Molecular Mechanisms Of Tetrandrine On The TGF-?-induced Smad Signal Transduction Pathway In Hypertrophic Scar Fibroblasts

Background:The scar is the natural outcome of wound healing, including inflammatory response, hyperblastosis and remodeling, which is based on a series of activities of inflammatory cells and repair cells. In the whole process of wound healing, a variety of cells and proteins and extrcellular matrix (ECM) were finely regulated by body in the wound microenviroment. Cell migration and proliferation, and synthesis and metabolism of ECM affect wound healing by a variety of cytokines in different phase. Regulated balance is broken, which could lead to scar hyperplasia and scar formation.Hypertropic scar is common complication of severe trauma, burns and surgical operation, resulting in dysfunction and deformity of corresponding parts for patients, which cause patients to a series of serious obstacles of function, appearance, and psychology. Although, with rapaid development of cytobiology and molecular biology, the study on scar was pushed deeply, the pathogenesis is not completely elucidated. So prevention and treatment of hypertrophic scars has become one of urgent and troublesome problems to be solved. Main treatments of hypertrophic scars in the present are surgery, machanical pressure, drugs, radiotherapy and laser, which are used with single or combination therapy. But the effect is still not ideal. It was belived that the characters of hypertrophic scar include abnormal proliferation of fibroblasts and abnormal deposition of collagen and extracellular matrix.It is believed that transforming growth factor ?1 (TGF-?1) plays a central role in wound healing and hypertrophic scar formation, both as a potent regulator of cellular proliferation and a modulator of the interaction of cells with ECM, although wound healing and scar formation is orchestrated by many growth factors and cytokines. Elevated levels of TGF-?1 were found in the serum of recovering burn patients and hypertrophic scar fibroblasts (HSFs) stain more intensely for this cytokine than do normal skin samples or mature scars. Abnormally elevated TGF-?1 level and altered intracellular signaling bear a close relation to fibroblasts remodelling and cell population, which result in abnormal accumulation of ECM, along with fibrosis and scar formation. Smad proteins as intracellular effectors of TGF-? signaling, which are the only downstream substrates of TGF-? receptors (T?R) known so far, are central to intracellular signaling of TGF-?1. Smads translocate into the nucleus after being activated by receptors to regulate transcription. Although this pathway is inherently simple, combinatorial interactions in the heteromeric receptor and Smad complexes, receptor-inter-acting and Smad-interacting proteins, and cooperation with sequence-specific transcription factors allow substantial versatility and diversification of TGF-? family responses. Therefore, it could be a key point to inhibit fibrosis wih the regulation of TGF?/Smads signal pathway to block TGF-?1 biological effects.Tetrandrine, a bisbenzylisoquinoline alkaloid, derived from S. tetrandra. Previous pharmacological and clinical studies have shown that tetrandrine is effective in anti-inflammatory, immune-modulation, reversion of cardiac and vascular remodeling, inhibition of pulmonary vessels and airway smooth muscle contraction, suppression of tumor proliferation and multi-drug resistance. Previous studies provided evidence that tetrandrine inhibits hepatic stellate cells of rats by up-regulation of Smad7 which in turn blocks TGF-?1 expression and its downstream signaling. Our previous studies have shown that tetrandrine inhibited proliferation of HSFs and synthesis of collagen significantly and decreased expression of DNA. It suggests that tetrandrine also possesses a potent protective effect against hypertrophic scarring formation. However, these studies are still limited to a preliminary observation. Pharmacological molecular mechanisms of tetrandrine exerting anti-hpertrophic scar from cellular signal transduction is still un-reported by now, and it is still unknown which target points are tetrandrine exerting anti-hpertrophic scar in molecular level, and how?To solve these problems, TGF-?/Smad signaling transduction pathway is applicated for the main line in whole study. We test effects of tetrandrine on TGF-?1-mediated cell signaling and analysis functional changes of HSFs affected by TGF-?/Smads signaling with RNAi interference technology, reverse transcription PCR (RT-PCR), Western blot, flow cytometry, immunocytochemistry and HSFs cultured in vitro, and gene chip technology is used to check different expressed genes of HSFs between before and after tetrandrine intervention to explore the relation between the Gene Expression Profiling and the changes of TGF-?/Smad signal transduction pathway and collagen metabolism after tetrandrine intervention, and other signal crosstalk. Those studies are attempted to deeply elucidate the molecular mechanisms of tetrandrine anti-scar formation, to provide new ideas for prevention and treatment of hypertrophic scars and provide a theoretical basis and experimental basis for further clinical application.Objection:1. To investigate and establish an easy and short cycle culture method of hypertrophic scar fibroblasts in vitro, and to choose optimal concentration of tetrandrine for_follow-up experiments.2. To evaluate in vitro effects of tetrandrine on the TGF-?-induced smad signal transduction pathway and proliferation and collagen metabolism altered by tetrandrine in HSFs.3. To evaluate in vitro effects of tetrandrine on the expression changes of TGF-?1 and its downstream signal proteins-Smad proteins by tetrandrine in Smad7-siRNA HSFs.4. To find relations of between the Gene Expression Profiling and the changes of TGF-?/Smad signal transduction pathway and collagen metabolism after tetrandrine intervention and other signal crosstalk.Methods and materials1. Hypertrophic scar tissues was obtained from severe burn patients who were not received scar drug therapy in recently and were not associated with tumor or other serious diseases. HSFs were established as a primary cell line with the method which combined tissue explants adherent and enzyme digestion. HSFs were passaged and frozen. The morphological changes of fibroblasts were observed under inverted microscope, fibroblasts were identified with immunocytochemistry, and proliferation assay was performed with growth curve. MTS was used to choose optimal concentration of tetrandrine, and inhibition ration of HSFs was computed at different concentration of tetrandrine.2. HSFs were exposed to four different treatments (control, tetrandrine, TGF-?1 and mixture). After 48h culture, expression of TGF-?1, Smad2, Smad7, T?R ? and T?R ? were examined using reverse transcription PCR and Western blotting. To evaluate cellular function alteration morphological changes of HSFs were observed under an inverted microscope, expression of type ? and ? collagen were tested by immunocytochemistry, and cell cycles were analyzed by flow cytometry.3. The 4th-6th passaged HSFs were used to follow-up experiment. To choose suitable carrier for transfection, transfection efficiency was evaluated for HSFs with siRNA-NC-FAM. The specific siRNA was chosen in accordance with the AmbionSiRNA design principles and analysis of known gene sequence and secondary structure. Smad7-siRNA was builded with chemical synthesis, was tansfected to HSFs, and then total RNA was extracted to test the change of Smad7 expression, which was to confirm that target gene was silent. True nuclear expression vector lipofectamine TM2000 was used to transfect Smad7-siRNA into HSFs. Smad7 silented HSFs was were exposed to three different treatments (Control, Smad7-siRNA, Tetrandrine+Smad7-siRNA). After 48h cultured, Smad2, Smad7, T?R ?,T?R ? and TGF-?1 mRNA expressions were tested with RT-PCR. Ptoteins of TGF-?1 and Smad2 were tested with west-blot.4. The 4th-6th passaged HSFs were used to follow-up experiment. HSFs was treated with tetrandrine, total RNA was extracted and purified and denatured with formaldehyde, which was inspected with gel electrophoresis. The RNA was labeled with fluorescent, hybrided, and washed. Differentially expressed genes were checked with LuxScan 3.0 image analysis software after fluorescence signal scanned, and were classified with statistical analysis of the Go classification. The microarray results were verified with Real time PCR.Statistical analysisAll data were expressed as X ±SD Statistical significance for the difference between the groups was assessed using one-way ANOVA test. Values of P<0.05 were considered to indicate a significant difference.Results:1. HSFs were established as a primary cell line with the method which combined tissue explants adherent and enzyme digestion. For 48h culture, HSFs grew out scar specimens for 48h, grew to local fusion for 7days and grew to full bottom of flask about 14days. The vimentin protein expression was positive in fibroblasts cultured. Fibroblasts which were passed grew fusion for 5 days and cells stayed in the mitotic index between 1st and 5th days. MTS results showed that HSFs were significantly inhibited between 2.5?g/ml-12.5?g/ml of tetrandrine, and showed a dose-response relationship. Inhibition rate was 50.72% at 5?g/ml of tetrandrine.2. Expression of Smad7 increased while expression of Smad2 and TGF-?1 mRNA decreased in HSFs with tetrandrine. In addition, type ? and ? collagen was suppressed and S phase of the cell cycle markedly shortened in HSFs by tetrandrine. The number of HSFs was descreased, cells became round and small, and cellular synapse became short and disappears. In TGF-?1 group, expression of Smad7 decreased while expression of Smad2 and TGF-?1 mRNA increased, expression of type ? and ? collagen was increased, and S phase of the cell cycle markedly lengthened. Cellular number was increased, cells became bigger and cellular synapse became longer. The mixed results were similar to tetrandrine group. T?R ? and T?R ? expression was no significantly different in those groups.3. In results of transfection efficiency, Lipofectamin2000 was suitable for HSFs and that 0.5?l of Lipo reversed 10 pmol of siRNA-NC-the FAM can can achieve good results. Expression of type I collagen was increased after Smad7 of HSFs being silent, and expression of Smad7 mRNA was increased while expression of TGF-?1 mRNA was decreased and other gene expression was no significantly different in Smad7 silent HSFs with tetrandrine.4. Compared different genes in HSFs with tetrandrine with control, there is 202 different expressed genes,184 upregulated genes, and 18 down-regulated genes. According to the classification of Pathway,25 signal pathways related with TGF-? signal pathway could be classified, which involved in protein translation, energy synthesis and apoptpsis. Those included TGF-beta signal pathway, Toll-like receptor signaling pathway, PPAR signaling pathway, Wnt signaling pathway, MAPK signaling pathway, mTOR signaling pathway and p53 signaling pathway, et al. There were 31 upregulated genes (such as DCN) and 5 down-regulated genes (such as JUN). The result of Real time PCR showed that JUN expression was down-regulated in HSFs treated with tetrandrine, which was constant to gene chip.Conclusion1. The more stable fibroblasts were cultured more easily and quickly with the method combining the tissue explant adherent and trypsin digestion, and the optimal concentration of tetrandrine was chosen, which could provid experimental basis for mechanism and prevention of hypertrophic scar.2. These results suggest that tetrandrine inhibits HSFs at least partially through induction of Smad7 and decrement of Smad2 resulting in inhibition of TGF-?1 transcription and its intracellular signaling, which led to reduction of type I and ? collagen production and suppression of cell reproductive activity.3. These results suggest that efficiency of Smad7 negatively regulating TGF-?/Smads signal pathway was decreased in Smad7 silent HSFs. While Smad7 silent HSFs was treated with tetrandrine, although Smad7 mRNA expression was increased, increased Smad7 could not negatively regulating TGF-?/Smads signal pathway, and inhibition effection of HSFs and collagen production were not significant.4. Many signal pathways such as TGF-beta signal pathway, Toll-like receptor signaling pathway, PPAR signaling pathway, Wnt signaling pathway, MAPK signaling pathway, mTOR signaling pathway and p53 signaling pathway, et al. were changed engaged in anti-fibrosis in HSFs treated with tetrandrine, which indicated that tetrandrine possessed characteristics of multi-effect ways and multi-targets, and also indicated that signal pathways showed network and was complex in hypertrophic scar formation.