| ObjectiveTo study the correlation of Coronin-1 and mycolic acid(MA)-induced foam cell formation of macrophages and to explore its possible mechanism.Methods1. According to the difference of Coronin-1 expression level, the experiment was divided into 3 groups: RAW264.7-Cor.Plus, RAW264.7and RAW264.7-Cor.Minus group. The 3 groups cells were co-cultured with microspheres coated with 0, 25, 50, 75 and 100 μg/mL MA for 24, 48,72 h, 5 and 8d, then the cell morphology of each group was observed under microscope after Oil Red O staining, and the level of total cholesterol(TC)and free cholesterol(FC) was tested by TC enzyme kit and FC enzyme kit respectively; then the cholesterol ester(CE) and the CE/TC ratio was used to quantitatively evaluate the level of foam cell formation.2. After the polystyrene microspheres coated with 100μg/mL MA being swallowed by 3 groups cells for 24 hours respectively, the level of Coronin-1 in each group was detected by RT-PCR and Western-blotting.3. After treated with 2 μmol/L cytochalasin D(ctyD), the 3 groups cells were co-cultured with microspheres coated with 0, 25, 50, 75 and 100μg/mL MA for 24, 48, 72 h, 5 and 8d respectively, then their level of foam cell formation were quantitatively evaluated again.4. The ctyD treated cells(RAW264.7-ctyD and RAW264.7-ctyD-MA)and their control groups were stained with FITC-phalloidin, then the percentage of F-actin rearrangement was calculated.5. After the 3 groups cells treated with 0, 25, 50, 75 and 100μg/mL MA for 24, 48, 72 h, 5 and 8d respectively, the calcineurin(CaN) activity was detected by enzyme reaction phosphorus measurement, and the protein level of Ca N of the groups treated with 100μg/mL MA for 24 h was detected by Western-blot.Results1. The level of foam cell formation of macrophages in each group with different Coronin-1 level was proportional to the MA coating concentration and its phagocytic time. The highest coronin-1 expression group(RAW264.7-Cor.Plus) had the highest foam cell formation level, and the lowest coronin-1 expression group(RAW264.7-Cor.Minus) had the lowest foam cell formation level, and there was significant difference between 3groups(RAW264.7-Cor.Plus>RAW264.7>RAW264.7-Cor. Minus).2. After MA induction, the coronin-1 level was significantly higher than that of the corresponding control group, and there was also significantdifference between 3 groups, with the trend: RAW264.7-Cor.Plus >RAW264.7 > RAW264.7-Cor.Minus.3. Compared with the untreated group, the level of foam cell formation of ctyD treatment group was significantly lower, but its trend is still as RAW264.7-Cor.Plus>RAW264.7>RAW264.7-Cor.Minus. The inhibition of F-actin by CtyD significantly decreased the foam cell formation induced by MA, but the inhibition of F-actin had no significant impact on the positive correlation between Coronin-1 and the foam cell formation induced by MA.4. After phalloidin staining, the F-actin rearrangement of MA treated cells(RAW264.7-ctyD and RAW264.7-ctyD-MA) was significantly higher than that of non-MA treated control cells.5. Under same MA coating concentration and same effect time, the activity of Ca N of the MA treated cells keeps the trend as RAW264.7-Cor.Plus>RAW264.7>RAW264.7-Cor.Minus, but within 5~8d phagocytic time, the Ca N activity of 3 groups cells was stable at a relatively low level,which has no significant difference between 2 groups, and the western-blot results of 3 groups treated with MA for 24 h was consistent with the Ca N activity.Conclusion1. The MA could induce the expression of coronin-1 of macrophage.2. The Coronin-1 level was positively correlated with the foam cellformation induced by MA.3. The F-actin was involved in the process of lipid accumulation in MA treated macrophages, but the F-actin was not the key or the only way in coronin-1 regulating the foam cell formation induced by MA, which may be related to calcineurin and so on. |
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