2,5-Hexanedione Induces Apoptosis In PC12Cells By Altering Expression Of Bax, Bcl-2and Caspase-3

Background: N-Hexane is widely used as an organic solvent in adhesivepreparation, oil and fat extraction, decontamination, oil-based paint industries, shoemanufacturing, and shoe repair. If the protection is inappropriate n-hexane will enterthe body through respiratory tract, skin, digestive tract among which inhalation is themain route of occupational exposure.Long-term exposure to low levels of n-hexaneleads to neuropathy and the clinical manifestations are legs numbness, Muscleweakness, muscle atrophy. Metabolism studies have demonstrated that the neuropathyinduced by n-hexane is mediated by the main active metabolite,2,5-hexanedione (2,5-HD).The mechanism of2,5-HD-induced neuropathy is still not completely understood.Research shows that,2,5-HD can lead to nerve cell and axonal injury and nerve cellsloss and2,5-HD exposure can regulating the protein associated with apoptosis in ratnerve tissue.Moreover, acrylamide and other chemical toxicant exposure cancause nerve cell apoptosis. Which remind us, the damage effect of2,5-HDon neurosystem may associated withapoptosis. Some studies have shown that Bax, Bcl-2and Caspase-3are involved in apoptosis of nerve cells caused by variousof factors.So, whether2,5-HD-induced neuropathy associated with apoptosis andwhether Bax, Bcl-2and Caspase-3are involved in the mechanism of neurotoxicityof2,5-HD? These scientific problems brought to our attentionObjective: To investigate the2,5-HD induced apoptosis of PC12cells and therelated mechanism and provide the basis for elucidating the mechanismof neurotoxicity induced by2,5-HD.Methods: WithPC12cells as the model of neurons,different concentrations of2,5-HD(0、10、20、40mM) were added into the culture of PC12cells for12h、24h、 36h. The cell viabiblity was tested with MTT; HE staining to observe the morphologychange of PC12cells; Annexin V-FITC/PI double staining flow cytometry to detectapoptosis rate;transmission electron microscope to observe the ultrastructural changesof cell nucleus; Hoechst33342fluorescent staining to observe the morphologychange of PC12cells;Real-time PCR to detect the expression of Bax,Bcl-2andCaspase-3on mRNA level;Western blot to detect the expression of Bax,Bcl-2andCaspase-3on protein level.Results: MTT indicated that with the increase of2,5-HD concentrations andthe prolongation of time, the cell viability decreased(P<0.05).HE staining observedthat the PC12cells were obviously changed after treatment of2,5-HD.Hoechst33342fluorescent staining observed characteristic apoptotic nucleus. Transmissionelectron microscope observed that after the treatment of2,5-HD36h40mM, thenucleolus disappeared, chromatinaggregationmobile, have characteristic apoptotic bodyformation. Annexin V-FITC/PI double staining flow cytometry detected the apoptosisrate increased with the increased dose of2,5-HD, shows a dose responserelationship.Real Time PCR indicates that with2,5-HD dose increased and2,5-HDexposure prolonged, the expression of Bax and Caspase-3mRNA increased, theexpression of Bcl-2mRNA decreased.Western blot indicates that with2,5-HD doseincreased and2,5-HD exposure prolonged, the expression of Bax protein increased, theexpression of Bcl-2protein decreased.