The High Methionine Diet On The Expressions Of CCL5 And CCR5 In Aorta Smooth Muscle Cells Of Rats And Its Mechanism

BackgroundAs the main pathology of ischemic heart disease the incidence of which is rising year after year, atherosclerosis (AS) has become the leading cause of death in developed countries and China, and it makes serious harm to people’s health. In recent years, plasma homocysteine (Hcy) level, as a new proposed and key non-traditional risk factor of AS, has got people’s attention increasingly. But the exact pathogenesis is unclear. Therefore, further investigation of the mechanism of Hyperhomocysteinemia (HHcy) induced by long-term high methionine (Met) diet on AS, can provide a theoretical basis and new treatment ideas for the treatment of AS through decreasing plasma Hcy levels in future.ObjectivesTo explore the correlation and pathogenic mechanisms of HHcy with AS, we obverse whether long-term high methionine diet could induce HHcy and whether HHcy could induce the expression of CC chemokine ligand 5 (CCL5) and its receptor chemoattractant cytokines receptor 5 (CCR5) by nuclear transcription factor-KB (NF-κB) pathway in rat aortic smooth muscle cells. To provide a theoretical basis for clinical intervention, therapeutic effect of Folic acid combined with VitB12 and rosuvastatin is also obserbed.MethodsForty of clean grade, healthy,8-week-old male SD rats, each of them weighing 180～ 220g, adaptively fed for one week, randomly divided into four groups including normal control group, high methionine diet group, folic acid combined with VitB12 treatment group and rosuvastatin treatment group.The control group were fed with normal food; those of high methionine diet group were raised with normal food and 1.7% methionine; those in rosuvastatin treatment group were raised with high methionine diet for four weeks. Then rosuvastatin was administrated intragastricly daily at 5 mg/kg, at the same time high methionine group diet was provided. The rats of folic acid combined with VitB12 treatment group were fed with the same food as that of high methionine diet group for four weeks, then folic acid combined with VitB12 were intragastricly administrated everyday at 3 mg/kg and 15ug/kg respectively, at the same time high methionine group diet was given.Twelve weeks later, the serum triglyceride and cholesterol concentrations were detected with enzyme method (Step to the end of colorimetric method). The Hcy concentrations in the plasma of rats were determined with high performance liquid chromatography. The pathological morphology of thoracic aortas stained with hemotoxylin and eosin (HE) and oil red "O" were examined with microscopy. And the ultra structures of the thoracic aortas were detected with transmission electron microscope. The expressions of CCL5 and CCR5 proteins in aortic smooth muscle cells were determined with immunohistochemistry. The mRNA expressions of CCL5, CCR5 and nuclear factor-kappaB (NF-κB) were detected with Reverse Transcription-Polymerase Chain Reaction (RT-PCR).Results1. Concentrations of lipid in the serum There is no significant difference of total cholesterol and triglyceride concentrations in the serum of rats between different groups (P>0.05).2. Concentrations of homocysteine in the plasma Hcy concentrations in the plasma of group with high methionine diet (80.79±23.86mmol/L) were significantly higher than those in the normal control group (9.47±4.55 mmol/L, P<0.05) and treatment groups (53.97±12.24 mmol/L,37.88±4.84mmol/L, P<0.05). Between the two treatment groups, Hcy concentrations in the folic acid combined with VitB12 group (37.88±4.84mmol/L) were lower than those in the groups treated with rosuvastatin significantly (53.97±12.24 mmol/L, P<0.05).3. Pathological changes of the thoracic aorta in rats ①HE staning observation (light microscope):The endothelial cells of thoracic aorta in the control group were lined up in order, the internal elastic membranes were integral, and smooth muscle cells in the medial membrane were lined up normally. In high methionine diet group, the endothelial cells were necrotic and thrown off, and endomembrane was injuried partly. Some medial smooth muscle cells were loosen and disordered. Foam cells could be seen under endothelium. The alterations of endothelial cells and smooth muscle cells in folic acid combined with VitB12 and rosuvastatin treatment group were between those of the control group and the high methionine diet group.②Oil red "O" staning observation (light microscope):The thoracic aorta of high methionine diet group were stained with Oil Red "O" positively, the color was red, mainly in the endothelium, but those of the other three groups were stained negatively.③ Ultrastructure observation (electron microscopy):The endomembranes of thoracic aorta were injuried partly in high methionine diet group, and increasing bubbles were seen in the plasma of the endothelial cells. The smooth muscle cells were lined up in disorder, and the mitochondria were severely swollen. Many medial smooth muscle cells were more visible from contraction type to synthesis type. There were no alterations of endothelial cells and smooth muscle cells mentioned above in the control group. The alterations of endothelial and smooth muscle cell in folic acid combined with VitB12 and rosuvastatin treatment group were between the control group and the high methionine diet group.4. Immunohistochemistry The CCL5 and CCR5 proteins were stained as uneven brown granules and located in the cytoplasm. The expressions of CCL5 protein in the high methionine diet (139.50±15.40) were significantly higher than those of the normal control group (112.25±10.75, P<0.05) and treatment group (114.05±10.90,125.89±11.89, P<0.05). The expressions of CCL5 protein in the folic acid combined with VitB12 treatment group (125.89±11.89) were significantly higher than those in the rosuvastatin treatment group (114.05±10.90, P<0.05). The expressions of CCR5 protein in the high methionine diet group (139.12±7.71) were much significantly than those in the normal control group (125.41±9.93, P<0.05) and two treatment groups (121.11±9.15,130.59±8.29, P<0.05). The expressions of CCR5 protein in the folic acid combined with VitB12 treatment group (130.59±8.29) were significantly higher than that of the rosuvastatin treatment group (121.11±9.15, P<0.05).5. RT-PCR The expressions of CCL5 mRNA in the high methionine diet group (0.93±0.02) were significantly higher than those in the normal control group (0.45±0.00, P<0.05) and treatment group (0.69±0.01,0.87±0.00, P<0.05). The expressions of CCL5 mRNA in the folic acid combined with VitB12 treatment group(0.87±0.00) were significantly higher than those in the rosuvastatin treatment group(0.69±0.01, P<0.05). The expressions of CCR5 mRNA in the high methionine diet group (1.97±0.36) were significantly higher than those in the normal control group(1.02±0.04, P<0.05) and treatment group (1.12±0.23,1.53±0.41, P<0.05). The expressions of CCR5 mRNA in the folic acid combined with VitB12 treatment group(1.53±0.41) were significantly higher than those in the rosuvastatin treatment group (1.12±0.23, P<0.05). The expressions of NF-κB P56 in the high methionine diet group (2.16±0.43) were significantly higher than those in the normal control group(1.35±0.06, P<0.05) and treatment group (1.39±0.18, 1.73±0.26, P<0.05). The expressions of NF-κB P56 in the folic acid combined with VitB12 treatment group(1.73±0.26) were significantly higher than those in the rosuvastatin treatment group (1.39±0.18, P<0.05).Conclusions1. HHcy can induce high expressing levels of chemokine CCL5 and its receptor CCR5 through NF-κB pathway in arterial smooth muscle cells, and then promote the occurrence and development of atherogenesis.2. Both rosuvastatin and folic acid combined with VitB12 both can reduce the lesions of atherosclerosis induced by HHcy. But in the terms of chemokine inhibition, rosuvastatin is better than folic acid combined with VitB12.