Effects Of Blood Glucose Fluctuation On Cognitive Function And P38 MAPK Pathway In Type 2 Diabetic Rats

Objective By comparing the effects of intermittent high glucose and constant high glucose on oxidative stress,apoptosis and p38 MAPK pathway related proteins in hippocampus of rats,we hope to investigate the mechanism of blood glucose fluctuation aggravating cognitive impairment in type 2 diabetic rats.Methods 1.Type 2 diabetic model:after adaptive feeding and screening,rats were randomly divided into control group（C）and type 2 diabetes mellitus group（T2DM）.Rats were fed with different diets for 4 weeks:C:maintain diet,unlimited time and supply,T2DM:high-sugar and high-fat diet,unlimited time and supply.The latter was intraperitoneally injected low-dose streptozotocin（STZ）to induce T2DM.2.Blood glucose fluctuation model:T2DM rats were randomly divided into intermittent high glucose（IHG）and constant high glucose group（CHG）.C and CHG continued previous feeding,while IHG adjusted the feeding mode:limited time（twice one day,9:00-10:00,15:00-16:00）and unlimited supply,high-sugar and high-fat diet.3.Data collection:（1）General condition:at the 7^thand 13^thweek,recording the general state and body weight and metabolism;（2）Model evaluation:at the 7^thweek,evaluating T2DM model by measuring two different-day fasting blood glucose（FBG）;at the 13^thweek,evaluating blood glucose fluctuation model by measuring glucose values at 9 time points;（3）At the 17^thweek,evaluating the spatial learning and memory function by Morris water maze test;（4）Detecting oxidative stress level of hippocampus by Elisa;（5）Observing neuronal number and morphology in hippocampal CA1 region by HE staining;（6）Observing neuronal apoptosis in hippocampal CA1 region by Nissl staining;（7）Detecting expression of p38 MAPK pathway proteins in hippocampus by Western blotting.（8）Analysing data by SPSS26.0 software.Results 1.General situation:（1）At the 7^thweek,compared with C,general state of T2DM rats was poor,and their body weight and metabolism were significantly increased,which indicated that they were already obese.（2）At the 13^thweek,IHG and CHG occurred in diseases or even died,showing"three more,one less"symptoms of diabetes.2.Model evaluation:（1）At the 7^thweek,twice FBG in T2DM increased significantly on different days,suggesting that the model of T2DM rats was successful.（2）At the 13^thweek,the results of repeated measurement variance showed that there were differences in glucose levels among three groups at 9 time points during one day（P<0.05）,which is glucose levels in IHG changed significantly,while CHG and C changed less,and there was a peak value of blood glucose in IHG after two meals,suggesting that the blood glucose fluctuation model could be successfully established by changing the daily feeding habits of rats.3.Spatial learning and memory ability:（1）Localized navigation test:The escape latency among three groups was different in day1-day4by repeated measurement variance（P<0.05）,and decreased in groups with days going on,and in the same day,the escape latency of IHG was longer than CHG than C.（2）Spatial exploration test:crossing the platform times in IHG was less than CHG than C（P<0.05）.The ratio of target quadrant activity time in IHG and CHG was less than C（P<0.05）.Which suggested that spatial learning and memory function in IHG and CHG were impaired,and which in IHG was more serious.4.Oxidative stress level:Malondialdehyde（MDA）:IHG was higher than CHG than C（P<0.05）;Total superoxide dismutase（T-SOD）and glutathione peroxidase（GSH-Px）:IHG was lower than CHG than C（P<0.05）.which suggested that the oxidative stress reaction in hippocampus of rats in IHG and CHG were increased,and IHG was more serious.5.Histopathological staining:results of HE and Nissl staining were almost the same:compared with C,the number of neurons in IHG and CHG decreased,the arrangement was disordered and paramorphia.Nissl bodies decreased and the boundaries were blurred,especially in IHG.The number of surviving neurons in CA1 area:IHG and CHG were less than C（P<0.05）;Pathological section score of CA1 area:IHG was higher than CHG than C（P<0.05）.These results suggested that neurons in CA1 area of hippocampus in IHG and CHG were damaged,and which in IHG was more serious.6.Expression of p38 MAPK pathway proteins:（1）Expression of inflammation-related proteins:P-p38MAPK,P-NF-κB proteins:IHG was higher than CHG than C（P<0.05）;TNF-αand IL-1βproteins:IHG and CHG were higher than C（P<0.05）;（2）Expression of cognitive-related proteins:P-Tau-ser199 and P-Tau-ser202 proteins:IHG was higher than CHG than C（P<0.05）;P-Tau-ser396 protein:IHG was higher than C（P<0.05）;（3）Expression of apoptosis-related protein:Caspase-3 protein:IHG was higher than CHG than C（P<0.05）.These results suggested that the inflammatory pathway of p38 MAPK in IHG and CHG rats were activated,which induced increased Tau protein phosphorylation and apoptosis signal,and the above changes were more obvious in IHG.Conclusion Compared with constant high glucose,the cognitive impairment of rats with intermittent high glucose is more serious.The molecular mechanism may be that blood glucose fluctuation activates p38 MAPK inflammatory pathway（p38MAPK/NF-κB/TNF-α/IL-1β）and Tau phosphorylation to a deeper degree by intensifying oxidative stress in hippocampus,forming a vicious circle involving neurofibrillary tangles and inflammatory processes,leading to more neuronal apoptosis,and finally aggravating cognitive impairment.