Research On The Mechanism Of UVB Affecting Tyrosine-mediated Melanin Synthesis

ObjectiveIt has been clinically found that the onset and condition of some patients with vitiligo occur in summer and autumn when the sun is strong;and discoloration.Spots may occur on the skin of the UV burned part.The UVB irradiation may promote the senescence of melanocytes.This is because when melanocytes as functional cells synthesize a large amount of melanin,overload work will lead to premature senescence.And the ability to synthesize melanin will be reduced or the loss,it may lead to aggravate the occurrence or development of vitiligo,suggesting that strong ultraviolet irradiation may be one of the factors inducing or aggravating vitiligo.At the same time,it has been found that UV can enhance the activity of tyrosinase in melanocytes,increase the amount of melanin and accelerate the migration;Which indicates that there is a dose threshold for the effect of UV on melanin synthesis.Therefore,it is of great significance to clarify the mechanism of UVB.Method1.Primary melanocyte culture:take adult male foreskin samples,soak in 70%ethanol for disinfection,wash out ethanol with PBS solution;cut off fat and subcutaneous tissue,cut skin into 5 mm wide strips;add 0.25%trypsin solution,and incubate in 4℃ refrigerator for 18 h.The dermis and epidermis were separated with a blade,the dermis was discarded,and the epidermis was transferred to a centrifuge tube containing PBS solution,and incubated in a shaking table at room temperature;at room temperature,the cell suspension was washed and centrifuged,and cultured in M254 medium at 37℃ and 5%CO2 for 48-72 h;the culture medium was replaced by centrifugation,and cultured twice a week.2.Ultraviolet irradiation:SH4B-T ultraviolet phototherapy instrument is used to irradiate according to the required UVB dose of each group in the experimental group.After irradiation,continue to culture in a constant temperature cell incubator at 37℃,5%CO2 until the time required by the experiment.3.NaOH were used to detect the melanin content.The activity of tyrosinase,the rate limiting enzyme of melanin synthesis,and the expression of MSH protein were measured by biochemical enzyme method and Western blot.4.Detection of cell premature senescence:observe cell morphology by Giemsa staining,detect cell proliferation by MTT test and clone formation test,observe the positive rate of premature senescence by β-galactosidase staining,detect the change of lysosome at different time and dose by lyso tracker fluorescence probe test,and detect cell migration energy by scratch test change in force.5.Western blot was used to detect the expression changes of P62 and GATA4 protein in Hacat cells and primary melanocytes irradiated with different doses of UVB for 72 hours.Then,after treatment of Hacat cells and primary melanocytes with ATM inhibitor Ku55933,ATR inhibitor VE-821 and P53 inhibitor Nutlin-3,Western blot was used to detect the expression changes of GATA4 protein.6.Statistical methods:SPSS 23 statistical software was used for data processing,the data accorded with normal distribution,and the two groups were compared with independent samples,single factor analysis of variance,with α=0.05 as the test level,with P<0.05 as the difference of statistical significance.Result1.After irradiation of MC cells and Hacat cells with 20,50,80,100,150 and 200 mJ/cm2 UVB for 72 hours,when the irradiation dose was less than 80 mJ/cm2,the melanin content increased gradually with the increase of irradiation dose,reached the peak value at 80 mJ/cm2,and decreased gradually at 100 mJ/cm2,with statistical significance(P<0.05).2.Compared with 0 mJ/cm2,the tyrosinase activity of primary melanocytes and Hacat cells increased 72 hours after UVB irradiation of 20,50,80 and 100 mJ/cm2(P<0.05),and the expression of MSH protein increased with the increase of irradiation dose(P<0.05).3.When the MC cells and Hacat cells were irradiated with different doses of UVB,the cell morphology changed,the cell clone formation rate and survival fraction decreased,the positive rate of premature senescence in β-galactosidase staining experiment increased,lyso tracker fluorescence probe lysosomal fluorescence increased,the number increased,and the cell migration ability decreased(P<0.05),UVB induced premature senescence of MC cells and Hacat cells.4.After UVB irradiation of 20,50,80 and 100 mJ/cm2 for 72 hours,the expression of P62 in primary melanocytes and Hacat cells was inhibited,and the expression of GATA4 was increased;after treatment with ATM inhibitor KU55933 and ATR inhibitor VE-821,the expression of GATA4 was not significantly increased;after treatment with Nutlin-3,the expression of GATA4 was increased.ConclusionUVB irradiation affected the expression of melanin in primary melanocytes and Hacat cells.Further study found that UVB increases melanin synthesis by activating tyrosinase activity and inducing high expression of MSH protein;meanwhile,it is found that after UVB irradiates primary melanocytes and Hacat cells,there is premature senescence,and P53,P16 and P62 premature failure pathways are activated;in-depth study of radiation-induced P62 signaling pathway,UVB activates P62-GATA4 early by P62 mediated selective autophagy senescence pathway,which can induce premature senescence of melanocytes and keratinocytes and then inhibit the synthesis of melanin,may be one of the mechanisms of excessive ultraviolet irradiation to make vitiligo patients develop or worsen.