The Molecular Mechanisms Of β-hydroxybutyrate Protecting Against Amyloid-beta Peptide-mediated Apoptotic And Oxidative Stress On Neurons

As an endogenous histone deacetylase (HDAC) inhibitor, β-HB can suppressoxidative stress by promoting the histone acetylation of FOXO3A and MT2at thepromoters, which are two genes encoding oxidative stress resistance factors. Inaddition, β-HB was proved to be protective on H2O2-induced cell injury in vitro. Theaccumulation and deposition of amyloid-beta peptide (Aβ) could causeoverproduction of ROS in neuron, further leading oxidative stress and cell apoptosis.The cytotoxicity of Aβ on neuron is considered to be a key step in the pathogenicmechanism of Alzheimer’s disease (AD). Currently, there is less reports about whetherβ-HB has effects on Aβ-induced cytotoxicity. In the present study, an Aβ-intracranialinjection animal model and Aβ-exposed PC12cell model were used to investigatedthe role of β-HB in neuronal cells.To investigate the protective role of β-HB against apoptosis and cell injury, MTTassay and apoptosis assay (AO/EB staining and Annexin V/PI double staining) wereperformed in pc12cells. The results showed that1.0-4.0mMβ-HB pretreatmenteffectively inhibited Aβ-treated pc12cell death on a dose-dependent manner. Inaddition, the β-HB pretreated-cells also showed less damaged cells and apoptotic cellsthan the Aβ-exposed cells, indicating that Aβ-induced cell injury and cell apoptosiswere effectively prevented by treatment with β-HB.In order to explore whether oxidative stress participated in the role of β-HBagainst apoptosis and cell injury, several oxidative/antioxidative indexes weredetected, including the levels of intracellular reactive oxygen species (ROS) and Ca2+,the relative expression of mRNA and the activities of antioxidant enzymes superoxidedismutase (SOD) and catalase (CAT), as well as the content of malonaldehyde (MDA)and nuclear Nrf2level. From the results of in vitro experiments, β-HB was found to be able to protect against Aβ-induced oxidative stress by inhibiting intracellular ROSand Ca2+levels, decreasing MDA level, recovering antioxidant enzymes and nuclearNrf2expression. These studies indicated that β-HB protects neurons against oxidativestress via the induction of Nrf2, which is responsible for cellular antioxidativeabilities.Then, the levels of apoptosis-regulated proteins, including p53activation, Cyt crelease, caspase activation, regulation of Bax/Bcl-2, AIF and Endo G activation wereexamined by Western blots, in PC12cell. The results of in vitro experiments showedthat β-HB effectively prevented Aβ-induced p53activation, Cyt c release, caspaseactivation, up-regulation of Bax/Bcl-2ration, AIF and Endo G activation, indicatingthat β-HB effectively suppressed apoptosis-regulated proteins activation.In addition, the protective effects of β-HB were examined in the Aβ-intracranialinjection animal model. The results of in vivo experiments showed that Aβ exposurecaused increased numbers of TUNEL-positive nuclei and Aβ-injected rats displayedpathological features as expected, including nucleoli ambiguity, circumscription(between nuclei and cytoplasm), obfuscation and cytoplasm diffusion. Aβ exposurealso caused oxidative stress and apoptosis-regulated proteins activation inhippocampus of rats. β-HB pretreatment were proved to prevent these eventseffectively, which is consistent with the results of in vitro experiments.Based on the results of in vivo and in vitro experiments, the present studyprovides novel findings that β-HB offers protection against oxidative stress andapoptosis in Aβ-injected rat hippocampus and in Aβ-treated PC-12cells by promotingNrf2induction, recovering cellular antioxidative activities and suppressing apoptoticproteins activation. These results support the clinical application of β-HB in thetreatment of Alzheimer’s disease.