Possible Effect Of Pentose Phosphate Pathway On Epileptogenesis And Its Mechanisms

Part OneUntargeted metabolomics identifies possible metabolites and metabolic pathways associated with epileptogenesisObjective:Epilepsy is among the most common neurologic diseases.Epileptogenesis is a latent period where brain will be transformed into an epileptic one.Mechanisms of epileptogenesis remain unclear.In recent years,with the development of technology,omics studies have been utilized to explore molecular mechanisms of epileptogenesis in a bid to clarify new possible mechanisms and identify potential biomarkers,with most of them concerning alteration of gene expression and very limited of them reporting metabolomic alteration.Herewith,without prior hypothesis about particular metabolites of interest,we aim to provide information of metabolic changes related with epileptogenesis in two kindling models,applying liquid chromatography couple to mass spectrometry（LC-MS）.Materials and Methods:We constructed two kinds of chemical kindling models using pentylenetetrazol（PTZ）and coriaria lactone（CL）,which have long been used as models of epileptogenesis.30 rats were randomly divided into three groups.Subthreshold doses of PTZ（30 mg/kg）and CL（1.75 mg/kg）were administrated intraperitoneally every other day.Two kindling and one control groups were all subjected to structural imaging acquisition and cognitive tests after successfully kindled.Voxel-based morphometry,a postprocessing method for brain imaging data,was used to segment and extract hippocampal gray matter volume for subsequent integrative analysis.LCMS based metabolomic analysis was applied to identify distinct hippocampal metabolic profiles between kindling and control groups.Further,we regress hippocampal structural indices on lipids to identify those metabolites associated with both epileptogenesis and brain structural changes.Results:Seizure scores in both epileptogenesis groups were significantly higher than in control group.In open field test,PTZ group showed higher anxiety level than control group（P=0.008）.In contextual fear conditioning,PTZ group spent less time freezing than control（P=0.033）,suggesting compromised contextual memory.For untargeted lipidomics,a total of 638 lipids were detected in all three groups.Among them were 98 individual lipids,showing significant alterations,in particular lipid class of phosphatidylethanolamine（PE）,glucosylceramide and phosphatidylcholine.Hippocampal gray matter volumes were found significant different between groups（P<0.05）.After combining brain imaging data,we demonstrate several individual PE,namely PE（O-18:1₂2:3）and PE（O-18:1₂2:6）are associated with both epileptogenesis and hippocampal gray matter volume.For untargeted metabolomics,we report 3464 metabolites detected among all samples.Among them were 190 metabolites,including 106 for positive mode and 84 for negative mode,contributing to epileptogenesis according to results of PLS-DA modelling.Further GO and KEGG analysis found that metabolites in pentose phosphate pathway（PPP）change in both epileptogenesis groups,suggesting possible correlation between PPP and epileptogenesis.Conclusion:This study has the following main findings:（1）metabolic pathway of PE might be involve in epileptogenesis.Particularly,content of PE（18:0₂0:3）and PE（18:1₁8:1）might contribute to epileptogenesis;（2）for the first time,we link level of PE with structural brain imaging indices,in an attempt to potentiate the futuristic application of noninvasive brain imaging techniques to identify epileptogenesis in its latent period;（3）PPP might be involved in epileptogenesis.Part TwoExploring the specific relationship between pentose phosphate pathway and epileptogenesisObjective:As Part One of this study shows multiple metabolites in pentose phosphate pathway（PPP）change as the result of epileptogenesis,suggesting the possible correlation between PPP and epileptogenesis.Yet there are possibilities that it is neuronal loss and/or gliosis that cause metabolites in PPP to change,rather than epileptogenesis.And on the other hand,glucose uptake could also influence the concentration of metabolites in PPP.As a result,whether PPP is disrupted in epileptogenesis and whether stimulating the activity of PPP could impede epileptogenesis remains unknown.The specific relationship between PPP and epileptogenesis is yet to be found.Fructose-1,6-diphosphate（FDP）is intermediate metabolite in glycolysis and administration of FDP is believed to be able to promote the activity of PPP.Thus,in this study,we explore whether FDP could ameliorate epileptogenesis and also measure glucose uptake and neuronal loss and gliosis in a bid to find out the specific relationship betwee PPP and epileptogenesis.Materials and Methods:We randomly seperated 40 rats to four groups:Control group（0.9%NaCl+0.9%NaCl）,PTZ group（0.9%NaCl+PTZ）,FDPPTZ group（FDP+PTZ）,FDP group（FDP+0.9%NaCl）.FDP injection was conducted one hour before PTZ injection.After PTZ injection,seizure scores were rated based on Racine Rating.Open field and contextual fear conditioning were used to measure cognitive changes.Hippocampus were seperated and used to measure metabolites associated with glucose metabolism based on targeted metabolomics.Immunofluorescence was used to detect neuron loss and gliosis.Small animal PET/CT scanner was administrated to measure glucose uptake in a longitudinal manner.Results:Seizure score of FDPPTZ group was significantly lower than that of PTZ group at the 9th injection（P=0.0352）,the 13th injection（P=0.0317）,the 14th injection（P=0.0127）and the 15th injection（P=0.0352）of PTZ.Open field test showed that rats in PTZ group spent more time in central area than those in control group（P=0.041）and those in FDP group（P=0.042）,suggesting FDP administration improve anxiety.Targeted metabolomic data showed that FDP concentration in FDP group was significantly higher than that in control group（P=0.0197）.The content of erythritose4-phosphate（P=0.048）and citric acid（P<0.05）in FDPPTZ group were also significantly higher than in control group.After controlling the impact of time on 18F-FDG uptake,it was found that the glucose uptake decrease as the result of FDP in multiple brain regions associated with epileptogenesis,including hippocampus,amygdala,entorhinal cortex,insular cortex,etc.There are no significant difference in neuronal loss and gliosis found between groups.Conclusion:The main findings of this study are:（1）FDP did show antiepileptogenic effect;（2）after administration of FDP,glucose uptake decreased in hippocampus;（3）even though glucose uptakes decrease,the metabolites in PPP pathway still increased after administration of FDP,suggesting that FDP promote PPP metabolism.（4）PPP might have a protective effect against epileptogenesis.Part ThreeExploring the possible mechanisms of fructose-1,6-diphosphate against epileptogenesisObjective:In the previous part,we explored the specific relationship between pentose phosphate pathway（PPP）and epileptogenesis,and suggesting that promoting the activity of PPP using fructose-1,6-diphosphate（FDP）might has an effect against epileptogenesis.Yet how does FDP impact on epileptogenesis still remains unclear.It is hypothesized that the downstream of PPP produce antioxidant effect so as to protect from epileptogenesis.However,some evidence do not support this hypothesis.Thus,in this study,we try to explore the possible mechanism of FDP against epileptogenesis from the perspectives of transcriptomics and proteomics.Materials and Methods:Rats were divided into three groups randomly:control group,PTZ group and FDPPTZ group,with 10 rats in each group.After model construction,hippocampus were isolated and used for omics analysis（transcriptomics and proteomics）.Animal model construction and hippocampal isolation/preservation are the same as the second part of this paper.Western blot was used to validate the possible associated proteins.Results:Transcriptomic data showed that there were significant changes in mRNA related to glucose metabolism between PTZ group and control group,such as downregulation of glycogen synthase kinase（Gsk3a）and up regulation of citrate synthase（Cs）.Compared with FDPPTZ group,Gsk3a was also down regulated in PTZ group.In addition,Gpx4 related to antioxidation and Bdhl related to ketone metabolism were also changed.No change of Gsk3a and Bdhl was found in FDPPTZ vs control group.Proteomic data showed that compared with the control group,proteins related to mitochondrial oxidative phosphorylation in PTZ group were down regulated,including ndufa2,ndufaf2 and mitochondrial ribosomal protein.Up regulation of protein includes G6PD.These results indicate that epileptogenesis itself can affect these proteins.Compared with FDPPTZ group,PTZ group showed up regulations in some proteins related with mitochondrial oxidative phosphorylation.However,no protein related to glucose metabolism was found to change between these two groups.Compared with the control group,FDPPTZ group also showed up regulation of protein related to mitochondrial oxidative phosphorylation.Down regulated proteins include Mdh1,which is related to TCA metabolism,and Bdhl,which is related to ketone metabolism.Interestingly,the expression of G6PD protein was up-regulated both in PTZ group and FDPPTZ group compared with control group.Pathway analysis found that several signal transduction pathways including mitochondrial oxidative phosphorylation,pentose phosphate pathway and glutathione metabolism may be involved in the pathogenesis of epileptogenesis and/or the antiepileptogenic mechanism of FDP.Conclusion:The main findings of this study are:（1）FDP might impact epileptogenesis by regulating ketone metabolism as Bdhl change in both mRNA and protein level.（2）Mitochondrial also play a part on the mechanisms of FDP against epileptogenesis.（3）It is still possible that FDP promote the activity of PPP to produce antioxidant effect against epileptogenesis...