Effect Of Na₂SiO₃ On ROS Production By Peripheral White Blood Cells Of The Rabbits With Experimental Hyperlipemia

Objective: Animal experiment studies had confirmed that Na₂SiO₃ could inhibit the formation and developement of atherosclerosis (AS). The purpose of this paper is to observe the effect of Na₂SiO₃ on ROS produced by the peripheral white blood cells under the stimulating of oxidized low density lipoprotein (ox-LDL) and hyperlipemia, and to provide further information for the anti-AS mechanism.Methods:The First part1 Isolation identification and oxidation of low-density lipoproteinThe chemical precipitation was used to extract low-density lipoprotein. Determine its concentration using Coomassie brilliant blue law. LDL was puted in the solution containing CuSO₄ on the concentration of 10μmol/L at 37℃, avoding from the light and oxidized for 24 h, the PH7.4 PBS was used to dialysis it for 24 h, then filtrated, and finally got and preservated it at 4℃.2 Isolation of peripheral white blood cellsThe peripheral blood was extracted from rabbit, added to the solution containing 3.8% natrium citricum prepared before, and then the anticoagulated blood was got. The same volume of Hank's solution was added and mixed for using. Lymphocyte isolation solution with the capacity of two times volume of the blood was added into another glass tube. 2 ml of lympholyte isolation solution was added to the centrifuge tube. Using a pipette, 2ml of the cell suspension was carefully layered above the lympholyte isolation solution at the interface as littlly mixing as possible. Since lympholyte isolation solution was of greater density than the cell suspension, a distinct interface would be formed, centrifuged for 20 minites at 2000rpm at room temperature. After centrifugation, there would be a well-defined lymphocyte layer at the interface. The white blood cells were removed and transfered from the interface to a new centrifuge tube carefully, then diluted with Hank's and centrifuged at 2000rpm for 15 minites to pellet the white blood cells, the supernatant was discarded, the white blood cells washed 2-3times with the Hank's before further processing. Then cells were randomly divided into normal control group, ox-LDL group, 68.5mg group, 34.2mg group, 17.1mg group, 8.5mg group.The Second part1 Animal groupThe male New Zealand White rabbits were divided into normal, model and Na₂SiO₃ groups.2 Detected of the targetsROS production was measured by DCFH-DA probe. Morphological changes of cells were observed under the electron microscopes. The expressions of CD36, TLR4, TLR2, gp91phox mRNA by white blood cells were detected by semi-quantitative RT-PCR.Results:The First part1 Electrophoresis results of rabbit blood serum, LDL, ox-LDL The agarose gel electrophoresis results of rabbit blood serum, LDL, ox-LDL showed that rabbit blood serum and LDL were at the same location and displayed a single strap each of them; the agarose gel electrophoresis result of ox-LDL was also a single strap and the speed of ox-LDLwas hardly more twice times than LDL. And all above showed that the isolation and oxidation of Low-density lipoprotein were succeeded.2 The effect of Na₂SiO₃ on the production of ROS by peripheral white blood cells stimulated by ox-LDLThe level of ROS in ox-LDL group was significantly increased compared with the control group (P <0.01). The level of ROS in 8.5mg group was decreased compared with the ox-LDL group (P <0.01) The Second part 1 The effect of Na₂SiO₃ on the level of ROS produced by the peripheral white blood cells of the rabbits with experimental hyperlipemiaThe production of ROS by white blood cells in model group was significantly higher than that of the control group (P <0.01) and Na₂SiO₃ decreased the production of ROS (P <0.01).2 Electron microscope observationIn model group the cell surface was covered lipid, shrinkaged and deformated. Cell surface structure was unclear, cell adhesion was enhanced. The state of cells in Na₂SiO₃ group had been markedly improved observed by the electron microscopy.3 The effect of Na₂SiO₃ on expression of CD36 mRNA by white blood cellsThe expression of CD36 mRNA in model group was higher than that in control group (P <0.01). And it was decreased evidently in Na₂SiO₃ group (P <0.01).4 The effects of Na₂SiO₃ on expression of TLR4 mRNA by white blood cellsThe expression of TLR4 mRNA in model group was higher than that in control group (P <0.01). And it was decreased evidently in Na₂SiO₃ group (P <0.01).5 The effects of Na₂SiO₃ on expression of TLR2 mRNA by white blood cellsThe expression of TLR2 mRNA in model group was higher than that in control group (P <0.01). And it was decreased evidently in Na₂SiO₃ group (P <0.01).6 The effects of Na₂SiO₃ on expression of gp91^phox mRNA by white blood cellsThe expression of gp91^phox mRNA in model group was higher than that in control group (P <0.01). And it was decreased evidently in Na₂SiO₃ group (P <0.01). Conclusion:Na₂SiO₃ inhibits the production of ROS by white blood cells stimulated by ox-LDL; Na₂SiO₃ decreases the expressions of CD36, TLR4, TLR2, gp91phox mRNA and inhibits the priming and activation of white blood cells in hyperlipemia, the effect of Na₂SiO₃ is not achieved by lowing blood lipid.