Abstract
To investigate the molecular mechanisms of polypeptide from Chlamys farreri (PCF)'s anti-apoptotic effect, HaCaT cells were exposed to 20 mJ/CM(2) UVB, with or without pretreatment of TGF-β1 antagonist SB431542, inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea sulfate (SMT), nitric oxide scavenger carboxy-PTIO, or 1.42, 2.84, and 5.69 mM PCF, or iNOS transfection (without UVB exposure). Apoptosis was confirmed with Hoechst 33258 staining; RT-PCR and western blot were used to determine the expression levels of iNOS and TGF-β1 signaling pathway. Both UVB exposure and iNOS transfection-induced apoptosis in UVB-exposed HaCat cells, while PCF, SB431542, SMT, and carboxy-PTIO all inhibited UVB-induced apoptosis. TGF-β1, Smad4, and Smad7 mRNA levels were all altered, similarly, iNOS, TGF-β1, and pSmad2/3 protein levels were all altered in UVB-exposed HaCaT cells. In pretreated cells, SB431542, SMT, carboxy-PTIO, and 1.42-5.69 mM PCF all inhibited UVB-induced apoptosis. Moreover, PCF treatment inhibited the expression levels of iNOS, TGF-β1, pSmad2/3, and Smad4, while increased the expression level of Smad7. SB431542 did not significantly alter iNOS expression, while SMT and carboxy-PTIO significantly altered TGF-β1 signaling level. The anti-apoptotic effect of PCF in UVB-exposed HaCaT cells involves the inhibition of iNOS expression and subsequently inhibition of TGF-β1 signaling pathway.
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