H₂O₂ Induced Keratinocytes Death Through A Cyclophilin D Intrinsic Signaling Pathway

Objective H₂O₂-induced skin cell death involves the opening of mitochondrial permeability transition pore（m PTP）,which leads to both apoptotic and necrotic cell death. Cyclophilin D（Cyp-D） translocation to the inner membrane of mitochondrion acts as a key component to open the m PTP. We detect the expression of Cyp-D of H₂O₂-induced primary cultured human skin keratinocytes and Ha Ca T cell line by Western-Blot.We created a stable Cyp-D deficiency primary cultured human skin keratinocytes by expressing Cyp-D-sh RNA through lentiviral infection and detect Cyp-D and viable cells after H₂O₂ treatment.We dectect Cyp-D and viable cells in Cs A cotreatment primary cultured human skin keratinocytes.At last,we detect viable cell in primary keratinocytes over-expressed Cyp-D.In the current study, we are set to understand the potential role of Cyp-D in H₂O₂-induced skin cell damage.Methods 1、We cultured primary cultured human skin keratinocytes and in Ha Ca T cell line.The cells were divided into four groups:primary cultured human skin keratinocytes with 500 μmol/L H₂O₂ induced,Ha Ca T cell line with 500 μmol/L H₂O₂ induced,primary cultured human skin keratinocytes control group,Ha Ca T cell line control group. Cells pretreated with N-Acetyl-Cysteine（NAC） were treated with H₂O₂, detect Cyp-D.with Western blot. 2、primary cultured human skin keratinocytes were pretreated with Cyclosporin A,set control group,detect Cyp-D by Western blot and detect viable cell by MTT stain and PI stain. 3、We created a stable Cyp-D deficiency primary cultured human skin keratinocytes by expressing Cyp-D-sh RNA through lentiviral infection. We have sh RNA group,Green fluorescent proteins group as control group. primary cultured human skin keratinocyteswere transfected with p Super-puro-Cyp-D or p Super-puro,Stable cells were selected by puromycin.We detect viable primary cultured human skin keratinocytes treated with H₂O₂ by MTT stain and PI stain.Results 1、H₂O₂ induced a significant Cyp-D upregulation in primary cultured skin keratinocytes and Ha Ca T cell line. Anti-oxidant N-Acetyl-Cysteine（NAC） significantly inhibited H₂O₂-induced Cyp-D expression. 2、We successfully created a stable primary skin keratinocytes line expressing Cyp-D sh RNA.The number of viable skin keratinocytes after H₂O₂ treatment increased from 46.0 ± 2.6% to 79.1 ± 3.9% in Cyp-D deficient cells（P < 0.05）, while the percentage of PI positive cells decreased from 32.1 ± 3.2% to 18.4 ± 3.7%（P < 0.05）. 3、Cs A didn’t affect H₂O₂-induced Cyp-D expression. The number of viable skin keratinocytes after H₂O₂ treatment increased from 48.2 ± 3.4% to 72.3 ± 5.3% with Cs A co-treatment（P < 0.05）, while the percentage of PI positive cells decreased from 28.2 ± 3.7% to 16.3 ± 2.3%（P < 0.05）.Conclusion 1、H₂O₂-induced skin keratinocytes show cyclophilin D（Cyp-D） Upregulation; 2、H₂O₂-induced skin keratinocytes death is diminished after Cyp-D silencing; 3、Cyclosporine A（Cs A） inhibited UVB/H₂O₂-induced skin keratinocytes death; 4、Overexpression of Cyp-D caused spontaneous cell death in primary cultured keratinocytes.