Objective: To investigate the influence of antisense phosphorothioate oligonucleotides on peroxisome proliferator-activated receptors (PPARbeta) in the TNF-alpha mediated apoptosis of HaCat cells.
Methods: HaCat cells were resuscitated and randomly divided into normal control (without transfection), sham (merely with liposome transfection), scrODN (with transfection of 4 micromol/L PPARbeta scrODN), asODN (with transfection of 4 micromol/L PPARbeta asODN), TNF-alpha with transfection of 10 micromol/L TNF-alpha), scrODN + TNF-alpha with 10 micromol/L TNF-alpha stimulation after transfection of 4 micromol/L PPARbeta scrODN), asODN + TNF-alpha with 10 micromol/L TNF-alpha stimulation after transfection of 4 micromol/L PPARbeta asODN) groups. The mRNA and protein levels of PPARbeta were determined with RT-PCR and Western blotting, respectively. The changes in cell morphology were observed with Hoechst 33258 fluorescent staining to quantitate apoptotic rate of nuclei. The effect of PPARbeta asODN on HaCat cell viability was assayed with MTT method. Activation of caspase-3 was evaluated with caspase colorimetric analysis kit.
Results: The mRNA and protein expression of PPARbeta in normal control, sham, scrODN groups were similar, but it decreased obviously in asODN group. The nuclear apoptotic rate in normal control, scrODN and asODN groups were rather low, and the caspase-3 activity in these groups was also low. After 24 hours of culture, the nuclear apoptotic rate in TNF-alpha and scrODN + TNF-alpha groups were (33.1 +/- 2.7)% and (32.9 +/- 3.0)%, respectively, while that in asODN + TNF-alpha group was obviously increased (58.8 +/- 4.6)%, with the caspase-3 activity significantly higher, but the number of live cells markedly lower than that in the former 2 groups (P < 0.05).
Conclusion: PPARbeta expression can promote the apoptosis of HaCat cells mediated by TNF-alpha.