| Objective: This study used mass spectrometry-based acetylation modification omics technology to screen out differentially acetylated proteins in hippocampus of rats with diabetic cognitive dysfunction.The major functions and involved signal pathways of these differentially acetylated proteins were analyzed,and the role of histone acetylation modification in diabetic encephalopathy was explored in preliminary.It can provide a good resource and idea for studying the pathogenesis of diabetic encephalopathy.Methods: 1.Morris water maze was used to screen 30 rats of SD with normal cognitive function and divided them into control group and diabetic group randomly(n = 15),the diabetic group were modeled by using one-time intraperitoneal injection of streptozotocin,and the blood glucose were tested in every week.2.The change of their cognitive function were tested by using morris water maze after the diabetic model was replicated twelve weeks.3.HE staining was used to observe the pathological changes of hippocampus 4.Three rats in the control group with normal cognitive function and in the diabetic group with cognitive impairment were selected randomly.Based on mass spectrometry,we used quantitative acetylation modification omics to accurate these differentially acetylated proteins in identification and quantitative analysis in the hippocampus of these rats.The bioinformatics methods such as GO analysis,subcellular localization analysis,COG functional classification,KEGG pathway enrichment were selected for further analysis.5.Then detected some of differentially histone acetylation modification sites such as H4K8 ? H4K12 and H4K16 by western blot.6.The mouse hippocampal neuron HT-22 cells were cultured in vitro,the cells were treated with different concentrations of sugar medium: 25,35,45,55,65,75mmol/L for 24 h,48h and 72 h,respectively.The reference concentration of 25mmol/L was used as the control group,and the other groups were the experimental group.The cell activity was measured by cell counting kit(CCK)-8 and the optimal action time was screened.7.The morphological changes of cells in each group were observed by light microscope.8.The lactate dehydrogenase(LDH)test was selected to detect LDH release rate in cell supernatant.9.The apoptosis of cells in each group was detected by flow cytometry10.And then the expression of apoptosis-related proteins Bax and Bcl-2 was detected by Western blot,and finally,determined the optimal high glucose concentration.11.Under the condition of optimal high glucose action time and concentration,western blot was used to detect the expression of H4K8 ac,H4K12ac and H4K16 ac.Results: 1.We successfully screened out 30 rats of SD with normal cognitive function and successfully replicated the diabetic rat model.Compared with the control group,the fasting blood glucose level in diabetic group was significantly increased,which was basically maintained above 20 mmol / L(P <0.01).2.Compared with the control group,the escape latency of rats in diabetic group increased significantly,and the number of crossing the platform decreased significantly(P <0.01),which proved that its learning and memory abilities were impaired.3.HE staining results showed that compared with the control group,the hippocampus of the diabetic group was disordered,the cells were arranged loosely and irregularly,the nuclei were lightly stained,the cells shrank,and the number were reduced.4.In the control group and diabetic group,we take the 1.5 times as the change threshold,and identified that the levels of 66 acetylation sites on 61 proteins have changed,of which 44 acetylation sites on 43 proteins were significantly up-regulated,and 22 acetylation sites on 18 proteins were significantly down-regulated by mass spectrometric analysis(P <0.05).Also,in diabetic group,the levels of quantitative acetylation of H4K8,H4K12 and H4K16 was decreased significantly(P<0.05).We also used bioinformatics analysis methods such as GO analysis,and found that these differentially acetylated proteins were mainly combined with binding and catalytic functions,and mainly involved in cellular process,metabolic process and biological regulatory process.The subcellular structure localization analysis found that most proteins were predicted to be localized in nucleus and cytoplasm.COG function classification showed that the mainbiological functions of these differentially acetylated modified proteins were focused on signal transduction mechanisms,post-translational modifications,protein folding,molecular chaperones,energy generation and conversion,chromatin structure dynamics,amino acid transport and metabolism,and other aspects.KEGG pathway enrichment analysis found that they were mainly involved in metabolism and endocrine-related pathways,such as cysteine and methionine metabolism,niacin and nicotinamide metabolism,thyroid hormone synthesis,and c GMP-PKG signaling pathway,etc.5.Western blot showed that the expression of H4K8 ac,H4K12ac and H4K16 ac of hippocampus in diabetic group were decreased significantly(P <0.01).6.The results of CCK8 assay showed that high glucose can inhibit the viability of HT-22 cells in a dose-and time-dependent manner.When the high glucose action time is 48 h,the cell viability?80% can meet the requirements of subsequent experiments.So,we determined 48 h as the best action time.7.Observed the changes of cell morphology by light microscope.We found that compared with the control group,the cells in high glucose showed number decrease,large body,nucleus dissolve,synaptic rupture,cell debris and other changes.8.And the release rate of LDH in other experimental groups also increased.Among them,the 55,65,and 75 mmol / L high-glucose groups were the most significantly(P <0.05).9.Flow cytometry showed that compared with the control group,the apoptosis rate of cells in other experimental groups increased significantly(P<0.05).10.Western blot found that the expression of Bax increased and Bcl-2 decreased in the high-glucose group at 55,65,and 75 mmol / L(P <0.05).Based on the above cell experiments,we determined that 55 mmol / L was the optimal concentration for subsequent experimental research.11.Compared with the control group,the expression of H4K8 ac,H4K12ac and H4K16 ac in high glucose group were reduced significantly(P<0.01).Conclusion: 1.Compared with the rats of normal cognitive function,we found that a variety of proteins in the hippocampus of diabetic cognitive dysfunction rats showed significant acetylation modification.Besides,histone acetylation may has a certain relationship with the pathogenesis of diabetic encephalopathy.2.It was determined that the high glucose concentration of 55 mmol /L for 48 h in mouse hippocampal neurons HT-22 cells was the best model condition.Based on the above,the levels of histone H4K8,H4K12,and H4K16 acetylation modifications reduced significantly. |
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