| Background & ObjectiveAtherosclerosis (AS) is a chronic progressive inflammatory response within the artary wall and is the common pathological foundation of many cardiovascular diseases, which seriously threatens people's health. Three important aspects in formation of atherosclerosis plaque are the proliferation of vascular smooth muscle cells (VSMCs),injury of vascular endothelium cells, and macrophages thransforming into foam cells which is the first step in development of AS. A large number of researches at home and abroad indicate that hypercholesterolemia, hypertension, diabetes mellitus and hyperhomocysteinemia are pathogenic risk factors of AS. Asymmetric dimethylarginine (ADMA) is closely related with these risk factors, and is considered as a new risk factor of cardiovascular diseases. ADMA is a competitive inhibitor of endothelium drived nitric oxide synthase (NOS). In normal circumstances ADMA in human plasma has no effect on the activity of NOS, but under the stress status and the injury site, the concentration of ADMA in plasma is obviously increased. High concentration of ADMA plays an important role in inhibiting the synthasis of nitric oxide (NO), injuring the vascular endothelium and inducing VSMCs. However, the study is relatively few about the effect of ADMA on the process of macrophages thransforming into foam cells, and the mechainism is not clear. NOS is the key rate-limiting enzyme of NO biosynthesis, including constitutive nitric oxide synthase (cNOS) and inducibe nitric oxide synthase (iNOS). cNOS is known in a wide variety of cells and tissues. Normally, there is minimal or no expression of iNOS, but iNOS is induced to express by a variety of stimulus. Lectin like oxidized low density lipoprotein receptor-1 (LOX-1) is the specific receptor of oxidized low density lipoprotein (oxLDL). LOX-1 is different in structure from the classical scavenger receptors, belonging to scavenger receptor type E. Macrophages ingest a great quantity of oxLDL via scaverger receptors when there is no a negitive feedback, which leads to accumulation of lipid in the cytoplasm of macrophages and formation of foam cells. Researches show that ADMA enhances the expression of LOX-1 and its mechanism is not very clear yet. The promotor regions of iNOS gene containing an active nuclear factory kappa B (NF-κB) binding site, which can specially combine to nucleoprotein and regulate transcription of down-stream responsive genes. NF-κB is a ubiquitous intracellular transcription factor, taking part in regulateing gene transcription. In the present study, we adopted molecular biological techniques to explore the mechanism of ADMA inducing to express iNOS and LOX-1 during development of AS in cell level and tissue level, and whether activation of NF-κB has a relationship with the mechanism.Methods1. Study in vitroFifty-Healthy, male wistar rats were enrolled. Celiac macrophages of rats were gathered and cultured. Groups and interference:①Control group:macrophages were incubated with 10% FBS DMEM.②oxLDL group:macrophages were treated by oxLDL (final concentration 50mg/L) for 48 hours.③A+O group:macrophages were pretreated with ADMA (final concentration 15umol/L) for 2 hours, then co-incubated with oxLDL (final concentration 50mg/L) for 48 hours;④P+A+O group:macrophages were pretreated with PDTC (final concentration 25umol/L) for 30 minutes, then co-incubated with ADMA (final concentration 15umol/L) for 2 hours, finally co-incubated with oxLDL (final concentration 50mg/L) for 48 hours. Each group was repeated for 3 times. Macrophages and medium were respectively collected. Intracellular cholesterol level, NO level, iNOS activity were detected separately. The total RNA, total proteins and nucleoprotein were extracted from macrophages respectively. Real time fluorescent quantitative PCR (real-time PCR) was used to detected the expression of mRNA, including iNOS mRNA and LOX-1 mRNA. The expressions of iNOS and LOX-1 protein were measured by Westen blotting, using rat actin as an internal standard. NF-κB activity was detected with gelelectrophorestic mobility shift assay (EMSA) and enhanced chemiluminescent (ECL) technique.2. Study in vivoFifty-Healthy, male wistar rats were enrolled and randomly divided into four groups.①Control group (n=10):Fed with standard diet.②H group (n=12):Fed with high fat diet.③A+H group (n=4):Fed with high fat diet, and administered intergastrically with ADMA [0.2mg/(kg-d)] once a day.④P+A+H group (n=14):Fed with high fat diet, administered intergastrically with ADMA [0.2mg/(kg-d)] and given interperitoneal injection with PDTC [40mg/(kg-d)], respectively once a day. Both control group and H group were administered intergastrically and injected interperitoneally with the same volume of normal saline. A+H group was injected interperitoneal with the same volume of normal saline.18 weeks later, the rats were anesthetized, rat blood and aortas were gathered. Serum NO level and iNOS activity were measured respectively. Collected thoracic aortas were made pathology slices and observed the histological structure under microscope. The total RNA, total protein and nucleoprotein were separately extracted from rat aortas. The expressions of iNOS mRNA and LOX-1 mRNA were detected with real time fluorescent quantitative PCR. Westen blotting was used to examinate the expression of iNOS and LOX-1 protein, using rat actin as an internal standard. EMSA and ECL technique was used to detect NF-κB activity.Results1. Study in vitro①Intracellular cholesterol level in oxLDL group and A+O group were higher than that in control group (P<0.05,either), and compared with oxLDL group, intracellular cholesterol level in A+O group significantly increased (P<0.05), but intracellular cholesterol level of P+A+O group was lower than that of A+O group (P<0.05).②Compared with control group and oxLDL group, NO level in A+O group was significantly lower (P<0.05,either), and NO level of P+A+O group was much higher than that of A+O group (P<0.05).③The activity of iNOS in A+O group significantly higher than that of in control group and that of in oxLDL group (P＜0.05,either), but in P+A+O group, iNOS activity significantly decreased than that in A+O group (P<0.05). The correlation analysis indicated that NO level had a obvious negitive correlation with iNOS activity (r=-0.773,P<0.05).④Compared with control group and oxLDL group, the mRNA and protein of iNOS expression in A+O group significantly increased (P<0.05,either), and in P+A+O group,it obviously decreased than that in A+O group (P＜0.05).⑤The mRNA and protein of LOX-1 expression in A+O group significantly increased than that in control group and oxLDL group (P<0.05,either), but decreased in P+A+O group compared with A+O group (P<0.05).⑥In A+O group, the NF-κB activity markedly increased compared with that in control group and oxLDL group (P<0.05,either), and decreased in P+A+O group compared with A+O group.⑦Correlation analysis showed that the NF-κB activity took on a significant positive correlation with the expression of iNOS mRNA and protein (r=0.82,r=0.81,respectively;P<0.05), and the NF-κB activity took on a significant positive correlation with the expression of LOX-1 mRNA and protein (r=0.82,either;P<0.05).2. Study in vivo①Pathological examination:the paraffin-cut slices of thoracic slices were stained with HE and inspected under optic microscope. In control group the intima was smooth and integrated, without the formation of vacuoles. In H group, the intima contained a small amount vacuoles. In A+H group, the intima and elastic fibers ruptured, there were proliferation and irregular assay of VSMCs and a lot of vacuoles in intima and media. Compared with A+H group, the intima was integrated, the degree of VSMCs proliferation and the amount of vacuoles was decreased in P+A+H group.②Sesum NO level and iNOS activity in A+H group were obviously higher than those in control group and H group (P<0.05,either), and which in P+A+H group was lower than in A+H group (P＜O.05). Correlation analysis indicated that serum NO level was positively associated with iNOS activity (r=0.68,P<0.05).③The mRNA and protein of iNOS expressions in A+H group increased compared with those in control group and H group (P<0.05,either), and which in P+A+H group decreased than those in A+H group (P＜O.05).④The mRNA and protein of LOX-1 expressions in A+H group increased compared with those in control group and H group (P<0.05,either), and which in P+A+H group decreased than those in A+H group (P<0.05).⑤The NF-κB activity was significantly higher in A+H group than that in control group and H group (P<0.05,either), and was lower in P+A+H group than that in A+H group.⑥Correlation analysis showed that the NF-κB activity was significantly and positively correlated with the iNOS mRNA and protein expression (r=0.85, r=0.87, respectively; P＜O.05, either),and which was also significantly and positively correlated with the LOX-1 mRNA and protein expression (r=0.79, r=0.81, respectively; P＜O.05, either).ConclusionADMA up-regulates the expression of iNOS and LOX-1 mainly through the NF-κB pathway, which promotes the occurrence and development of atherosclerosis.
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