Studies Of The Effect And Mechanism Of Pyrodoxine Against Atherosclerosis

Background Atherosclerosis is a slow pathological process of major vascular remodeling, including endothelial cells damage, the invading of smooth muscle cells in vascular intima and vascular smooth muscle cells proliferation, which is main reason of human death around world. There are lots of pathogenic factors contribute to atherosclerosis development, such as inflammation, endothelial dysfunction, oxidative stress, etc.In atherosclerosis progression, oxidized low density lipoprotein (ox-LDL) is being increasingly focused due to its special and complicated function during lipid metabolism in arterial intima. ox-LDL is multimolecular structural complex composed by hydrophilic surface and hydrophobic core, and it comes from low density lipoprotein (LDL) which accumulated in subendothelium and oxidized by free radicals. The accumulation of uptake ox-LDL in macrophage may lead to transdifferentiation to foam cells.ox-LDL receptors on cell membrane play an important role in this macrophage phagocytic progression. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is one of key receptors for ox-LDL uptake. Besides, other scavenger receptors like scavenger receptors type 1 (SR-A1), cluster of differentiation 36 (CD36) also participate in this progression.Pyridoxine is main component of vitamin B6, as coenzyme of homocysteine (hcy), can lower blood hcy to anti-AS. Some studies claimed pyridoxine probably could protect endothelium in AS progression, but they know little about its mechanism-it may be concerned with ox-LDL receptors such as CD36. In recent years, our lab did lots of work in pursuing this mechanism. The results reveals in HUVEC cells induced by ox-LDL, pyridoxine could effectively ameliorate the ox-LDL caused endothelium impairment by increasing endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) level. Nevertheless, whether pyridoxine is effective to foam cell formation is still unknown.This research is aimed to explore whether pyridoxine could reduce macrophage uptake ox-LDL and ameliorate foam cells development. Furthermore, we will study its detailed mechanism.Materials and Methods Cell model construction Incubated RAW264.7 cell till fill 60-70% of six well plate, divide into 4 groups randomly:18 h control group,18 h ox-LDL group,24 h control group,24 h ox-LDL group, co-incubated with 50mg/L ox-LDL every 6 h. After incubation for 18 h and 24 h, used oil-red O staining to detect intracellular cholesterol ester, DHE to detect oxidative stress and Western Blot for LOX-1 protein level. Pyridoxine mechanism Incubated RAW264.7 as above, divide into 5 groups randomly:control group, ox-LDL group,10 umol/L pyridoxine+ ox-LDL group、1 umol/L pyridoxine+ox-LDL group、100 nmol/L pyridoxine+ ox-LDL group. Added pyridoxine 15min before ox-LDL. After 24 h incubation, used oil-red O staining to detect intracellular cholesterol ester, DHE to detect oxidative stress, Real-time PCR to detect LOX-1, SR-A1, CD36 mRNA level and Western Blot for LOX-1 protein level.Results Cell model construction The results show that in oil-red O staining, RAW264.7 had more intracellular cholesterol ester in 24 h group compared to 18 h group, which reveals 24 h group has better transdifferentiation rate. Meanwhile, the DHE results show that in 24 h group, oxidative stress level is much higher than 18 h group, also Western Blot results performed same results that LOX-1 protein level is significant higher than control group (P<0.05), and 18 h group has no significance. Pyridoxine mechanism The results reveals in oil-red staining, all pyridoxine group could down regulated RAW264.7 cells uptake cholesterol ester, and this effect is concentration-dependent. Oxidative stress is also inhibited by pyridoxine with concentration-dependent. In mRNA level, LOX1 and CD36 up regulated significantly after adding ox-LDL (P<0.05), but SR-A1 has no change; only highest concentration (10 umol/L) could inhibitor LOX-1 mRNA expression (P<0.05), and even this concentration failed to inhibit CD36 mRNA expression. In protein level, all pyridoxine group could significantly down regulated LOX-1 protein compare to ox-LDL group (P<0.05), and 10 umol/L and 1 umol/L is more effective compare to 100 nmol/L group (P<0.05).Conclusion All results above indicate that pyridoxine could inhibit the oxidative stress, decrease vascular inflammation and inhibit lipid deposition induced by ox-LDL. Pyridoxine could improve endothelial function, therefore reduce the atherosclerotic plaque formation, slower atherosclerotic progression, which may provide a new target for pharmacy and clinical research.