Effects Of Vernonia Anthelmintica Combinding With Ultraviolet Irradiation On The Secretion Of Bfgf, Et-1 And Scf From Hacat Cell And The Proliferation And The Migration Of A375 Melanoma Cell

Vitiligo is a kind of disease characterized by the depigementation of the skin and hair because of acquired melanocytes damage. Vitiligo can be cosmetically disfiguring and it is a stigmatizing condition, leading to serious psychologic problems in daily life. At present, the pathogensis of vitiligo may be related with heredity, immunity and nerval elements. In recent years, more and more studies find that the cytdokines participate in the development of vitiligo. Human keratinocytes have been shown to synthesize and secrete basic fibroblast growth factor (bFGF), endothelin-1(ET-1) and stem cell factor (SCF) in culture. Recently it has been shown that bFGF is a potent mitogen for melanocytes,and ET-1 is able to stimulate melanocytes proliferation and tyrosinase activity via a receptor-mediated signal transduction pathway, SCF has been reported to stimulate the proliferation of human melanocytes in culture and in xenografts of normal human skin. For these reasons, it is important to study the relationship between bFGF, ET-1, SCF which secreted by keratinocytes and activation of melanocytes.Many treatments have been used to stop progression and achieve repigmentation in order to repair the morphology and functional deficiengcies of the depigmented. Both VW plus ultraviolet A or B irradiation had been used for the clinical treatment of vitiligo and had achieved better therapeutic efficacy. However, the mechanisms of these modalitys in repigmentation are not thoroughly clarified. In addition to the action on the melanocyte directly, the melanocyte features for stimulated proliferation, melanogenesis and migration in lesional epidermis led us to assume the possibility that there are some paracrine factors produced and secreted by stimulated keratinocytes, which after the interaction with melanocytes, leads in turn to the accentuated function of melanocytes.ObjectiveTo investigate effects of secretion of bFGF, SCF and ET-1 from HaCaT cells by VW combined with irradiation of ultraviolet B or A and effects of culture supernatants on the proliferation and migration of A375 cells. Methods(1) Cells culture HaCaT cells and A375 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum and were plated in 6-well and 96-well plate with the same number of cells.(2) Ultraviolet irradiation The culture medium was replaced by fresh medium without fetal calf serum 24h before each experiment. The time and dosage of UVB or UVA irradiation were conducted according to the experiment design. VW were added into the medium before irradiation according to designed concentration.(3) Collection of culture supernatanants Culture supernatants were collected, centrifuged at 1000g for 5 min, and stored at -70℃.(4) Enzyme-linked immunosorbent assay Enzyme-linked immunosobent assay (ELISA) was performed to detect the concentration of bFGF, ET-1 and SCF in culture supernatanants.(5) Cell proliferation assay The effects of conditioned medium on A375 cells'proliferation were detected by MTT method. (6) Melanocyte migration assy The conditioned medium was also assessed in terms of its effect on A375 cells'migration by micropore filtration.(7) Sastistical analysis The experimental data were analyzed with SPSS. P values less than 0.05 were considered to be statistically significant.Results:1. Effects of VW combinding with UVB or UVA, VW, UVB and UVA on the secretion of bFGF,ET-1 and SCF from HaCaT cellThe secretion of bFGF by HaCaT cells were significantly higher in both VW combined with UVB ( 119.430±6.828ng/L ) and UVB ( 102.839±6.826ng/L )than that in blank controls(62.982±8.250ng/L) (P<0.05)and no significant differences were observed between two groups(P>0.05). ET-1 secreted by HaCaT cells were significantly higher in both VW combined with UVB (4.470±0.192 g/L) and UVB (4.233±0.116 g/L) than that in blank controls (3.820±0.212 g/L) (P<0.05) and no significant differences were observed between two groups (P>0.05). SCF secreted by HaCaT cells were significantly higner in VW combined with UVB (128.27±3.35ng/L) than that in blank control (103.60±8.67ng/L) (P<0.05). SCF secreted by HaCaT cells was not significantly higner in UVB(113.64±7.39ng/L) than that in blank control (P<0.05). Besides, the secretion of bFGF, ET-1 and SCF by HaCaT cells were not significantly higher in VW combined UVA, UVA and VW than that in blank controls(P>0.05).2. Effects of HaCaT cells'supernatuants on the proliferation of A375 melanoma cells which harvest after treated by VW combinding with UVB or UVA, VW, UVB and UVA.Culture supernatants of HaCaT cell which treated by VW combined with UVB and UVB can significantly stimulate A375 cells proliferation. The A value in VW combined with UVB (0.582±0.0554) and UVB (0.509±0.060) were significantly higher than that of the blank control (0.332±0.070) (P<0.05). There was no significant difference observed between two group (P>0.05). There were not significant differences observed in proliferation of A375 cells which were stimulated by conditioned medium of VW plus UVA, VW and UVA in contrast to the blank groups (P>0.05).3. Effects of HaCaT cell's supernatuants on the migration of A375 melanoma cell which harvest after treated by VW combinding with UVB or UVA, VW, UVB and UVACulture supernatants of HaCaT cell which treated by VW combined with UVB and UVB can significantly stimulate A375 cells'migration. A375 cells passed through the micropore filter of transwell chanmber in VW combined UVB (72±6/ per field of vision×40 times) and UVB (60±5/ per field of vision×40 times) is more than that in blank groups (42±13/per field of vision×40 times) (P<0.01 and P<0.05). There was a synergistic effect in stimulating A375 cell migration by VW and UVB. There was no significant difference observed on migration of HaCaT cells which were stimulated by conditioned medium of VW combined with UVA, VW and UVA in contrast to blank groups and between UVB combined VW and UVB(P>0.05).Conclusions1. Both VW combined with UVB and UVB can upregrate the secretion of bFGF and ET-1 in HaCaT cells, the proliferation and migration of A375cell. These results suggest that the therapeutic effect of VW combined with UVB and UVB may be related to promoting ET-1 and bFGF secretion of keratinocytes and stimulating the proliferation and migration of melanocytes.2. VW combined with UVB can upregulat the secretion of SCF in HaCaT cells, but UVB can not upregulate the secretion of SCF in HaCaT cells. These may partially account for the therapeutic mechanisms of clinical evidene that the efficacy of VW combined with UVB was superior to that of UVB on vitiligo.3. A significant increase was not noticed in the conditioned medium of HaCaT cells for it's secretion of bFGF, ET-1 and SCF and ability to promote A375 cells'proliferation and migration which treated by VW combined with UVA, VW and UVA. All theseshow there remain other mechanisms involved in the process of repigmenta- tion in patients with vitiligo by theses therapeutic modalities.