| Atherosclerosis and its related cardiovascular diseases have seriously threatened human health. Concerning the pathogenesis of atherosclerosis, there are several theories including lipid infiltration theory, thrombosis theory, smooth muscle cell clone theory and inflammation theory. The most recognized theory is the inflammation theory proposed by ROSS, and he considered that in the stimulation of various factors, the injured arterial intima will secrete a variety of inflammatory factors which can further impaire endothelial function. And the inflammatory factors also cause the imbalance of proliferation and apoptosis of vascular smooth muscle cells which promote the formation of atherosclerotic plaques.Renin Angiotensin System (RAS) as an important part of the regulation of human body system, is involved in regulating blood pressure, water and electrolyte in order to keep the stability of the environment in the human body. The excessive activation of the RAS can lead to a variety of cardiovascular diseases, such as hypertension, atherosclerosis, myocardial hypertrophy and heart failure. Angiotensin Ⅱ (AngⅡ) is the main active component of RAS, and the binging to its receptor can elicit a series of effects including proliferation, fibrosis and proinflammatory response which cause the damage of target organs. AngⅡ also plays a key role in the formation of atherosclerosis because it can promote the proliferation and migration of vascular smooth muscle cells.Prorenin is mainly synthesized in the kidney in which prorenin is converted into the active renin. Proenin has been considered as the inactive renin precursor, but in the last century80’s, a report showed that plasma prorenin levels increased in the diabetic patients. Subsequent studies have shown increased plasma prorenin that occurred before the occurrence of microalbuminuria in diabetic patients is associated with microvascular complications. And increased prorenin levels along with glycated hemoglobin can be used to predict the onset of microalbuminuria. So the increased prorenin levels may be associated with various diseases, and even can be used as an indicator to predict some disease.In2002, Nguyen firstly proposed the functional (pro)renin receptor (PRR), and detected its distribution in the heart, brain, placenta, kidney and liver. Then researchers found that the binding of prorenin to PRR in the nanomolar level can active the signal pathway to elicit a series of profibrotic and inflammatory effects in both AngⅡ-dependent and AngⅡindependent way. The AngⅡ-independent role of PRR makes people have a new understanding of RAS. The blockade of the RAS is considered to be beneficial and to reduce cardiovascular risks, despite the fact that its inhibition cannot completely inhibit the development of these diseases. The answer to this question might reside in the AngⅡ-independent effects induced by the binding of prorenin with PRR. Based on this, we invetigated the Angll-independent role of prorenin and PRR on the proliferation and apoptosis in human umbilical artery smooth muscle cells (HUASMCs).Atherosclerosis is a chronic inflammatory process, and the important sign of inflammation is reactive oxygen species (ROS) production. Oxidative stress is a pathological condition in which excessive production of ROS damage the intracellular antioxidant system. A lot of studies have confirmed that ROS play an important role in the pathophysiology of cardiovascular disease. ROS produced by inflammatory cells and various types of cells in vascular wall can promote cells proliferation, apoptosis, migration and increase expression of proinflammatory genes. The damage and functional changes of cell induced by ROS constitute the pathological basis of atherosclerosis. Angiotensin Ⅱ can activate NADPH oxidase system that is the main source of the vessel wall ROS. The production of ROS can further cause the abnormal proliferation and migration of vascular smooth muscle cells by impairing endothelial function, accelerating the degradation of NO, reducing the endothelial contractile function, and promoting the phenotype transformation of smooth muscle cells. The molecular mechanism of PRR is still not completely clear, and whether the PRR can like AngⅡ through oxidative stress induce the changes which is responsible for atherosclerosis in smooth muscle cells is not defined. On basis of this, we invetigated the AngⅡ-independent role of prorenin and PRR on the production of ROS in HUASMCs.Mitogen activated protein kinase (MAPK) is an important enzyme in signal transduction, including ERK, JNK, p38MAPK and ERK5four subfamilies, and ERK1/2signal transduction pathway is closely related to the proliferation and differentiation of cells. ERK1/2activation by a variety of stimuli can promote cells proliferation and increase the inflammatory factor expression. The binding of AngⅡ to its receptor can promote the proliferation of smooth muscle cells through the phosphorylation of ERK1/2. In rat cardiomyocytes, AngⅡ can induce the phosphorylation of ERK1/2through oxidative stress. So, we explored the role of ERK1/2signal pathway in effects induced by prorenin and PRR, and study the relationship between ERK1/2and ROS.Ojective:To observe the distribution of PRR in HUASMCs. To observe the effect of prorenin on proliferation and apoptosis protein Bcl-2, Bax expression in HUASMCs. To explore the role of ERK1/2and ROS in prorenin-induced effects.Methods:1.We used fluorescence immunohistochemistry to determine the distribution of PRR in HUASMCs.2.In the presence of Valsartan and PD123319, we used prorenin of different concentration (0.1nM,1nM,10nM) to stimulate HUASMCs for24-48h and evaluated the proliferation by CCK-8.3.In the presence of Valsartan and PD123319, we used prorenin different concentration (0.1nM,1nM,10nM) to stimulate HUASMCs for24h and evaluated the expression of Bcl-2and Bax by western blot and real-time PCR.4.HUASMCs were transfected with PRR siRNA respectively24,48and72hours, and the mRNA and protein of PRR expression was detected by real-time PCR and western blot. 5.After transfection of PRR siRNA and preincubation with Valsartan and PD123319for30min, we used prorenin of concentration (10nM) to stimulate HUASMCs for24h and evaluated the proliferation by CCK-8and evaluated the expression of Bcl-2and Bax by western blot and real-time PCR.6. In the presence of Valsartan and PD123319, we used prorenin of different concentration (0.1nM,1nM,10nM) to stimulate HUASMCs for24h and measured the production of ROS on a flow cytometer.7. After transfection of PRR siRNA and preincubation with Valsartan and PD123319for30min, we used prorenin of concentration (10nM) to stimulate HUASMCs for24h and measured the production of ROS on a flow cytometer.8. In the presence of Valsartan and PD123319, we used prorenin of concentration (lOnM) to stimulate HUASMCs and evaluated the phosphorylation of ERK1/2by western blot.9. After the preincubation with DPI for30min, we used prorenin of concentration (10nM) to stimulate HUASMCs for0-60min and evaluate the phosphorylation of ERK1/2by western blot.10. After the preincubation with PD98059and DPI respectively for30min, we used prorenin of concentration (10nM) to stimulate HUASMCs and evaluated the proliferation by CCK-8and evaluated the expression of Bcl-2and Bax by western blot and real-time PCR.Results:1.We determined the distribution of PRR in HUASMCs by fluorescence immunohistochemistry and detected the cytoplasmic expression of PRR.2. Different concentrations of prorenin can independently of AngⅡ promote the proliferation in HUASMCs and the effect of prorenin (10nM) was most significant.3. Prorenin (10nM) for24-48h, as stimulation time extended the effect of prolifertion in HUASMCs was more significant.4. Different concentrations of prorenin can independently of AngⅡ upregulate the expression of Bcl-2and downregulate the expression of Bax in HUASMCs and the effect of prorenin (10nM) was most significant.5.After transfection with a PRR siRNA, the effects that prorenin elicited on the proliferation of HUASMCs was significantly attenuated, and the upregulated expression of Bcl-2and the downregulated expression of Bax were also altered.6. Prorenin can independently of AngⅡ promote the production of ROS and the effect of prorenin (10nM) was most significant. Transfection with a PRR siRNA significantly decreased the production of ROS.7. Prorenin can independently of AngⅡ induce the phosphorylation of ERK1/2, and the effect at15min-30min was most significant.8. DPI (NADPH oxidase inhibitor) and PD98059(the ERK1/2inhibitor) respectively attenuated the phosphorylation of ERK1/2induced by prorenin.9. DPI and PD98059respectively attenuated the prorenin-induced proliferation of HUASMCs.10. DPI and PD98059respectively attenuated the prorenin-induced up-regulation of Bcl-2at mRNA and protein levels in HUASMCs.11. DPI and PD98059respectively attenuated the prorenin-induced down-regulation of Bax at mRNA and protein levels in HUASMCs.Conclusions:1. There were the expression of PRR in all the cytoplasm of HUASMCs.2. The binding of prorenin to PRR can promote the proliferation and upregulate the anti-apoptotic protein Bcl-2and downregulate the pro-apoptotic protein Bax independently of AngⅡ in HUASMCs.3. The binding of prorenin to PRR can independently of AngⅡ promote the production of ROS in HUASMCs.4. Prorenin can independently of AngⅡ induce the phosphorylation of ERK1/2through the production of ROS in HUASMCs.5. Inhibition of the production of ROS and the phosphorylation of ERK1/2respectively attenuated the prorenin-induced proliferation and regulation of apoptosis factors in HUASMCs. |
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