Study On The Anti-neuroinflammatory Effect Of A Novel Phosphodiesterase 4/5 Dual-target Inhibitor

Neuroinflammation is a common pathological mechanism of a variety of neurodegenerative diseases,and drug research targeting neuroinflammation can provide a candidate strategy for the development of neurodegenerative disease drugs.Cyclic nucleotides,as second messengers,regulate various physiological processes in the central nervous system(CNS),among which cyclic adenosine monophosphate(c AMP)and cyclic guanosine monophosphate(c GMP)can play a neuroprotective role by participating in the regulation of neuroinflammation.Phosphodiesterases(PDEs)are the only key enzymes that can catalyze the hydrolysis of c AMP and c GMP in vivo,with PDE4-specific hydrolysis of c AMP and PDE5-specific hydrolysis of c GMP.The design and synthesis of novel PDE4/5 dual-target inhibitors can regulate a variety of interrelated pathological pathways,and provide personalized treatment schemes for neurodegenerative diseases with complex mechanisms.Based on the above theories,the research contents and specific results of this dissertation are as follows:1.The combination of PDE4 and PDE5 inhibitors synergistically antineuroinflammation.Transcriptome sequencing(RNA-seq)analysis showed that the combination of Rolipram(a PDE4-specific inhibitor)and Tadalafil(a PDE5-specific inhibitor)could regulated gene expression associated with c AMP,c GMP,and inflammatory signaling pathways.The results were verified by enzyme-linked immunosorbent assay(ELISA),and the results showed that the combination of Rolipram and Tadalafil increased the content of c AMP,c GMP,and reduced interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNFα)levels in BV2 cells activated by Lipopolysaccharide(LPS).The results of Chou-Talalay showed that the combination index(CI)of the combination of Rolipram and Tadalafil inhibited the production of IL-1β,IL-6 and TNFα was less than 0.8,indicating that simultaneous inhibition of PD4/5could synergistically regulate inflammatory response.The Morris water maze(MWM)experiment confirmed that the combination of Rolipram and Tadalafil could improve the learning and memory of amyloid precursor protein/presenilin-1(APP/PS1)mice.ELISA and Western blot(WB)experiments showed that the combination of Rolipram and Tadalafil increased the content of c AMP and c GMP,decreased the levels of IL-1β,IL-6and TNFα,and promoted the phosphorylation of c AMP-response element binding protein(CREB)and up-regulated the expression of brain-derived neurotrophic factor(BDNF)in APP/PS1 mouse cortex and hippocampus.These results indicating that combination of drugs significantly inhibited the inflammatory response and had the potential for neuroprotective effects in the brain of APP/PS1 mice.Based on this,PDE4/5 dual-target inhibitors are good therapeutic drug for neuroinflammation as the research hypothesis in this dissertation.2.Design and synthesis of novel PDE4/5 dual-target inhibitors.By analyzing the structural characteristics of PDE4 and PDE5 proteins,and using the skeletal transition strategy,the structure of benzimidazole was determined to be the central structure of the newly designed and synthesized PDE4/5 dual-target inhibitor.In the transformation of the branched chain,the rigid acetamide and methyl-linker are selected from strong to weak to the final ethyl-linker.The results of PDE4/5 activity inhibition assay showed that the flexibility of the ethyl-linker improved the inhibitory activity of the compound against PDE4/5,and with the druggability prediction,B-2-3,C-2-4,C-3-1 and F-3-1 with balanced and strong inhibitory activity against PDE4/5 in this series were selected for further activity screening finally.3.Study on the anti-neuroinflammatory effect by novel PDE4/5 dual-target inhibitors.The results of Cell Counting Kit-8(CCK-8)showed that C-3-1 could significantly improve the viability of BV2 cells stimulated by LPS at concentrations of10 μM and 20 μM.C-3-1 has the long-term drug safety and can penetrate the blood-brain barrier.MWM experiments showed that C-3-1 could significantly reverse the escape latency time,number of virtual platform crossings and target quadrant residence time in APP/PS1 mice,indicating that C-3-1 could improve the learning and cognitive ability of APP/PS1 mice.The results of ELISA,WB,immunofluorescence(IF),transmission electron microscopy(TEM),and Hematoxylin-eosin staining(HE)of neurons in the brain showed that C-3-1 could increase the c AMP and c GMP content and significantly upregulated the expression of p-CREB and BDNF in BV2 cells induced by LPS and brain of APP/PS1 mice.These results indicated that C-3-1 activates the downstream CREB signaling pathway by regulating c AMP and c GMP content.C-3-1 significantly reduced the levels of pro-inflammatory factors IL-1β,IL-6 and TNFα,indicating that C-3-1significantly inhibited the neuroinflammatory response in BV2 cells and the brain of APP/PS1 mice.C-3-1 significantly inhibited the activation of microglia and astrocytes in the brain of APP/PS1 mice.C-3-1 depolymerizes highly ordered amyloid β-protein(Aβ)plaques into aggregates that have no stable structure and can be metabolized normally.C-3-1 also increased the number of Nissl and neurons and inhibited neuronal apoptosis,suggesting that C-3-1 has neuroprotective effects.4.Study on the anti-neuroinflammatory mechanism of novel PDE4/5 dual-target inhibitors.RNA-seq analysis results show that C-3-1 regulated the expression of genes related to the nuclear factor-κB(NF-κB)/c-Jun N-terminal kinase(JNK)signaling pathway.The above results were verified by WB,and the results showed that C-3-1inhibited the expression of PDE4 and PDE5 in BV2 cells and HT22 cells after LPS induction,and leads to the inactivation of the NF-κB inhibitor α(IκBα)/NF-κB signaling pathway,and blocked the c-Jun/JNK signaling pathway by inhibiting the phosphorylation of c-Jun and JNK.These results suggest that C-3-1 inhibits neuroinflammation by targeting the NF-κB/JNK signaling pathway,thereby exerting neuroprotective effects.In summary,this dissertation demonstrated that simultaneous inhibition of PDE4/5can reduce neuroinflammation.Alzheimer’s disease(AD)was used as a disease model to evaluate the therapeutic effect of inhibition of PDE4/5 in reducing neuroinflammation,and the results showed that there was a synergistic effect of simultaneous inhibition of PDE4/5 compared with inhibition of PDE4 or PDE5 alone in the process of regulating neuroinflammation.Therefore,it is of scientific value to design lead compounds targeting PDE4/5.Accordingly,this dissertation designed and synthesized a novel PDE4/5 dualtarget inhibitor to evaluate its efficacy and safety in regulating neuroinflammation,and used RNA-seq to explore the mechanism of action network of the new PDE4/5 dual-target inhibitor,so as to provide data support for the development of innovative AD therapeutic drugs as a lead compound.