The Effects Of Tranilast On Biological Active Factors Production And Type Ⅰ/Ⅲ Procollagen MRNA Expression Of Keloid Fibroblasts

Keloids are considered to be the result of abnormal wound healing. They are characterized by the proliferation of dermal fibroblasts and excessive accumulation of extracellular matrix including collagen. Despite of the various incidences in the races, Asian individuals are more likely to develop keloids. The pathogenetic mechanisms that cause keloids remain unknown, and current therapeutic approaches are difficult to resolve the problem of recurrence. In this study, the production of some major biological active factors and the synthesis of type â…  / â…¢ procollagen mRNA were compared between normal and keloid fibroblasts. Furthermore, we investigated the effects of tranilast with various concentrations on fibroblasts proliferation, biological active factors production and type â…  / â…¢ procollagen mRNA expression. The possible involved mechanisms of tranilast were discussed.Firstly, primary fibroblasts of normal skin and keloid were established from the specimens by standard tissue culture techniques. Both were cultured in serum-free media, and the growth curves of them demonstrated exponential growth. The population double time of keloid fibroblasts (42 hours) and normal fibroblasts (57.6 hours) had significant difference.Secondly, normal and keloid fibroblasts were propagated in a serum-free in vitro model, then exposed to tranilast with various concentrations for 24, 72, 96 hours. Levels of TGF-β₁, bFGF and IL-6 in the supernatants were determined by double antibody sandwich ABC-ELISA method. Result: (1) keloid fibroblasts secreted more TGF-β₁ than normal fibroblasts, and tranilast could inhibit the expression. Compared with the control group, keloid fibroblasts treated with 25μg/ml tranilast at 24, 96 hours and 50, 250μg/ml tranilast at all time points had statistical significance in reducing the production of TGF-β₁ (P<0.05). (2) only slightly elevated production of bFGF was seen in keloid fibroblasts, and tranilast could increase the expression, especially at 24,72hours. Compared with the control group, keloid fibroblasts treated with 50μg/ml tranilast at 24, 72 hours and 250μg/ml tranilast at all time points had statistical significance in increasing the production of bFGF (P<0.05). (3) keloid fibroblasts induced more IL-6 than normal fibroblasts at 72,96 hours, and tranilast could inhibit theexpression. Compared with the control group, keloid fibroblasts treated with 10,25,50μg/ml tranilast at 96 hours and 250μg/ml tranilast at 72,96 hours had statistical significance in reducing the production of IL-6 (P<0.05). In addition, Tranilast had the similar effects on normal fibroblasts. Concentration*time, Concentration*cell and time*cell demonstrated various interaction effects on TGF-β₁, bFGF and IL-6 expression by using multifactor analysis of variance (MNOVA).Finally, the effects of tranilast on fibroblasts proliferation and the expressions of type â…  and type â…¢ procollagen mRNA were studied. Tranilast had the similar effects on the morphology of normal and keloid fibroblasts. With the intervention of high concentration tanilast, fibroblasts with obvious vacuoles in the cytoplasm were found, which became more and more when the drug concentration increased or the action time prolonged. These fibroblasts grew slowly, eventually shrinked, fell off or became anoikis. In the condition of low concentration, vacuolization was not obvious, but with the action time prolonged, the intercellular space became wider and the profile of fibroblasts changed a little. MTT test demonstrated significant decreases of OD value in normal and keloid fibroblasts when added with various concentrations of tranilast. Compared with the control group, the difference had statistical significance (P<0.05). With the increase of tranilast concentration, OD value gradually decreased, which suggested the inhibition effects on fibroblasts growth.Consequently, the levels of mRNA for procollagen type â…  and type â…¢ were determined by RT-PCR. It was found that keloid fibroblasts expressed more type â… /â…¢ procollagen mRNA than normal fibroblasts. The levels of type I were about 1.28 fold increased while the levels of type â…¢ were about 1.54 fold. Tranilast could reduce the synthesis of procollagen â… /â…¢ mRNA in normal and keloid fibroblasts. The inhibition ability became more evident when the concentration was increased. Compared with the control group, 250μg/ml and 500μg/ml tranilast could low down the expression of procollagen â… /â…¢ mRNA in keloid fibroblasts, with statistic significance (P<0.05). It suggested that tranilast had the capability of inhibiting collegen production.In conclusion, keloid fibroblasts were different from normal ones in proliferation, biological active factors autocrine and type â…  / â…¢ procollagen mRNA expression. Certain concentration of tranilast could elevate the expression of bFGF, inhibit the expression of TGF-β₁ and IL-6, which demonstrated its anti-fibrotic capability. With the intervention of tranilast, the proliferation of fibroblasts was inhibited and theproduction of type â…  / â…¢ procollagen mRNA was also reduced. Tranilast had the potentials in the treatment of keloid, and more animal or clinical studies would be needed to evaluate its clinical effect, so as to develop the application in future.