Effects Of Genistein On Hyperplastic Scar Fibroblasts And Study Of Cell Signal Mechanisms

The nature of hyperplatic scar(HS) is abnormal proliferation of fibroblasts and excessive deposition of extracellular matrix, which can cause severe damage on the patients'life and health not only in appearance but functions. Up to now,there has no ideal and satisfactory method to treat it in spite of lots of researches on either mechanism or therapy. Genistein, as a generally known phytoestrogen, has multiple biological activities. It is a high-performance and specific inhibitor to tyrosine protein kinase(TPK), and has been proved to inhibit many kinds of tumors and some fibrotic diseases with diversified experiments or epidemiology. It has been used in clinic to treat cancers with satisfactory results. Genistein may have the analogous effects on the prevention or therapy for hyperplastic scar.ObjectiveThe study is to observe the effects on the proliferation and function of HS fibroblasts by Genistein and to explore the cell singnal transduction mechanism initially. Under the cultured human HS fibroblasts model in vitro, the effects of Gensitein on cells including proliferation, apoptosis and some biological functions were detected, as well as the alteration of correlative proteins'expression in TPK singnal pathway. The results is to verify the biological effects of Genistein on HS and illuminate the possible signal transduction mechanisms, both of which can provide ideal experimental foundations for the therapy of HS with Genistein.Methods1. HS fibroblasts were isolated and cultured from hyperplastic scars, then different concentration of Genistein were added to them. The cells'growth and proliferation were observed directly and detected with MTT assay. The PCNA expression was measured with immunocytochemistry and the cell cycle were detected with FCM. The cells'apoptosis was measured by TUNEL with the apoptosis correlated proteins Bcl-2,Bax,Caspase-3 detected by Western Blot.2. The synthesis of collagen was measured with H³-proline incorporation and their mRNAs'transcriptin were semi-quantitated with RT-PCR. The expression of TGF-β1 was analyzed by Western Blot. The concentration of Ca²⁺ was observed under Laser Scanning Confocal Microscope.3. The activity of TPK was measured with[γ-³²p]ATP substrate incorporation. The phosphorylation level of some important signal molecules in two main TPK signal pathway Ras-MAPK and PI3K-Akt (including c-Raf,MEK1/2,ERK1/2,p38,JNK,Akt) were tested to evaluate the effects of Genistein on the activation of signal transduction by Western Blot.Results1. The cells'growth was inhibited by Genistein with an appearance of decreased cell population and deformed shape. The growth curve was declined following the drug dose and cultured time. Under the influence of Genistein, the cell lost characteriastic of fibroblasts with apophysis disappeared, arrangement disordered and inter-touch decreased.2. As showed by MTT assay, Genistein can inhibit the proliferation of HS fibroblasts both dose-dependently and time-dependently. Compared with control group, the l00μmol/L group has the most significant inhibitory effect.3. The PCNA expression in experimental groups was inhibited by Genistein.4. After con-cultured with Genistein, the cell cycle analysis showed the amount of cells in G0-G1 phase decreased whereas those of G2-M phase increased. The number of S phase cells rose in l00μmol/L group.5. The apoptosis of HS fibroblasts increased with a higher expression of Bax,Caspase-3 protein and a lower expression of Bcl-2 protein under Genistein's action.6. With the disposal of Genistein, the synthesis of collagen was partially blocked. The transcriptions ofâ… orâ…¢precollagen mRNA were reduced either.7. The expression of TGF-β1 protein was down-regulated by Genistein.8. The concentration of Ca²⁺ in HS fibroblasts lessened when mixed with Genistein while increased with bFGF. Genistein could inhibit the positive effect of bFGF on the Ca²⁺ in HS fibroblasts.9. Genistein reduced the activity of TPK in HS fibroblasts and inhibited the positive effect of bFGF on TPK's activity in HS fibroblasts.10. Treated with Genistein 30 miniters later, the c-Raf,MEK1/2,ERK1/2,p38,Akt phosphorylation protein were reduced with an obviously alteration on c-Raf,ERK1/2,Akt. There has no effect on JNK's phosphorylation by Genistein. It showed the same tendency in Genistein's effects on these signal proteins when added with bFGF.Conclusions1. Genistein can inhibit the proliferation of HS fibroblasts and induce apoptosis. The mechanism may be the cell cycle block and the regulation of apotosis correlative protein expression.2. Genistein can affect some HS fibroblasts'important biological functions including collagen synthesis, secretion of TGF-β1 and the concentration of Ca²⁺.3. Genistein can inhibit the tyrosine protein kinase singnal pathway, most likely the Ras→Raf→MEK→ERK/p38 and PI3K→Akt signal transduction pathway to affect HS fibroblasts.