Polypeptide From Chlamys Farreri Inhibits Murine Thymocytes Radiative Damage Induced By UVB

AIM The study establishs the apoptotic models of thymocytes caused by UVB radiation in vitro to investigate the inhibit effects and mechanism of PCF (polypeptides from Chlamys farreri) on thymocytes from the mouse damaged by UVB radiation. MATHODS Using 45mJ/cm2 UVB to radiate mouse thymocytes, UVB-induced apoptotic model of thymocytes is established by orthogonal design and apoptotic rate is determined by flow cytometry PI staining. Cells are divided into six groups: control group, UVB model group, UVB+5.69 mM PCF group, UVB+2.84 mM PCF group, UVB+1.42 mM PCF group, UVB+5.69 mM Vitamine C group. First, the study investigates the effect of PCF on redox state in mouse thymocytes after UVB radiation. The anti-hydroxy radical, anti-superoxide anion level, activity of xanthine oxidase(XOD)and NADPH oxidase in thymocytes is detected after UVB radiation. Effect of PCF on UVB-induced production of ROS is detected by 2', 7'-Dichlorofluorescin diacetate (DCFH-DA). Second, the study investigates the effect of PCF on apoptosis signal transduction pathway from mitochondria and cell surface receptor in mouse thymocytes induced by UVB radiation.The mitochondria membrane potential and the free calcium variation on thymocytes are tested using Rhodamine 123 and Fluo-3-AM fluorescein stain respectively. Bcl-xl and Bid proteins are examined by immunocytochemical technica. The caspase-3 activity is measured by Flow Cytometry (FCM). Western blot analysis is performed to determine the release of cytochrome c, expressing levels of FADD and caspase-8. The effects of PCF and caspase-8 inhibitor z-IETD-fmk on UVB-induced apoptosis of thymocytes are investigated by electron microscope. Expressing levels of Fas mRNA are detected by RT-PCR. And last, the effect of PCF on the different of gene expression profile in thymocytes after UVB radiation are studied by gene chip technique. The activation of thioredoxin (Trx) and protein kinase B (Akt/PKB) is investigated by western-blot. ASK1, JNK activation, DNA ladder and Mitochondria memberine potential are also investigated after pretreatment with or without PI3K/Akt pathway inhibitor LY294002. P53 protein is examined by immunocytochemical technica. The p38mRNA and P21 mRNA expressions are detected through the technique of in situ hybridiation. The expression of c-fos and c-jun is observed by RT-PCR. RESULTS Results of orthogonal experiment suggest that 45mJ/cm2 UVB and 2 h incubation after radiation make up the best apoptotic model. 1.42~5.69 mM PCF increases the anti-hydroxy radical and anti-superoxide anion level, decreases the activity of xanthine oxidase(XOD), NADPH oxidase and the ROS accumulation in thymocytes. PCF can modulate the expression of molecular that related to apoptosis signal transduction pathway from mitochondria and cell surface receptor. PCF can stabilize the mitochondria membrane potential, decrease free calcium, increase the expression of Bcl-xl, reduces the expression of Bid and the releasing of cytochrome c and caspase-3 activity. PCF can inhibit the expression levels of FADD and caspase-8 that related to apoptosis signal tranduction pathway from cell surface receptor. The ultrastructural of thymocytes is less damaged by UVB when pretreatment with the caspase-8 inhibitor z-IETD-fmk and PCF. The ruslts of genechip indicate that 104 genes which have identified functions show a differential expression in PCF group, with 56 genes up-regulated and 48 genes down-regulated when compared with model group. Western-blot shows that PCF can active Trx and Akt, inhibit the activation of the ASK1-JNK/p38 apoptosis pathway. When pretreatment with PI3K/Akt pathway inhibitor LY294002 with PCF, the fragment of DNA ladder is increased while the mitochondria memberine potential is decreased. PCF can elevate the expression of p53 protein, p21 mRNA which related to cell cycle, cut down the expression of transcription factors c-fos and c-jun mRNA. CONCLUSION PCF can reverse the cell damage radiated by UVB. PCF fundamentally depresses active oxygen free radical product, function as a good antioxidant reagent from ocean. PCF can inhibit apoptosis signal transduction pathway from mitochondria and cell surface receptor, increase antioxygen protein and genes which related to survival, inhibit the activation of the ASK1-JNK/p38 apoptosis pathway, cut down the expression of transcription factors. PCF exerts its antioxidation, anti-apoptosis and anti-UVB radiative damage by modulate the expression of numerous genes that related to cellular oxidoreduction, apoptosis, signal transduction, cell cycle and transcription regulation. These results show that PCF is a good preventive regimen from ocean against UVB radiation.