FPS-ZM1 Attenuates Advanced Glycation End Products Induced Inflammation And Oxidative Stress In Rat Primary Microglia

[Background]Alzheimer's disease(AD)is a neurodegenerative disease characterized by progressive cognitive impairment and affects a large proportion of aged population.The major clinical features of those affected by AD are progressive loss of cognitive function leading to an inability to perform routine activities of daily living.The main pathological characteristics are senile plaques and neurofibrillary tangles and subsequent inflammation.The complexity of the pathogenesis of AD is not fully clear.It's has been well appreciated that chronic inflammation caused by neural microglia activation in the brain plays a key role in the pathogenesis of AD.It has been indicated that advanced glycation endproducts(AGEs),which was a irreversible polymer generated by a non-enzymatic reaction between reducing sugar and protein amino.It was found that there is a large amount of AGEs assemble in the surrounding of senile plaques and tangles,participating in the occurrence and development of AD.It has been proved that AGEs which can promote themodification of senile plaques substantially enhance microglia activation and intensify inflammatory response,but the reaction mechanism is not clear.AGEs can directly damage the structure of many proteins by glycosylation.Furthermore,AGEs can interact with its receptor RAGE leading to the transformation of downstream multiple signaling pathways and inducing a series of biological effects.RAGE is a member of immunoglobulin superfamily that can bind with a broad repertoire of ligands,such as AGEs,A?,the S100/calgranulin,HMGH1.It has been demonstrated previously that interaction between AGEs and its receptor RAGE activate some signaling pathways,such as ERK1/2,JNK and P38 in neurocytes,and participate in AD development.The interaction between AGEs and its receptor RAGE not only lead to endoplasmic reticulum stress,but also promote A?generated and abnormal tau protein phosphorylation.The pathological changes can obviously improved by decreasing the expression of RAGE and blocking the combination of RAGE and ligand,Thus,targeted pharmacological interventions using AGEs-inhibitors,RAGE-antibodies or RAGE-antagonists may be promising therapeutic strategies to slow down the progression of recognition decline in AD.Previous research data have clarified that anti-RAGE antibodies,sRAGE and RAGE gene knockout.However,the front two of these agents cannot modify disease progression effectively because they do not cross the blood-brain barrier(BBB).Thus,the development of small molecule RAGE-antibodies become the new development trend.A type of small molecule antagonist of RAGE(designated FPS-ZM1)has been developed for the first time by Deane in 2012 and tested in experimental models.FPS-ZM1 has the advantages of inhibiting the generation of A? in the brain of AD mouse and inflammatory response,significantly improving cognitive function,low toxicity,and crossing the blood-brain barrier easily.But the neural protect mechanism by FPS-ZM1 need further research.Another purpose of this study is discussing the protective effect on microglia activation induced by AGEs and oxidative stress by FPS-ZM1,and the mechanism of action in vitro.[Object]1.To explore the effect of AGEs on microglia activation,oxidative stress and cytokines and the possible mechanism in vitro.2.To explicit the protective effect of a new type of small molecules FPS-ZM1 on the oxidative stress and inflammatory response caused by AGEs-induced microglia activation and the possible mechanism.[Methods]1.The mixed cultivation,separation and purification of microgliaThe co-cultivation cultures were from 8 Wistar mice within 3 day-old newborn after beheaded in a sterile environment and then separated and purified microglial cell from the co-cultivation.2.The observation for morphology of the cell change by MTT,cultured cell model of the original generation of microglia cells intervened by AGE-BSA.In order to observe the influence of AGEs to the microglial cell,protein solutions of increasing doses(0,50,100,200,300,400?g/mL)were added in cultures separately.In order to ensure the best concentration and action time by AGEs-BSA,the cell survival rate was measured by the method of MTT.This experimental results showed that AGEs-BSA(300?g/mL)caused the microglia cells vigor to drop to about 50%comparing to control group 48 hours later.Different doses of BSA did not have obvious influence to the microglia cells vigor.3.Identification of microglial cell by immunocytochemical methodsThe identification of microglia is confirmed by specific antigen CDllb,by immunocytochemistry labeling CDllb positively identify microglia and observe its morphology.Brown cells are positive cells.4.Grouping and interventionMicroglia were plated onto 6-well plates in culture medium(4x105 cells/mL,1ml per well).Cells were divided into five groups:(1)BSA group(Control group),(2)AGEs-BSA group(300 g/mL AGEs-BSA for 24 hours),(3)AGEs+F1 group(25 nM FPS-ZM1 for 1 hour,then 300 ?g/mL AGEs-BSA for 24 hours),(4)AGEs+F2 group(50 nM FPS-ZM1 for 1 hour,then 300 ?g/mL AGEs-BSA for 24 hours),(5)AGEs+F3 group(100 nM FPS-ZM1 for 1 hour,then 300 ?g/mL AGEs-BSA for 24 hours).Cells were treated with or without AGEs-BSA(300 ?g/mL)in the absence and presence of FPS-ZM1(25,50 or 100 nM)for 24 hours.After treatment,cells were collected for immunocytochemistry and ELISA.5.Cell oxidative damage and oxidation index detectionReactive oxygen species(ROS)fluorescence quantitative(DCFH-DA)test is used by the way of thiobarbituric acid method which MDA kits are used to determine the content of intracellular MDA;Disulfide dinitrodiphenyl formic acid colorimetric method is applied to detect intracellular GSH with GSH kits;Take xanthine oxidase method with SOD kits to detect the content of SOD in the cell.At the same time using coomassie brilliant blue method to test protein concentration in microglia in order to correct content of each index in the cell.6.ELISAThe level of inflammatory mediators TNF-?,IL-1?,COX-2,PGE2,p-NF-?Bp65 and NO were quantified using specific ELISA kits for rats according to the manufacturer's instructions.7.Western blotTo test expressions of NOX2,p47phox,p67phox,inducible NO synthase(iNOS),Nrf2 and HO-1 with western blot method.[Statistical analysis]All data were shown as mean ± standard error of the mean(SEM).Statistical analysis of the results was performed using one-way ANOVA followed by a Tukey-Kramer test for multiple comparisons.The acceptable statistical significance was the conventional p<0.05 level.All statistical analyses were performed using SPSS software,versionl6.0.[Result]1.AGEs-BS A affects primary microglia vitality of reproductionTake microglia for the model,respectively add different doses(0,50,100,200,300,400?g/mL)ofAGEs-BSA for 24,48 and 72 hours.With the increased AGEs-BSA dose,microglia reduction rate gradually decreased.After 24h,48h,above the AGEs-BSA dose of 200?g/mL,microglia vitality had a significantly reduction.The difference was statistically significant(P<0.01);300?g/mL,after 48 and 72 h,a significant reduction of 50%in the cell vitality was found.The difference was statistically significant(P<0.01).As shown in figure 22.FPS-ZM1 affects BSA microglia activationResults showed that BSA control group mostly had been dormant state microglia,cells were small and circular,and a few cells protrusion was branching out long curved.Microglia in AGEs group and different doses of FPS-ZM1 groups showing amoeboid,became hypertrophy,large,round nucleus;Some cells became spindle with increasing intracytoplasmic particles,in which,the AGEs group was the most obvious,while,the FPS-ZM1 group showed a lower degree of microglia activation than AGEs group.As shown in figure 33.FPS-ZM1 affects oxidative stress of microglia and has influence on antioxidant capacityThis experiment selected the ROS,MDA test to assess the oxidative damage to microglia by AGEs.After AGEs-BSA(300?g/mL)cultured for 24hours,microglia intracellular ROS,MDA were higher than in BSA cotrol group(P<0.01,P<0.01).Compared with AGEs-BSA group,AGEs+Fl groups AGEs+F2 group?AGEs+F3 group,were of obvious declined concentrations of ROS,MDA,with significant difference(P<0.01,P<0.01).Different doses of FPS-ZM1 groups had a higher ROS,MDA level than BSA control group(P<0.05).As shown in figure 4,5Select SOD activity and GSH content determination to test radical scavenging activity of microglial cell.Treated by AGEs-BSA(300?g/mL)for 24 h,SOD activity of AGEs group significantly decreased than control group(P<0.01,P<0.01).Compared with AGEs-BSA group,AGEs+F1 group,AGEs+F2 group,AGEs+F3 group,with different concentrations could improve the level of SOD activity significantly(P<0.01,P<0.01).SOD activity of different doses of FPS-ZM1 groups were still lower than the BSA control group(P<0.05).Intracellular GSH content significantly decreased than BSA control group(P<0.01);GSH content in different FPS-ZM1 groups increased than AGEs group(P<0.05).As shown in figure 6,74.FPS-ZM1 affects microglia in iNOS expression and NO contentAfter cultured 24 h,NO produced by AGEs group of microglia is significantly higher than the control group(P<0.01),while AGEs+F1 group,AGEs+F2 group,AGEs+F3 group had a lower level of NO than AGEs group(P<0.01),with significant difference(P<0.01,P<0.01).Using Western blot test iNOS expression,according to the results,iNOS protein expression of AGEs group was obviously higher than that of control group.Compared with AGEs group,different doses of FPS-ZM1 after intervention could obviously inhibit the expression of iNOS,the difference was statistically significant(P<0.01).As shown in figure 85.FPS-ZM1 reduced the influence on microglia,which caused by AGEs induced NADPH oxidase related subunit.By Western blot,testing three NADPH oxidase sub-units NOX2,p47phox and p67phox,found in the primary microglia,FPS-ZM1 can not only decrease the amount of NOX2,can also inhibit p47phox and p67phox from cell membrane to extranuclear transportation.As shown in figure 96.FPS-ZM1 elevates antioxidative Nrf2 and HO-1 productionWe examined the protein levels of Nrf2 and HO-1 by Western blot.AGEs significantly decreased the expression of Nrf2 and HO-1(P<0.01,P<0.01),however,compared with AGEs group,this downregulation was recovered by FPS-ZM1 treatment in a dose-dependent manner.These results suggested that Nrf2 and HO-1 elevation might also be related to the protective mechanisms of FPS-ZM1.As shown in figure 107.FPS-ZM1 attenuates AGEs-induced activation of NF-?B p65 and inflammatory markers(TNF-? and IL-1?)in primary microgliaBy comparing proteins extracted from cytoplasm and nuclei,we found that AGEs treatment increased NF-?B p65 nuclear translocation from cytoplasm(P<0.01,P<0.01).However,this effect was markedly decreased by FPS-ZM1 in a dose dependent manner(25nM,50nM?100nM)(P<0.01,P<0.01).As shown in figure 11AGEs treatment significantly elevated expressions of TNF-?,IL-1? in primary microglia compared with the control group(P<0.01,P<0.01).Similarly,the presence of FPS-ZM1 significantly attenuated the upregulation of these pro-inflammatory biomarkers induced by AGEs(P<0.01,P<0.01).As shown in figure 128.FPS-ZM1 attenuated the AGEs mediated cox-2/PGE2 signaling pathways in primary microglia.The experiment results showed that cox-2/PGE2 of AGEs group was obviously higher than that of control group(P<0.01,P<0.01);compared with AGEs group,different doses of FPS-ZM1 can obviously inhibit the produce of cox-2/PGE2,the difference was statistically significant(P<0.01,P<0.01).As shown in figure 13[Conclusion]AGEs can upregulate expression of NADPH oxidase and its subunit NOX2 in microglia,promote transportation of p47phox and p67phox from cell membrane to extranuclear,inhibit Nrf2-HO-1 channel,promote the ROS,increases the production of MDA and start the oxidative stress reaction,at the same time,inhibit oxidation resistance related SOD activity and GSH production,involved in oxidative stress injury.FPS-ZM1 is a specifically RAGE receptor blockers that can block AGEs-RAGE pathway,inhibit NADPH oxidase and related subunits,activate Nrf2-HO-1 pathway,improve the oxidation resistance and inhibit generation of oxidation product.AGEs can activate iNOS in microglia,promote NO production,FPS-ZM1 can block AGEs-RAGE pathway,inhibiting activation of iNOS and excessive generation of NO.Microglia is activated by AGEs-RAGE pathways,promoting NF-?B transfer from cytoplasm to nucleus;activate the expression of a large amount of inflammatory factors,which lead to inflammation.FPS-ZM1 can restrain AGEs-RAGE pathway,inhibit transfer or activation of NF-?B,reduce expression of cytokines and attenuate chronic inflammation.Our research results indicate that,FPS-ZM1 through direct block AGEs-RAGE pathway protects neurons from AGEs-RAGE induced oxidative stress and inflammatory cell damage.This multifunctional receptor in AD treatment has a very broad application prospects.