[Changes in expression of autophagy-related proteins, Beclin-1 and LC3, and effects of rapamycin on their expression in hypoxic-ischemic hippocampus].

OBJECTIVE: To observe changes in the expression of autophagy-related proteins, Beclin-1 and LC3, in the hippocampal tissue of neonatal rats with hypoxic-ischemic brain damage (HIBD) at different time points, and to investigate the effect of rapamycin (Ra) on the expression of the above two proteins. METHODS: A total of 108 7-day-old Sprague-Dawley rats were randomly divided into sham, HIBD, and Ra groups (n=36 each). The HIBD model was established using the modified Rice method. For sham rats, only the left common carotid artery was separated without ligation or hypoxic treatment. For Ra-treated rats, 0.5 mg/kg Ra was administered by an intraperitoneal injection 1 hour before model establishment. The rats were anesthetized and sacrificed to collect brain tissues at 0, 6, 12, 24, 48, and 72 hours after model establishment. Changes in the expression of Beclin-1 and LC3 proteins in rat hippocampus were examined by Western blot. RESULTS: The expression level of Beclin-1 in HIBD rats began to increase at 0 hour, peaked at 24 hours, and then declined thereafter, similar as those of Beclin-1 and LC3-II in Ra-treated rats. The expression level of LC3-II in HIBD rats began to increase at 0 hour, peaked at 12 hours, and then declined thereafter. At all time points, both Beclin-1 and LC3-II expression levels were significantly higher in HIBD and Ra-treated rats than in sham rats (P<0.05); except LC3-II at 12 hours, Beclin-1 and LC3-II expression levels were significantly higher in Ra-treated rats than in HIBD rats (P<0.05). CONCLUSIONS: Hypoxia-ischemia activates autophagy in rat hippocampal cells, while Ra enhances the expression process of autophagy.

Autophagy-regulated AMPAR subunit upregulation in in vitro oxygen glucose deprivation/reoxygenation-induced hippocampal injury.

Autophagy has been implicated to mediate experimental cerebral ischemia/reperfusion-induced neuronal death; the underlying molecular mechanisms, though, are poorly understood. In this study, we investigated the role of autophagy in regulating the expression of AMPAR subunits (GluR1, GluR2, and GluR3) in oxygen glucose deprivation/reperfusion (OGD/R)-mediated injury of hippocampal neurons. Our results showed that, OGD/R-induced hippocampal neuron injury was accompanied by accumulation of autophagosomes and autolysosomes in cytoplasm alongside a dramatic increase in expression of autophagy-related genes, LC3 and Beclin 1 and increased intracellular Ca2+ levels. Pre-treatment with autophagy inhibitor 3-methyladenine (3-MA) significantly reduced this effect. Moreover, the OGD/R-induced upregulation of mRNA and protein expressions of GluR1, GluR2, and GluR3 were also effectively reversed in cells pretreated with 3-MA. Our findings indicate that OGD/R induced the expression of GluRs by activating autophagy in in vitro cultured hippocampal neurons, which could be effectively reversed by the administration of 3-MA.

Histone acetyltransferase inhibitor II induces apoptosis in glioma cell lines via the p53 signaling pathway.

BACKGROUND: Histone acetyltransferase (HAT) inhibitors can inhibit proliferation and induce apoptosis in cancer cell lines. The novel cell-permeable p300/CREB-binding protein (CBP)-selective HAT inhibitor HATi II can reduce histone H3 acetylation and induce chromatin condensation in HeLa cells. Here, we examined the effects and mechanism of action of HATi II in glioma cell lines. METHODS: Cell viability was assessed using the CCK-8 assay. Cell cycle analysis was performed using flow cytometry. Apoptosis was evaluated using Annexin V staining and flow cytometry, Hoechst 33342 staining and the TUNEL assay. Expression and cleavage of caspase-3, caspase-9 and poly ADP-ribose polymerase (PARP) were assessed by Western blotting. Statistical analysis was performed using two-tailed Student's t-tests. The gene expression profiles of U251 glioma cells treated with HATi II or DMSO were analyzed using the Arraystar Human 8 x 60 K LncRNA/mRNA expression array; data was analyzed using MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profiles (≥2-fold) derived from the cluster analyses were subjected to gene ontology and pathway analysis. RESULTS: HATi II inhibited the proliferation of U251, U87, HS683 and SHG44 cells in a dose-dependent manner. HATi II induced cell cycle arrest at the G2/M phase, and induced significant levels of apoptosis, apoptotic body formation and DNA fragmentation in HATi II-treated U251 and SHG44 cells. HATi II induced cleavage of caspase-3, caspase-9 and PARP in U251 and SHG44 cells. In HATi II-treated U251 cells, 965 genes were upregulated, 984 genes were downregulated and 3492/33327 lncRNAs were differentially expressed. GO analysis showed the differentially expressed genes with known functions are involved in a variety of processes; alcoholism, p53 signaling pathway, cytokine-cytokine receptor interaction and transcriptional mis-regulation in cancer were the four most significant pathways. Upregulation of p53 signaling pathway-related genes in HATi II-treated cells was confirmed by quantitative RT-PCR and Western blotting. CONCLUSIONS: HATi II inhibits proliferation and induces apoptosis via the caspase-dependent pathway in human glioma cell lines, possibly by activating the p53 signaling pathway. HATi II deserves further investigation as a novel treatment for glioma.