Inhibitory Effect Of β-sitosterol In Vitro Cultured Human Keloid Fibroblasts

Background:Research shows that, β-sitosterol is able to inhibit the proliferation of breast cancer cells, colon cancer cells, oral cancer and esophageal cancer cells;β-sitosterol also can enhance the activity of Caspase-3, Caspase-8, caspase-9 by exogenous and endogenous pathways to induce cancer cell apoptosis. Resent study shows that the aqueous extract from deied powdered rhizomes of Typhonium giganteum has an inhibitory effect on keloid fibroblasts proliferation,after 24 hours,it can significantly promote the apoptosis rate at 72%.However,the components of Typhonium rhizome is complex, the water extract is also a mixture, and its exact mechanism of action is not clear. Typhonium Giganteum stem block contains β-sitosterol and β-sitosterol-D- glucoside. Therefore,β-sitosterol may affect the biological characteristics of keloid fibroblasts. In oder to understand the mechanism of β-sitosterol that whether it promoted cell apoptosis or inhibitory proliferation, we cultured fibroblast keloid in vitro and took β-sitosterol as the study drug with different concentration, as a depth study of the scar treatment by Typhonium Giganteum.Objective:To study the influence of β-sitosterol on apoptosis proliferation and of KFB in vitro.Methods:1, we used ear keloids as our specimen, and all of the keloid were caused by the damagement of skin puncture for about 3 months. The patients had undergo no operation, without any drug injection and physical therapy. The tissue transported to thelaboratory under 4 degrees Celsius.And primary keloid fibroblasts cells were cultured by tissue culture method. 2,we used the the third generation as the selected cells, and count cells after digestion and centrifugation; collecting one million cells and observing cell nuclei, cytoplasm, Golgi apparatus and endoplasmic reticulum ultrastructure under transmission electron microscope after the dehydration of ethanol fixed, then identified the cells.3, We selected the the logarithmic growth phase cells and cultured them for 24 hours with medium incubated different concentration of β-sitosterol,20 uM,30uM,40 uM,50uM; In oder to determine the effects of β-sitosterol on cell viability for 24 hours,we used Cell Counting Kit8 cell proliferation assay, and detected the absorbance with a microplate assay at a wavelength of 450 nm after 4 hours. We did the experiments for three times, and get effective concentration gradient. 4, We selected cells in the third generation,then counted cells after digestion and centrifugation. The control group was treated with medium containing solvent and experimental group were treated with different concentrations of β-sitosterol, the final concentration was 30umol/L,40umol/L and 50umol/L.24 hours later,after incubation, centrifugation and resuspended, we joined500 ul Binding Bμffer liquid gently, then add 5ul FITC-Annexin V and 10 ul PI. Then detected the apoptosis rate in corresponding concentration.Results:β-sitosterol is able to inhibit the proliferating activity of KFB in a dose dependent manner during 24 hours,when the concentrations is up to 30 uM.However there is no influence when the concentrations under 20 uM and the culture medium containingβ-sitosterol could increase the apoptosis rate of the KFB compared with blank control group(p<0.05).The apoptosis rate is 8.5%,18.0% and 33.2%,according to the concentrations of 30 um,40um and 50 um.Conclusion:β-sitosterol could significantly inhibit the proliferating activity of KFB and can significantly induce apoptosis to KFB in 24 hours.