Pterostilbene Alleviates Diabetic Cognitive Impairment By Suppressing TLR4/NF-kB Pathway Through Microbiota-Gut-Brain Axis

Diabetic cognitive impairment(DCI)is a serious complication caused by damage to the central nervous system of diabetic patients.Although the incidence of DCI is gradually increasing,there is no effective intervention strategy for it.Pterostilbene is an active ingredient in Dracaena cochinchinensis,which has many pharmacological effects,such as ameliorating diabetes,neurological diseases,intestinal diseases and so on.Modern pharmacological studies have shown that the pathogenesis of DCI is closely related to the "microbiota-gut-brain axis".Therefore,this study is based on the TLR4/NF-κB signaling pathway and "microbiota-gut-brain axis" to explored the mechanism of pterostilbene improve cognitive dysfunction in DCI mice.The specific research contents are as follows:1.Exploration of the protective effects of pterostilbene on DCI miceDiabetic cognitive impairment(DCI)model was established by high-fat diet combined with injections of STZ.After 10 weeks of pterostilbene intervention,cognitive function was measured by Morris water maze.The blood glucose and blood lipid levels were tested by automatic biochemical analyzer.HE,Nissl staining and Congo red staining were used to evaluate brain pathology,and HE and AB-PAS staining were used to evaluate colon pathology.The results showed that pterostilbene could improve the cognitive dysfunction of DCI mice,regulate glucose and lipid metabolism disorders,improve neuronal damage in brain tissue,reduce the accumulation ofβ-amyloid,improve colon injury,and thus play a role in protecting DCI.2.Pterostilbene alleviates DCI through regulating TLR4/NF-κB pathwayTo further explore the mechanism of pterostilbene in improving cognitive dysfunction in diabetes,the levels of ACh,MDA,and SOD in brain,and the content of AGEs in plasma and brain were detected,indicating that pterostilbene could reduce the neurotransmitter deficiency and oxidative stress in DCI mice.Fluorescent labeling was used to test dopaminergic neurons,GFAP and Iba-1 related to neuroinflammation in brain,indicating that pterostilbene could inhibit the loss of dopaminergic neurons and the increase of GFAP and Iba-1 in DCI mice.ELISA kits were used to measure the content of inflammatory factors(IL-6,IL-1 β)and LPS in colon and brain,indicating that pterostilbene can reduce the accumulation of inflammatory factors and LPS in colon and brain.Using Western bloting technology to observe the expression of inflammation-related pathway proteins in colon and brain,indicating that pterostilbene can regulate the TLR4/NF-κB signaling pathway in colon and brain.Pearson correlation analysis was used for intestinal inflammation and neuroinflammation.The result showed that intestinal inflammation and neuroinflammation were closely related in DCI mice.3.Pterostilbene improves intestinal barrier,blood-brain barrier and microbial dysbiosis in DCI miceTo deeply investigate the correlation of intestinal inflammation and neuroinflammation and the mechanism of pterostilbene intervention in cognitive dysfunction in diabetes,using immunofluorescence technology,the expression of tight junction protein ZO-1 in the colon was observed,suggesting the intestinal barrier of mice has been damaged,and pterostilbene can improve the intestinal barrier damage.Using Western blotting technology to characterize the tight junction proteins ZO-1 and Occludin in the intestinal barrier and blood-brain barrier,the results show that pterostilbene can improve the tightness of the gut and brain.Connexin expression.HPLC testing and RT-qPCR technology were used to detect short-chain fatty acids and their receptors,respectively,the results showed that pterostilbene could inhibit the reduction of short-chain fatty acids and their receptors in DCI mice.16S rDNA sequencing technology was used to detect each group microbial changes,the diversity analysis,differential species analysis,species composition analysis,and KEGG pathway analysis were carried out,and the results showed that pterostilbene can improve the microbial dysbiosis.Furthermore,gut microbes are closely related to intestinal barrier,blood-brain barrier,and intestinal inflammation and neuroinflammation.4.Exploration of beneficial effects of pterostilbene on DCI by untargeted metabolomics analysis.To verify whether there is systemic inflammation in DCI mice and the mechanism of rosewood in improving diabetic cognitive impairment by regulating the "microbiota-gut-brain axis",differential metabolites in plasma of DCI mice were screened and analyzed by a non-targeted metabolomics method,and their association with inflammation was analyzed.The results showed that a total of 23 potential biomarkers were detected in the plasma of normal group and DCI mice,involving 7 related metabolic pathways.After the intervention of pterostilbene,17 potential biomarkers were recalled to the normal group.These potential biomarkers were closely related to metabolic pathways and inflammation,indicating that pterostilbene could significantly improve systemic inflammation in DCI mice.The results of correlation analysis showed that the screened potential biomarkers and enriched related metabolic pathways were closely related to intestinal inflammation and neuroinflammation.In total,pterostilbene can improve the microbial dysbiosis in DCI mice,reduce the intestinal barrier injure,and inhibit the production of intestinal-derived harmful substances(LPS,inflammatory factors,harmful bacteria,etc.),increase the content of short-chain fatty acids and their receptors mRNA expression,and prevent intestinal-derived harmful substances from entering the body through the intestinal barrier,thereby inhibit the damage of blood-brain barrier and the activation of the gut-brain TLR4/NF-κB signaling pathway,reduce intestinal inflammation and neuroinflammation,and finally improve the cognitive dysfunction of DCI mice through the "microbiota-gut-brain axis".