Protective Effect And Mechanism Of Ketogenic Diet On Hypoglycemia-induced Brain Injury In Mice

Hypoglycemia is a metabolic disease with low blood glucose concentration caused by many reasons,and is a common complication in diabetic patients during insulin therapy.Hypoglycemia leads to neuronal death in different areas of the brain,impairs the cognition of diabetic patients and is associated with the development of dementia.In addition,hypoglycemia is closely related to many animal diseases.Lactating cows have a negative energy balance due to lactation needs,resulting in the decline of glucose,loss of appetite,decrease of milk production and even death.Prevention and control of the occurrence and development of hypoglycemia is beneficial to alleviate its serious consequences.Ketogenic diet(KD)is a formula diet with high fat,low carbohydrate and medium protein.KD using fat as raw material to generate ketone bodies,especially the increase of the concentration ofβ-hydroxybutyrate(BHBA),which provides energy source for the brain.KD plays an anti-inflammatory and anti-apoptotic role by reducing the accumulation of reactive oxygen species(ROS)and inhibiting the inflammasome formation.As a classical dietary pattern,KD can regulate gut microbiota and their metabolites,affecting human health in many aspects such as energy homeostasis,nutrient metabolism and immune regulation.However,the effects of KD and gut microbiota in brain injury induced by hypoglycemia and its related molecular mechanisms have been seldom reported.Our research adopted 16S r RNA sequencing to monitor the changes of gut microbiota in KD mice and used insulin to establish mice models of hypoglycemia.By means of immunofluorescence,western blotting,real-time quantitative PCR,flow cytometry,and other methods,the potential mechanism of KD on hypoglycemia brain injury and neuroinflammation in mice were approached in vivo and in vitro.The results are as follows:(1)After 20-30 min of insulin injection,hypoglycemia occurred in mice,and at 3 h the survival rate was 55%.KD could alleviate hypoglycemia,so at 3 h the survival rate was 95%.Evans blue staining showed that blood-brain barrier(BBB)permeability increased in hypoglycemia mice.KD could increase the expression of Podxl and ZO-1,which was able to alleviate BBB injury induced by hypoglycemia.KD reduced brain edema induced by hypoglycemia by decreasing AQP4 content and maintaining its polarized expression.(2)16S r RNA sequencing results showed that KD reduced the Chao 1 index of gut microbiotaα-diversity.In theβ-diversity analysis,significant separation was found between the control and KD groups.KD increased the relative abundance of Firmicutes and Proteobacteria and decreased that of Bacteroidetes.Moreover,KD increased the relative abundance of Pseudoflavonifractor positively associated with weight loss and decreased Lactobacillus associated with immunity and energy metabolism.(3)Open field test found that single severe hypoglycemia had no significant effect on depressed behavior in mice,while KD mice without gut microbiota were depressed due to the fear of new open environment,which was related to the decrease of TH and TPH2.Elevated plus maze and Morris Water Maze test results showed that KD alleviates anxiety-like behavior and cognitive impairment in hypoglycemic mice.Further observations of dendritic spines and synaptic structures revealed that KD increased the proportion of Mushroom dendritic spines and decreased the proportion of Stubby dendritic spines in hippocampus of hypoglycemia mice.KD regulated synaptic plasticity by increasing the levels of SPN,PSD95 and SYP synaptic related proteins.Mechanism studies showed that KD and gut microbiota could increase the density of newborn neurons(Brdu~+DCX~+cells)and surviving/differentiated neurons(Brd U~+Neu N~+cells)by inhibited the activation of IRE1-XBP1 and ATF6 endoplasmic reticulum stress pathway and reduced the levels of cleaved caspase-3/caspase-3 in hypoglycemia mice.(4)KD and gut microbiota increased the levels of SOD and GSH-PX and decreased the m RNA levels of TNF-α,IL-1βand IL-6 induced by hypoglycemia in the brain.Hippocampal microglia branch endpoints and dendrite complexity were increased by hypoglycemia.KD inhibited the activation of microglial induced by hypoglycemia and the activation of TLR4/p38 MAPK/NF-κB signaling pathway.As a result,KD and gut microbiota could alleviate neuroinflammation induced by hypoglycemia.(5)In vitro,by the low-glucose cell models constructed by PC12 cells and primary neurons,we found that BHBA inhibited intracellular ROS production induced by low glucose,mediated p38 MAPK signaling pathway and caspase-3 apoptosis cascade and activated ERK phosphorylation to inhibit primary neuronal apoptosis.These results suggested that KD alleviated hypoglycemia-induced adult neurogenesis disorder and neuroinflammation by increasing BHBA levels and regulating intestinal flora.This study provides new ideas and directions for optimizing the treatment strategy of neuron damage caused by hypoglycemia.