The Study On The Role Of Rab23 In Regulation Of Melanogenesis In PIG1 Cells Induced By UVB Irradiation And Its Mechanism Involved

Objection: The physiological degree of melanin synthesis can protect the skin from the external harmful environment.Repeated exposure to one or more stimulating conditions will cause abnormal melanin synthesis.UV is one of the most common factors which can cause skin pigmentation.Repeated exposure to the UV can cause skin erythema,hyperpigmentation,inflammatory response,etc.UVB has the property of inducing melanin synthesis,but it’s mechanism has not been elucidated.Genetic and biochemical analysis of gene products responsible for mammalian pigmentation disorders in the past decades have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin.Studies have shown that after irradiated with UVB,the expression of Rab23 in keratinocytes significantly increased,strongly indicated the critical role of Rab23 in the physical and pathological process of keratinocytes.Thus,we speculate that Rab23 may be involved in the regulation of UVB induced melanogenesis.Therefore,this study focused on the regulatory effects of Rab23 on melanogenesis in melanocyte induced by UVB.Methods: We established a cell model of melanogenesis through irradiate PIG1 cells with UVB.The PIG1 cells were irradiated with UVB at 0 mJ/cm2,25 mJ/cm2,50 mJ/cm2 and 100mJ/cm2,then western blot was performed to determine the expression of Rab23 and MITF in PIG1 cells.Then PIG1 cells were treated with UVB at 50 mJ/cm2,extraction of protein was carried out respectively after 0h,0.5h,1h,2h,6h,12 h,then western blot was conducted to measuring the expression of Rab23,MITF and gp100.Transfected PIG1 cells of RNA interference knockdown of Rab23 were treated with UVB at 50 mJ/cm2,extraction of protein was carried out after1 h,6h,12 h,then western blot was conducted to measuring the expression of Rab23,MITF and gp100.Results1.UVB induced the expression of Rab23 and melanogenesis in a dose-dependent manner: The results of Western Blot showed that,the higher irradiation dose of UVB,the higher expression of gp100 when the dose of UVB is 100 mJ / cm2 blow,which is one of the index of melanagenesis.At the same time the Rab23 changed parallelly with melanogenesis.2.UVB induced the expression of Rab23 and melanogenesis in a time-dependent manner: Because the toxic effect of 100mJ/cm2 UVB irradiation,we chose 50mJ/cm2 UVB to stimulate melanogenesis.The results showed that melanin content increased gradually with the prolongation of the time of UVB irradiation,and the expression level of melanin-related protein gp100 gradually increased,and Rab23 also had the same change trend,which strongly indicate that Rab23 involved in melanogenesis induced by UVB.3.The downregulation of Rab23 alleviate the melanogenesis induced by UVB: Western blot analysis and melanin content assays were used to detect the level of melanin synthesis at 6h after irradiation with UVB at the dose of 50mJ/cm2.The results showed that declined expression of Rab23 significantly reduced the expression of gp100 and melanin content.4.The downregulation of Rab23 relieve the time trend of melanin synthesis induced by UVB: The results of Western Blot showed interference of Rab23 expression significantly reduced the ascent trend of melanogenesis after irradiated with UVB,demonstrateing that Rab23 involved in UVB-induced melanogenesis.5.UVB induced the expression of MITF: The expression of MITF was detected by Western Blot analysis after UVB irradiation.The results showed that UVB could induce the expression of MITF,and the expression level of MITF increased with the prolongation of irradiation time.6.The downregulation of Rab23 alleviate the expression of MITF: Western blot analysis were used to detect the expression of MITF in PIG1 cells at 6h after irradiation with UVB at the dose of 50mJ/cm2.The results showed that declined expression of Rab23 significantly reduced the expression of MITF.7.The downregulation of Rab23 relieve the time trend of MITF: The results of Western Blot showed interference of Rab23 expression significantly reduced the ascent trend of expression of MITF after irradiated with UVB,supporting that MITF is participate in Rab23 regulatory effect of UVB-induced melanogenesis.Conclusions: In this study,we found that UVB could promote melanogenesis in a dose-and time-dependent manner,and Rab23 is involved in melanogenesis via regulation of MITF expression in melanocyte.