| Inflammatory immune mechanisms play a central role in the causation of Alzheimer's disease. Tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine and a "master regulator" of the immune response, is the key initiator of immune-mediated inflammation in multiple organ systems, including the brain. To simulate neuroinflammation and neurotoxicity in-vitro, PC12 (rat pheochromocytoma) cells were stimulated with Lipopolysaccharide (LPS). The effect of Pentoxifylline, a phosphodiesterase E4 inhibitor, on LPS injured PC12 cells was investigated. To elucidate the protective role of Pentoxifylline against the neurotoxic and neuroinflammatory effect of LPS, the cells were pretreated with varying doses of Pentoxifylline (0.1--1mM) for 2 hours before stimulating the cells with 4 microg/ml of LPS for 48 hours. Cell viability assay was performed using MTT (3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium bromide) at a concentration of 0.5 mg/ml, and the assay results showed a maximal increase in cell viability on pre-treatment of LPS treated PC12 cells with 1 mM pentoxifylline, thus confirming its protective effect. Furthermore, western blot analysis showed the reduction in the expression of TNF alpha, with increasing doses of pentoxifylline. This proved that the protective effect of pentoxifylline is due to the inhibition of TNF alpha. In parallel, agarose gel electrophoresis demonstrated that neurotoxicity induced with LPS trigerred apoptosis (characterized by DNA fragmentation), and pentoxifylline exerted its protective effect through inhibition of the apoptotic pathway (reduction in DNA fragmentation). This anti-apoptotic effect of Pentoxifylline was confirmed by the decrease in the expression of pro-apoptotic protein Bad, and by the increase in the expression of the antiapoptotic protein Bcl2 on western blot analysis. Furthermore, LPS induced an increase in the caspase 3 levels, which were reduced by Pentoxifylline. The present study indicates that Pentoxifylline may be a promising approach for the treatment of Alzheimer's disease and other neurodegenerative diseases, through inhibition of apoptosis. |
