Parkinson disease(PD) is a chronic neurodegenerative disorder caused by progressive dopaminergic neuronal death in the substantia nigra pars compacta within the midbrain.There still is no cure,effective treatments for...Parkinson disease(PD) is a chronic neurodegenerative disorder caused by progressive dopaminergic neuronal death in the substantia nigra pars compacta within the midbrain.There still is no cure,effective treatments for PD,available therapies are only capable of offering temporary and symptomatic relief to the patients.There are certain patents that claim phosphodiesterase(PDE) inhibitors as possible anti-PD drugs,PDE4 is a promising target for the treatment of PD and the underlying mechanism has not yet been well elucidated.PDE4 is an enzyme that specifically hydrolyzes intracellular cyclic adenosine monophosphate(cAMP)throughout the body,including the brain.Most of the available PDE4 inhibitors exert unpleasant and serious side effects,such as emesis and nausea,which hinder its clinical application.Therefore,more efforts are needed before PDE4 inhibitors with high therapeutic indices are available for treatment of PD.FCPR16 is a novel PDE4 inhibitor with little emetic potential,which exhibits excellent enzyme inhibition activity(IC50=90 nmol·L^(-1)).METHODS SH-SY5 Y cell was induced with 1-methyl-4-phenylpyridinium(MPP+)to mimic PD cell injury in vitro,and CCK-8 assay was used to investigate the viability effects of different concentration of FCPR16(3.1-50 μmol·L^(-1)) on MPP+-injured SH-SY5 Y cells.Detection of apoptosis was performed by flow cytometry.The level of ntracellular reactive oxygen species was detected with the fluorescent probe DCFH-DA,and the mitochondrial membrane potential of cells in different experimental groups was detected with the JC-1 fluorescent probe.AO staining and Lysotracker Red staining were used to detect the intracellular antophagy changes.The expression of apoptosis related proteins,autophagy and other related signal molecules were demonstrated by Western blotting.Different cellular signaling pathway inhibitors were used to invesitigate the specific cellular mechanisms of FCPR16 protecting MPP+-induced cell injury.RESULTS FCPR16(12.5-50 μmol·L^(-1)) dose-dependently reduced MPP+-induced decline of cell viability,accompanied by reductions in nuclear condensation and lactate dehydrogenase release.The level of cleaved caspase 3 and the ratio of Bax/Bcl-2 were also decreased after treatment with FCPR16 in MPP+-treated cells.Furthermore,FCPR16(25 μmol·L^(-1)) significantly suppressed the accumulation of reactive oxygen species(ROS),prevented the decline of mitochondrial membrane potential(Δψm) and attenuated the expression of malonaldehyde level.Further studies disclosed that FCPR16 enhanced the levels of cA MP and the exchange protein directly activated by cA MP(Epac) in SHSY5 Y cel s.Western blotting analysis revealed that FCPR16 increased the phosphorylation of c AMP response element-binding protein(CREB) and protein kinase B(Akt)down-regulated by MPP+in SHSY5 Y cells.Moreover,the inhibitory effects of FCPR16 on the production of ROS and Δψm loss could be blocked by PKA inhibitor H-89 and Akt inhibitor KRX-0401.CONCLUSION The novel PDE4 inhibitor FCPR16 can protect against damaging pathways including oxidative stress,mitochondrial dysfunction and apoptosis in SH-SY5 Y cells.FCPR16 preventes MPP+-induced neurotoxicity through activation of cAMP/PKA/CREB and Epac/Akt signaling pathways.These may lead to develop mechanism based therapeutics and improved pharmacotherapy for PD.It is reasonable to assume that FCPR16 is a potential candidate for the prevention and treatment of PD.展开更多
Luteolin is an active ingredient found early from Fofium perillae and Flos Ionicerae, and has a specific inhibition on phosphodiesterase 4 （PDE4） activity in vitro. Researches show luteolin has pharmacological effec...Luteolin is an active ingredient found early from Fofium perillae and Flos Ionicerae, and has a specific inhibition on phosphodiesterase 4 （PDE4） activity in vitro. Researches show luteolin has pharmacological effects of anti-inflammation, anti-anaphylaxis, antitumor, antioxidant, protection of nervous system and so on, and has mainly been used for the treatment of respiratory inflammatory diseases, cancer and cardiovascular disease in clinic. PDE4, specific to hydrolyze cyclic AMP （cAMP）, is considered to be a new anti-inflammatory target due to the decisive role on cAMP signal in inflammatory cells such as neutrophils. In order to explore the anti-inflammatory mechanism, we further studied the effects of luteolin on the activity and expression of PDE4, the expression of lymphocyte function-associated antigen-1 （LFA-1） and macrophage-1 （MAC-l） in neutrophils, and the adhesion of neutrophils and endothelial cells. The results showed that luteolin had a dose-dependent inhibition on both bare PDE4 activity and PDE4 in cultured neutrophils, and had an obviously promotive effect on gene expressions of PDE4A, 4B and 4D in later period. Luteolin had a significant inhibitory effect on neutrophils adhesion and LFA-1 expression in early stage, and had no obvious effect on MAC-1 expression. Therefore, luteolin can inhibit LFA-1 expression of neutrophils, then inhibit the adhesion of neutrophils and endothelial cells, and the mechanism is at least related with the inhibition of PDE4 activity.展开更多
Our previous studies showed that the anti-inflammatory effects of Paeonia lactiflora roots extract may be mediated, at least in part, through its gallic acid content, and this effect may be regulated in part by an inh...Our previous studies showed that the anti-inflammatory effects of Paeonia lactiflora roots extract may be mediated, at least in part, through its gallic acid content, and this effect may be regulated in part by an inhibition on c AMP-phosphodiesterase（PDE）. To explore the anti-inflammatory effect and mechanism, the influence of gallic acid on neutrophils PDE4 activity and expression, TNF-α and IL-6 content and rat arthritis model were further studied. PDE4 activity and gene express were calculated respectively by substrate c AMP change examined with HPLC and real-time RT-PCR. The concentration of IL-6 and TNF-α in supernatant were assayed by ELISA method. Model of rat arthritis was caused by complete Freund＇s adjuvant. Results showed that gallic acid had a dose-dependent restraint on PDE4 activity of neutrophils in vitro, promoted significantly PDE4 A expression（P〈0.01）, and had no influence on the expressions of PDE4 B and 4D. However, PDE4 C expression was not detected. Gallic acid could promote IL-6 release（P〈0.05）, and inhibit TNF-α release of neutrophils（P〈0.05）. The experiment in vivo showed that gallic acid had obvious restraint on local inflammation of animal model（P〈0.05）. Therefore, the anti-inflammatory effect of gallic acid may be mediated in part through an inhibition on PDE4 activity and further an increase of IL-6 and a decrease of TNF-α of neutrophils, and this effect seemed to have no relationship with PDE4 expression.展开更多
AIM To investigate the association of urinary chemokines with the treatment response in chronic prostatitis/chronic pelvic pain syndrome(CP/CPPS) patients.METHODS Between 2007-2011,18 out of 21 male CP/CPPS patients m...AIM To investigate the association of urinary chemokines with the treatment response in chronic prostatitis/chronic pelvic pain syndrome(CP/CPPS) patients.METHODS Between 2007-2011,18 out of 21 male CP/CPPS patients met the exclusion/inclusion criteria of the 16 wk longitudinal study on twice daily oral treatment with Phosphodiesterase 4 inhibitor called Apremilast for 12 wk. Symptom scores and urine specimen were collected at baseline and every visit at 4 wk interval from CP/CPPS patients who completed at least 8 wk of drug treatment. Urine collected at each visit was frozen and then analyzed together after thawing for chemokines and growth factors using MILLIPLEX? MAP immunoassay. Cross sectional association of Chronic Prostatitis Symptom Index(CPSI) and visual analog scale(VAS) with chemokine levels in urine collected at baseline was assessed in 18 CP/CPPS patients relative to 10 asymptomatic male subjects. Longitudinal association between urine chemokine levels and symptom scores was assessed in 8 treatmentadherent CP/CPPS patients at baseline and at 4,8,12 and 16 wk.RESULTS Urine chemokines levels of CXCL-1(GRO-a),CXCL-8(IL-8),CXCL-10(IP-10) and CCL5(RANTES) in CP/CPPS patients at baseline were significantly elevated relative to asymptomatic subjects,whereas levels of s IL-1RA in CP/CPPS were significantly lower compared to controls(P <0.05). Quantitatively,urine levels of CXCL-10 were higher than other chemokines in CP/CPPS,but its fold change of5 relative to controls was lower than the 20 fold change noted for CXCL-8. The mean age of enrolled patients who completed at least 8 wk of treatment(n = 8) was 46.5± 9.4 years and analysis found that elevation of CXCL-8and CCL5 increased the odds for higher score of CPSI by54% and 25%,respectively(F test,P = 0.00007). Urine levels of CCL2(MCP-1) and CXCL-10 together explained approximately 85% of variance in longitudinal data on multivariate analysis. Bivariate analysis of 5 patients who fully complied and completed the assigned dose regimen,showed strong linear correlation of reduced urine levels of CXCL-10,CXCL-8,CCL5,CCL2 and PDGF with improvement in clinical activity as measured by pain VAS and CPSI(Pearson r = 0.83-0.97; P < 0.05).CONCLUSION Urine levels of CXCL-10,CCL2 and PDGF can be sensitive,objective and non-invasive markers of response to new therapeutic intervention in CP/CPPS patients.展开更多
Background:Phosphodiesterase 4(PDE4)is a promising target for the treatment of Parkinson’s disease(PD).However,the underlying mechanism has not yet been well elucidated.Additionally,most of current PDE4 inhibitors pr...Background:Phosphodiesterase 4(PDE4)is a promising target for the treatment of Parkinson’s disease(PD).However,the underlying mechanism has not yet been well elucidated.Additionally,most of current PDE4 inhibitors produce severe nausea and vomiting response in patients,which limit their clinical application.FCPR16 is a novel PDE4 inhibitor with little emetic potential.In the present study,the neuroprotective effect and underlying mechanism of FCPR16 against cellular apoptosis induced by 1-methyl-4-phenylpyridinium(MPP+)were examined in SH-SY5Y cells and primary cultured neurons.Methods:CCK-8 assay,Hoechst staining,lactate dehydrogenase release and flow cytometry were used to study the protective effect of FCPR16 against cell damage caused by MPP+.Mitochondrial membrane potential(Δψm)was measured by JC-1 staining.The extent of oxidation was evaluated using Cell ROXs Deep Red Reagent and malonaldehyde(MDA)kit.Pretreatments with various pathway inhibitors were used to investigate the possible pathways involved in the protection of FCPR16.The phosphorylated and total levels of various proteins were analyzed by Western blot.Results:FCPR16(12.5-50μmol·L-1)dose-dependently reduced MPP+-induced loss of cell viability,accompanied by reductions in nuclear condensation and lactate dehydrogenase release.The level of cleaved caspase 3 and the ratio of Bax/Bcl-2 were also decreased after treatment with FCPR16 in MPP+-treated cells.Furthermore,FCPR16(25μmol·L-1)significantly suppressed the accumulation of reactive oxygen species(ROS),prevented the decline ofΔψm and attenuated the expression of MDA level.Further studies disclosed that FCPR16 enhanced the levels of cAMP and the exchange protein directly activated by cAMP(Epac)in SH-SY5Y cells.Western blotting analysis revealed that FCPR16 increased the phosphorylation of cAMP response element-binding protein(CREB)and protein kinase B(Akt)down-regulated by MPP+in SH-SY5Y cells.Moreover,the inhibitory effects of FCPR16 on the production of ROS andΔψm loss could be blocked by PKA inhibitor H-89 and Akt inhibitor KRX-0401.We also found that MPP+induced a dose-dependent apoptosis in cultured neurons,and 500μmol·L-1 MPP+caused an approximately 50%loss of cortical neurons,while treatment with FCPR16 reversed the toxic effect of MPP+and enhanced the cell viability in a dose-dependent manner.Conclusion:These results suggest that FCPR16 attenuates MPP+-induced dopaminergic degeneration via lowering ROS and preventing the loss ofΔψm in SH-SY5Y cells.Mechanistically,cAMP/PKA/CREB and Epac/Akt signaling pathways are involved in these processes.展开更多
Objective To investigate the effects of novel selective phosphodiesterase4 (PDE4) inhibitors,Ariflo and SB242126A, on the endothelin-1 (ET-1) - induced contractility occurring in nonpregnant human myome-trium specimen...Objective To investigate the effects of novel selective phosphodiesterase4 (PDE4) inhibitors,Ariflo and SB242126A, on the endothelin-1 (ET-1) - induced contractility occurring in nonpregnant human myome-trium specimens. Methods Contractile responses to Ariflo and SB242126A were recorded cumulatively on isola-ted human longitudinal myometrium specimens obtained through surgical operations. Results Ariflo andSB242126A could inhibit both the frequency and amplitude of spontaneous contractions of myometrium (pD2 =8. 6and 7. 6,n =4) and ET-1 -induced contractions in a concentration-dependent manner (pD2 = 7. 7 and 8. 1 ,n =5) ,with a potency similar to that of Rolipram. Conclusion Ariflo and SB242126A have an obvious inhibitory effecton endothelin-1-induced contractility of isolated human myometrium. The finding suggested that PDE4 inhibitorsmight have clinical potential in treating preterm labour and dysmenorrhoea.展开更多
Pulmonary hypertension (PH) is a fatal disorder characterized by pulmonary vascular remodeling and obstruction. The phosphodiesterase 4 (PDE4) family hydrolyzes cyclic AMP (cAMP) and is comprised of four subtypes (PD...Pulmonary hypertension (PH) is a fatal disorder characterized by pulmonary vascular remodeling and obstruction. The phosphodiesterase 4 (PDE4) family hydrolyzes cyclic AMP (cAMP) and is comprised of four subtypes (PDE4A–D). Previous studies have shown the beneficial effects of pan-PDE4 inhibitors in rodent PH;however, this class of drugs is associated with side effects owing to the broad inhibition of all four PDE4 isozymes. Here, we demonstrate that PDE4B is the predominant PDE isozyme in lungs and that it was upregulated in rodent and human PH lung tissues. We also confirmed that PDE4B is mainly expressed in the lung endothelial cells (ECs). Evaluation of PH in Pde4b wild type and knockout mice confirmed that Pde4b is important for the vascular remodeling associated with PH. In vivo EC lineage tracing demonstrated that Pde4b induces PH development by driving endothelial-to-mesenchymal transition (EndMT), and mechanistic studies showed that Pde4b regulates EndMT by antagonizing the cAMP-dependent PKA–CREB–BMPRII axis. Finally, treating PH rats with a PDE4B-specific inhibitor validated that PDE4B inhibition has a significant pharmacological effect in the alleviation of PH. Collectively, our findings indicate a critical role for PDE4B in EndMT and PH, prompting further studies of PDE4B-specific inhibitors as a therapeutic strategy for PH.展开更多
基金NationalNatural Science Foundation of China (81773698)Funding from Guangzhou Science and Technology Department (2015B020211007,201604020112).
文摘Parkinson disease(PD) is a chronic neurodegenerative disorder caused by progressive dopaminergic neuronal death in the substantia nigra pars compacta within the midbrain.There still is no cure,effective treatments for PD,available therapies are only capable of offering temporary and symptomatic relief to the patients.There are certain patents that claim phosphodiesterase(PDE) inhibitors as possible anti-PD drugs,PDE4 is a promising target for the treatment of PD and the underlying mechanism has not yet been well elucidated.PDE4 is an enzyme that specifically hydrolyzes intracellular cyclic adenosine monophosphate(cAMP)throughout the body,including the brain.Most of the available PDE4 inhibitors exert unpleasant and serious side effects,such as emesis and nausea,which hinder its clinical application.Therefore,more efforts are needed before PDE4 inhibitors with high therapeutic indices are available for treatment of PD.FCPR16 is a novel PDE4 inhibitor with little emetic potential,which exhibits excellent enzyme inhibition activity(IC50=90 nmol·L^(-1)).METHODS SH-SY5 Y cell was induced with 1-methyl-4-phenylpyridinium(MPP+)to mimic PD cell injury in vitro,and CCK-8 assay was used to investigate the viability effects of different concentration of FCPR16(3.1-50 μmol·L^(-1)) on MPP+-injured SH-SY5 Y cells.Detection of apoptosis was performed by flow cytometry.The level of ntracellular reactive oxygen species was detected with the fluorescent probe DCFH-DA,and the mitochondrial membrane potential of cells in different experimental groups was detected with the JC-1 fluorescent probe.AO staining and Lysotracker Red staining were used to detect the intracellular antophagy changes.The expression of apoptosis related proteins,autophagy and other related signal molecules were demonstrated by Western blotting.Different cellular signaling pathway inhibitors were used to invesitigate the specific cellular mechanisms of FCPR16 protecting MPP+-induced cell injury.RESULTS FCPR16(12.5-50 μmol·L^(-1)) dose-dependently reduced MPP+-induced decline of cell viability,accompanied by reductions in nuclear condensation and lactate dehydrogenase release.The level of cleaved caspase 3 and the ratio of Bax/Bcl-2 were also decreased after treatment with FCPR16 in MPP+-treated cells.Furthermore,FCPR16(25 μmol·L^(-1)) significantly suppressed the accumulation of reactive oxygen species(ROS),prevented the decline of mitochondrial membrane potential(Δψm) and attenuated the expression of malonaldehyde level.Further studies disclosed that FCPR16 enhanced the levels of cA MP and the exchange protein directly activated by cA MP(Epac) in SHSY5 Y cel s.Western blotting analysis revealed that FCPR16 increased the phosphorylation of c AMP response element-binding protein(CREB) and protein kinase B(Akt)down-regulated by MPP+in SHSY5 Y cells.Moreover,the inhibitory effects of FCPR16 on the production of ROS and Δψm loss could be blocked by PKA inhibitor H-89 and Akt inhibitor KRX-0401.CONCLUSION The novel PDE4 inhibitor FCPR16 can protect against damaging pathways including oxidative stress,mitochondrial dysfunction and apoptosis in SH-SY5 Y cells.FCPR16 preventes MPP+-induced neurotoxicity through activation of cAMP/PKA/CREB and Epac/Akt signaling pathways.These may lead to develop mechanism based therapeutics and improved pharmacotherapy for PD.It is reasonable to assume that FCPR16 is a potential candidate for the prevention and treatment of PD.
基金financial support of the Beijing Natural Science Foundation, China (6112007)the National Natural Science Foundation of China (31101851)+1 种基金the Funding Project for Academic Human Resources Development in Institutions of Higher Learning under the Jurisdiction of Beijing Municipality, China (PHR201107134)the Comprehensive Reforming Project to promote talents training of Beijing University of Agriculture, China (BNRC&GG201404)
文摘Luteolin is an active ingredient found early from Fofium perillae and Flos Ionicerae, and has a specific inhibition on phosphodiesterase 4 （PDE4） activity in vitro. Researches show luteolin has pharmacological effects of anti-inflammation, anti-anaphylaxis, antitumor, antioxidant, protection of nervous system and so on, and has mainly been used for the treatment of respiratory inflammatory diseases, cancer and cardiovascular disease in clinic. PDE4, specific to hydrolyze cyclic AMP （cAMP）, is considered to be a new anti-inflammatory target due to the decisive role on cAMP signal in inflammatory cells such as neutrophils. In order to explore the anti-inflammatory mechanism, we further studied the effects of luteolin on the activity and expression of PDE4, the expression of lymphocyte function-associated antigen-1 （LFA-1） and macrophage-1 （MAC-l） in neutrophils, and the adhesion of neutrophils and endothelial cells. The results showed that luteolin had a dose-dependent inhibition on both bare PDE4 activity and PDE4 in cultured neutrophils, and had an obviously promotive effect on gene expressions of PDE4A, 4B and 4D in later period. Luteolin had a significant inhibitory effect on neutrophils adhesion and LFA-1 expression in early stage, and had no obvious effect on MAC-1 expression. Therefore, luteolin can inhibit LFA-1 expression of neutrophils, then inhibit the adhesion of neutrophils and endothelial cells, and the mechanism is at least related with the inhibition of PDE4 activity.
基金financial support of the Beijing Natural Science Foundation of China (6112007)the Funding Project for Academic Human Resources Development in Institutions of Higher Learning under the Jurisdiction of Beijing Municipality,China (PHR201107134)2012 Scientific Research Quality Raising Funds of Beijing University of Agriculture,China (PXM2012_014207_000010/ PXM2012_014207_000013)
文摘Our previous studies showed that the anti-inflammatory effects of Paeonia lactiflora roots extract may be mediated, at least in part, through its gallic acid content, and this effect may be regulated in part by an inhibition on c AMP-phosphodiesterase（PDE）. To explore the anti-inflammatory effect and mechanism, the influence of gallic acid on neutrophils PDE4 activity and expression, TNF-α and IL-6 content and rat arthritis model were further studied. PDE4 activity and gene express were calculated respectively by substrate c AMP change examined with HPLC and real-time RT-PCR. The concentration of IL-6 and TNF-α in supernatant were assayed by ELISA method. Model of rat arthritis was caused by complete Freund＇s adjuvant. Results showed that gallic acid had a dose-dependent restraint on PDE4 activity of neutrophils in vitro, promoted significantly PDE4 A expression（P〈0.01）, and had no influence on the expressions of PDE4 B and 4D. However, PDE4 C expression was not detected. Gallic acid could promote IL-6 release（P〈0.05）, and inhibit TNF-α release of neutrophils（P〈0.05）. The experiment in vivo showed that gallic acid had obvious restraint on local inflammation of animal model（P〈0.05）. Therefore, the anti-inflammatory effect of gallic acid may be mediated in part through an inhibition on PDE4 activity and further an increase of IL-6 and a decrease of TNF-α of neutrophils, and this effect seemed to have no relationship with PDE4 expression.
文摘AIM To investigate the association of urinary chemokines with the treatment response in chronic prostatitis/chronic pelvic pain syndrome(CP/CPPS) patients.METHODS Between 2007-2011,18 out of 21 male CP/CPPS patients met the exclusion/inclusion criteria of the 16 wk longitudinal study on twice daily oral treatment with Phosphodiesterase 4 inhibitor called Apremilast for 12 wk. Symptom scores and urine specimen were collected at baseline and every visit at 4 wk interval from CP/CPPS patients who completed at least 8 wk of drug treatment. Urine collected at each visit was frozen and then analyzed together after thawing for chemokines and growth factors using MILLIPLEX? MAP immunoassay. Cross sectional association of Chronic Prostatitis Symptom Index(CPSI) and visual analog scale(VAS) with chemokine levels in urine collected at baseline was assessed in 18 CP/CPPS patients relative to 10 asymptomatic male subjects. Longitudinal association between urine chemokine levels and symptom scores was assessed in 8 treatmentadherent CP/CPPS patients at baseline and at 4,8,12 and 16 wk.RESULTS Urine chemokines levels of CXCL-1(GRO-a),CXCL-8(IL-8),CXCL-10(IP-10) and CCL5(RANTES) in CP/CPPS patients at baseline were significantly elevated relative to asymptomatic subjects,whereas levels of s IL-1RA in CP/CPPS were significantly lower compared to controls(P <0.05). Quantitatively,urine levels of CXCL-10 were higher than other chemokines in CP/CPPS,but its fold change of5 relative to controls was lower than the 20 fold change noted for CXCL-8. The mean age of enrolled patients who completed at least 8 wk of treatment(n = 8) was 46.5± 9.4 years and analysis found that elevation of CXCL-8and CCL5 increased the odds for higher score of CPSI by54% and 25%,respectively(F test,P = 0.00007). Urine levels of CCL2(MCP-1) and CXCL-10 together explained approximately 85% of variance in longitudinal data on multivariate analysis. Bivariate analysis of 5 patients who fully complied and completed the assigned dose regimen,showed strong linear correlation of reduced urine levels of CXCL-10,CXCL-8,CCL5,CCL2 and PDGF with improvement in clinical activity as measured by pain VAS and CPSI(Pearson r = 0.83-0.97; P < 0.05).CONCLUSION Urine levels of CXCL-10,CCL2 and PDGF can be sensitive,objective and non-invasive markers of response to new therapeutic intervention in CP/CPPS patients.
文摘Background:Phosphodiesterase 4(PDE4)is a promising target for the treatment of Parkinson’s disease(PD).However,the underlying mechanism has not yet been well elucidated.Additionally,most of current PDE4 inhibitors produce severe nausea and vomiting response in patients,which limit their clinical application.FCPR16 is a novel PDE4 inhibitor with little emetic potential.In the present study,the neuroprotective effect and underlying mechanism of FCPR16 against cellular apoptosis induced by 1-methyl-4-phenylpyridinium(MPP+)were examined in SH-SY5Y cells and primary cultured neurons.Methods:CCK-8 assay,Hoechst staining,lactate dehydrogenase release and flow cytometry were used to study the protective effect of FCPR16 against cell damage caused by MPP+.Mitochondrial membrane potential(Δψm)was measured by JC-1 staining.The extent of oxidation was evaluated using Cell ROXs Deep Red Reagent and malonaldehyde(MDA)kit.Pretreatments with various pathway inhibitors were used to investigate the possible pathways involved in the protection of FCPR16.The phosphorylated and total levels of various proteins were analyzed by Western blot.Results:FCPR16(12.5-50μmol·L-1)dose-dependently reduced MPP+-induced loss of cell viability,accompanied by reductions in nuclear condensation and lactate dehydrogenase release.The level of cleaved caspase 3 and the ratio of Bax/Bcl-2 were also decreased after treatment with FCPR16 in MPP+-treated cells.Furthermore,FCPR16(25μmol·L-1)significantly suppressed the accumulation of reactive oxygen species(ROS),prevented the decline ofΔψm and attenuated the expression of MDA level.Further studies disclosed that FCPR16 enhanced the levels of cAMP and the exchange protein directly activated by cAMP(Epac)in SH-SY5Y cells.Western blotting analysis revealed that FCPR16 increased the phosphorylation of cAMP response element-binding protein(CREB)and protein kinase B(Akt)down-regulated by MPP+in SH-SY5Y cells.Moreover,the inhibitory effects of FCPR16 on the production of ROS andΔψm loss could be blocked by PKA inhibitor H-89 and Akt inhibitor KRX-0401.We also found that MPP+induced a dose-dependent apoptosis in cultured neurons,and 500μmol·L-1 MPP+caused an approximately 50%loss of cortical neurons,while treatment with FCPR16 reversed the toxic effect of MPP+and enhanced the cell viability in a dose-dependent manner.Conclusion:These results suggest that FCPR16 attenuates MPP+-induced dopaminergic degeneration via lowering ROS and preventing the loss ofΔψm in SH-SY5Y cells.Mechanistically,cAMP/PKA/CREB and Epac/Akt signaling pathways are involved in these processes.
基金Supported by the grant of Sino-France Cooperation of INSERM and SSMU.
文摘Objective To investigate the effects of novel selective phosphodiesterase4 (PDE4) inhibitors,Ariflo and SB242126A, on the endothelin-1 (ET-1) - induced contractility occurring in nonpregnant human myome-trium specimens. Methods Contractile responses to Ariflo and SB242126A were recorded cumulatively on isola-ted human longitudinal myometrium specimens obtained through surgical operations. Results Ariflo andSB242126A could inhibit both the frequency and amplitude of spontaneous contractions of myometrium (pD2 =8. 6and 7. 6,n =4) and ET-1 -induced contractions in a concentration-dependent manner (pD2 = 7. 7 and 8. 1 ,n =5) ,with a potency similar to that of Rolipram. Conclusion Ariflo and SB242126A have an obvious inhibitory effecton endothelin-1-induced contractility of isolated human myometrium. The finding suggested that PDE4 inhibitorsmight have clinical potential in treating preterm labour and dysmenorrhoea.
基金This work was supported by Beijing Natural Science Foundation[Z220019 to Jing Wang,China]National High Level of Hospital Clinical Research Funding[2022-PUMCH-D-002 to Jing Wang,China]+3 种基金National Key Research and Development Program of China Grants[2019YFA0801703 and 2019YFA0801804 to Jing Wang]Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences[2022-I2M-JB-007 to Chen Wang,2021-I2M-1-016 to Hongmei Zhao,2021-I2M-1-049 to Jing Wang,2021-I2M-1-005 to Yanjiang Xing,China]Haihe Laboratory of Cell Ecosystem Innovation Fund[22HHXBSS00010 to Jing Wang,China]National Natural Science Foundation of China[82241004 to Jing Wang].
文摘Pulmonary hypertension (PH) is a fatal disorder characterized by pulmonary vascular remodeling and obstruction. The phosphodiesterase 4 (PDE4) family hydrolyzes cyclic AMP (cAMP) and is comprised of four subtypes (PDE4A–D). Previous studies have shown the beneficial effects of pan-PDE4 inhibitors in rodent PH;however, this class of drugs is associated with side effects owing to the broad inhibition of all four PDE4 isozymes. Here, we demonstrate that PDE4B is the predominant PDE isozyme in lungs and that it was upregulated in rodent and human PH lung tissues. We also confirmed that PDE4B is mainly expressed in the lung endothelial cells (ECs). Evaluation of PH in Pde4b wild type and knockout mice confirmed that Pde4b is important for the vascular remodeling associated with PH. In vivo EC lineage tracing demonstrated that Pde4b induces PH development by driving endothelial-to-mesenchymal transition (EndMT), and mechanistic studies showed that Pde4b regulates EndMT by antagonizing the cAMP-dependent PKA–CREB–BMPRII axis. Finally, treating PH rats with a PDE4B-specific inhibitor validated that PDE4B inhibition has a significant pharmacological effect in the alleviation of PH. Collectively, our findings indicate a critical role for PDE4B in EndMT and PH, prompting further studies of PDE4B-specific inhibitors as a therapeutic strategy for PH.
