| Objective: To explore whether curcumin(CMN) could induce the expression of heme oxygenase-1(HO-1) safely and persistently in rat's vascular smooth muscle cell(VSMC) and play an anti-atherosclerosis(AS) role mediated by HO-1. Methods: The healthy aged of 3 months male Wistar rats were taken as the investigated objects.There were 46 rats divided into 5 groups randomly,including CMN group(group A,n=8), Zinc protoporphyrinⅨ( ZnPPⅨ) group(group B,n=8), CMN + ZnPPⅨgroup(group C,n=8), high-fat diet group(group D,n=14), and control group(group E,n=8), the totle experimental time was 14 weeks.There were 4 steps in the experiment: 1.Establish the AS model. Rats in group E was given normal diet ,but rats in group A,B,C and D were given high fat diet and intra-peritoneal injected with vitamin D 700,000 U/kg for once. 2 rats from group D were executed at the end of 6,10,14 weeks respectively,took the 1-2 cm initial segment of descending aorta to detect the pathological changes and the AS degree. 2. The research about drugs affecting HO-1 expression and its activity. Rats in group A were intra-peritoneal injected with CMN 3mg/kg 1 time every 2 days; Rats in group B were intra-peritoneal injected with ZnPPⅨ7.5mg/kg 1 time every 2 days ;Rats in group C were intra-peritoneal injected with CMN 3mg/kg + ZnPPⅨ7.5mg/kg 1 time every 2 days. The experiment time is 14 weeks. At the end of the experiment, took part of the initial segment of descending aorta tissue from rats in each group to detect the expression and distribution about HO-1 protein by immunocytochemistry (IHC).The positive cells showed brown granule in cytoplasmic. 5 campus visualis were selected at random to detect the optical density (OD) value of HO-1 protein under light microscope with magnification×200;the average of the OD value represented the amount of HO-1. The HO activity of descending aorta tissue were detected.According to the principle that heme can be degraded to bilirubin by HO-1,so we could measure the output of bilirubin in the reaction system to represent the HO-1 activity. 3.The research of anti-atherosclerosis role and its mechanisms about inducing and inhibiting the expression of HO-1. At the end of experiment, descending aorta tissue were taken from all rats and then fixed in 10% neutral buffered formaldehyde solution,after conventional paraffin sections and Hematoxylin and Eosin(HE)staining,the pathological changes inclued smooth muscle cells,foam cells,the aortic tunica intima and tunica media were observed under light microscope. The thickness about aortic tunica intima, tunica media and its ratio were also measuremented by multi-media graphic analysis system. 4. The blood was drawn via heart from all rats in each groups and used to detect the liver and renal function,blood lipids,C-reactive protein(CRP), interleukin-6 (IL-6), malondialdehyde (MDA) ,oxidized low-density lipoprotein(ox-LDL). Further more, the rat's kidney of group A and group E were taken to detect the pathological changes and the results combined with the renal functions conduced to the evaluation of the safety of CMN. Results:1.The rat model of AS was established successfully. Compared with group E,rats in group D showed that the aortic tunica intima thickening apparently and outstanding to the lumen, a large number of foam cells collected below the tunica intima,and the smooth muscle cells malalinement, polarity disappeared,the thickness ratio of intima/media increased significantly(P<0.05) at 14th weekend. 2. The HO-1 expression and its activity increased significantly and the degree of AS decreased markedly in group A Compared with that in group B,C and D. Rats in group B got much lower HO-1 expression and its activity than any other group and with the most severe degree of AS. Group C and group B had a similar expression and activity of HO-1(P>0.05),and the degree of AS of group C was similar to group D (P>0.05) but lighter than group B(P<0.05).3.The biochemical indicators such as blood lipid, liver function, CRP, IL-6, MDA and ox-LDL were significant difference between each group.all the biochemical indicators in group B and C were higher than that in group D(P<0.05) but were lower than that in group A(P<0.05). 4. The renal functions and the renal pathological changes had no significant difference between group A and group E. Conclusions : 1.High fat diet combined with vitamin D intra-peritoneal injection could make rate AS model successfully.2.CMN could induce HO-1 protein overexpression and increase its activity in rats VSMC, however, this induction were blocked significantly by ZnPPⅨ.3.Inducing HO-1 expression might be the main anti-AS mechanism of CMN.4. CMN perhaps existence other anti-AS mechanisms in addition to the roles of inducing HO-1 expression.5.CMN administration persistently was security, no toxic or side effects were observated in this experiment. |
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