Protective effect of tubuloside B on TNFalpha-induced apoptosis in neuronal cells

Aim: To investigate the neuroprotective effect of tubuloside B, one of the phenylethanoids isolated from the stems of Cistanche salsa, on tumor necrosis factor-alpha (TNFalpha)-induced Apoptosis in SH-SY5Y neuronal cells.

Methods: Cell viability was analyzed using MTT assay. Apoptotic cells were detected using Hoechst33342 staining, and confirmed by DNA fragmentation and flow cytometric analysis. The activity of Caspase-3 was measured with special assay kit. The concentration of free intracellular calcium was determined with the probe Indo-1 by spectrometer. The level of intracellular Reactive Oxygen Species and the potential of mitochondrial membrane were determined by laser scanning confocal microscopy (LSCM) combined with fluorescence probe H2DCFDA or JC-1 respectively.

Results: SH-SY5Y cells treated with TNFalpha 100 microg/L for 36 h showed typical morphological changes of Apoptosis. DNA ladder could be observed by Agarose gel electrophoresis. The highest percentage of apoptotic cells accumulated to 37.5 %. Following 36 h treatment with TNFalpha, accumulation of intracellular ROS and [Ca2+]i and decrease in mitochondrial membrane potential were observed, and Caspase-3 activity increased by about five-fold compared with controls. However, pretreatment with tubuloside B (1, 10, or 100 mg/L) for 2 h attenuated the TNFalpha-mediated Apoptosis. The antiapoptotic action of tubuloside B was partially dependent on an anti-oxidative stress effects, maintain of mitochondria function, decrease of concentration of free intracellular calcium and inhibition of Caspase-3 activity.

Conclusion: Tubuloside B has the neuroprotective capacity to antagonize TNFalpha-induced Apoptosis in SH-SY5Y cells and may be useful in treating some neurodegenerative diseases.