Effects Of Paeoniflorin On Proliferation And Biological Behavior Of Hypertrophic Scar Fibroblasts In Vitro

Background Hypertrophic scarring remains a major problem for patients who have suffered deep burns. The undesirable physical properties of hypertrophic tissue are associated with an abnormal proliferation of fibroblasts and overproduction of collagen and extracellular matrix(ECM). Transforming growth factor β1(TGF-β1) plays a central role in hypertrophic scar formation. TGF-β1, a multifunctional cytokine, has the capacity to regulation cell growth, differentiation, migration, apoptosis of fibroblasts through intracellular signal transduction pathways. In addition it is one of the main collagen-stimulating factors, especially type I in fibroblasts. It also inhibits different MMPs, which further promotes the accumulation of collagen fibers. Paeoniflorin, the major active ingredient of paeonia lactiflora pallas which is a kind of traditional Chinese herb, that inactivates the effect of TGF-β1 and therefore displays a beneficial effect of antifibrosis in various tissues, such as liver, kidney, lung. Hypertrophic scarring is a fibroproliferative disorder of the dermis that occurs following wounding. However, little is known about the paeoniflorin in hypertrophic scars.Objective To investigate the effects of paeoniflorin on biological behavior in hypertrophic scar fibroblasts.Methods(1) The fibroblast was established as a primary cell line from normal skin or hypertrophic scar tissue obtained from patients recovering from severe burns.(2) Hypertrophic scar fibroblasts and normal skin fibroblasts were cultured in the absence(0 μM) or presence of various concentrations of paeoniflorin(200, 400, 800 μM) for 24, 48 and 72 h, using MTS to monitor growth.(3) Hypertrophic scar fibroblasts and normal skin fibroblasts were treated with paeoniflorin(200, 400, 800 μM) or without paeoniflorin addition for 48 h, cell cycle and apoptosis were examined by flow-cytometric analysis.(4) Fibroblasts were cultured in growth medium containing 0, 200, 400, 800 μM of paeoniflorin, after a 48 h incubation, TGF-β1 and type I collagen levels in supernatants were measured by ELISA.(5) The expression of α-SMA protein were dectected by Western Blot after a 48 h incubation with various concentrations of paeoniflorin(0, 200, 400, 800 μM).Result(1)Hypertrophic scar fibroblasts and normal skin fibroblasts proliferation was significantly inhibited by paeoniflorin at concentrations of 400 and 800 μM, after 24, 48, 72 h incubation.(2) Hypertrophic scar fibroblasts and normal skin fibroblasts treated with 200, 400, 800 μM of paeoniflorin for 24 h were blocked mainly at the G2 phase of the cell cycle, little apoptosis in normal and hypertrophic scar fibroblasts treated with paeoniflorin.(3) The TGF-β1 concentration per cell was significantly lower in hypertrophic scar fibroblasts treated with 400 800 μM of paeoniflorin compared with these fibroblasts without paeoniflorin addition. There was no significant difference in TGF-β1 production per cell between normal skin fibroblasts without paeoniflorin and the fibroblasts treated with 200, 400, 800 μM of paeoniflorin.(4) The type I collagen concentration was significantly lower in normal or hypertrophic scar fibroblasts treated with 200, 400, 800 μM of paeoniflorin compared with corresponding fibroblasts without paeoniflorin addition.(5) α-SMA protein expression in hypertrophic scar and normal fibroblasts was significantly inhibited by paeoniflorin at all concentrations(200, 400, 800 μM).Conclusion These results suggest that paeoniflorin has a down-regulatory effect on cell proliferation, type I collagen synthesis,and α-SMA protein expression in hypertrophic scar fibroblasts and normal skin fibroblasts. Paeoniflorin can reduce TGF-β1 production of hyperplastic scar fibroblasts, but does not affect the normal skin fibroblasts. Improved understanding of such a regulatory mechanism may eventually be of therapeutic significance in the control of hypertrophic scarring.