Effect Of Oleanolic Acid On Heme Oxygenase-1 Expression And The Related Signaling Mechanism In Rat Vascular Smooth Muscle Cells

BackgroundCoronary heart disease is not only the most common heart disease that is dangerous to health of people around the world now, but also the leading causes of hospitalization and mortality in China. Atherosclerosis is involved in coronary heart disease and the development of atherosclerosis, especially in which plaques rupture, is the leading“fuse”of acute coronary events, such as acute coronary syndrome (ACS), the death of coronary heart disease. Vascular smooth muscle cells (VSMCs) are the majority of cells in vessel walls. And migration, proliferation and apoptosis of VSMCs in pathological states are considered to play an important role in the progress of atherosclerosis. Therefore, how to effectively regulate the physiological functions of VSMCs has became a concerned question now.Oleanolic acid (OA) is a triterpenoid compound naturally occurring in many plant foods, which is used for the treatment of acute and chronic hepatitis in China. In addition, accumulating evidences demonstrate a significant vasoprotective effect of OA, such as protection against myocardial ischemia-reperfusion injury, anti-hyperlipidemia, anti- atherosclerosis and so on. However, the underlying molecular mechanisms of such protective effect of OA on vascular system are not fully understood. Heme oxygenase-1 (HO-1) is one of the most important antioxidant enzymes. It is considered as the stress-response gene. And the expression of HO-1 is generally not detected or low in normal conditions, but under stress conditions, the expression of it can be upregulated. It has been reported that HO-1 may be involved in pathophysiology of cardiovascular system and protect against damage induced by a variety of adverse stimuli. Reported recently, HO-1 is also involved in the protective effects of some drugs in treatment of cardiovascular diseases. Therefore, using primary rat vascular smooth muscle cells as the target of OA, we examined whether OA leads vasoprotective response against damage induced by oxidative stress through up-regulation of HO-1 expression and elucidated the related upstream signaling pathways.MethodsPart I. Effects of OA on the expression of HO-1 and its relationship with PI3K or MAPK pathways in VSMCs.Rat aortic smooth muscle cells were obtained from the thoracic aorta of male Sprague-Dawley rats. Isolated VSMCs were cultured in DMEM at 37°C in a humidified atmosphere. The purity and identity of VSMCs were verified using a monoclonal antibody against smooth muscle?-actin. Cells from passages 4-8 were used in all experiments. VSMCs were divided into four groups:?control group,?10?M OA group,?2 5?M OA group,?50?M OA group. After cells were treated for 24 h, RT-PCR and western blot were used for detecting the expression of HO-1; spectrophotometer was used for determining the HO-1 activity. And then we used the optimal concentration of OA for the next experiments. After cells were treated for indicated time, the protein levels of HO-1 were determined by western blot analysis.VSMCs were divided into control group and various indicated time groups. The expressions of P-Akt and P-MAPKs were determined by western blot analysis. By this way, we determined which kinases could be activated by OA. In the next experiments, VSMCs were divided into control group, OA group and OA + various inhibitors groups. Western blot analysis was used for determing the expression of HO-1.Part II. The Role of Nrf2 in the OA-induced HO-1 expression in VSMCs. VSMCs were divided into control group, 10?M OA group, 25?M OA group and 50?M OA group. After cells were treated for 4 h, the nuclear proteins of cells were extracted and used for determing the expression of Nrf2 by western blot analysis. And then VSMCs were divided into control group, OA group and OA + various inhibitors groups. After cells treated for indicated time, the nuclear proteins were subjected to EMSA for the measurement of Nrf2-ARE binding.In the next experiments, VSMCs were transfected with Nrf2 siRNA-expressing lentiviral vector. Western blot analysis used to determine the expression of Nrf2. And then VSMCs were divided into the control siRNA group, OA + control siRNA group, Nrf2 siRNA group and OA + Nrf2 siRNA group. Western blot analysis was used to determine the expression of HO-1.Part III. Protective effects of OA against VSMCs damage induced by oxidative stress and the related mechanism.First MTT was used to determine the optimal concentration of H₂O₂. And then VSMCs were divided into control group, H₂O₂ group, OA + H₂O₂ group, OA + H₂O₂ + ZnPP group and OA + H₂O₂ + various inhibitors groups. The apoptosis and death of VSMCs were determined by MTT, Hoechst 33342 or flow cytometry. VSMCs were divided into control group, H₂O₂ group, 10?M OA + H₂O₂ group, 25?M OA + H₂O₂ group and 50?M OA + H₂O₂ group. The contents of GSH, NADPH and SOD were determined by corresponding kits.Results(1) OA treatment dose-dependently increased the activity, mRNA and protein levels of HO-1 and reached the maximum effect at dose of 50?M. So 50?M OA was used in next experiments.(2) When VSMCs were treated with OA at 50?M, HO-1 protein was increased up to 6 hours, and then decreased thereafter.(3) OA can activate Akt, ERK and p38 in VSMCs. Pretreatment of VSMCs with wortmannin or PD98059 effectively inhibited the OA-induced HO-1 expression, wheras SB203580 had no effect.(4) Treatment of cells with OA for 4 h, Nrf2 translocation to the nucleus increased in a dose-dependent manner. The OA-induced increase in Nrf2-ARE DNA binding activity was attenuated by wortmannin or PD98059.(5) VSMCs were transiently transfected with either control or Nrf2 siRNA. Nrf2 siRNA significantly inhibited Nrf2 protein expression compared to the control. And the OA-induced upregulation of HO-1 was reduced significantly by transfection with the Nrf2-specific siRNA, whereas there was no change in VSMCs transfected with the control siRNA.(6) A significant decline in cell viability was observed after 24 h of 400?M H₂O₂ incubation. Compared with H₂O₂ group, the contents of GSH, SOD and NADPH increased in various doses of OA groups. Compared with OA + H₂O₂ group, cell death was increased greatly in presence of ZnPP, wortmannin or PD98059. Conclusion(1) OA can increase the mRNA and protein levels of HO-1, accompanied by enhanced HO-1 activity in rat VSMCs.(2) OA induced HO-1 expression through Akt and ERK pathways in rat VSMCs.(3) OA activated Nrf2 through Akt and ERK pathways in rat VSMCs.(4) Nrf2 is involved in OA-induced HO-1 expression in rat VSMCs.(5) OA protected rat VSMCs against H₂O₂-induced damage by increasing the antioxidants, such as HO-1.