| Objective:1、To investigate the learning and memory abilities of experimental type 2diabetic mice by Morris water maze test and to detecte the effect of trigonelline up-regulating neuritin on learning and memory of experimental type 2 diabetic mice.2、To observe the effects of trigonelline on hippocampal neuritin,astrocyte marker GFAP,SYN and Tomosyn and JAK2/STAT3 signaling protein expression of experimental type 2 diabetic mice by Immunofluorescence and western blot.3、To observe effects of trigonelline up-regulating neuritin expression on neuronal damage repair in experimental type 2 diabetic mice by Nissl staining.To investigate the relationship among cognitive impairment of type 2diabetes mellitus and up-regulated neuritin by trigonelline and JAK2/STAT3signaling pathway to inhibit astroglial reactive glia and block hippocampal neuronal synaptic plasticity in type 2 diabetic cognitive dysfunction.Methods:1、Breeding of transgenic mice and model miceLoxp transgenic mice were crossed with Emx1 transgenic mice to obtain Loxp/Emx1 transgenic mice which contain both Loxp and Emx1 genes.Then Loxp/Emx1 transgenic mice were crossed with heterozygous db/m mice to obtain homozygous transgenic mice with Loxp,Emx1 and db/db.In large numbers of heterozygous db/m mice are breeded to obtain homozygous db/db mice be model mice.2、Experimental group and drug treatmentSix wild mice were selected as the normal control group,homozygous db/db mice as model mice of type 2 diabetes,and the other experimental groups include Loxp/Emx1（transgenic high expression neuritin mice）group,Loxp/Emx1/db group（transgenic high expression of neuritin type 2 diabetic mice）,Loxp/Emx1/db with trigonelline(50 mg·kg-1),Loxp/Emx1/db with metformin(50 mg·kg-1),model group with trigonelline(50 mg·kg-1)and model group with metformin(50 mg·kg-1).The mice in the normal control group,model group and Loxp/Emx1 group were given gavage orally once a week for8 weeks.The fasting blood glucose（Evacuated food the day before）and body weight was measured every 2 weeks,according to the weight changes to adjust the drug dose.3、Tissue preparationAfter 8-week treatment,the mice were administered with Morris water maze about learning and memory ability.The mice were anesthetized by intraperitoneal injection of 10%chloral hydrate(100 mg·kg-1)After the mice were thoracotomy,blood samples were taken to a 0.5 mL syringe from the abdominal vena cava for determination of biochemical parameters.After the blood sample was collected,the hippocampus was used to extract the protein for Western Blot experiments and used 4%paraformaldehyde to fix it used to make frozen sections.4、Total cholesterol,triglyceride and glycosylated hemoglobin A1cc were measured by commercial kit according to the experimental procedure and the weight of brain tissue were weighed.5、The repair of hippocampal neurons and the integrity of the hippocampus were observed by Nissl staining.6、To investigate the expression of neuritin,astrocyte marker GFAP,hippocampal synaptophysin（SYN）and Tomosyn,hippocampal Inflammatory factors（TNF-α,IL-6,IL-1β）and JAK2/STAT3 signal pathway related proteins（JAK2,p-JAK2,STAT3 and p-STAT3）by Immunofluorescence and Western Blot in model group and others.7、Statistical analysisAll experimental data are carried out using the SPSS17.0 software and presented as mean±standard.Behavioral and biochemical data were analyzed by one-way analysis of variance,the other data were compared using t test.The difference was statistically significant when P<0.05.Results:1、Reproduction and identification of transgenic miceThe Loxp transgenic mice,Emx1 transgenic mice and heterozygous db/m mice were crossed to obtain the homozygous Loxp/Emx1/db genotype mice.Gene band of the genotypes of homozygous Loxp/Emx1/Loxp mice identified by the method of genotype identification were positive.That is,Loxp gene band,Emx1-cre gene band and db/m gene band were all positive.2、The results of Morris water maze behavior testThe locomotion path and latency of mice in each group were shortened gradually during the continuous training of Morris water maze.And there were significant differences in the distance,time and the number of acrossing the safety platform.Compared with those in the normal control group,the latency of mice was significantly longer and there were significant decreased in the distance and time and the number of acrossing the safety platform in the model group(###P<0.001).Compared with those in the model group,the latency of mice in Loxp/Emx1 group,Loxp/Emx1/db group,Loxp/Emx1/db with trigonelline group,Loxp/Emx1/db with metformin group and model with trigonelline was significantly shortened and there were significant increased in the distance and time and the number of acrossing the safety platform(*P<0.001,**P<0.001,***P<0.001),Loxp/Emx1/db with trigonelline group were more significant particularly.However,there was no significant difference in the latency and the number and in the distance and time and the number of acrossing the safety platform in model with metformin group（P>0.05）.3、The levels of body weight and blood glucose and blood lipidsBefore administration,there was significantly different in the the body weight of each group mice（P<0.05）.Compared with those in the normal control group,the boody weight of model mice was significantly different(###P<0.001).Compared with those in the model group,the body weight of Loxp/Emx1 Group,loxp/Emx1/db Group,Loxp/Emx1/db with metformin Group,Loxp/Emx1/db with trigonelline and model with trigonelline gourd was significantly decreased(***P<0.001).After administration,the weight of each group mice increased slightly.Compared with those in the model group,the Model with metformin and the Model with trigonelline gourd had no statistical difference in body weight（P>0.05）,the body weight of Loxp/Emx1group,Loxp/Emx1/db group,Loxp/Emx1/db with metformin group and Loxp/Emx1/db with trigonelline gourd was significantly decreased(*P<0.05,***P<0.001).Before the administration,compared with thosee normal control group,the levels of blood glucose value in the model group was significantly increased(###P<0.001);Compared with those model group,the levels of blood glucose in the Loxp/Emx1 group decreased significantly(***P<0.001),and the blood glucose in the other groups decreased but no statistically significant（P>0.05）.At the end of 2 weeks,compared with those in the normal group,the levels of blood glucose in the model group were significantly increased(###P<0.001);Compared with those model group,the levels of blood glucose in the Loxp/Emx1 group and Loxp/Emx1/db with trigonelline gourd was significantly decreased(*P<0.05,***P<0.001),The levels of blood glucose in the other groups were significantly decreased but no statistically significant（P>0.05）.At the end of 4 weeks,6 weekend and 8 weekend after administration,compared with those in the normal group,the levels of blood glucose in the model group was increased significantly(###P<0.001);Compared with those in the model group,the levels of blood glucose in Loxp/Emx1/db group was slightly reduced but no statistically significant（P>0.05）,the levels of Loxp/Emx1 group,Loxp/Emx1/db with Metformin group,Loxp/Emx1/db with trigonelline group,model with metformin group and model with trigonelline gourd had a significant decrease(*P<0.01,**P<0.01,***P<0.001),but at the end of 6 weeks after administration,compared with those model group,the levels of blood glucose in the model with metformin group was slightly decreased but no statistically significant（P>0.05）.Compared with those in the normal control group,the levels of HbA1c,TG and TC in model group were significantly increased(###P<0.001);Compared with those in the model group,the HbA1c level of model with metformin group and model with trigonelline gourd was decreased but no statistically significant（P>0.05）;The level of HbA1c in the Loxp/Emx1 group,Loxp/Emx1/db group,Loxp/Emx1/db with metformin group and Loxp/Emx1/db with trigonelline gourd was significantly decreased(*P<0.05,***P<0.001).Compared with those in the model group,the levels of TG in the Loxp/Emx1 group,Loxp/Emx1/db group,Loxp/Emx1/db with trigonelline and Loxp/Emx1/db with metformin group were significantly decreased(***P<0.001),and the level of TG in the Model with metformin group was not statistically significant（P>0.05）.Compared with those in the model group,the levels of TC in the Loxp/Emx1 group,Loxp/Emx1/db with trigonelline gourd and model with trigonelline gourd were significantly decreased(***P<0.001),and there was no statistically difference in levels of TC in the Loxp/Emx1/db with metformin group and model with metformin group.4、Brain tissue weightCompared with those in the normal control group,the weight of brain tissue was significantly lower in the model group(###P<0.001);Compared with the model group,the weight of brain tissue was significantly increased in the Loxp/Emx1 group,Loxp/Emx1/db with metformin group and Loxp/Emx1/db with trigonelline(**P<0.01,***P<0.001),but,there were no obvious difference（P>0.05）in brain weight in other group.5、Observation of hippocampal neurons injury repair and hippocampus integrityTo observe and analyze the effects of neuritin up-regulated by trigonelline on neuronal injury repair and hippocampus integrity in type 2diabetic model group and others by Nissl staining.Compared with those in the normal control group,there was obvious loss of neurons and Incompleteness of hippocampus in the model group and the model with metformin group as shown by the arrow in the figure;Compared with those in the model group,the hippocampal regions showed no significant difference in the loss of neurons in the model with metformin groups,Loxp/Emx1 group,Loxp/Emx1/db group,Loxp/Emx1/db with metformin group,Loxp/Emx1/db with trigonelline group and model with trigonelline group,at the same time,neurons arranged closely,neuronal damage reduced and the hippocampus relatively intact.6、The expression levels of neuritin,GFAP,synaptophysin-related proteins Tomosyn and SYN in hippocampusCompared with those in the normal control group,the expression of neuritin and Tomosyn and SYN protein were significantly decreased(#P<0.05,##P<0.01,###P<0.001)in the model group;Compared with those in the model group,there were no significant difference（P>0.05）in the expression of neuritin and Tomosyn protein in the model with metformin,but were significantly increased in the Loxp/Emx1 group,Loxp/Emx1/db group,Loxp/Emx1/db with metformin group and Loxp/Emx1/db with trigonelline group(*P<0.05,**P<0.01,***P<0.001).Compared with the model group,the expression of SYN protein were also significantly increased in each group mice(***P<0.001).Compared with the normal control group,the GFAP protein expression was significantly increased(###P<0.001)in the the model group.Compared with those model group,the expression of GFAP protein was significantly decreased(*P<0.05,**P<0.01,***P<0.001)in the mice of each group.7、The expression levels of TNF-α、IL-1β及IL-6 in hippocampusCompared with those in the normal control group,the expression of TNF-α,IL-1βand IL-6 were significantly increased(##P<0.001,###P<0.001)in model group.Compared with the model group,The levels of TNF-α,IL-1βand IL-6 in the Loxp/Emx1 group,Loxp/Emx1/db group,Loxp/Emx1/db with metformin group and Loxp/Emx1/db with trigonelline group were significantly decreased(*P<0.05,**P<0.01,***P<0.001),but there was no significant difference in the model with metformin and model with trigonelline（P>0.05）.8、The expression levels of JAK2/STAT3 signal pathway related proteins in hippocampusCompared with those in the normal control group,the expression level of JAK2 protein in model group and each group had no statistical significance（P>0.05）.The expression level of p-JAK2 protein was obviously increased in the model group(##P<0.01).Compared with the model group,the expression level of p-JAK2 in model with metformin group and model with trigonelline group had no significant difference（P>0.05）,but The expression level of p-JAK2 were significantly decreased in the Loxp/Emx1 group,Loxp/Emx1/db group and Loxp/Emx1/db with metformin group and Loxp/Emx1/db with trigoneine group(*P<0.05,**P<0.01).Compared with the normal control group,the expression level of STAT3protein in model group and each group had no statistical significance（P>0.05）.The expression level of p-STAT3 protein was significantly increased in the model group(###P<0.001).Compared with the model group,the expression level of p-STAT3 in model with metformin group and model with trigonelline group had no statistical significance（P>0.05）,but the expression of p-STAT3were significantly decreased in the Loxp/Emx1 group,Loxp/Emx1/db group and Loxp/Emx1/db with metformin group and Loxp/Emx1/db with trigonelline group(**P<0.01,***P<0.001).9、Immunofluorescence of neuritin and GFAP in hippocampusAccording to the fluorescence intensity,the expression of neuritin in the hippocampus was significantly higher in the normal group and the Loxp/Emx1group than in the model group,and hippocampal neuritin was also significantly higher in the Loxp/Emx1/db with metformin group and the Loxp/Emx1/db with trigoneine group particularly.Compared with those in the normal control group,the expression of neuritin in the model group was significantly lower(###P<0.001).Compared with the model group,the expression of neuritin in the Loxp/Emx1 group and the Loxp/Emx1/db with trigonelline were increased significantly(***P<0.001).The expression of neuritin in the Loxp/Emx1/db group and the Loxp/Emx1/db with metformin were increased but had no statistical significance（P>0.05）.There was no significant difference in the expression of neuritin in the model with metformin group and model with trigonelline group（P>0.05）.According to the fluorescence intensity,the expression of GFAP in the hippocampus was significantly lower in the normal group and the Loxp/Emx1group than in the model group,and hippocampal GFAP was also significantly higher in the Loxp/Emx1/db with metformin group and the Loxp/Emx1/db with trigoneine group particularly.Compared with those in the normal control group,the expression of GFAP in the model group was significantly higher(###P<0.001).Compared with the model group,the expression levels of neuritin in the Loxp/Emx1 group and the Loxp/Emx1/db with trigonelline were decreased significantly(***P<0.001).The expression levels of GFAP in the Loxp/Emx1/db group and the Loxp/Emx1/db with metformin were decreased but had no statistical significance（P>0.05）.There was no significant difference in the expression of GFAP in the model with metformin group and model with trigonelline group（P>0.05）.Conclusion1、The learning and memory function of model mice with type 2 diabetes mellitus was decreased.High expression of neuritin and up-regulation of neuritin expression by trigonelline in the hippocampus improved learning and memory function.2、Compared with the normal control group,the mice with type 2 diabetes had higher body weight,higher blood glucose level,higher glycated hemoglobin level and higher blood lipid levels（TC and TG values）.Both metformin and trigonelline decreased body weight and glycosylated hemoglobin levels;trigonelline also reduced the TC and TG values in type 2 diabetic mice,but metformin did not reduce the level of TC and TG.3、Hippocampal neurons in type 2 diabetic mice are impaired and lost,and the hippocampus is relatively incomplete,and the weight of brain tissue was reduced slightly.Transgenic high expression of neuritin and up-regulated neuritin by trigonelline can promote hippocampal neuron regeneration and repair of hippocampal damage and reduce the relative integrity,reduce brain atrophy.4、Glial reactive hyperplasia and the expression of GFAP was significantly increased in astrocytes of type 2 diabetic mice,which resulted in that the expression of proinflammatory cytokines,such as TNF-α,IL-1βand IL-6,were increased and the expression of hippocampal neuritin was decreased.The levels of hippocampal synaptic plasticity protein Tomosyn and SYN were decreased and hippocampal JAK2/STAT3 signaling pathway was activated.The high expression of neuritin and the neuritin up-regulated by trigonelline can inhibit the activation of astrocytes and the proliferation of astrocytes,prevent the proliferation of astrocyte,TNF-α,IL-1βand IL-6;After high expression of neuritin and neuritin up-regulated by trigonelline,a large number of neuritin a were associated with specific receptors in the cell membrane of astrocytes to inhibit the activation of JAK2 by the combination of cytokines and the specific receptors on the membrane,thereby inhibiting STAT3 phosphorylation,So that the two-body form into the nucleus and the specific DNA components,the initiation of reactive glial hyperplasia target gene transcription,so as to regulate reactive glial hyperplasia,so as to regulate the formation of astrocytes scar on synaptic plasticity,thus improving learning cognitive function. |
PDF Full Text Request