The Antioxidation Effect Of Persimmon Tannin On Intracellular Low Density Lipoprotein Of Macrophages

Objective:This study is aimed to explore the antioxidation effect of persimmon tannin on intracellular LDL of Macrophages.Methods: 1.Inflammatory peritoneal macrophages were obtained by stimulating the mice with 3% fluid thioglycollate medium and lavaging its enterocoelia with serum free RPMI 1640 culture,and then were cultivated in RPMI l640 culture containing 10% fetal bovine serum.The cells were identified by cytomorphology observation,cell vitality determination using trypan blue staining and purity calculation using Wright's staining.2.Macrophages were divided into 6 groups.First culture the cells in the medium containing LDL for 24 h,then add Cu²⁺ and persimmon tannin with the final concentration of 10 ?g/m L,20?g/m L,40?g/m L respectively.The effect of Persimmon tannin on macrophage survival was detected by CCK-8 test;The level of MDA?T-SOD?GSH-PX was determinated by the corresponding kits;Ox-LDL was measured by using ELISA kit;lipid droplets distribution in macrophages was observed by oil red O staining;the level of cytosolic ROS was detected by using fluorescent probe DCFH-DA.3.RT-PCR technique was applied to detect the mRNA expression level of NADPH oxidase subunits p47 phox,p22phox,p40 phox,p67phox,gp91 phox.Results: 1.Morphology of cultured cells: after adherently culturing for 24 h,we observed that the cells had diverse shapes?circular,oval,spindle,irregular?,large volume,abundant cytoplasm,and visible hairy small bumps?pseudopodia?;Trypan blue staining showed that macrophage survival rate was up to 95%;The result of Wright's staining showed that the purity of macrophages is more than 95%.2.When compared with LDL group,the level of MDA and Ox-LDL in Cu²⁺ group significantly increased?P<0.05?,and the level of T-SOD and GSH-PX in Cu²⁺ group significantly decreased?P<0.05?;When compared with Cu²⁺ group,MDA and Ox-LDL levels decreased significantly?P < 0.05?in persimmon tannin intervention group,while T-SOD and GSH-PX increased with different degrees.The result of oil red O staining showed that macrophages had a bright glossy colour without lipid accumulation in normal group,and a little of lipid accumulation in LDL group;in Cu²⁺ group,a lot of lipid accumulated in the cells and even gathered into droplets;in persimmon tannin intervention group,lipid infiltration significantly reduced in the macrophage.The result of ROS determination by DCFH-DA fluorescent probe showed that the level of the Cu²⁺ group significantly increased?P < 0.01?compared with the LDL group,the ROS level in intervention group significantly reduced?P < 0.01?compared with the LDL group.3.After oxidative modification of LDL by Cu²⁺,the mRNA expression of NOXs' subunits p22 phox,p47phox,p67 phox,gp91phox and p40 phox increased to different extent,and the intervention of persimmon tannin reduced the mRNA expression of p67 phox,gp91phox and p40phox?P < 0.05?.Moreover,gp91 phox,p47phox mRNA expression had a very significant?P < 0.01?reduction.Conclusions:Persimmon tannin has antioxidation effect on intracellular LDL in macrophages.It reduces the production of ROS which induced by NOXs,and inhibits the oxidative modification of LDL,thus reducing the degree of foaming in macrophages.