Neuroprotection Of PDE4 Inhibitor FCPR16 Against Cerebral Ischemia-reperfusion Injury And Its Underlying Mechanisms

Objective:FCPR16 is a novel phosphodiesterase-4(PDE4)inhibitor synthesized in our laboratory with potential anti-inflammatory potential.In this study,we investigated the effects of FCPR 16 on ischemia-reperfusion injury and its possible potential mechanism using oxygen glucose deprivation(OGD)model in SH-SY5Y cells or middle cerebral artery occlusion(MCAO)model in rats.Methods:1.OGD model was established by exposure to Na2S2O4 in SH-SY5Y cells.Then,the effect of FCPR 16 on the viability of SH-SY5Y cells after OGD modeling was detected by CCK-8 assay.We used flow cytometry assay,TUNEL staining,and mitochondrial membrane potential assay to access the effect of FCPR 16 on apoptosis after OGD model.The expressions of apoptosis-related proteins were detected by Western bolt.The mitochondrial protein and cytoplasmic protein were isolated and the effects of FCPR16 on mitochondrial and cytoplasmic Cyt C protein were detected by Western blot.Then,we assessed the effects of FCPR 16 on CREB and p-CREB after OGD model.2.MCAO model was established in rats with an intraluminal silicon-coated filament.After 2 h of ischemia,rats were received a single intraperitoneal injection of 2.5,5,or 10 mg/kg FCPR 16.Then after 24 h of reperfusion,neurological deficit scores were measured and the infarct volume was evaluated by TTC staining.H&E staining was performed for morphological analysis.The TNF-?,IL-6 and IL-? levels in serum and ischemic hemisphere were detected by ELISA and Western bolt.We evaluated the expression of Iba-1 and GFAP using immunohistochemistry and Western blot.Effect of FCPR16 on apoptosis after MCAO was detected by TUNEL staining and the expressions of apoptosis-related proteins were detected by Western bolt.In the end,we evaluated the intracellular concentrations of cAMP and CREB phosphorylation by ELISA and Western bolt.Result:In the first experiments,we found that:1.OGD model was established by exposure to 10 mmol/L Na2S2O4 for 4 h and re-culture 24 h in SH-SY5Y cells.After 5 ?M,10 ?M and 20 ?M FCPR16 treatment,the viability of SH-SY5Y cells was increased in a concentration dependent manner after OGD model.2.The results of flow cytometry assay,TUNEL staining showed that cell apoptosis was increased after OGD,while 5 ?M,10 ?M and 20 ?M of FCPR16 treatment could decrease the cell apoptosis.3.The mitochondrial membrane potential of SH-SY5Y cells was decrease after OGD,and FCPR16 could resist such decrease.We also found that FCPR16 could reduce the protein levels of caspase-3,increase the ratio of Bcl-2/Bax and the phosphorylation of Akt,and decrease the release of Cyt C from mitochondria to cytoplasm.4.The results showed that FCPR16 increased the expression of p-CREB in OGD model.In vivo experiments,we found that:1,2.5,5,10 mg/kg of FCPR16 treatment could improve neurological function,reduce cerebral infarct volume,and attenuate brain histological changes in MCAO rats.2.Levels of proinflammatory cytokines TNF-?,IL-6 and IL-1? in serum and ischemic hemisphere were increased after MCAO,FCPR16 decreased the levels of TNF-a,IL-6 and IL-1?.3.The expressions of Iba-1 and GFAP were decreased in MCAO rats after FCPR16 treatment detected by immunohistochemistry and Western blot.4.TUNEL staining showed that FCPR16 could decrease the cell apoptosis in rats after MCAO.We also found that FCPR16 could reduce the protein levels of caspase-3,increase the ratio of Bcl-2/Bax and the phosphorylation of Akt.5.FCPR16 increased the concentrations of cAMP and CREB phosphorylation.Conclusion:1.PDE4 inhibitor FCPR16 could increase the viability of SH-SY5Y cells after OGD,reduce apoptosis inducing by mitochondria apoptosis pathway.These effects might be exerted through CREB signal pathway.2.FCPR16 could improve neurological function,reduce cerebral infarct volume,and attenuate brain histological changes in MCAO rats.The underlying mechanisms might involve in decreasing levels of proinflammatory cytokines,inhibiting activation of astrocyte and microglial cell,reducing apoptosis of neural cell and activating cAMP/CREB pathway.