Protective Effects And Mechanism Of Dendrobium Nobile Lindl.Alkaloids On Learning And Memory Impairment Induced By High Methionine Diet

Objective:To explore the protective effect and mechanism of Dendrobium nobile Lindl.alkaloids（DNLA）on learning and memory impairment in mice induced by high methionine（Met）diet.Method:8-week-old male C57 BL/6J mice were adaptively housed for 2 weeks and randomly divided into control group,DNLA20 control group,Met group,DNLA10 group and DNLA20 group,among which control group and DNLA20 control group were given normal diet,other groups were given 2%Met diet.After 11 weeks of diet,DNLA20 control group（20 mg/kg）,DNLA10（10 mg/kg）group and DNLA20（20 mg/kg）group were orally administrated with DNLA for 3 months,and the other groups were given the same volume of 1%Tween 80 by orally administration.Nesting test,the Y maze test,and the Morris water maze test were used to detect the motor ability,social behavior,learning and memory abilities of the mice.After the behavioral test,the mice were euthanized（Orbital blood collection）and the materials were collected.HE and Nissl stainings were used to detect the damage of hippocampus dentate gyrus（DG）and cortex neurons regions in mice.Immunofluorescence staining was used to detect the levels of 5-methylcytosine（5-m C）,β-amyloid 1-40(Aβ_1-40)andβ-amyloid 1-42(Aβ_1-42)in the hippocampus（DG）and cortex regions.ELISA detected the levels of homocystein（Hcy）,Aβ_1-42 in serum and S-adenosine methionine（SAM）,S-adenosine homocysteine（SAH）in the liver;Western blot（WB）was used to detect the Aβproduction-related proteins amyloid precursor protein（APP）,presenilin-1（PS1）andβ-secretase 1（BACE1）expression levels,Aβmetabolism-related proteins insulin-degrading enzyme（IDE）and neprilysin（NEP）expression levels,Aβ_1-40 and Aβ_1-42 expression levels,DNA methyltransferases（DNMTs）related proteins DNA methyltransferase1（DNMT1）,DNA methyltransferase3a（DNMT3a）,and DNA methyltransferase3b（DNMT3b）expression levels.Methylation-specific PCR（MSP）detected whether there was methylation on the cytosine-phosphate-guanine（CPG）islands of APP,PS1,BACE1,and NEP genes.Methyl Target method detected the CPG islands methylation level APP,PS1,BACE1 and NEP gene.Results:1.Behavioral test:（1）Nesting building test:Compared with the control group,the nesting scores of the Met group mice trend to reduce（p>0.05）.DNLA treatments trend to improve the nesting scores of the Met group mice（p>0.05）;（2）Y maze test:Compared with the control group,the correct alternation percentage of the Met group trend to decrease（p>0.05）.DNLA treatment trend to increase the correct alternation percentage of the Met group（p>0.05）;（3）Morris water maze test:Compared with the control group,the escape latency of the Met group mice was significantly increased on the fourth day（p<0.05）and the number of crossing platforms was significantly reduced on the fifth day（p<0.05）.DNLA treatment can significantly reduce the escape latency and increase the number of crossing platforms（p<0.05）of the Met group.2.Pathology test:（1）HE staining and Nissl staining:Compared with the control group,the hippocampal DG and cortex regions neurons of the Met group were damaged,the cell arrangement was disordered,and the number of normal neurons was significantly reduced（p<0.05）.DNLA treatment can improve the damage,the disorder of cell arrangement,and increase the number of normal neurons（p<0.05）of hippocampal DG and cortex regions neurons in the Met group;（2）Immunofluorescenc staining:Compared with the control group,the level of 5-m C in the hippocampal DG（p<0.05）and cortex regions（p<0.001）increased;hippocampal DG（p<0.05）and cortex regions（p<0.05）Aβ_1-40 level increased;hippocampal DG（p<0.01）and cortex regions（p<0.001）Aβ_1-42 level increased of mice in the Met group.DNLA treatment can reduce 5-m C levels in hippocampal DG（p>0.05）and cortex regions（p<0.05）,Aβ_1-40levels in hippocampal DG（p<0.05）and cortex regions（p>0.05）,hippocampus DG（p<0.05）and cortex regions（p<0.05）Aβ_1-42 levels in the Met group.3.ELISA:（1）Serum:Compared with the control group,the levels of Hcy（p<0.001）and Aβ_1-42（p<0.05）in the serum of the Met group increased significantly.DNLA treatment can significantly reduce the Hcy（p<0.05）and Aβ_1-42（p<0.05）levels of serum in the Met group.（2）Liver:Compared with the control group,the levels of SAM（p>0.05）,SAH（p>0.05）,and SAM/SAH（p<0.05）in the liver of the Met group increased,after DNLA treatment the levels of SAM（p>0.05）,SAH（p>0.05）,and SAM/SAH（p<0.05）in the liver of the Met group decreased.4.WB:（1）Cortex:Compared with the control group,APP（p<0.05）,PS1（p<0.05）,BACE1（p<0.05）,DNMT1（p<0.05）proteins expression were up-regulated in the Met group mice,and NEP（p<0.001）,DNMT3a（p<0.05）、DNMT3b（p<0.01）proteins expression down-regulated,Aβ_1-40（p<0.01）and Aβ_1-42（p<0.01）levels increased but there was no change in IDE protein expression（p>0.05）.DNLA treatment down-regulated APP（p<0.05）,PS1（p>0.05）,BACE1（p<0.05）,DNMT1（p<0.05）proteins expression,and up-regulated NEP（p<0.05）,DNMT3a（p>0.05）,DNMT3b（p<0.05）proteins expression,reducing Aβ_1-40（p<0.05）and Aβ_1-42 levels（p<0.05）,but there was no change in IDE protein expression（p>0.05）.（2）Hippocampus:Compared with the control group,APP（p<0.05）,PS1（p<0.05）,BACE1（p<0.01）,DNMT1（p<0.01）proteins expression were up-regulated in the Met group.IDE（p<0.01）,NEP（p<0.001）,DNMT3a（p<0.05）,DNMT3b（p<0.05）proteins expression were down-regulated,and Aβ_1-40（p<0.05）and Aβ_1-42（p<0.05）levels increased.DNLA treatment down-regulated PS1（p<0.05）,BACE1（p<0.05）,DNMT1（p<0.05）proteins expression,and up-regulated IDE（p<0.05）,NEP（p<0.05）,DNMT3a（p<0.05）,DNMT3b（p>0.05）proteins expression,reducing Aβ_1-40（p<0.05）and Aβ_1-42（p<0.05）levels,but there was no change in APP protein expression（p>0.05）.5.Methylation level detection:（1）MSP:The CPG islands of APP,PS1,BACE1,and NEP genes in each group were all methylated.（2）Methyl Target detection:Compared with the control group,the methylation levels of APP（p<0.05）and BACE1（p<0.05）genes CPG islands in the Met group decreased,and DNLA treatment increased APP（p<0.05）and BACE1（p<0.05）genes CPG island methylation level,but PS1 and NEP genes CPG island methylation level did not change in each group.Conclusion:DNLA can improve learning and memory impairment in mice induced by high Met diet via DNA methylation pathway.