The Mechanism Research Of Hsp27 Regulating Apoptosis Through P21 In Photodamaged Cutaneous Keratinocytes

Background:Ultraviolet?UV?is undoubtedly an important one of all the environmental factors affecting the skin.The skin is exposed to UV irradiation almost every day,thus the potential risk of UV-induced photodamage is easily neglected.Long-term repeated UV exposure can cause chronic photodamage of the skin,which called skin photoaging.In previously study,the skin of the young,middle and old aged exposed parts and non-exposed parts were taken as the research object.The protein was extracted from keratinocytes for 2-DE separation.MALDI-TOF mass spectrometry and database query were used to find differentially expressed proteins.The research found that heat shock protein?Hsp?27 and p21^WAF1/CIP1?p21?were highly expressed in the exposed group in the same age group,which suggested that Hsp27 and p21 may be closely related to skin photoaging,but its exact function is for further study.Objectives:In this study,based on the photoaging model of human skin keratinocytes?HaCaT?induced by Ultraviolet B?UVB?irradiation at30mJ/cm²,the effect of Hsp27 on apoptosis and the possible protective mechanism between Hsp27 and p21 were detected,which to further explore the mechanism of photoaging and provide new ideas for preventing and alleviating photoaging and photodermatosis.Methods:In this study,to establish efficient skin photoaging cell model,HaCaT cells were irradiated with UVB at 30mJ/cm².We interfered with the regulation of the Hsp27 gene to assess its downstream effectors,apoptosis and cell proliferation ability.The cell proliferation ability was detected by CCK-8 assay.The cell cycle phase and apoptosis were detected by flow cytometry.The percentage of positive cells was detected by SA-?-gal staining.The expression of mRNA was detected by qRT-PCR.The expression of protein was detected by Western-blot.The subcellular localization was elucidated by immunofluorescence.Results:1.UVB irradiation?30mJ/cm²?inhibited proliferation activity and increased G2 phase arrest.The positive rate of SA-?-gal staining was also significantly higher than control group.The expression of p53 and p16which were the marker proteins of senescence-associated pathway were significantly increased,and the expression of p21 was decreased,but UVB irradiation caused significant nuclear translocation of p21.2.Knockdown of Hsp27 decreased cell viability and increased the incidence of UVB-induced apoptosis in photoaging HaCaT cell.3.Hsp27 was always mainly localized in the cytoplasm while p21 was detectable in the nuclei UVB irradiation,the co-localization of Hsp27 and p21 was not observed.4.The protein and mRNA expression of p21 was unaffected by Hsp27inhibition,but knockdown of Hsp27 induced p21 protein accumulation in the nucleus until 72 h post-UVB.Meanwhile,the nuclear localization of p21 was significantly translocation to the cytoplasmat at 72 h after UVB irradiation in the shNC group.4.Knockdown of Hsp27 decreased the activation of p-Akt and induced the nuclear accumulation of p21.5.Knockdown of Hsp27 increased the protein of p53,the Bax:Bcl-2ratio,and activating caspase-3.Conclusions:The above results suggested that UVB irradiation at30mJ/cm² successfully construct skin photoaging model in HaCaT cell.The mechanism of senescence may induced by regulating nuclear localization of p21 and p16-dependent pathway.Hsp27 plays a photoprotective role through the inhibition of apoptosis by modulating Akt/p21-dependent pathways and inhibition of the p53/Bax/Bcl-2-dependent mitochondrial apoptotic pathway,which might implicate a potential therapeutic target for treatment of photoaging and photodermatosis.