The Function And Mechanism Of Melatonin In The Therapeutic Effects Of Cognition Dysfunction In Type 2 Diabetic Mice

BackgroundDiabetes is a serious global public health problem,of which about 90%is type 2 diabetes,and its worldwide prevalence is increasing yearly.It is one of the most common metabolic diseases;it can extensively damage a variety of organs and tissues,including the heart,kidney,retina,and central nervous system,and cause serious complications.Cognitive impairment is a common but severely underestimated complication of diabetes.Impaired cognitive function in diabetes is manifested by many aspects,including learning and memory,executive ability,attention,and emotion.However,the clinical manifestations and pathogenesis of diabetes-related cognitive dysfunction are not well understood,and the clinical treatment effect of this complication is poor.Therefore,deciphering the mechanism and finding new strategies to delay or reduce the occurrence of cognitive dysfunction is crucialNeuroinflammation is closely related to cognitive dysfunction.Microglia are immune cells in the brain that act as important regulators of neuroinflammation.Activated microglia can secrete multiple proinflammatory mediators,including tumor necrosis factor α,interleukin-6,and reactive oxygen species,which have toxic effects on neurons and further activate microglia,thereby aggravating neuroinflammation.Neuroinflammation caused by microglial overactivation is the main feature of neuropathology.Thus,microglial activation must be properly and strictly regulated to maintain normal physiological homeostasis.A growing body of evidence has demonstrated that autophagy can regulate inflammation and apoptosis by regulating the activation of microglia.The activation of autophagy inhibits the expression of IL-6 and the death of lipopolysaccharide(LPS)-stimulated microglial cellsMelatonin(MLT)is the main secretory product of the pineal gland.It acts as a regulator of the circadian rhythm.It also acts on energy expenditure,glucose metabolism,inflammatory factor secretion regulation,and anti-apoptosis.However,patients with type 2 diabetes have decreased serum melatonin level and disordered circadian rhythm.A small dose of melatonin can improve cognitive function,and exogenous MLT can penetrate the blood-brain barrier and play a role in different brain regions.Moreover,a recent study demonstrated that MLT can alleviate memory and cognitive impairment.MLT has been shown to inhibit microglial activation and reduce proinflammatory cytokine levels in many experimental models,including Alzheimer’s disease.Notably,the administration of MLT significantly suppresses the expression of toll-like receptor 4.However,the therapeutic effects and mechanism of MLT in diabetes-associated cognitive decline remains unknown.PurposesTo investigate the effect and mechanism of melatonin on cognitive dysfunction of T2DM.Methods1.In vivo:T2DM mice model was induced by a high-fat diet combined with STZ.The mice were randomly divided into four groups:control group,melatonin group,T2DM group,and T2DM+melatonin group.Melatonin(10mg/kg)was injected intraperitoneally.After one month of continuous intervention,the Morris water maze test was performed to test the learning and memory ability.Intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were performed to detect the islet function.All mice were killed,and the left hemispheres were fixed.HE and Nissl staining were used to detect the neuronal apoptosis,TUNEL staining was used to detect the microglial apoptosis,and the immunofluorescence staining was used to detect the expression of Iba1,GFAP,TLR4,and MAPLC3B in the hippocampus,Western blot and RT-qPCR were used to detect the levels of apoptosis,inflammation,autophagy,and TLR4/Akt/mTOR pathway-associated proteins and mRNA of the right hippocampus.2.In vitro,mouse microglial BV-2 cells were used to detect the mechanism.0.4mmol/L palmitic acid(PA)and 100nmol/L melatonin were used as stimulations.They were divided into four groups:control group,melatonin group,PA group,and PA+melatonin group.After intervention with PA and melatonin for 24h,RNA and protein were extracted and detected by RT-qPCR and Western blot respectively.The autophagy inhibitor 3-methyladenine(3-MA,5mmol/L),TLR4 inhibitor TAK-242(10umol/L),and Akt inhibitor MK-2206(10umol/L)were administered to examine the effects of autophagy and TLR4/Akt/mTOR pathway on the improvement of melatonin on microglial apoptosis and inflammation.Results1.IPGTT and IPITT showed that melatonin improved glucose tolerance and insulin sensitivity in T2DM mice.2.The cognitive function of T2DM mice treated with melatonin has been improved obviously,the learning and memory ability was improved and the learning retention was enhanced.3.The neurons of the hippocampal CA1 region in type 2 diabetic mice exhibited pathological features,such as irregular arrangement,widened intercellular space,reduced cell volume,and nuclear condensation.Nissl staining showed that the number of Nissl bodies in neurons was decreased,suggesting the apoptosis of neurons.TUNEL staining showed that the apoptosis of microglia was increased and the expression of apoptosis indicators was increased.Apoptosis of neurons and microglia was decreased after melatonin treatment.The astrocytes and microglia in the hippocampus of T2DM mice were activated,the expressions of inflammatory cytokines were increased,and the NF-κB signaling pathway was activated,suggesting neuroinflammation.Melatonin decreased the activation of microglia,the expression of inflammatory factors,and the NF-κB pathway,suggesting that melatonin could inhibit neuroinflammation by inhibiting the activation of microglia.4.Melatonin improved the autophagy defect in the hippocampus of T2DM mice,which increased the expression of autophagy formation markers and decreased the expression of autophagy degradation markers5.In vitro,melatonin could improve apoptosis and inflammation of BV-2 cells induced by PA,while autophagy inhibitor 3-MA reversed the anti-inflammatory and anti-apoptosis effects of melatonin6.TLR4/Akt/mTOR signaling pathway was activated in the hippocampus of T2DM mice and PA-induced BV-2 cells.Melatonin treatment inhibited the activation of the TLR4/Akt/mTOR signaling pathway7.TLR4 inhibitor TAK-242 and Akt inhibitor MK-2206 ameliorated PA-induced autophagy defects,thus improving inflammation and apoptosisConclusionMLT could improve learning and memory in type 2 diabetic mice by activating autophagy via the TLR4/Akt/mTOR pathway,thereby inhibiting neuroinflammation and microglial apoptosis.