Curcumin Inhibits AngⅡ-induced Proliferation And Oxidative Stress In VSMCs

Background and ObjectiveWith lifestyle changes, the incidence of atherosclerosis(AS) has been risen for recent years. AS is an important basical pathology of cardiovascular disease.Vascular smooth musclr cells(VSMCs), play a significant role in the pathological changes, are the important components of blood vessel walls.Therefore, to investigate the physiological pathology function of VSMCs is of great significance, is vital for AS prevention and treatment.Angiotension Ⅱ(Ang Ⅱ),as an important factor in reninangiotensin system(RAS),is one of the important biochemical factor in the proliferation of VSMCs.Its effect of promoting hormone growth can stimulate smooth muscle cell proliferation,a dose depend,and stimulate the DNA and protein synthesis in VSMCs.Thus,the inhibition of Ang Ⅱ induced the excessive proliferation of VSMCs can be used to treat AS.Studies have showed that oxdative stress(OS) is one of the important factors to promote the development process of AS. OS, when the body in a harmful situation, is the definition of the generation rate of reactive oxygen spices(ROS) in tissue is greater than elimination rate, to lead ROS accumulate in body. When the production of ROS is greater than the clear, the excessive ROS will directly damage the vascular endothelia cells, and induce the vascular smooth cells migration, proliferation and the excessive expression of matrix composition to aggravate the pathological progress of AS.We ourselves have the ability to scavenge the excessive free radicals,dependingon free radical scavenge system which include superoxide dismutase(SOD),glutathione peroxidase(Gpx) and hydrogen peroxide enzyme.As a result,antioxidant drugs play an important role in the treatment of AS.Curcumin(Cur), as the main active ingredient of curcuma, found that has many medical function, such as antiprofliration, antioxidant, scavenge of oxygen free radical, anti-inflammtory, hypolipidemic, anti-cancer and other pharmacolic effect.Furthermore, it has a significant cardiovascular protection activity. In recent years,epidemiological invistagition demonstrated that the intake of Cur negatively correlated to incidence of AS.This experiment is to observe the Cur’s effect about the Ang Ⅱ induced proliferation and oxidant stess in VSMCs. The research object is vascular smooth muscle cells separated from rat aorta. By the Ang Ⅱ induced, using the modern biological technology, such as MTT, Reverse Transcription–Polymerase Chain Reaction(RT-PCR), Western blot to obseve the effection of Cur about Ang Ⅱ induced proliferation and oxidative stress in VSMCs.MethodsPrimary rat VSMCs were cultured and divided into control group, AngⅡ group,Ang Ⅱ+Cur 5μmol/L group, Ang Ⅱ+Cur 10 μmol/L group, Ang Ⅱ+Cur 20 μmol/L group, and Cur 20 μmol/L group. The proliferation of Ang Ⅱ-induced VSMCs was measured by MTT assay. The m RNA and protein expressions of inducible nitric oxide synthase(iNOS) and p47 phox were detected by real-time PCR and Western blot.Nitric oxide(NO) production was measured by Griess reaction. Production of intracellular ROS was measured by DCFH-DA staining and the activities of SOD and Gpx were detected by xanthine oxidase assay and visible spectrophotometer. Small interfering RNA(si RNA) was used to silence the expression of p47 phox, to further explore the inhibitory effect of Cur on the proliferation of Ang Ⅱ-induced VSMCs and oxidative stress mechanism.Results1. We found that the proliferation of VSMCs had been increased apparently by stimulating Ang Ⅱ(10-7mmol/L); compared to different concentration of Cur pretreatment 1 h,the proliferation of VSMCs were inhibited and showed a dose dependent;2.RT-PCR and Western blot analysis the expression of iNOS, p47 phox m RNA and protein by stimulating Ang Ⅱ(10-7mmol/L) in VSMCs has been increased(p<0.01);using different concentration of Cur to pretreatment,we found that with the increasing concentration of Cur, the expression of iNOS, p47 phox m RNA and protein showed a declining trend(P<0.05);3. The effect of Cur in AngⅡ induced the expression of ROS,SOD and Gpx:under the stimulation of Ang Ⅱ((10-7mmol/L) in VSMCs,the content of ROS has been increased and the activity of SOD,Gpx has been decreased; in the contrary,giving different concentration of Cur,can effectively inhibited Ang Ⅱ induced the expression of ROS and enhanced the activity of SOD、Gpx(P<0.05);4. This experiment has proved that Cur can inhibite Ang Ⅱ induced the expression of p47 phox m RNA and protein in VSMCs(P<0.05). To further use small interfering RNA(si RNA) was used to silence the expression of p47 phox, the result demonstrated that the content of ROS in VSMCs is decreasing. Therefore, Cur has the antioxidant effect.ConclusionsVSMCs activities were not significantly affected by Cur at concentration between 0 to 80μmol /L. Cur(5、10、20μmol /L)significantly inhibited Ang Ⅱ-induced proliferation of VSMCs. Cur had an inhibitory effect on over-expression of NO、iNOS、p47phox、ROS in VSMCs and upregulated the activity of SOD and Gpx, in a concentration-dependent manner. Ang Ⅱ-induced ROS production in VSMCs was significantly attenuated by pretreatment with p47 phox specific si RNA.