| Objective To investigate the epigenetic effects of nerve injuries induced by hyperbilirubinemia, primary neuronal culture was introduced in this study to build bilirubin-induced neurotoxicity model. Following the observation of neuronal structure abnormity and detection of cell dysfunctions, the alteration in some intracellular endpoints at molecular biological level were determined inculding intracellular calcium concentration, caspase-3 activity and total Tau microtubule-associated protein, and the global DNA methylation was subsequently detected in order to explore the epigenetic molecular mechanism of hyperbilirubinemia.Methods (1) Health neonatal SD rats (24h or less) were chosen to establish a cortical neurons primary system with serum-free medium Neurobasal Medium (Invitrogen, USA). Cells were maintained at 37 ℃ in a humidified atmosphere of 5% CO2.Neuron-special enolase (NSE) immunofluorescence was used to identify cell types. (2) Cell cultured for 3 days (D3) were exposed to bilirubin at different concentrations of 25,50,100 μM in culture medium for 4,12,24,48h, and then morphological pathological changes were observed by HE staining. (3) Cell viability was measured by MTT assay after bilirubin exposure. (4) The apoptosis induced by bilirubin was observed by fluorescence microscope following Hoechst33342 labeling. (5) Ca2+ content in cellular plasma was determined by a fluorescent staining using Fluo-3 as a probe. (6) Caspase-3 activity assay kit (Beyotime,China) was used to estimate the alteration in Caspase-3 activity with by microplate reader. (7) The expression of T-Tau protein was detected by using an Elissa T-Tau protein quantification kit (Shanghai Crystal Day Biotech, China). (8) By use of an immune-fluorescence staining approach, the level of global DNA methylation was explored in neurons nuclei of cultured cells treated by bilirubin at concentrations of 0,25,50 and 100μM for 24h.Results (1) A primary culture system of cortical neurons in rat was successfully established with serum-free medium, and the cultured cortical neurons were identified by NSE-specific immunofluorescence assay. (2) Bilirubin could lead to shrinked cell bulk, karyopyknosis, shortened neurite and decrease in the number of nerve nodes after bilirubin exposure. (3) Bilirubin caused the decrease in cell viability with a negative dose-effect. The similar negative time-effect relationship was observed in the indicator of the cell activity. (4) The rate of apoptosis significantly increased at different doses of bilirubin exposure, just as the extension of exposure time. (5) Bilirubin destroyed intracellular Ca2+homeostasis, and led to intracellular Ca2+ concentration increasing after exposure at various bilirubin doses. The similar positive time-effect relationship was observed. (6) The results also showed that bilirubin enhanced the intracellular Caspase-3 activity with positive dose-effect and time-effect relationships. (7) The content of intracellular Tau microtubular-associated protein increased with increasing exposure doses. When exposure time was extended to 12h, it showed a significant difference(F3,20=13.391,p=0.002). However, Tau protein content began to drop down after 48h exposure(F3,20=13.313,p=0.002). (8) The global DNA methylation significantly increased at each dose exposure of bilirubin for 24h(F3,28=11.218,p=0.001).Conclusion (1) Hyperbilirubinemia can lead to neurotoxicity manifested as that morphological alteration in cultured neurons, decreasing cell viability and increase in the rate of apoptosis. (2) Bilirubin also affects intracellular Ca2+homeostasis and activates Caspase-3. In addition, Bilirubin may causes increasing the content in neuron cytoplasm, which may be the outcome of bilirubin-induced insult on the microtubule system. (3) Hyperbilirubinemia may induce increase in DNA methylation under this condition of our experiment study, which offers a foundation to further study epigenetic mechanisms of hyperbilirubinemia-induced neural toxicity. |
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