Effect Of Ferulic Acid On Anti-oxidization And Apoptosis In Epidermal Melanocytes Based On Caspase-3、Bcl-2、Bax

ObjectiveThis project intends to oxygen free radical regulation of melanogenesis as a starting point,studying the regulation of ferulic acid on the normal human epidermal melanocytes as a whole through the experiment in vitro.Further studies were carried out on the influence that ferulic acid has on antioxidant effect and apoptosis of the normal human epidermal melanocytes at the cellular level.The purpose is to clarify and reveal parts of melasma of effect that ferulic acidin has on the treatment of Dermatosis of Dyspigmentation, providing experimental data for traditional Chinese medicine to be used in the treatment of Dermatosis of Dyspigmentation and a new approach for the clinical treatment of Dermatosis of Dyspigmentation.Methods1. Subculture and identification of melanocytes The introduction of the melanocyte cell lines subculture, identified by DOPA staining cells were cultured normal human epidermal melanocytes. 2. Determination of the ROS, SOD, MDA levels in melanocytes Reactive ROS levels in cultured melanocytes were evaluated by lasers canning confocal microscope after using different concentrations of ferulic acid.SOD activities was measured by WST-1 method and MDA levels was measured by TBS method. 3. Determination of caspase-3 protein、Bcl-2 protein、Bax protein expression levels Using Western blot to analysis the effect of different concentrations of ferulic acid on the expression levels of Caspase-3 protein 、 Bcl-2 protein 、 Bax protein in cultured melanocytes. 4. Use SPSS17.0 to perform Statistical analysis, representing with the mean ± standard deviation(),using One-Way ANOVA analysis between groups, pairwise comparison method when using SNK. It is a statistically significant difference that P <0.05.Results1. Normal human epidermal melanocytes after L-DOPA staining was positive, showing thecytoplasm and dendrites of each cell tan or black. 2. The number of cells containing blue-green fluorescence in the control group is largest. And it was decreasing with the concentration of Ferulic acid in the experimental group, reflecting the level of ROS decreased with the concentration of Ferulic acid. 3. Compared with the control group, SOD activity in the experimental group was significantly increased(p <0.05); whick MDA activity was significantly decreased(p <0.05). 4. Expression levels of caspase-3 protein in control group were higher than those in experimental groups(p<0.05).With the concentration of ferulic acid increases,the expression levels of caspase-3 protein decreased gradually,which difference had statistically significance(P <0.05). 5. Expression levels of Bcl-2 protein in control group were lower than those in experimental groups(p<0.05).With the concentration of ferulic acid increases,the expression levels of caspase-3 protein increased gradually,which difference had statistically significance(P <0.05). 6. Expression levels of Bax protein in control group were higher than those in experimental groups(p<0.05).With the concentration of ferulic acid increases,the expression levels of Bax protein decreased gradually,which difference had statistically significance(P <0.05).Conclusions1. Different concentrations of ferulic acid can reduce the ROS levels in melanocytes at different extents. 2. Ferulic acid may reduce ROS levels within the normal humanmelanocytes,therefore reduce the production of MDA by increasing the activity of SOD. 3. Ferulic acid increses expression levels of Bcl-2 protein within melanocytes and decreases the expression levels of caspase-3 protein and Bax protein. Thus inhibiting apoptosis of normal human epidermal melanocytes. 4. Ferulic acid can reduce ROS levels within melanocytes and inhibit the apoptosis of melanin. It did not rule out ferulic acid anti- apoptotic mechanisms of melanocyte cells may be related to its antioxidant effect.