ABSTRACT
AIMS: Inflammation is strongly associated with the mechanism of ß-cell failure in type 2 diabetes mellitus (T2DM). Blockade of the key proinflammatory cytokine IL-1ß has been implicated as a promising therapeutic strategy for the prevention and treatment of type 2 diabetes. In this study, we developed an IL-1ß-targeted therapeutic vaccine consisting of an IL-1ß epitope peptide (A1ß) and assessed its efficacy on a diabetic KK-Ay mouse model. MAIN METHODS: KK-Ay mice were immunized with A1ß for three injections at a 2-week interval. The induced antibody titers, body weights and blood glucose levels were monitored every two weeks. Then the intraperitoneal glucose tolerance test and insulin tolerance test were performed. The ß-cell mass, ß-cell apoptosis and proliferation were evaluated by immunofluorescence. IL-1ß gene expression in islets was also measured by quantitative RT-PCR. KEY FINDINGS: A1ß immunization induced robust antibody responses, reduced body weight gain, improved glucose tolerance and insulin sensitivity in KK-Ay mice. Moreover, A1ß restored ß-cell mass, inhibited ß-cell apoptosis, enhanced ß-cell proliferation and downregulated IL-1ß expression. SIGNIFICANCE: The novel IL-1ß-targeted epitope vaccine has the therapeutic potential for T2DM.
Subject(s)
Blood Glucose/metabolism , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/therapy , Interleukin-1beta/immunology , Vaccines/therapeutic use , Animals , Antibodies/analysis , Apoptosis , Body Weight , Cell Proliferation , Diabetes Mellitus, Type 2/genetics , Disease Models, Animal , Down-Regulation/genetics , Down-Regulation/immunology , Epitopes/immunology , Glucose Tolerance Test , Immunoglobulin G/analysis , Immunoglobulin G/biosynthesis , Insulin Resistance , Insulin-Secreting Cells/metabolism , Male , Mice , Mice, Inbred C57BL , Vaccines/immunologyABSTRACT
We investigated whether GABA activates phospholipase A2 (PLA2) during acrosomal exocytosis, and if the MEK-ERK1/2 pathway modulates PLA2 activation initiated by GABA, progesterone or zona pellucida (ZP). In guinea pig spermatozoa prelabelled with [14C]arachidonic acid or [14C]choline chloride, GABA stimulated a decrease in phosphatidylcholine (PC), and release of arachidonic acid and lysoPC, during exocytosis. These lipid changes are indicative of PLA2 activation and appear essential for exocytosis since inclusion of aristolochic acid (a PLA2 inhibitor) abrogated them, along with exocytosis. GABA activation of PLA2 seems to be mediated, at least in part, by diacylglycerol (DAG) and protein kinase C since inclusion of the DAG kinase inhibitor R59022 enhanced PLA2 activity and exocytosis stimulated by GABA, whereas exposure to staurosporine decreased both. GABA-, progesterone- and ZP-induced release of arachidonic acid and exocytosis were prevented by U0126 and PD98059 (MEK inhibitors). Taken together, our results suggest that PLA2 plays a fundamental role in agonist-stimulated exocytosis and that MEK-ERK1/2 are involved in PLA2 regulation during this process.
Subject(s)
Acrosome Reaction/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Phospholipases A/metabolism , Progesterone/metabolism , Spermatozoa/metabolism , Zona Pellucida/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Arachidonic Acid/chemistry , Arachidonic Acid/metabolism , Aristolochic Acids/metabolism , Diglycerides/metabolism , Enzyme Activation , Exocytosis/physiology , Female , Guinea Pigs , MAP Kinase Signaling System/physiology , Male , Phospholipases A/antagonists & inhibitors , Phospholipases A2 , Protein Kinase C/metabolism , Spermatozoa/chemistryABSTRACT
In the present study, double fluorescence staining combined with confocal laser scanning microscopy analysis were used to examine the effects of melatonin on ischemia-induced neuronal DNA strand breaks and its possible mechanisms in a transient middle cerebral artery (MCA) occlusion model. Results showed that melatonin dose-dependently reduced infarct areas and decreased both DNA double and single strand breaks (DSB and SSB) and enhanced cell viability in the peri-ischemic brain regions. Furthermore, Bcl-2 induction in the ischemic brain was further enhanced by melatonin treatment. Double staining analysis indicated that the cells costained for Bcl-2 and TdT-mediated-deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL), a DSB marker, displayed a relative regular morphology compared with the cells only stained with TUNEL. Transient ischemia induced an expression of excision repair cross-complementing factor 6 (ERCC6) mRNA, a gene essential for the preferential repair of nuclear excision repair, in the injured neurons. Double labeling showed that ERCC6 only co-localized with proliferating cell nuclear antigen (PCNA), a member of the nuclear excision repair complex, but not with TUNEL. Melatonin further and statistical significantly up-regulated ERCC6 mRNA expression in the peri-ischemic region of rat brains. The results suggest that neuroprotection by melatonin against ischemic injury may be related to modulation of apoptosis and DNA repair capacity.
