Gasotransmitter Hydrogen Sulfide（H₂S）:Development Of H₂S Fluorescent Probe And H₂S-Releasing Hybrid Drugs

This thesis mainly deals with two parts of work.(1)A serial of novel fluorescent probes for the selective determination of hydrogen sulfide(H2S),and its application in H2S-related biological research.(2)Design,synthesis and antiinflammation activities study of genistein-based hydrogen sulfide-releasing derivatives.Part 1:Hydrogen sulfide plays important roles in biological signaling and metabolic process.Fluorescent probe is preferable for the real-time and quantitative analysis of endogenous H2S in complex biological samples.(1)Two naphthalimide-based ratiometric fluorescent probes,NAP-1 and RHP-2,have been developed for the selective and sensitive detection of H2S.Advantages of both probes include low detection limit,good selectivity,high sensitivity,excellent photostability and low cytotoxicity.A linear relationship between the emission intensity ratios and H2S concentrations were observed in PBS buffer and bovine serum,indicating that both probes are suitable for the quantitative analysis of endogenous H2S.Our probes facilitate ratiometric determination and imaging of endogenous H2S in living MCF-7 cells.Furthermore,these probes were successfully applied to the measurement of endogenous H2S in human plasma and mouse hippocampus.A significant reduction in H2S levels and CBS mRNA expression were observed in the hippocampus of mouse models of lipopolysaccharide-induced neuroinflammation-related diseases and the chronic unpredictable mild stress(CUMS)-induced depression,suggesting that decreased levels of endogenous H2S might be involved in the pathogenesis of neuroinflammation-related neurodegenerative diseases and chronic stress depression.(2)We prepared two near infrared(NIR)fluorescent probes for H2S,SCY-1 and SCY-2,which feature low detection limit(30-40 nM),specific selectivity,high sensitivity and rapid response(15s-30s).The rapid response could meet the requirement of real-time detection of H2S.Notably,SCY-1,the water-soluble and cell transmembranous probe,can be utilized to fluorescence image of endogenous H2S in living cells in 100%aqueous solution.In conclusion,these probes have the potential to be a useful tool in H2S-related medical research.Part 2:Neuroinflammation and synaptic plasticity impairment have been implicated as important pathologic features in various neurodegenerative diseases,such as Alzheimer’s disease(AD)and Parkinson’s disease(PD).H?S is emerging as an important endogenous modulator as well as a neuroprotectant.The neuroprotection by H2S appears to be involved in various mechanisms,including antioxidative,anti-inflammatory and antiapopotic effects.Genistein,a typical flavonoid,has been reported to possess anti-inflammatory and neuroprotective activities.Our previous studies showed that genistein improved the synaptic plasticity impairment in the mouse models of lipopolysaccharide(LPS)-induced neuroinflammation-related diseases.Accordingly,we design and synthesize hydrogen sulfide-releasing derivatives of genistein.LPS-induced neuroinflammation in BV2 microglia was selected as the neuroinflammation-related disease model in vitro.The anti-neuroinflammation activities of target compounds were evaluated.The levels of H2S generated by target compounds were detected via ratiometric fluorescent probe NAP-1.We investigated synergistic effects and structure-activity relationship.The experimental results in vitro showed that:(1)The anti-neuroinflammation activities of all target compouds are better than genistein,its related H2S donor and genistein+H2S donor;Among them,GHS-2 has the best anti-neuroinflammation activities;(2)The anti-neuroinflammation activities and H2S levels produced by target compounds were positively correlated,H2S plays an important role in improving anti-neuroinflammation activities;(3)Genistein derivatives has a synergistic effect with H2S,which contributed to the remarkably enhancement of anti-neuroinflammation activities.The neuroprotective activity of GHS-2 was explored form synaptic plasticity and neuroinflammation.We selected LPS-induced neuroinflammation as the disease model.The effect of GHS-2 on cognitive dysfunction in LPS-induced mice was detected by Morris water maze and step-through test.To evaluate the anti-neuroinflammation activities of GHS-2,inflammatory factors mRNA of CDllb,GFAP,TNF-α and IL-1β,contents of TNF-α and IL-1β,protein contents of p-NF-κB were detemined by RT-PCR,Western blotting and ELISA,respectively.The effect of GHS-2 on synaptic plasticity in LPS-induced mice was investigated by detecting the protein contents of SYN and PSD-95.The levels of H2S released from the targeted compound were determined by the H2S fluorescence probe NAP-1,according to our previous report.The experimental results in vivo showed that;(1)GHS-2 could improve the learning and memory impairment in LPS-induced cognitive decline in mice;(2)GHS-2 could significantly improve the expression of SYN and PSD-95 in hippocampus,consequently improve the synaptic plasticity impairment in LPS treated mice;(3)GHS-2 could significantly decrease GFAP mRNA and CD11b mRNA expression in hippocampus of LPS treated mice,and inhibition of glial cell abnormal activation;(4)GHS-2 could inhibit the phosphorylation of NF-κB,and consequently significantly decrease the inflammatory factors mRNA level and protein expression in hippocampus of LPS treated mice;(5)GHS-2 could significantly increased H2S content,levels of CBS mRNA in the hippocampus of LPS treated mice.In conclusion,the neuroprotection of GHS-2 appears to be involved in various mechanisms,including inhibition of phosphorylation of NF-κB,regulation of CBS/H2S,inhibition of glial cell activation,decrease TNF-α and IL-1β expression,significantly improving the expression of SYN and PSD-95.Importantly,GHS-2 has better anti-neuroinflammation and neuroprotective activities than genistein and ibuprofen.Therefore,GHS-2 is expected to become a promising drug candidate for the treatment of neuroinflammation-related neurodegenerative diseases.