Molecular Mechanism Of Ketogenic Diet Improving Cognitive Dysfunction In Db/db Mice

Objective: By observing the ketogenic diet(KD)intervention in cognitive impairment to improve the effect of diabetic mice,investigate the possible molecular mechanism.To provide a theoretical basis for the clinical application and promotion of KD in diabetic cognitive impairment(DCI).Methods: There were 36 male SPF mice aged 8 weeks,including 12wild-type m/m mice and 24 db/db mice.After adaptive feeding for one week,the mice were divided into 3 groups with 12 mice in each group,m/m mice were used as normal control group(MA group),and db/db mice were divided into experimental control group(DA group)and KD intervention group(DS group).The MA and DA groups were routinely fed,and the DS group was fed with ketogenic diet with the ratio of 70% coconut oil,20% casein,5.5% cellulose and4.5% premix for 8 weeks.During the period,weight and Fasting blood glucose were measured once a week and blood ketone was measured every two weeks.After 8weeks,the cognitive ability of the three groups of mice was tested.The spontaneous activity and new object recognition experiments were used to detect the autonomous exploratory behavior and non-spatial short time memory.The spatial learning and memory abilities were tested by directional navigation and spatial exploration experiments.Furthermore,the three mice form each group were perfused by 4% paraformaldehyde in the heart.Besides,the HE staining was used for observing the arrangement of the hippocampus which was taken by the whole brain after the perfusion.In addition,the immunofluorescence was applied for the detection of fluorescence intensity in hippocampus of Amyloid β-protein(Aβ)、microglia marker "Ionized calcium binding adaptor molecule-1(Iba-1)" and astrocyte marker " Glial Fibrillary Acidic Protein(GFAP)".Lastly,the remaining9 mice were sacrificed and collected,and the hippocampal tissue was detected by Western blot which included the protein expressions of Advanced glycation end-product(AGE)、 Receptor for advanced glycation end-products(RAGE)、nuclear factor-kappa B(NF-kappa B)、 Interleukin-6(IL-6)and Tumor necrosis factor-α(TNF-α).Results:1.General indexBody weight: all groups mice in 8W was obviously higher than that in 0W(P< 0.01 or P < 0.001)in the same group.While the weight data in three groups presents huge difference,the body weight of the MA group was obviously lower at 0w than that of the DA group(P < 0.001),but there was no obvious distinction between the DS group and DA g roup.Additionally,the body weight in MA group and DS group were obviously lower than that in DA group(P < 0.01 or P < 0.001)at 8W.Fasting blood glucose: there was no visible difference between 8W and 0W in MA group,while it increased between 8W and 0 W in DA group(P < 0.05),and decreased between 8W and 0W in DS group(P < 0.01).in addition,MA group was obviously lower than that in DA group at 0 W(P < 0.001),while there was no visible distinction between DS group and DA group.Both MA group and DS groups was obviously lower than that in the DA group(P < 0.001)at 8W.Blood ketone: there is no obvious change in DA group in each week.But there was visible increase in DS group from 0W to 8W(P < 0.01 or P < 0.001).In addition,it is in the normal range in the DA group all the time and it is in the range of nutritional ketone in the DS group all the time.2.Cognitive abilitySpontaneous activity: The Percentage of active time has obvious increased in MA group and DS group than in DA group(P < 0.01 or P < 0.001).New object recognition: It takes more for percentage of new object exploration time in MA group and DS group than that in DA group(P < 0.05).Directed navigation: The escape latency of all tested mice has successfully decreased after the training.The escape latency in DA group is much higher than that in both MA group and DS group during the 4-day directional navigation(P <0.01 or P < 0.001).Space exploration: T he swimming speed,target quadrant exploration time percentage,number of crossing platforms have been collected and compared among all experimental mice.It indicated that the mice in MA group and DS group reflect more excellent performance than other mice in DA group.3.Morphological indexHE staining results: pyramidal cells in hippocampal CA1 region in DA group were arranged loosely and disorderly,with fewer cell layers and uneven nuclear size.While The pyramidal cells in the MA group and the DS group were arranged neatly and closely,with more cell layers and uniform nuclear size.The py ramidal cells are much less in DA group compared to MA group and DS group which count in the same field of vision(P < 0.05).Immunofluorescence results:Results of Aβ fluorescence intensity: the expression in the MA group and DS group was obviously down-regulated than DA group(P < 0.001).Results of Iba-1 fluorescence intensity: the expression in the MA group and DS group was obviously down-regulated than DA group(P < 0.001).Results of GFAP fluorescence intensity: the expression in MA group and DS group was obviously down-regulated than DA group(P < 0.01 or P < 0.001).4.Western blot index of hippocampal tissueResults of AGE protein expression: the gray value expression in MA group and DS group was obviously lower than DA group(P < 0.05 or P < 0.01).Results of RAGE protein expression: the gray value expression in MA group and DS group was obviously lower than DA group(P < 0.001).Results of NF-κB protein expression: the gray value expression in MA group and DS group was obviously lower than DA gro up(P < 0.05 or P < 0.01).Results of IL-6 protein expression: the gray value expression in MA group and DS group was obviously lower than DA group(P < 0.05 or P < 0.01).Results of TNF-α protein expression: the gray value expression in MA group and DS group was obviously lower than DA group(P < 0.01 or P < 0.001).Conclusion:1.KD can improve diabetic symptoms by reducing the body weight and blood glucose of db/db mice and putting them in a state of nutritional ketosis;2.DCI was significantly improved in db/db mice treated with KD,which may be related to the optimization of pyramidal cell arrangement in hippocampal CA1 region,the down-regulation of inflammation-related indexes,and the change of AGE/RAGE/NF-κB signaling pathway.