| Objective(1) To observe the characteristic changes of mPGES-1-PGE2-EP1-4 signaling pathway in primary cultured rat hippocampal neurons induced by aluminium.(2) To observe the effects of EP1-4 agonist and antagonist on aluminum overload primary cultured rat hippocampal neurons.Methods(1) The purity of primary cultured hippocampus neurons was identified by immunohistochemical staining on day 7 after primary culture. The aluminium overload group was treated with Al(mal)3(100μM), The blank control group was treated with PBS, and the control group was treated with maltol (300μM). The neuron survival rate was detected by MTT assay. LDH leakage was detected by LDH assay. Neuronal PGE2 concentration was detected by ELISA. The mRNA and protein expression of neuronal mPGES-1 and EP1-4 were measured by RT-PCR and western blot, respectively.(2) The control group was treated with maltol(300μM), and the aluminium overload group was treated with Al(mal)3(100μM). Seven EP1-4 intervention groups were induced with aluminium followed by EP recpetor agonists or antagonists. Intervention groups were treated with agonists of EPl(17-phenyl trinor Prostaglandin E2 ethyl amide), EP2(Butaprost), EP3(Sulprostone), EP4(CAY 10598) or antagonists of EP1(SC-19220), EP2(AH6809), EP4(L-161982), respectively. Each intervention group was treated with gradient concentrations of agonists or antagonists(10,1,0.1, 0.01μM). The neuron survival rate was detected by MTT assay. LDH leakage was detected by LDH assay.(3) The control group was treated with maltol(300μm), and the aluminium overload group was treated with Al(mal)3(100μm). The intervention group was treated with Al(mal)3(100μm) followed by EP1 agonist(17-phenyl trinor Prostaglandin E2 ethyl amide, 10μM), EP3 agonist(Sulprostone, 10μM), EP2 antagonist AH6809, 10μM), EP4 antagonist(L-161982, 10μM), respectively. The morphological changes of primary cultured hippocampus neurons were identified by HE staining. Neuronal Ca2+ concentration was detected by Calcium Kit.Results(1) Compared to control group, Al(mal)3(100μm) caused a significant damage to primary cultured rat hippocampus neuron. PGE2 level significantly increased in aluminium overload group compared to the control group. The treatment of Al(mal)3 caused significant increase of mRNA and protein levels of mPGES-1, EP1 and EP2, and decrease of mRNA and protein levels of EP3. However, only the mRNA expression of EP4 significantly increased in aluminium-treated group.(2) Compared to aluminium overload group, EP3 agonist(Sulprostone) exerted neuroprotective effects, while EP1 agonist(17-phenyl trinor Prostaglandin E2 ethyl amide), EP2 antagonist(AH6809) and EP4 antagonist(L-161982) exerted neuronal damage to primary hippocampal neurons. EP1 antagonist(SC-19220), EP2 agonist(Butaprost) and EP4 agonist(CAY10598) had no effects on injury of primary cultured rat hippocampus neuron induced by aluminium overload. Neuronal Ca2+ increased significantly in EP1 agonist-treated group and decreased significantly in EP3 agonist treated group. EP2 antagonist and EP4 antagonist had no effected on Ca2+ levels in aluminium-overload primary cultured rat hippocampus neurons.ConclusionsAluminium overload caused an imbalance of mPGES-1-PGE2-EP1-4 signaling pathway in primary cultured rat hippocampal neurons, which may be involed in the mechanisms of aluminium overload induced neuronal injury in primary cultured hippocampal neurons. |
