Dan Li,Jiangping Xu,Yunyun Qin,Ningbo Cai,Yufang Cheng,Haitao Wang
Roflupram (ROF) is a novel phosphodiesterase 4 inhibitor. We previously found that ROF suppressed the production of pro-inflammatory factors in microglial cells; however, the underlying mechanisms are largely unknown. The present study aimed to elucidate the underlying molecular mechanisms of the anti-neuroinflammatory effects of ROF in lipopolysaccharide (LPS)-activated microglial cells and LPS-challenged mice. Treatment with ROF suppressed LPS-induced expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in BV-2 microglia cell line. Immunofluorescence and Western blotting analysis showed that ROF significantly inhibited the activation of microglia, as evidenced by decreased expression of ionized calcium binding adaptor molecule-1 (Iba1). Similar results were obtained in primary cultured microglial cells. ROF induced the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of Sirtuin 1 (Sirt1). Interestingly, the AMPK inhibitor, compound C, blocked the role of ROF in both the phosphorylation of AMPK and the expression of Sirt1 in BV-2 cells stimulated with LPS. More importantly, the Sirt1 inhibitor, EX527, abolished the inhibitory role of ROF on the production of pro-inflammatory factors, and reactivated BV-2 cells. In mice challenged with LPS, ROF improved cognition and decreased the levels of IL-6 and TNF-α in both the cortex and hippocampus. In contrast, EX527 weakened the effects of ROF on cognitive enhancement and reduction of pro-inflammatory factors in the cortex and hippocampus. Furthermore, EX527 blocked the inhibitory role of ROF in the activation of microglial cells in both the hippocampus and cortex. Taken together, our results indicated that ROF attenuated LPS-induced neuroinflammatory responses in microglia, and the AMPK/Sirt1 pathway is essential for the anti-inflammatory effects of ROF.
We have previously shown that roflupram (ROF) protects against MPP + -induced neuronal damage in models of Parkinson’s disease (PD). Since impaired degradation of α-synuclein (α-syn) is one of the key factors that lead to PD, here we investigated whether and how ROF affects the degradation of α-syn in rotenone (ROT)-induced PD models in vivo and in vitro. We showed that pretreatment with ROF (10 μM) significantly attenuated cell apoptosis and reduced the level of α-syn in ROT-treated SH-SY5Y cells. Furthermore, ROF significantly enhanced the lysosomal function, as evidenced by the increased levels of mature cathepsin D (CTSD) and lysosomal-associated membrane protein 1 (LAMP1) through increasing NAD + /NADH and the expression of sirtuin 1 (SIRT1). Pretreatment with an SIRT1 inhibitor selisistat (SELI, 10 μM) attenuated the neuroprotection of ROF, ROF-reduced expression of α-syn, and ROF-increased expression levels of LAMP1 and mature CTSD. Moreover, inhibition of CTSD by pepstatin A (20 μM) attenuated ROF-reduced expression of α-syn. In vivo study was conducted in mice exposed to ROT (10 mg·kg −1 ·d −1 , i.g.) for 6 weeks; then, ROT-treated mice received ROF (0.5, 1, or 2 mg·kg −1 ·d −1 ; i.g.) for four weeks. ROF significantly ameliorated motor deficits, which was accompanied by increased expression levels of tyrosine hydroxylase, SIRT1, mature CTSD, and LAMP1, and a reduced level of α-syn in the substantia nigra pars compacta. Taken together, these results demonstrate that ROF exerts a neuroprotective action and reduces the α-syn level in PD models. The mechanisms underlying ROF neuroprotective effects appear to be associated with NAD + /SIRT1-dependent activation of lysosomal function.
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