PED5 Inhibitors Inhibit Microglial Activation Induced By LPS

Phosphodiesterases are intracellular enzymes that catalyze the breakdown of 3',5'-cyclic nucleotides such as cyclic guanosine monophosphate (cGMP), into inactive 5'-monophosphate nucleotides. PDE5 hydrolyzes cGMP and is found in several parts of the body such as the lungs, platelets, various forms of smooth muscle and several brain regions.PDE5 inhibitors (sildenafil, vardenafil and tadalafil) were approved for the treatment of erectile dysfunction and pulmonary hypertension. However, PDE5 inhibitors exert significant neuroprotective effects in improving cerebral ischemia, memory enhancement,neurogenesis and antinociception, which were associated with microglia activation.Therefore, the potential role of PDE5 inhibitors in preventing or treatment of the diseases in the central nervous system (CNS) may be concerned with inhibition of microglia.Microglia activation is associated with numerous central nervous system diseases such as cerebral ischemia, brain injuries and neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD). It has been reported that dipyridamole, a PDE5 inhibitor, appears to block the activation of microglia induced by A(3 or by lipopolysaccharide (LPS). In recent years lines of experimental evidence has shown that PDE5 inhibitors also exert many neuroprotective effects. However, there are no reports showing whether PDE5 inhibitors exert these neuroprotective effects through suppression of microglia activation. Accordingly, in the present study, we adopt Confocal microscopy,High content screening(HCS),ELISA?RT-PCR and Western-blot to conform the possible mechanisms of PDE5 inhibitors on inhibition of microglia activation.In the first part of this research, our results showed that both sildenafil and yonkenafil blocked NO, TNF-a and IL-1? production induced by LPS in microglial cells. In addition,we investigated whether the inhibition of microglial activation by sildenafil and yonkenafil can protect dopaminergic neurons. When conditioned media from LPS-stimulated N9 microglia was added to cultured SH-SY5Y cells, neuronal cell death, as measured by the MTT assay was significantly increased after 24 h. These results indicated that both sildenafil and yonkenafil inhibited microglial activation and protected neuron damaged by microglial activation.Next, the present study showed that the mRNA and protein expression of PDE5 was up-regulated by LPS in N9 microglial cells. Besides, it can be antagonized by PDE5 inhibitor, sildenafil, antioxidant compound, NAC, NADPH oxidase inhibitor, DPI, and NF-?B inhibitor, PDTC. Moreover, H2O2 and TNF-?, which can activate NF-?B signaling pathway, also increased the protein expression of PDE5 in N9 cells. These results demonstrated that the up-regulation of PDE5 induced by LPS in N9 microglia may be partly act through the NF-?B/NADPH/ROS pathway.Third, the possible mechanism of sildenafil and yonkenafil on the production of pro-inflammatory mediators in LPS-activated N9 microglial cells were further investigated.The results showed that both sildenafil and yonkenafil markedly decreased iROS production and the expression of NADPH oxidase induced by LPS. Moreover, both sildenafil and yonkenafil blocked the phosphorylation p38, ERK and JNK mitogen-activated protein kinases (MAPKs). Besides, sildenafil blocked IRAK/TAK1/IKK pathways and antagonized I?B? phosphorylation and degradation, suppressed nuclear factor-?B (NF-?B) activation. In summary,these data suggest that both sildenafil and yonkenfil exert their anti-inflammatory effects in LPS-activated N9 microglial cells by blocking NF-?and MAPKs activation,which may be partly due to its potent down-regulation of the NADPH-derived iROS production.Finally, 8-bromo cGMP (8-Br cGMP) and dibutyryl-cAMP (dbcAMP), the cell-freely permeable cGMP and cAMP analogues, respectively, blocked the release of NO, TNF-? and iROS stimulated by LPS (2.5 ng/ml) in N9 microglia. However, sGC inhibitor ODQ, PKG inhibitor KT5823, and PKA inhibitor KT5720 antagonized the inhibitory action of sildenafil on LPS-induced microglia activation. These results suggested that sildenafil may act through the GCs/cGMP/PKG-dependent pathway or cAMP/PKA-dependent to reduce inflammation in microglia.In conclusion, our research suggested for the first time that both sildenafil and yonkenfil exerts their anti-inflammatory effects in LPS-activated N9 microglial cells by blocking NF-?B and MAPKs activation, which may be partly due to its potent down-regulation of the NADPH-derived iROS production. Furthermore, it may concern with cGMP and cAMP pathway. Besides, our study reported for the first time that the up-regulation of PDE5 induced by LPS in N9 microglia may be partly acted through the NF-?B/NADPH/ROS pathway. Therefore, our research will declare that PDE5 may be the target for the treatment of neuroinflamation related CNS diseases accompanied by microglial activation.