Roles Of Bachl In Pterostilbene-mediated Protection Against UV-induced Acute Photodamage In Human Keratinocytes

[Background]The human skin, as the largest organ, is exposed to a variety of environmental attacks, especially ultraviolet (UV). UV is classified in three bands according to wavelength: UVA (320-400 nm), UVB (280-320 nm) and UVC (200-280 nm).Acting on the surface of the human body is UVA and UVB. Long term or a large amount of UV radiation can cause skin damage, such as acute sunburn, chronic actinic dermatitis, photoaging and skin cancer. The incidence of such dermatological disorders is increasing because of the environmental pollution. The damage mechanism induced by UV irradiation is complex. UV irradiation directly damages DNA causing dimeric photoproducts formation, more important is the oxidative stress leads to excessive reactive oxygen species (reactive oxygen species, ROS) which can damage protein, DNA, lipid and many kinds of photodermatology. Scavenging excess ROS, enhancing the activity of antioxidant enzymes, clarifing the skin defense mechanism of antioxidant and exploring effective UV protective agent have become the research focus.Nuclear factor erythroid 2 - related factor 2(Nrf2) signaling pathway is one of the most important antioxidant stress pathways. When excessive ROS production, Nrf2 and Kelch ECH protein 1 (Keap1) uncouples and translocates into the nucleus, then Maf recognition element (MAREs) and the transcription factor BTB-CNC homology-1 (Bachl) in the nucleus uncouples, Bachl translocated to the cytoplasm. After translocation, Nrf2 binds to the MAREs inorder to regulate the phase ? enzymes, and antioxidants genes, playing antioxidant effect.Bachl that was competing with the Nrf2 binding sites of ARE gene expression plays against oxidation negative role;however recent research found that Bachl can directly regulate the redox, expression of cell cycle related target genes, resulting in the occurrence of oxidative stress reaction and a variety of diseases. It is expected to become the new therapeutic targets for diseases associated oxidative stress.Recently, various botanical ingredients, such as pterostilbene, tea polyphenols,curcumin, lycium barbarum polysaccharide, sulforaphane, resveratrol have been showed antioxidant activity. Pterostilbene has been shown the properties of preventive and therapeutic in numerous cancer cells due to its ability to activation of Nrf2 pathway. However, the photoprotective effects and the mechanism underlying the action of pterostilbene against UV-induced damage remains unclear. Therefore, This study was designed to investigate the protective effect and the regulation of Bachl on UV induced by human keratinocytes (HaCaT), and to provide a theoretical basis for the development of new type of protective agent.[Object]1. To investigate the protective effect of pterostilbene from UVA/UVB induced photodamage in HaCaT cells.2. To explore potential underlying mechanisms of pterostilbene against UVA/UVB induced photodamage in HaCaT cells.3. To explore the role in pterostilbene-mediated protection against UVA/UVB induced photodamage in HaCaT cells.[Method]1. For photodamage cells model, HaCaT cells were exposed to UVA (30 J/cm2) and UVB (300 mJ/cm2) respectively. Then the cell viability and morphology were detected by MTT and microscope respectively.2. After treatment with various concentrations of pterostilbene, cell viability and morphology were determined using the CCK-8 assay and microscopy respectively.3. The cell viability and DNA damage after exposure to UVA/UVB were detected by MTT and comet assay respectively.4. Intracellular SOD activity and ROS level were measured by WST-1 and DCFH-DA respectively.5. The levels of Nrf2 and Bachl protein in cytosol and nuclear extracts after treatment with pterostilbene were investigated by using Western Blot.6. The effect of genistein on cytosol and nucleus distribution of Nrf2 and Bachl protein were measured by using Western Blot.7. The effects of genistein on protection of pterostilbene against UVA/UVB induced cell viability were detected by CCK-8 assay.8. To get Nrf2 knockdown cell (Nrf2KD cells), Nrf2 was silenced by transduction HaCaT cells with Nrf2-targeted small hairpin RNA (shRNA).9. The qRT-PCR was used to detect mRNA expression of some known Bachl target genes.[Results]1. UVA (30 J/cm2) and UVB (300 mJ/cm2) irradiation significantly reduced cell viability (P< 0.05), and increased the cell volume and intracellular particles.2. After treatment with various concentrations of pterostilbene, cell viability was determined by CCK-8 assay. Non cytotoxic effect concentration was 5 ?M.3. UVA/UVB irradiation significantly reduced cell viability (P< 0.05), whereas cells pretreated with pterostilbene showed significantly increased viability. UVA/UVB irradiation increased the nucleus of tail length, tail moment, tail DNA content and percentage of tail DNA significantly (P < 0.05), while pterostilbene pretreatment apparently reduced the tail length, tail moment, tail DNA content and percentage of tail DNA (P< 0.05).4. Compared with the untreated control cells, UVA/UVB irradiation increased SOD activity slightly. However, pretreatment with pterostilbene followed by UVA/UVB exposure pronounced increased SOD activity. The ROS level was dramatically increased in the acute UVA/UVB irradiated cells. Interestingly, the ROS levels were significantly reduced in cells pretreated with pterostilbene followed by UV exposure.5. Using Western blot to detect the distribution of Nrf2/ Bachl protein in the cytoplasm and nucleus, the Nrf2 in the nucleus increased, Bachl decreased.6. Pretreated with genistein followed by pterostilbene incubation resulted in accumulation of nuclear Bachl and had no obvious effect on Nrf2.7. Genistein had no obvious effect on HaCaT cell viability (P > 0.05). However,pretreatment with genistein abolished the protective effect of pterostilbene in HaCaT cell viability (P < 0.05).8. The transduction of Nrf2-targeted shRNA led to reduction of the Nrf2 protein level (>80%).9. Compared with the untreated control cells (non- genistein), pretreatment with genistein prior to pterostilbene incubation increased the genes expression of bachl,bcl2l11,ftl,hmga2,hmoxl ,mafg,mapt,mmp13,mmp9,sqstm1,tfe3,vrnal-1 and vrnal-2 (P< 0.05) in HaCaT cells. However, in Nrf2KD cells, pretreatment with genistein resulted the down-regulation of gclc, gclm, hmga2, hmoxl, itpr2, mel and vrnal-2 (P< 0.05), and up-regulation of mafg, mapt and vegf (P< 0.05).[Conclusion]1. Pterostilbene has attractive photoprotective activity against acute UVA/UVB irradiation induced photodamage in HaCaT cells.2. Pterostilbene effectively protected against UVA/UVB induced photodamage by increasing cell viability and reducing DNA damage through activation of anti-oxidative pathway.3. Bachl plays the important role in pterostilbene-mediated protection against UV-induced acute photodamage in HaCaT cells partially through coordinated regulation of gclc, gclm, hmga2,hmoxl, itpr2,mel and vrnal-2 with Nrf2, and accompanied with regulation of mafg, mapt and vegf alone.