The results showed that treatment with MDMA for 18, 24 and 48 h significantly induced a time-dependent increase in ration of LC3B-II to LC3B-I to 1 1.3-fold, 2.2-fold, and 2.7-fold of control values, respectively (n?=?3, em p /em 0.05) (Fig. 3 times. Densitometric analysis of the immunoreactive bands was performed using ImageJ software (NIH). Lactate dehydrogenase (LDH) assay As an index of cell death, release into extracellular medium was measured using the LDH assay. Briefly, supernatants were collected at the times indicated and intact cells were lysed using Triton X-100-made up of lysis buffer. The amount of LDH release was decided spectrophotometrically at 492 nm using the LDH-Cytotoxicity Assay Kit (Sigma). Percent cell death was calculated using the formula: % cytotoxicity ?=? LDH release (OD492)/maximum LDH release (OD492), after correcting for baseline absorbance of the LDH release at 492 nm. EPZ004777 hydrochloride Statistical analysis The quantitative data are presented as mean S.D. The Student test was used to calculate values, with a value 0.05 defined as statistically significant and a em P /em 0. 01 as highly statistically significant. Results MDMA decreased neuronal cell viability in a dose-dependent manner We first examined the dose effect of MDMA on cell viability. Cortical neuron cultures were treated with different concentration of MDMA (0.5, 1, 1.5, 2 mM) for 48 h, and then the cell viability was determined by MTT assay. MDMA produced a concentration-dependent decrease in cell viability (Fig. 1). Treatment with 0.5 and 1 mM MDMA for 48 h did not have an obvious effect on the cell viability, as approximately 95% and 91% of the cell viability compared to normal control. When cells were treated with higher concentration of MMDA (1.5 and 2 mM), the cell viability was significantly decreased to 75% and 52%, respectively. Open in a separate window Physique 1 Effect of MDMA on cortical neuron viability.Cultured cortical neurons were treated with different concentration of MDMA for 48 h, and then cell viability was determined by MTT EPZ004777 hydrochloride assay. The data are expressed as percentage of untreated controls (mean S.D., n?=?3, quadruplicate wells for each condition). * em P /em 0.05; ** em P /em 0.01 vs control. MDMA induced autophagy and neurite degeneration in a dose- and time-dependent manner We investigated the dose-dependent effect of MDMA on autophagy activity and neurite outgrowth in cultured rat cortical neurons. Autophagy activity was assessed using immunoblotting and immunofluorescence to detect the essential autophagy protein LC3. Upon autophagy activation, the LC3-I protein localized in the cytoplasm is usually cleaved, lipidated, and inserted as LC3-II into autophagosome membranes. Thus, an increase in the amount of the smaller-molecular-weight LC3-II protein and an increase in the LC3-II/LC3-I ratio are a hallmark of autophagy and correlate with an increased number of autophagosomes . In the present study, cortical neurons were treated with different concentrations (0.5 mM, 1 mM, 1.5 LANCL1 antibody mM, 2 mM) of MDMA for 48 h and then performed western blot analysis using LC3 antibody. The results revealed a dose-dependent increase in ratios of LC3B-II/LC3B-I. Treatment with MDMA at 0.5, 1, 1.5, 2 mM significantly increased the ratios of LC3B-II/LC3B-I to 1 1.5-fold, 2.6-fold, 3.5-fold, and 4.2-fold of the control values, respectively (n?=?3, em p /em 0.05) (Fig. 2A and 2B). EPZ004777 hydrochloride Increase in ratio of LC3B-II/LC3B-I are known EPZ004777 hydrochloride to represent upregulation of autophagy. Beclin-1 expression are known to be involved in the formation of preautophagosomal structures. However, the expression level of beclin-1 was not affected by MDMA (Fig. 2A). We then performed double immunofluorescence to correlate the LC3 expression and neurite outgrowth using anti-MAP2 (a general neurite marker) and anti-LC3B antibodies after 48 h of MDMA treatment. As shown in Fig. 2C, in non-treated control neurons, the pattern of LC3B immunoreactivity was light, diffuse, and cytoplasmic within the cell body. At 500 M of MDMA treatment, anti-LC3B staining exhibited a more condensed diffuse distribution. After exposure to 1 mM MDMA, common cytoplasmic LC3B punctate was formed notably in almost all.