The Anti-neuroinflammation Effects In The Regulation Of Cognitive Dysfunction Of PDE4 Inhibitor Roflupram

Objective:Alzheimer’ s disease(AD)is a neurodegenerative disorder which is characterized by inreversible memory decline and dementia.AD brains show three major characteristic lesions:extracellular deposits of b-amyloid peptides,so called neuritic or senile plaques,the intracellular neurofibrillary tangles(NFT)of hyperphosphorylated tau protein,and the progressive loss of neurons.The exact mechanisms of AD pathogenesis remain unclear,many hypotheses have been proposed,but there is no mechanism to fully elucidated so far.Recent evidence suggests that inflammation may be a another important component which once initiated in response to neurodegeneration or dysfunction,may actively contribute to disease progression and chronicity,therefore the inflammation hypothesis in AD could not be ignored.Phosphodiesterase 4(Phosphodiesterase 4,PDE4)is a critical controller of intracellular cAMP concentrations.There has numbers studies have shown that compounds that selectivel inhibit PDE4 have prominent anti-inflammatory effects in vivo and in vitro,which also can improve cognition through promoting LTP by activation of the cAMP-PKA-CREB pathway,PDE4 inhibitiors therefore has a great potential to become the drug treatment of neuroinflammation-related diseases in the central nervous system such as Alzheimer’s.However,pharmacological therapies aimed at reducing PDE4,produce severe emetic side effects.We have recently synthesized PDE4s inhibitor structurally different from roflupram,endowed with no emetic potential verification by Beagles ig way,and it also can reverse rat cognitive disorder induced by scopolamine(data not shown).The aim of the present study was to investigate whether the PDE4 inhibitor Roflupram can ameliorate Aβ25-35 induced cognitive deficits through the anti-neuroinflammation effects,and further research whether it has anti-inflammatory and neuroprotective effects as well as its potential molecular mechanisms in LPS-induced microglial activation in BV2 cells,a in vitro model of neuroinflammation.Methods:1.To investigate whether roflupram,one identified PDE4 inhibitor,improves cognitive deficits through anti-neuroinflammation effects in Alzheimer’s disease.Alzheimer’s disease model was established by microinjection of amyloid-β(Aβ)25-35 into the bilateral hippocampal CAl area of male rats.Then the rats was randomly divided into sham-operated control group,Aβ25-35 microinjected group,Aβ25-35 microinjected followed by donepezil hydrochloride or rolipram treated group,Aβ25-35 microinjected combined Roflupram treated group with different dose.Treatment was given(i.g.)24 h after microinjection surgery operation for consecutive 23 days.Open field test,Morris water maze and passive avoidance test were performed to evaluate the behavior performance after drug treatments of 10,14 and 20 days.The level of NO、ROS and the enzymatic acitivity of iNOS in hippocampal were measured by commercial kits.Protein level of cAMP downstream signaling molecules(p-PKA、P-CREB),proinflammatory cytokines(iNOS,COX-2,TNF-α,IL-1β),microglia and astrocyte activation marker(Iba-1,GFAP)and inflammation related protein(nuclear NF-кB p65、p-p38)in hippocampus were detected by Western blotting.mRNA level of proinflammatory cytokines(iNOS,COX-2,TNF-a,IL-1β)were measured by semi-quantitative PCR.2.In this part,we investigated the anti-inflammatory effects of Roflupram against LPS(1μg/ml)-induced microglial activation in BV2 cells,a in vitro model of neuroinflammation.Firstly,we investigated the possible cytotoxic effects of Roflupram(6.25～200μM)and LPS(0.1～20μg/ml)treatment in BV2 cells by measuring the cell viability using the MTT,and determine the safety concentration of Roflupram and LPS treatment in BV2 cells.We aslo determine the concentration of LPS used in the follow-up experiment through investigating the effect of different concentrations of LPS treatment in BV2 cells on production of NO using Griess reagent,in addition,we determine the concentration of Roflupram used in the follow-up experiment through assess the inhibition effect of NO release of treatment of cells with different concentrations of Roflupram prior to LPS;Secondly,the effects of Roflupram on the level of reactive oxygen species(ROS)and enzymatic acitivity of iNOS in LPS induce BV-2 cell was determined using commercially kits.The mRNA level of proinflammatory cytokines(iNOS,COX-2,TNF-α,IL-1β)in LPS induce BV-2 cell were measured by semi-quantitative PCR,and the protein level of p-PKA,p-CREB,p-p38,NF-KBp65(nuclear),iNOS,COX-2,IL-1β and TNF-a in LPS induce BV-2 cell were detected by Western blotting.Therefore,we continued by investigating the relation between the anti-inflammatory effects of Roflupram and cAMP/PKA/CREB signaling pathway using PKA selective inhibitor H89.Finally,to validate whether inhibition of the inflammatory response by Roflupram in microglia could protect the neurons,conditional media of LPS-treated BV-2 cells with or without Roflupram were collected to treat N2a cells,and determine the cytotoxicity and apoptosis of N2a cells by MTT and hoschest33342 fluorescence stainingResult:1.Roflupram ameliorated cognitive deficits and neuroinflammation in a rat model of Alzheimer’s disease(1)Roflupram ameliorated Aβ25-35induced cognitive deficits in rat.The open field test showed that there were no significant differences among groups both in number of crossings and number of ambulation in 5 min,indicating that surgery and drug treatments did not affect locomotor activity in rats.The Morris water maze test showed that the Aβ25-35 injected group show significantly longer escape latency in the place navigation test than the sham control group(p<0.01),which was reversed by rolipram donepezil hydrochloride,rolipram and Roflupram high dose treatments(p<0.05,p<0.01);During the probel trail the Aβ25-35 injected group spend less time in the target quadrant(the second quadrant)than the sham control group,donepezil hydrochloride group,rolipram group and Roflupram high dose group(p<0.05,p<0.01);the Aβ25-35 injected group has less entries in the target quadrant than the sham control group,donepezil hydrochloride group and Roflupram high dose group(p<0.05,p<0.01).The passive avoidence test show that rats spend same time to cross the passage during the habituation trial,in the retention test performed 3 h after training,latency was not significantly changed by each treatments,indicating short-term memory was not altered;The result of long-term memory test after 24h training indicate that rats injected with Aβ25-35 fibrils show significantly shorter 24h-retention lantency than the sham control group,donepezil hydrochloride group,rolipram group and Roflupram middle dose and high dose group(p<0.05,p<0.01);(2)Roflupram ameliorated Aβ25-35-induced neuroinflammation in rat The result of NO,ROS and enzymatic acitivity of iNOS showed that:the level of NO and enzymatic acitivity of iNOS in model group were higher than sham control group(p<0.01),and each treatments can reduce these indicators by varying degrees;The level of ROS have a tendency to increase,and each treatments can reduce these indicators by varying degrees.Results of Western blotting analysis revealed that the hippocampus protein level of p-CREB and p-PKA(p<0.01)of rats were significantly decreased induced by microinjection of Aβ25-35,while the level of NF-кBp65(nucleus),p-p38,iNOS,COX-2,TNF-α,IL-1β,GFAP and Iba-1(p<0.05,p<0.01)were significantly increased,these effects were reversed by each kinds of drug treatmentsSemi-quantitative PCR test showed that each treatments can reduce the mRNA level of proinflammatory cytokines(COX-2、iNOS、IL-1β and TNF-α)compared to the Aβ25-35 microinjection animal.2.Roflupram inhibited LPS induced BV-2 inflammation and its underline mechanisms(1)Establishment of LPS-induced activation of BV-2 cell modelAfter treatment with different concentration of Roflupram and LPS for 24 h,the MTT test showed that Roflupram(6.25-150μM)and LPS(0.1～μg/ml)had no effect on BV-2 cell viability,while Roflupram(200μM)and LPS(10μg/ml)had significant cytotoxic effects on BV-2 cell.Treatment of BV2 cells with different concentration of LPS(0.01～5μg/ml)for 24h significantly increased production of NO compared to the controls in a dose-dependent manner,and the differences of 0.1,1,5μg/ml reached a significant level,therefore,the concentration of LPS to establishment the in vitro model of neuroinflammation was determined at 1μg/ml.(2)Roflupram inhibited LPS induced BV-2 activation and its underline mechanismsThe result of commercially available kits showed that BV-2 cells were pretreated with Roflupram(10、20μM)for 1 h and then with LPS(1μg/ml)for 24 h can significantly inhibited the levels of NO and enzymatic acitivity of iNOS,in addition,pretreated with Roflupram(20μM)for 1 h and then with LPS(1μg/ml)for 24 h can significantly inhibited the levels of ROS.Effect of Roflupram on protein levels of p-PKA and p-CREB in LPS induced BV-2 cell:Results of Western blotting analysis revealed that LPS can induce the protein levels of p-PKA and p-CREB and Roflupram can further increased both levels in a dose-dependent manner when BV-2 cells were pretreated with Roflupram(10、20μM)for 1h and then with LPS(1μg/ml)for 30 min.Effect of Roflupram on protein levels of NF-кB p65(nuclear)and p-p38 in LPS induced BV-2 cell:Results of Western blotting analysis revealed that LPS can induce the protein levels of NF-kB p65(nuclear)and p-p38 while Roflupram can inhibited both levels in a dose-dependent manner when BV-2 cells were pretreated with Roflupram(10、20μM)for1 hand then with LPS(1μg/ml)for 1h.Effect of Roflupram on protein expression of IL-1β、TNF-α、COX-2 and iNOS in LPS induced BV-2 cell:Results of Western blotting analysis revealed that LPS can induce the protein expression of IL-1β,TNF-a,COX-2 and iNOS while Roflupram can inhibited these expression in a dose-dependent manner when BV-2 cells were pretreated with Roflupram(10、20μM)for1h and then with LPS(1μg/ml)for 24h.Effect of Roflupram on mRNA levels of IL-1β.TNF-α.COX-2 and iNOS in LPS induced BV-2 cell:Results of RT-PCR test revealed that LPS can induce the mRNA levels of IL-1β,TNF-a,COX-2 and iNOS while Roflupram can inhibited these levels in a dose-dependent manner when BV-2 cells were pretreated with Roflupram(10,20μM)for 1 h and then with LPS(1μg/ml)for 6h.Relation between the anti-inflammatory effects of Roflupram with cAMP/PKA/CREB signaling pathway:Roflupram increased LPS-induced p-PKA and p-CREB protein levels but that effects was not seen in the presence of PKA selective inhibitor H89,Roflupram inhibited LPS-induced p-p38,NF-кB p65(nuclear),TNF-a and iNOS but that effect was not seen in the presence of PKA selective inhibitor H89,these indicate that the anti-inflammatory effects of Roflupram are,at least partly,mediated by cAMP/PKA/CREB signaling pathway.(3)Effect of Roflupram on protection of N2a cell from Conditioned Media from LPS induced BV-2 cell.Compare to treatment with conditional media from LPS alone-treat BV-2 cell,N2a cell treatment with conditional media from LPS in the presence of Roflupram had higher cell viability and less apoptosis using MTT test and hoschest33342 fluorescence staining after treatments with conditional media from LPS-treated in the presence or absence of Roflupram BV-2 cells for 24h.Conclusion:1.The PDE4 inhibitor Roflupram can ameliorate cognitive impairment induced by Aβ25-35 infusion rat,which related to its antigliosis,inflammatory cytokines expression reducing and neuroinflammation amelioration effect.2.Roflupram can inhibited LPS-induced BV-2 microglia activation and its mediated inflammation,which related to its activation of cAMP/PKA/CREB pathway by inhibition of intracellular cAMP hydrolysi,increasing intracellular cAMP levels,further blocking the activation of NF-кB p65 and p-p38 signaling pathway and therefore inhibition of the expression of inflammation-related proteins and downstream inflammatory cytokines.