To Investigate The Effect Of Transient Receptor Potential Ankyrin 1 On Keratinocytes Phagocytosis

Background: Ultraviolet radiation(UVR)is one of the main causes of melasma and other pigmented skin diseases.It is also one of the inducements and treatments for vitiligo,other depigmentation skin and mucous membrane diseases.After exposed to UVR,the treatment strategy focused on proliferation of melanocytes and melanin synthesis in the downstream,and the initial step of targeted therapy is less.Transient receptor potential ankyrin 1(TRPA1)is a non-selective calciumpermeable ion channel to participate in various physiological activities.In recent years,studies have shown that UVA phototransduction in human melanocytes(MCs)activates TRPA1,leading to a rapid calcium influx,it is necessary for UVA-induced early melanogenesis.Meanwhile,Bellono et al shown that TRPA1 is essential for a unique only extraocular phototransduction pathway in mammalian.In the epidermal cell,MCs synthesize melanin and transferred to keratinocytes(KCs)through phagocytosis.In addition,the study shown that calcium plays an important role in phagocytosis.Therefore,it is important for studying the role and mechanism of TRPA1 phototransduction pathway in epidermal KCs,and understanding the pathogenesis and the upstream mechanism of melanosomes transport.Objective: 1.To study the phototransduction of TRPA1 in KCs.2.To investigate the effect of TRPA1 on KCs phagocytosis.Methods: 1.The expression of TRPA1 mRNA was detected by agarose gel electrophoresis(AGE)and real-time fluorescence quantitative PCR(q PCR)in human KC cell lines(HaCaT cells).2.The expression of TRPA1 protein was detected by Western Blot in HaCaT cells.3.Flow cytometry(FCM)was used to detect the retinal-dependent calcium influx in HaCaT cells after exposed to UVA and UVB,and the retinal-dependent calcium influx after activated or inhibited TRPA1 by agonist or antagonist.4.The KCs phagocytosis was detected by scanning laser confocal microscopy after exposed to UVA and UVB.Results: 1.The expression of TRPA1 gene and protein in HaCaT cells was detected by AGE,q PCR and Western Blot.2.After UVA or UVB exposure,the retinal-dependent calcium influx was significantly increased in HaCaT cells.3.When TRPA1 was activated in HaCaT cells,the calcium influx was significantly increased in HaCaT cells.When TRPA1 was inhibited in HaCaT cells,the retinaldependent calcium influx was significantly decreased after UVA or UVB exposure.4.When TRPA1 was activated in HaCaT cells,the number of fluorescent microspheres was significantly increased using FCM in HaCaT cells.When TRPA1 was inhibited in HaCaT cells,the number of fluorescent microspheres was significantly decreased using FCM after UVA or UVB exposure.5.When TRPA1 was activated in HaCaT cells,the number of fluorescent microspheres was significantly increased by scanning laser confocal microscopy in HaCaT cells.When TRPA1 was inhibited in HaCaT cells,the number of fluorescent microspheres was significantly decreased by scanning laser confocal microscopy after UVA or UVB exposure.Conclusion: 1.UVA and UVB can activate the TRPA1 pathway and induce retinal-dependent calcium influx in HaCaT cells.TRPA1 pathway has phototransduction in KCs.2.UVA and UVB enhanced the phagocytosis of HaCaT cells through the TRPA1 pathway.