Polypeptide From Chlamys Farrer Regulates Cathepsin B/Bid/Bax/Cyt C Signal Pathway Inhibiting UVB-induced Apoptosis In HaCaT Cells

OBJECTIVE To reproduce 20 m J/cm2 UVB-induced apoptosis model of Ha Ca T keratinocytes and explore the molecular mechanisms of Polypeptide from Chlamys farreri(PCF) protecting Ha Ca T cells from UVB-induced apoptosis, focusing on lysosomal Cathepsin B(Cat B)/Bid/Bax/Cyt c signaling pathway. METHODS 20 m J/cm2 UVB-induced apoptotic model of Ha Ca T cells were established by exposing cells to UVB lamp for 30 min, exposed cells were utilized for subsequent assays. Ha Ca T cells were treated as follows: control group, UVB group,UVB+1.42 mmol/L PCF group, UVB+2.84 mmol/L PCF group, UVB+5.69 mmol/L PCF group, UVB+5.68 mmol/L vitamine C group, control si RNA group, Bid si RNA group, Bax si RNA group and Cat B inhibitor CA-074Me(10 mmol/L) group. Hoechst 33258 staining was used to assess apoptosis rate in Ha Ca T cells 18 h after UVB exposure; RT-PCR was used to measure m RNA levels of Cat B, Bid and Bax at 0, 3, 6, 12,18 and 24 h after UVB radiation. At determined Cat B, Bid and Bax expression peak time points, RT-PCR and western blotting were used to assess the effects of PCF to the expression levels of Cat B, Bid and Bax. Furthermore, Cat B inhibitor CA-074 Me, Bid si RNA and Bax si RNA were used to treat Ha Ca T cells, and the protein expression of Cat B, Bid, Bax and Cyt c at peak time points were investigated with western blotting. One way analysis of variance(ANOVA) was used to analyze the data in SPSS 17.0. RESULTS Typical apoptotic cells were observed following UVB exposure. Hochest staining determined apoptosis rate of 35.9%. PCF pretreatment dose-dependently decreased apoptotic rate relative to UVB model group(P<0.05). Time course RT-PCR revealed a 3h peak of Cat B m RNA following UVB exposure(P<0.05). Double peak was observed for Bid at 3h and 18 h post UVB exposure(P<0.05). For Bax, a 6h peak was observed after UVB exposure(P<0.05). PCF and vitamine C pretreatment both significantly decreased the m RNA levels of Cat B, Bid and Bax(P<0.05) at peak time points as determined by RT-PCR. Western blotting revealed the conversion of Bid to its active form, t-Bid. Cat B, t-Bid and Bax protein expression levels were significantly decreased at peak time points by pretreatment of PCF or vitamin C. Cat B inhibitor CA-074 Me significantly decreased Cat B,t-Bid and Bax expression levels and the release of mitochondrial Cyt c relative to UVB model group(P<0.05). si RNA silencing of Bid significantly decreased the expression level of Bax and the release of mitochondrial Cyt c relative to UVB model group(P<0.05) without significant alteration in Cat B expression level. Silencing of Bax resulted in a significant decrease in the release of mitochondrial Cyt c relative to UVB model group(P<0.05) but not in Cat B and t-Bid. CONCLUSION Cathepsin B/Bid/Bax/Cyt c signaling pathway contributes to 20 m J/cm2 UVB-induced apoptosis of Ha Ca T, the cytoprotective effects of PCF is associated with inhibition of this signaling pathway.