The Role Of Heat Treatment In UVB-induced Injury In Human Melanocytesr And Its Molecular Mechanism

Vitiligo is an acquired, multifactorial, depigmenting disorder characterized by the appearance of white macules in the skin due to progressive loss of functional melanocytes in the epidermis. Recently the phototherapy with ultraviolet(UV) radiation of wavelengths between 280 and 320 nm(UVB) is wildly used in vitiligo, and effect is significant. Epidermal components, such as keratinocytes(KC) and melanocytes(MC) absorbe UVB and affected by its biological effects. In addition to positive effects, UVB has a series of negative effects, high doses of UVB radiation induce oxidative stress, cell damage, DNA mutations, eventually cause skin aging and tumor. Oxidative stress is considered as a major pathogenic mechanism of vitiligo and most patients show antioxidant defects. Thus high doses of UVB will accelerate damage of MC and will reduce the therapeutic effects in vitiligo. Heat treatment(42 ℃, 1h) is another stimulus which promote MC proliferation and melanin synthesis, moreover the heat shock proteins(HSPs) induced by heat treatment can enhance tolerance of cells against various environmental stimuli. It implied that heat treatment combine with UVB phototherapy would not only promote the repigment of vitiligo, but also reduce the UVB-induced injure of MC.Objectives: To observe the effects of heat treatment in UVB-induced injure in human MC. Explore distribution and expression of inducible hsp70 of MC in thermal stress and analyze the molecular mechanism behind the heat treatment protects human MC from UVB damage.Methods: Human primary MC were obtained from foreskin. After 3-4 passages, purified MC were classified into 4 groups: control group( incubated at 37 ℃), heat treatment group(incubated at 42 ℃, 1h), UVB group( exposed to UVB irradiation at 100mJ/cm2), combination group( first exposed to heat followed by a UVB irradiation at 100mJ/cm2), all group treated for a period of 3 days. Cell viability was monitored by MTT assays. Apoptosis rate of MC was measured with flow cytometry(FCM). DNA damage was detected by comet assay. The activity of superoxide dismutase(SOD) and concentration of malonaldehyde(MDA) were measured with biochemistry methods. Intracellular Reactive Oxygen Species(ROS) was observed by fluorescence microscopy and measured with FCM. MnSOD mRNA was detected by real-time PCR.To explore the relationship of between inducible Hsp70 expression and UVB damages, MCs were exposured to UVB(500mJ/cm2) after heating 0h, 2h, 4h, 6h, 8h, 10 h, 12 h, and Hsp70 expression and DNA damage were monitored by western blot and comet assay respectively at different time. Hsp70-specific siRNA transfection was used to silence Hsp70, in order to test the effects of Hsp70 against the UVB induced injure. Immunofluorescence was used to observe the Hsp70 distribution after heating and/or UVB radiation.Results:1 Exposure of human MC to UVB significantly damaged DNA(P<0.01) and reduced MnSOD mRNA expression(P<0.001) and SOD activity(P<0.01), while intracellular ROS(P<0.001) and MDA(P<0.001) increased, eventually induced apoptosis(P<0.001) and decreased MC viability(P<0.001).2 Pre-heating reduced UVB-induced DNA damage(P<0.05) and apoptosis(P<0.001), recovered MnSOD mRNA expression(P<0.05) and SOD activity(P<0.01), decreased the level of MDA(P<0.001) and ROS(P<0.001). Heat treatment improved tolerance of MC against UVB(P<0.001).3. There is negative correlation(r=-0.971, P<0.001) between Hsp70 expression level induced by heating and DNA damage induced by UVB. In addition, Hsp70-specific siRNA transfection can silence Hsp70 expression and abolishe the protection effects of Hsp70 against UVB(P>0.1).4. Even if the Hsp70 expression level not increased, Hsp70 translocates to the nuclei and nucleoli, which promoted by heat exposure, can also protected MC from DNA breaks(P<0.05); while these effects will be abolished by high doses of ATP(400μM) which disturbed Hsp70 nuclear translocation(P>0.1).ConclusionThis study demonstrates that the heat pre-exposed MC can be protected from UVB induced injure by stimulating Hsp70 translocation and upregulating Hsp70 expression. It implies that heat treatment will be a promising treatment when combinates with UVB phototherapy can protect the MC from various UVB induced damage, such as DNA break, oxidative stress and so on.