Effects Of Triamcinolone Acetonide And Propranolol On Proliferation Research Of Keloid Fibroblasts In Vitro

BackgroundKeloid(KD) is a kind of pathological scar which formed after skin injury. Pain, itching and other clinical abnormal sensations are the main symptoms. Progressive growth, gradual invasion to the surrounding normal tissue and a high rate of recurrence after cicatrectomy lead to new scar and it may exceed the original scar’s volume, all of which are similar to benign tumor [1]. In histology, the main characteristics of KD are the excessive proliferation of the fibroblast and excessive collagen accumulation of the extracellular matrix. Because after the skin trauma, normally quiescent fibroblasts are activated and proliferated. At the same time, a large number of fibroblasts synthesize massive collagen, but also can produce a variety of repair factors to promote wound healing and tissue repair. So the local tissue is excessively collagen fibrosis and the formation of special appearance looks like crab, which cause some psychological burden to patients. To an extent, KD formed in the joints can also lead to the dysfunction of the patients’ limbs[2].At present, the mechanisms of the treatment of keloid mainly include inhibition of fibroblasts’ proliferation, decrease of collagen synthesis of fibroblasts and inhibition of DNA synthesis, all of which are aimed to promote fibroblasts’ apoptosis and enhance the activity of collagenase so as to degrade collagen and so on[3].The research shows that during the formation, occurrence and developing process of the keloid, the expression of some cell factors and its receptors are abnormal. Such as vascular endothelial growth factor(VEGF), proliferating cell nuclear antigen(PCNA), transforming growth factor β 1(TGF-β 1) and so on. So far, the TGF-β 1 is the exact factor. After the trauma, the first and high expression of those factors in new blood vessels is TGF-β 1 which activate the surrounding fibroblast cells and make them highly expressed the TGF-β 1. Meanwhile, the TGF-β 1 can promote the proliferation of fibroblasts and produce a variety of protein secretion of extracellular matrix(ECM) in which most of them are collagen I, III. The excessive deposition of ECM results in the formation of keloid.In addition, keloid fibroblasts are able to synthesize and secrete collagen, and also could promote the expression of VEGF, Ang I and other vascular factor which enhance the vascular endothelial cells’ ability to induce and promote the formation of blood vessels, during the process of wound repair and under the hypoxic microenvironment. Those vascular growing factors may play an important role in the gradual invasion of keloid [4]. VEGF is currently known as the most active angiogenic factor, which can enhance endothelial cells’ activity and promote it’s proliferation, so as to promote the increase of the microvessel density of keloid’s internal tissue and further provide abundant blood supply for keloid to invade the normal tissue.At present, Triamcinolone acetonide(TA) is the first choice for the clinical treatment of keloid. Because it’s strong anti-inflammatory and it’s ability to inhibit the keloid fibroblasts’ proliferation are the pharmacological actions, TA can make the inflammatory and early tissue hyperemia, cell reaction and liquid exudation palliative, and inhibit the excessive growth of scar and the formation of granulation tissue in the late process of wound healing [5].Propranolol(P) as a common blockers is used for the treatment of cardiovascular diseases. In 2002, O ’Callaghan CJ had found that P had the effect of resisting fibrosis and inhibiting the formation of collagen and the mechanism of P was able to inhibit fibroblast’s biological activity, reduce the secretion of collagen and achieve the effect of anti fibrosis mainly through competitive inhibition of TGF-β 1 receptor [6]. In addition, P may reduce the secretion of proliferating hemangioma’s VEGF through inhibition of the ERK/MARK signaling pathway. During the late-stage treatment of hemangioma, P could inhibit the expression of VEGF, b FGF and promote the further regression of hemangioma through the down-regulation of MAPK signaling pathway [7][8]. The formation of keloid is closed to fibroblasts’ proliferation, collagen deposition and the rich blood supply. In 2010, DE Mesquita CJ put forward the hypothesis of treatment of keloid with P [9].At Present, researches on Propranolol’s treatment of keloid are rare. Although the TA for the treatment of keloid is preferred currently in clinical drug, but still there is no unified standard of concentration for TA. Objectives1. The aim of the experiment is to investigate the effects of different concentrations of P and TA that have an influence on keloid fibroblasts’ proliferation and to find out the differences of expression of TGF-β 1, VEGF and collagen I, III, by culturing keloid fibroblasts in vitro;2. Screening the effective drug concentration of P and TA and comparing the differences of the ability to inhabit the fibroblasts’ proliferation between P and TA.3. To provide the theoretical basis for the application of propranolol in the prevention and treatment of keloid. Methods1. Specimens came from clinical keloid patients, confirmed by department of pathology of the First Affiliated Hospital of Zhengzhou University.2. The keloid fibroblasts were extracted, cultured, subcultured and purified in vitro by using the two steps of trypsin digestion.3. The keloid fibroblasts that subcultured to third generation and grow well, by using immunohistochemical technology to detect the expression of CD73, CD90 and CD105 in mesenchymal stem cells’ surface markers and to identify keloid fibroblasts.4. Using different concentrations’(0mg/ml, 0.05mg/ml, 0.10mg/ml, 0.20mg/ml, 0.40mg/ml, 0.80mg/ml, 1.6mg/ml, 3.2mg/ml, 6.4mg/ml) propranolol and TA to deal with keloid fibroblasts at 24 h, 48 h and 72 h. There was no drug in negative control groups’ culture medium.5. Taking the measure of MTT to detect the effect of P and TA on keloid fibroblasts’ proliferation and vitality.6. Taking the measure of Western blot to explore the expression of TGF-β 1, VEGF and collagen I, III. The experimental data were analyzed by using SPSS17.0. Results1. Keloid fibroblasts’ identification.The cells can be seen under light microscope. The keloid fibroblasts’ growing characters and shapes are spindle shape, cells’ periphery has protrusions, oval nucleus is located in the middle of cell body and parallel and radial growth. Combined with fibroblasts’ morphology, growth characteristics, the expression of CD73, CD90, CD105 and clinical diagnosis(in reference to the 1992 Darzi’s diagnostic criteria), the cells cultured can be defined as keloid fibroblasts.2. Both TA and P’s effect on fibroblasts of keloid2.1 According to the MTT2.1.1. TA had the obvious ability to inhibit the proliferationin of keloid fibroblasts under a certain concentration and the inhibition depending on working time and concentration. In the range of 0.05mg/ml~6.4mg/ml, the concentration of TA was higher so as the growth rate for inhibition of the fibroblasts was also higher.(P < 0.05). With the increase of working time of the drug, the inhibition rate was higher(P < 0.05). At 24 h, 48 h, 72 h, IC50 were 5.2mg/ml, 3.88mg/ml, 1.91mg/ml, and selecting IC50 as the experimental drug’s concentration when the time was 72 h.2.1.2. P had the ability to inhibit the proliferationin of keloid fibroblasts under a certain concentration, and the inhibition depended on working time and concentration. In the range of 0.05mg/ml~6.4mg/ml, the concentration of P was higher so as the growth rate for inhibition of the fibroblasts was also higher.(P < 0.05). With the extension of working time of the drug, the inhibition rate was higher(P < 0.05). At 24 h, 48 h, 72 h, IC50 were 6.2mg/ml, 4.92mg/ml, 2.84mg/ml, and selecting IC50 as the experimental drugs’ concentration when the time was 72 h.2.2 According to the Western BlotBoth TA and P significantly inhibited the expression of collagen I, III, TGF- β 1 and VEGF derived from fibroblasts. Conclusions1. Both TA and P had definite inhibitory effect on keloid fibroblasts cultured in vitro, while the emphasis is different. Overall, TA had the more powerful inhibition on keloid fibroblasts than P, and the difference was statistically significant(P < 0.05).2. The expression of TGF-β 1, VEGF and collagenⅠ, III were inhibited by both TA and P. TA had more obvious inhibitory effect than the other three, except VEGF. There was significant difference between the two drugs(P < 0.05).3. Both TA and P had inhibitory effect on keloid fibroblasts proliferation and the effect depended on drugs’ working time and concentration.4. Compared with P, TA had the more powerful inhibitory effect on fibroblasts, we could predict that there might be other ways to inhibit the function of fibroblasts, except for TGF-β 1, VEGF and collagen I, III.