| Background: Several studies mentioned the association of oxidative stress and neuroinflammation with Parkinson's disease (PD). In the MPTP-injured Pheochromocytoma cell line (PC12 cells), a rat model of PD, MPTP induces neuronal cell death through the impairment of complex I activity in the mitochondria, increasing the production of reactive oxygen species (ROS) such as nitric oxide (NO), and pro-inflammatory mediators like Interleukin-1beta (IL-1beta) and TNF-alpha. Fisetin, a flavonoid, has been reported to exhibit various pharmacological activities including anti-oxidant and anti-inflammatory effects. Fisetin has also been reported to inhibit the production of NO and TNF-alpha in RAW 264.7 macrophages and mast cells, and inhibit the UV-induced oxidative stress and the activation of NF-kappaB and MAPK signaling in human lens epithelial cells. Ibuprofen has potent anti-fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils in vitro, and is also a commonly used NSAID that has been shown to reduce PD risks. This study, investigated the mechanisms underlying the neuroprotective effect of two anti-inflammatory drugs, Fisetin and Ibuprofen in MPTP-stimulated PC12 cells.;Methods: PC12 cells were pretreated with varying doses of fisetin (0.5 mug/mL, 1 mug/mL and 2 mug/mL) ibuprofen (2 mug/mL), or both drugs in combination for 2 hours, prior to MPTP (2 mM) stimulation for 48 hours. Cell viability was measured by MTT assay, IL-1beta levels, TNF-alpha levels and Cytochrome c levels were quantified by ELISA, alpha-Synuclein, TNF-alpha, NF-kappaB, pro-apoptotic Bax and anti-apoptotic Bcl-xL expression were assessed by western blot, and NO levels by colorimetric method were also evaluated. Quantitative detection of Caspase-3 activity in cellular lysates after induction of apoptosis was determined by Caspase-3 Colorimetric assay. Effect of fisetin on MPTP-induced apoptosis in differentiated PC12 cells was also evaluated by Immunofluorescence method.;Results: Pretreatment with fisetin produced a dose-dependent increase in cell viability, and a dose-dependent decrease in the expression of alpha-synuclein, pro-inflammatory mediators TNF-alpha, transcription factor NF-kappaB and pro-apoptotic Bax, and increased anti-apoptotic Bcl-xL expression in a dose-dependent manner. Ibuprofen at 2 mug/mL also decreased alpha-synuclein, TNF-alpha, NF-kappaB and Bax expression, and increased Bcl-xL expression in MPTP stimulated PC12 cells. The combination of both drugs showed a greater effect in inhibiting the expression of alpha-synuclein, TNF-alpha, NF-kappaB and Bax, while increasing the Bcl-xL expression. Fisetin also suppressed the production of NO, Cytochrome c, TNF-alpha and IL-1beta significantly, while Ibuprofen potentiated MPTP-induced Cytochrome c levels, and that both drugs combined showed a significant effect in suppressing the production of NO, TNF-alpha, IL-1beta and Cytochrome c. Fisetin reduced MPTP-induced apoptosis in differentiated PC12 cells as shown by Immunofluorescence method and this was further confirmed by the suppressive effect of fisetin on MPTP-induced caspase-3 activity as assessed by the caspase colorimetric assay. Combination of fisetin and ibuprofen increased cell viability as well as reduced inflammation and apoptosis caused by MPTP in differentiated PC12 cells. Along with our previous studies showing a synergistic effect of fisetin and Ibuprofen in inhibiting alpha-synuclein expression, our present data supports a therapeutic potential for fisetin and ibuprofen in the treatment of oxidative stress and neuroinflammation associated with Parkinson's disease. |
