim treatment at early stage was better than that at late stage (P<0.05). (10) In cultured neonatal rats cardiomyocytes stimulated with 1μg/ml lipopolysaccharide (LPS), The mRNA and protein expression levels of TNF-α, IL-6 and IL-10 increased in a time-dependent manner (P<0.05 or P<0.01). (11) The ROS level and NADPH oxidase activity in cardiomyocytes increased markedly in LPS group in comparison with that of control group (P<0.01), preceding the increase of expression of inflammatory cytokines. Pretreatment with NAC and diphenyleneiodonium (DPI), the NADPH oxidase inhibitor, inhibited the LPS-induced increase of protein content of TNF-αand IL-6 (P<0.01), but the IL-10 protein levels was not affected by NAC and DPI pretreatment. (12) Exogenous hydrogen peroxide (H2O2) also induced the increase of TNF-αand IL-6 protein level (P< 0.05), but not IL-10 protein level. (13) Pretreatment with Sim at concentrations of 0.1~10μmol/l attenuated the LPS-induced TNF-αand IL-6 mRNA and protein expression in a dose-dependent manner. The IL-10 level induced by LPS was not affected by treatment with Sim. Furthermore, mevalonate (MVA) pretreatment in combination with 1μmol/l Sim reversed the inhibitory effects of Sim on the expression of TNF-αand IL-6. Pretreatment with Sim or MVA alone didn't significantly affect the basal levels of these cytokines expression. (14) Pretreatment with 1μmol/l Sim had no effects on the H2O2-induced TNF-αand IL-6 protein expression (P>0.05). (15) Western blot indicated that stimulation with LPS for 1h significantly increased p38 MAPK activity (P<0.01), preceding the increase of cytokines expression. Pretreatment with NAC, DPI and Sim all attenuated p38 MAPK activation induced by LPS (P<0.01). The effect of Sim on p38 MAPK activation was reversed by concomitant pretreatment with MVA (P<0.01). (16) 5μmol/l SB203580, a p38 MAPK inhibitor, attenuated LPS-induced increase of TNF-αand IL-6 protein level (P<0.01), but not that of IL-10.Conclusion Hypertension can up-regulate the expression of proinflammatory cytokines such as TNF-αand IL-6. Although the expression of proinflammatory cytokines has no effect on the initiation of cardiac remodeling, it is involved in the formation and progression of cardiac remodeling and is significant correlation with matrix remodeling and diastolic dysfunction. The ROS signal pathway, at least in part, mediates the expression of proinflammatory cytokines in myocardium and cardiomyocytes. The p38 MAPK probably is the important downstream molecular in the ROS signal pathway. Sim can prevent and reverse the progress of cardiac remodeling, and the interventional effects of Sim treatment at early stage is better that those at late stage. The inhibition of NADPH oxidase activity and the subsequent inhibition of ROS signal pathway and the expression of proinflammatory cytokines is one of important mechanism of the effects of Sim on cardiac remodeling. In short, hypertension up-regulates the expression of proinflammatory cytokines, the latter promote the progression of cardiac remodeling. ROS signal pathway is involved in the mechanism of cytokines expression. Sim can inhibit cardiac remodeling by the effects on oxidative stress and expression of proinflammatory cytokines. These will provide novel strategy and theoretic evidence to prevention and treatment of hypertensive cardiac remodeling.