Study On The Protective Effects Of PPARγ Against Hydrogen Peroxide-induced Injury In Primary Cultured Cortical Neurons

ObjectiveTo observe the changes of PPARγ expression and activity induced by hydrogen peroxide(H2O2) in primary cultured cortical neurons, as well as the protective effects of neuronal PPARγ to H2O2-induced cellular injury. Methods1. Primary culture of cortical neurons: Cortical neurons from new born SD rats(within 24h) were cultured in vitro for 7 days. After the purity identification, neurons are used in the following experiments.2. H2O2-induced neuronal injury: H2O2 was added to neurons to elicit oxidative celluar insult. Neurons of d7 was exposed to H2O2 of different concentrations for 24 h or H2O2 of the indicated concentration for different times. Appropriate concentration and time course was selected for the following experiments.3. Morphological observation of neurons:The morphological damage was examined with an inverted phase contrast microscope. Immunofluorescent staining of MAP2 was also used to observe the gross morphological features of neurons.4. MTT assay: MTT assay was used to determine the cell viability after H2O2 exposure.5. Western blot analysis: Western blotting was chosen to observe the expressions of PPARγ, p-PPARγ, ERK1/2, p-ERK1/2, Bcl-2, cleaved caspase-3, and Nrf2 in neurons with different treatments.6. RT-PCR analysis:RT-PCR was used to detect mRNA expression of target genes of PPARγ in neurons with different treatments.7. ELISA assay: ELISA assay was applied to explore PPARγ-DNA binding activity in neurons with different treatments.8. The antisense oligonucleotides approach(asON): an asON-based strategy was used to downregulate PPARγ expression in neurons to assess the protective potential of neuronal PPARγ towards H2O2-induced injury.Results1. The primary cortical neurons were adhered to the dishes with a round or elliptical body and tiny neuritis when cultured for the first 2 days. When cultured for 7 days, the neurons were of plump size with plenty of neuritis interweaving to form a net. And the purity of neurons was up to 95% by immunofluorescent staining of MAP2, capable of being used in the follow-up experiments.2. Hydrogen peroxide can cause neuron damage in a concentration and time dependent manner: Cells lost the polygonal shape, some cells appeared swelling and others shrunken with surface blebbing. There was general loss of neurites, which were either irregular or fragmented. Hydrogen peroxide in the concentration of 500mM was selected as the injured model for the following observation.3. Hydrogen peroxide suppressed PPARγ protein level of neurons, and at the same time increased PPARγ inactivation. ERK1/2 activation inhibitor can block hydrogen peroxide-induced PPARγ inactivation.4. PPARγ activator Rosiglitazone(Ros) protected neurons against hydrogen peroxide injury, inhibited hydrogen peroxide-induced decrease in Bcl-2, Nrf2 expression and the increase in cleaved caspase-3 level. Furthermore, the above effects of Ros can be reversed by the PPARγ inhibitor GW9662.5. Neurons with PPARγ downregulation by antisense approach(asON) showed more damage to hydrogen peroxide and the enhancement in hydrogen peroxide-induced alterations in Bcl-2, Nrf2, and cleaved caspase-3 expression. Also, PPARγ agonist Ros based neuroprotective effect was reduced. Conclusion1. In primary cultured cortical neurons, hydrogen peroxide may induce cell injury by negative regulation of PPARγ.2. In primary cultured cortical neurons, PPARγ can protect neurons from hydrogen peroxide injury. The intrinsic PPARγ expression in neurons may be one of self-protective defense of neurons.3. PPARγ may exert its neuroprotective effects partly through upregulation of Bcl-2 and Nrf2, as well as downregulation of caspase-3.