The Effects Of Na₂SiO₃ On Inflammatory Reaction Of ECV304 Cell Line-Caused By Oxidized Low Density Lipoprotein

Objective:Animal experiment studyies had confirmed that Na₂SiO₃ could inhibit the formation and developement of AS on hyperlipoidema of animal through many component elements, which hints that Na₂SiO₃ is worth of being studied in anti-atherosclerosis. The purpose of this experiment is to observe the effect of Na₂SiO₃ on inflammatory reaction of the ECV304 cells damaged by oxidized low density lipoprotein (ox-LDL), and to elucidate the possible mechanism.Methods:1 Isolation, oxidation and identification of low-density lipoproteinThe chemical precipitation was used to extract low-density lipoprotein (LDL). LDL was oxidized in 37℃under 10μmol / L CuSO₄ for 24 h, then was dialyzed in the PH7.4 PBS for 24 h, in which bacterium was removed, and obtained ox-LDL and preservated at 4℃. Using agarose gel electrophoresis to identify and UV spectrophotometry to monitor the OD and the level of oxidation. Determined its concentration used by Coomassie brilliant blue law.2 Culture ECV304 cellsRoutine method cultured the ECV304 cells and divided five groups: control group, injury group, Na₂SiO₃ (Si 68.5 mg/L) high dosage group, Na₂SiO₃ (Si 34.2 mg/L) middle dosage group, Na₂SiO₃ (Si 17.1 mg/L) low dosage group. Observed the condition of each group cells.3 Detect the targetsThe levels of NO in the culture fluid and the activity of NOS were measured sepretallly by nitrate reductase and chemical colorimetry. The levels of IL-8 was measured by radio immunoassay. The expression of VCAM-1 protein was detected by flow cytometry. After extacting the totle RNA of cells, the expression of iNOS, IL-8, VCAM-1, LOX-1, NOX4 mRNA were detected by semi-quantitation RT-PCR. The expression of iNOS protein was detected by Western blot and the numbers of mononclercell were detected.Results:1 Extraction and oxidization of LDLThe agarose gel electrophoresis result of human blood serum, LDL, ox-LDL displayed that human blood serum and LDL were in the same line and was a single strap respectively; the agarose gel electrophoresis result of ox-LDL was also a single strap and the speed of ox-LDL was almost twice times than LDL, which showed that the isolation and oxidation of Low-density lipoprotein had succeed. The curve plot of oxidization of LDL showed that LDL was completely oxidized around 10 hours.2 The levels of NO in the culture fluidThe level of NO in injury group was lower than that in control group (P<0.01). the level of NO in Na₂SiO₃ groups were significantly increased compared with that in injury group (P<0.01).3 The activity of NOS,iNOS and the expression of iNOS protein in ECV304 cellsThe activity of NOS in injury group was lower while the activity of iNOS and the expression of iNOS protein were higher than that in control group (P<0.01). The activity of NOS in Na₂SiO₃ groups were increased while iNOS were decreased compared with that in injury group (P<0.01). The expression of iNOS protein in Na₂SiO₃ group was decreased compared with that in injury group (P<0.01).4 The expression of iNOS mRNA in ECV304 cellsThe expression of iNOS mRNA in injury group was higher than that in control group (P<0.01). And it was decreased evidently in 34.2μg/ml Na₂SiO₃ group (P<0.01).5 The adhesional numbers of mononudearcellThe adhesional numbers of mononudearcell in injury group were high compared with that in control group (P<0.01). The adhesional numbers of mononudearcell in Na₂SiO₃ groups were decreased than that in injury group (P<0.01).6 The levels of IL-8 in the culture fluid The level of IL-8 in injury group was higher than that in control group (P<0.01). the level of IL-8 in Na₂SiO₃ groups was decreased compared with those in injury group (P<0.01).7 The expression of VCAM-1 protein in ECV304 cellsThe expression of VCAM-1 protein in injury group was higher than that in control group (P<0.05). And it was decreased evidently in 34.2μg/ml Na₂SiO₃ group compared with that in injury group (P<0.05).8 The expression of IL-8 mRNA in ECV304 cellsThe expression of IL-8 mRNA in injury group was higher than that in control group (P<0.01). And it was decreased evidently in Na₂SiO₃ (Si 34.2 mg/L) middle dosage group (P<0.01).9 The expression of VCAM-1 mRNA in ECV304 cellsThe expression of VCAM-1 mRNA in injury group was higher than that in control group (P<0.01). The expression of VCAM-1 mRNA in Na₂SiO₃ groups was decreased compared with those in injury group (P<0.01).10 The expression of LOX-1 mRNA in ECV304 cellsThe expression of LOX-1 mRNA in injury group was higher than that in control group (P<0.01). The expression of LOX-1 mRNA in Na₂SiO₃ groups was decreased compared with that in injury group (P<0.01).11 The expression of NOX4 mRNA in ECV304 cellsThe expression of NOX4 mRNA in injury group was higher than that in control group (P<0.01). And it was decreased evidently in Na₂SiO₃ middle dosage group (P<0.01). Conclusion:Na₂SiO₃ has the effection of inhibiting the ininjury of ECV304 cells caused by ox-LDL, and also has the effection of anti-oxidative damage and relieving inflammatory reaction by decreasing the gene expression of IL-8, VCAM-1, iNOS and LOX-1, NOX4. This may be one of mechanism of Na₂SiO₃ anti-atherosclerosis.