Telomerase Activity-independent Function Of TERT Is Involved In Acrylamide-induced Neuron Damage

Objectives: The aim of this paper is to investigate the neurotoxicity of acrylamide（ACR）on rat’s cerebral neurons and its mechanism.Methods:1.In vivo:16male rats were randomly divided into four groups: control group,low-dose group, middle dose group and high-dose groups. The control group wasinjected with saline and the low, middle and high dose group were injected withdifferent concentrations of ACR (30mg/kg,40mg/kg,50mg/kg), respectively. After amonth，the apoptotic rate of cortical neurons and hippocampus neurons in SD rat braintissue was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL) assay. The expressions of cleaved Caspase-3, Bcl-2and TERT in SD ratbrain tissue were analyzed by Western blot analysis.2. In vitro: Primary cortical neurons were prepared from newborn rats and divided intocontrol group and the ACR-treated group. The ACR-treated cells were pretreated withACR (2.5mM/L，5mM/L，10mM/L) for24h. The cell viability was assessed by theMTT reduction assay and the apoptotic rate was determined by Flow cytometrymethod. The expressions of cleaved Caspase-3, Bcl-2and TERT were analyzed by themethod of Western blot. Isolation of cytosol and mitochondria was performedaccording to the instructions for the Mitochondria/Cytosol Isolation Kit. TERTexpression in cortical neurons and other fractions including cytosol and mitochondriawere detected by western blot. Meanwhile, TERT expression in cortical neurons wasmeasured by Real time PCR. Mitochondria was extracted according to the instructionsfor the Mitochondria/Cytosol Isolation Kit. The activities of Superoxide dismutase(SOD), glutathione (GSH), glutathione peroxidase (GSH-Px) and malondialdehyde(MDA) content were measured by kit assays according to the manufacturer’sinstructions. And total intracellular ROS levels were measured in cortical neuronsexposed to different concentrations of ACR using the DCFDA method. Results:1. In vivo: TUNEL-positive cells appeared occasionally in control group, diffusedwidely in control group, and were most frequently observed in those of high-dose group.Western blotting analysis revealed that Bcl-2and TERT expression was obviouslydecreased by ACR-treatment, whiles Cleaved caspase-3expression was increased in ratbrain tissue. Pretreatment with ACR (30mg/kg,40mg/kg,50mg/kg) for a month counlddown-regulated the expression of Bcl-2and TERT, but increased cleaved caspase-3expression compared to control group, which presented in a dose-dependent manner.2. In vitro: ACR inhibited the proliferation and induced apoptosis of cortical neurons.Data of MTT reduction assay and flow cytometry revealed that the viability reductionand the average apoptotic rate were obviously decreased in ACR-treated groupcompared with control group. Pretreatment of cortical neurons with ACR (2.5mM/L,5mM/L,10mM/L) for24h resulted in a dose-dependent decrease in viability andincrease in the apoptotic rate compared with control group (P<0.05or P<0.01).Western blotting analysis revealed that in rat cortical neurons Bcl-2expression wasobviously decreased by ACR-treatment, whiles Cleaved caspase-3expression wasincreased. Pretreatment with ACR (2.5mM/L,5mM/L,10mM/L) for24hdown-regulated the expression of Bcl-2, but increased cleaved caspase-3expressioncompared to control group, which presented in a dose-dependent manner. ACR inducedROS generation of cortical neurons, decreased the levels of SOD, GSH and GSH-Pxand increased the concentration of MDA in mitochondria. Compared to control group,the decreased of SOD, GSH-PX activities and GSH content and the increased of MDAlevel were observed significantly in ACR-treated group(P<0.01). Preincubation withACR(2.5mM/L,5mM/L,10mM/L)obviously decreased the activities of SOD andGSH-PX and GSH content, whiles increased MDA level significantly compared tocontrol group(P<0.05or P<0.01) in a dose-dependent manner. Compared with controlgroup, TERT expression was increased with ACR at the concentration of2.5mM to10mM (p<0.05or p<0.01). However, TERT mRNA and protein expression in cortical neurons significantly decreased at higher concentration of10mM compared to5mMACR-intervened group (p<0.05or p<0.01). We also studied TERT protein expression indifferent fractions including cytosol and mitochondria, it was found that the changingtrends of mt-TERT expression among ACR intervened groups was consistent with thatin cortical neurons, while there was no significance in the change of TERT expression incytosol.Conclusion:1. ACR inhibited the proliferation of cortical neurons in rat brain..ACR increased theexpression of Cleaved Caspase-3and decreased Bcl-2and expression in rat’s braintissue.3. ACR induced ROS generation of cortical neurons, decreased the activities of SODand GSH-Px, GSH content and increased MDA content in mitochondria of corticalneurons.4. In vivo,ACR inhibited the expression of TERT in rat brain; in vitro,TERT expressionwas increased with ACR at the concentration of2.5mM to10mM. However, TERTmRNA and protein expression in cortical neurons significantly decreased at higherconcentration of10mM compared to5mM ACR-intervened group.But there is nosignificance changes in cytosol. So ACR influenced TERT expression mainly inmitochondria.5. Above the all，our study suggested ACR that the enhanced mt-TERT expressionobserved in ACR-induced neurons might be a compensatory response involved in theattempt tocounteract the cytotoxic responses during ACR exposure.