Fig 1.
Neuroprotective effect of PRL against glutamate-induced excitotoxicity. Cell viability was assessed by the Syto-13 and propidium iodide (PI) assay and mitochondrial activity was assessed by the MTT reduction assay.
Cell cultures were treated with PRL or Glu alone, or both PRL (10 ng/mL for 72 h) and Glu (100 μM for 24 h). (A) Representative images from neurons stained with Syto-13 (green) and PI (red) in cultures exposed to the different treatments: a-c, Vehicle (saline solution). d-f, PRL (10 ng/mL). g-i, PRL/Glu (10ng/mL and100 μM respectively), j-l, Glu (100 μM). White arrows indicate red condensed nuclei indicative of dead cells. (B) Values are the mean ± SD (n = 4 independent experiments). (C) Mitochondrial activity was assessed by MTT reduction. (D) LDH activity in medium culture expressed by the Δ Abs at 340nm. Data were analyzed by one-way ANOVA followed by a Tukey´s post hoc test *p<0.05 vs Glu, **p<0.001 vs Glu. Scale bar = 100 μm.
Fig 2.
Effect of PRL on [Ca2+]i in a cultured of hippocampal rat neurons.
(A) Control, neurons stimulated with PRL (10 ng/mL for 6 min). (B) Neurons stimulated with Glu (100 μM for 5 min). (C) Neurons pretreated with PRL (10 ng/mL for 72 h) and then exposed to Glu (100 μM for 6 min). Each recording of [Ca2+]i represents an independent experiment. (D) Bars represent the mean ± SD [Ca2+]i from 4–9 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey´s post hoc test.** p<0.01 versus Glu.
Fig 3.
PRL prevented procaspase-3 cleavage in hippocampal neurons exposed to Glu.
(A) Results from Western blot and densitometry analyses are expressed as the relative ratio of cleaved caspase-3/GAPDH. Bars represent the mean ± SD from 4 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey´s post hoc test. ** p<0.01 vs Glu. Control (Saline Solution; Ctrl), PRL (10 ng/mL), PRL/Glu (10 ng/mL and 100 μM, respectively), Glu (100 μM), rat uterus in estrous (ERU).
Fig 4.
PRL decreased the proapoptotic ratio Bax/Bcl-2 in hippocampal neurons exposed with Glu.
The results of Western blot and densitometry analyses are presented as the relative ratio of protein/β-actin. (A) Bcl-2 protein content and Bax protein content. (B) Pro-apoptotic ratio of Bax/Bcl-2 proteins. Bars represent the mean±SD from 4 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey´s post hoc test. *p<0.05 PRL/Glu vs Glu, ** p<0.01 vs Glu. Control (Ctrl), PRL (10 ng/mL), PRL/Glu (10 ng/mL and 100 μM, respectively), Glu (100 μM).
Fig 5.
PRL induced NF-κB activation in hippocampal neurons. Nuclear translocation of NF-κB was assessed by immunochemistry.
Cell cultures were exposed to PRL (10 ng/mL for 72 h) and Glu (100 μM for 24 h), or were treated with PRL or Glu alone. Representative photomicrographs from primary cultures of rat hippocampal neurons: a-d, Vehicle. e-h, PRL (10 ng/mL). i-l, PRL/Glu (10ng/mL and 100 μM respectively), m-p, Glu (100 μM). Cytoskeleton was stained green with Phalloidin 488: nuclei were stained blue with Hoechst and in NF-κB protein was labeled in red. White arrows indicate nuclear translocation of NF-κB. Scale bar = 24.08 μm.
Fig 6.
Proposed molecular mechanisms involved in the PRL-mediated neuroprotective effect against Glu-induce excitotoxicity in primary cultures of rat hippocampal neurons, which includes.
a) Regulation of [Ca2+]i homeostasis, b) NF-κB activation and the c) Concomitant overexpression of anti-apoptotic proteins. Question marks and dotted lines indicate hypothetical relationships.