| More and more evidences indicate that phosphodiesterases（PDE） play a significant role in learning and memory. PDE inhibitors can improve learning and memory impairment induced by the degenerative diseases of the central nervous system through regulation of intracellular cyclic adenosine monophosphate（c AMP） levels. Finding selective PDE inhibitor is one of the major targets in many pharmacological laboratories. Our recent experimental results show that ferulic acid（FA） can inhibit PDE expression, activity and affect the metabolism of a variety of organisms. However, the regulation mechanisms of ferulic acid on phosphodiesterases and the inhibition of ferulic acid is specific or unspecific are still unknown. This project will reveal the inhibiting effect of FA on PDE subtypes and the influence of FA on the downstream signaling pathway of PDE. The in vitro study will verify the relationship between the PDE inhibition and the anti-Alzheimer’s disease（AD） effect of FA. These efforts will not only provide a safe and effective natural medicine for specific targeted therapy of AD, but also provide theoretical support for pharmacological effects and mechanism of FA.The project is divided into the following four parts.Firstly, molecular docking was used to identify the interaction between the PDE4 B and FA. It showed that FA interacts strongly with amino acid residues including Tyr233, His234, Met347, Asn395, Phe414, Gln443, and Phe446 at the FA-binding site of PDE4B2, the data also showed that FA could enter the binding cavity of PDE4B2 and form π? π interactions with amino acid residues Phe446 and Phe414. Hydrogen bonding can be found between FA and amino acid residues Gln443 and His234. The conformation of FA is located in the hydrophobic cavity area, it indicated that electrostatic and hydrophobic interactions existed between FA and PDE4B2, which contribute to the free energy of binding between FA and PDE4B2. It indicated that FA can be used as a basic structure to design PDE4 inhibitors.Secondly, the in vitro study, in order to clarify the neuroprotective effect of FA, which against Aβ25–35 and lipopolysaccharide（LPS）- induced PC12 cellular damage, the superoxide dismutase（SOD）activity and the levels of inflammatory factors（TNF-α and IL-1β） in supernatant of PC12 cells were investigated. We further investigated the effects of FA against LPS induced changes in intracellular levels of c AMP and free Ca2+ concentration([Ca2+]i), which suggested possible therapeutic uses of FA for the treatment of AD. Furthermore, we investigated the stimulating effects of FA on nerve growth factor（NGF） induced differentiation of FA.The results showed that FA significantly maintained the levels of SOD and inhibited production of TNF-α and IL-1β induced by Aβ25–35. Pretreatment with FA increased the intracellular levels of c AMP and decreased intracellular Ca2+ concentration([Ca2+]i) against LPS induced [Ca2+]i increasing. Treated with NGF and FA, the effect of differentiation induced by NGF was stimulated by FA. Taken together, our results suggested that one of the therapeutic effects of on AD was potentially mediated by inhibiting the PDE.Thirdly, we examined the effects of FA on expression and activity of LPS-induced up-regulation of phosphodiesterase 4B（PDE4B）, which regulates cellular cyclic adenosine monophosphate（c AMP） levels and c AMP/c AMP response element binding protein（CREB） pathway. Laser scanning confocal microscope was performed to measure the effect of FA against LPS-induced F-actin changes in PC12 cells. We further investigated the effects of FA against LPS induced changes in c AMP-specific PDE activity, reactive oxygen species（ROS） and pro-inflammatory cytokines. The m RNA expressions of PDE4 B were analyzed by real-time fluorescent quantitative reverse transcription polymerase chain reaction（Q-PCR）, the protein expressions of CREB and phosphorylation of CREB（p CREB） were determined by immunoblotting.FA exerts protection against LPS-induced damage on cytoskeletal protein F-actin, pretreatment with FA meliorated the structure and distribution of cytoskeletal protein F-actin. FA also significantly inhibited production of ROS, TNF-α and IL-1β induced by LPS in PC12 cells. Enzyme activity tests showed that FA markedly attenuated LPS-induced up-regulation of PDE activity in PC12 cells. Examination of PDE4 B m RNA of PC12 cells revealed that pretreatment with FA decreased PDE4 B expression induced by LPS, while immunoblotting showed up-regulation of CREB and p CREB with FA pretreatment.Fourthly, the purpose of this study is to investigate the protective effects of FA on LPS-induced memory deficit and hippocampal apoptosis in Sprague-Dawley（SD） rats and its potential mechanisms.In the present study, Morris water maze was performed to measure memory enhancing effect of FA against LPS-induced learning and memory deficit in rats, Hematoxylin-eosin（HE） staining was used to show the protective effects of FA on the histological changes of cortex and hippocampus of rats induced by LPS. Immunofluorescence staining was conducted to show the protective effect of FA against LPS-induced destruction on the cytoskeletal protein β-tubulin. In order to clarify the neuro-protective effects of FA against LPS-induced hippocampal neurons, SOD activities were investigated, we further investigated the effects of FA against LPS induced changes of m RNA expressions of IL-1β, caspase-1, Nod-like receptor protein 3（NLRP3） and Phosphodiesterase4B（PDE4B）, which were analyzed by quantitative real-time RT-PCR（Q-PCR）; the protein expression of PDE4 B, NLRP3, CREB and p CREBwere determined by immunoblotting. Furthermore, immunohistochemistry staining was used to identify the effects of FA on PDE4 B expression in CA1, CA2,CA3 regions of hippocampus of rats stimulated by LPS.Morris water maze tests have shown that LPS-induced cognitive dysfunction was attenuated by pretreatment with FA, HE staining showed that neurons in the cortex and hippocampus of the FA groups had much fewer karyopyknosis compared with LPS groups. Pretreated with FA showed an increase inβ-tubulin expression compared with LPS group in hippocampus of rats. FA significantly maintained the activities of SOD inhibited by LPS in cortex and hippocampus of rats. Increased m RNA levels of IL-1β, caspase-1, NLRP3 and PDE4 B induced by LPS were altered by different concentrations of FA pretreatment. Western blot analysis showed that FA pretreated groups increased the expression of CREB and p CREB as compared with LPS group, while consecutively pretreated with FA decreased LPS induced PDE4 B and NLRP3 expressions in hippocampus of rats. Immunohistochemistry staining showed that FA pretreatment markedly prevented LPS-induced up-regulation of PDE4 B expression in CA1, CA2 and CA3 regions of hippocampus.FA could exert protection against LPS-induced learning and memory deficit and hippocampus damage of rats. The beneficial property of FA might be conferred by inhibiting LPS-induced up-regulation of PDE4 B and stimulating CREB and p CREB protein expression, which might be a putative therapeutic intervention of neuro-inflammatory diseases such as AD. |
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