The results are expressed as a percentage of viability of the control culture
The results are expressed as a percentage of viability of the control culture. Lactate dehydrogenase assay. or trichostatin Eugenol A) also caused synergistic neuroprotection together with lithium. Moreover, combination of lithium and HDAC inhibitors potentiated -catenin-dependent, Lef/Tcf-mediated transcriptional activity. An additive increase in GSK-3 serine phosphorylation was also observed in mice chronically treated with lithium and VPA. Together, for the first time, our results demonstrate synergistic neuroprotective effects of Eugenol lithium and HDAC inhibitors and suggest that GSK-3 inhibition is a likely molecular target for the synergistic neuroprotection. Our results may have implications for the combined use of lithium and VPA in treating bipolar disorder. Additionally, combined use of both drugs may be warranted for clinical trials to treat glutamate-related neurodegenerative diseases. and (for review, see Chuang, 2004a). For example, pretreatment with lithium or VPA protects cultured brain neurons from glutamate-induced apoptosis (Nonaka et al., 1998; Hashimoto et al., 2002; Leng and Chuang, 2006). These two drugs have also been shown to display beneficial effects in cellular and animal models of neurodegenerative diseases such as stroke, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, spinal cord injury, spinal muscular atrophy, retinal degeneration, and human being immunodeficiency disease-1 illness (for review, observe Tariot et al., 2002; Chuang and Priller, 2006). Lithium is known to directly inhibit glycogen synthase kinase-3 (GSK-3) activity (Klein and Melton, 1996; Stambolic et al., 1996). GSK-3 is generally considered to have a proapoptotic part, and its inhibition results in cytoprotection (for review, see Bijur and Jope, 2003; Doble and Woodgett, 2003). Lithium also indirectly inhibits GSK-3 by triggering phosphorylation of GSK-3Ser21/Ser9 (Chalecka-Franaszek and Chuang, 1999; De Sarno et al., 2002; Zhang et al., 2003). VPA, also an anticonvulsant, has been reported to inhibit GSK-3 enzymatic activity and induce GSK-3Ser9 phosphorylation in some, but not all, neurally related systems (for review, observe Rowe et al., 2007). Conversely, VPA is definitely a direct inhibitor of histone deacetylase (HDAC) (G?ttlicher et al., 2001; Phiel et al., 2001). HDAC inhibitors, including phenylbutyrate (PB), sodium butyrate (SB), and trichostatin A (TSA), cause chromatin redesigning through histone hyperacetylation to regulate manifestation of neuroprotective/neurotrophic proteins and proapoptotic/proinflammatory proteins (for review, observe Langley et al., 2005). Several lines of evidence suggest that neuroprotective/neurotrophic effects of lithium and VPA may be related to their medical effectiveness. Long-term lithium treatment raises total gray matter content material (Moore et Eugenol al., 2000a) and enhances levels of (DIV), and then exposed to 50 m glutamate for 24 h to TGFB2 induce neurotoxicity. At the time of experimentation, >92% of cells were CGC neurons. Measurement of cell viability. To determine cell survival inside a quantitative colorimetric assay, the mitochondrial dehydrogenase activity that reduces 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) was assayed (Nonaka et al., 1998). CGCs cultured on 96-well plates were incubated with MTT (125 g/ml) added directly to the growth medium Eugenol for 1 h at 37C. The medium was then aspirated, and the formazan product was dissolved in dimethylsulfoxide and quantified spectrophotometrically at 540 nm. The results are indicated Eugenol as a percentage of viability of the control tradition. Lactate dehydrogenase assay. Cell viability was also quantified having a cytotoxicity detection kit that actions lactate dehydrogenase (LDH) launch according to the instructions of the manufacturer (Roche Applied Technology, Indianapolis, IN). Briefly, an aliquot of 100 l of tradition medium was taken from the CGC tradition grown on a 96-well plate and incubated with the substrate. Total cellular LDH was identified in lysed control cells and compared with LDH levels in treated cell lysates. LDH launch into the medium was indicated as a percentage of total LDH. Analysis of chromatin condensation. Chromatin condensation was recognized by staining of cell nuclei with Hoechst dye 33258. CGCs cultivated on six-well plates were washed with ice-cold PBS and fixed with 4% formaldehyde in PBS. Cells were then stained with Hoechst 33258 (5 g/ml) for 5 min at 4C. Nuclei were visualized under an inverted fluorescence microscope at a wavelength of 360 nm. Western blotting. CGC neurons cultured in six-well plates were detached by scraping and then sonicated for 30 s in lysis buffer, as explained previously (Leng and Chuang, 2006). Protein concentration was identified having a BCA protein assay kit (Pierce, Rockford, IL). Aliquots comprising equal amounts of protein (10 g) from each sample were mixed with an equal volume of SDS sample buffer, loaded into a 4C12%.