| Type 2 diabetes mellitus(T2DM)is the main clinical type of diabetes mellitus and is often associated with neuropsychiatric behavioural changes,mainly cognitive dysfunction.Epidemiological data suggest that T2 DM increases the risk of reduced cognitive function in patients.Therefore,exploring the mechanisms underlying neuropsychiatric impairment in T2 DM and developing effective therapeutic agents is an important area of research in the neuropsychiatric field.Impaired glucose and lipid metabolism is a major cause of T2 DM progression,and hyperlipidaemia is thought to be an independent risk factor for cognitive impairment in T2 DM.The results of our group’s previous study suggest that hyperlipidemic stimulation may be the main cause of neuropsychiatric impairment in the T2 DM state,but the specific mechanisms need to be further explored.In recent years,the function of long-stranded non-coding RNAs(Lnc RNAs)has been intensively studied.Lnc RNA TUG1,a taurine up-regulated gene,was one of the first genes identified to be associated with human diseases,and is involved in the development of various neurological diseases such as Parkinson’s and epilepsy through the regulation of cell proliferation,differentiation and apoptosis,as well as the regulation of glucolipid metabolism.In this study,we aimed to firstly observe and confirm the association between the alteration of Lnc RNA TUG1 abundance in hippocampal tissue of T2 DM mice induced by high-fat diet and the decrease of learning and memory ability in T2 DM mice,and then establish a model of HT-22 cell damage induced by high palmitic acid.The aim was to investigate the role of Lnc RNA TUG1 in the neuropsychiatric injury of T2 DM and its possible mechanisms.ObjectiveObserving the role of Lnc RNA TUG1 in HT-22 cell injury induced by high-fat stimulation and exploring its possible molecular mechanisms.Method1.A high-fat diet combined with intraperitoneal injection of streptozotocin was used to construct a T2 DM mouse model.The differential expression of Lnc RNAs in hippocampal tissues of T2 DM and normal mice was observed by gene sequencing and analyzed by GO and KEGG enrichment.q RT-PCR was used to verify the expression levels of Lnc RNA TUG1 and six other Lnc RNAs with the most significant differential expression.2.HT-22 cells were cultured in vitro,stimulated with gradient concentrations of palmitic acid,and the experimental conditions were determined by MTT and LDH release rate assays.After model validation,morphological observations were combined with MTT and LDH release assays to detect the extent of cell damage.q RT-PCR was used to verify the expression level of Lnc RNA TUG1.Western Blotting was used to detect the apoptosis-related proteins Bcl-2 and Cleaved-Caspase-3,the key proteins of synaptic plasticity regulation Synapsin-1and Synaptotagmin-1 and brain-derived neurotrophic factor BDNF,Akt/GSK-3βsignaling pathway related proteins Akt,p-Akt,GSK-3β,p-GSK-3β were expressed.On this basis,si RNA interference technology was used to reduce the expression level of Lnc RNA TUG1 in HT-22 cells.control(C),model(M),model+negative control(M+NC),model+si RNA(M+si RNA),normal+negative control(C+NC)and normal+si RNA(M+si RNA)groups were set up.LDH assay HT-22 cell damage effect,Western Blotting method together with flow cytometry assay method to detect HT-22 apoptosis level,Western Blotting method to detect alteration of synaptic plasticity regulatory key protein and Akt/GSK-3β signaling pathway,chemiluminescence method and ELISA method to detect lipid peroxidation indexes MDA,The levels of MDA,LPO,GSH/GSSG and 4-HNE were measured by chemiluminescence and ELISA,and the levels of ROS,an indicator of oxidative stress,were measured by immunofluorescence.3.On this basis,the ce RNA network involved in the regulation of Lnc RNA TUG1 was predicted according to bioinformatics software,and downstream regulated mi RNAs were identified.q RT-PCR method and dual luciferase reporter gene method were used for validation.The experiments were set up in control(C),model(M),model+negative control(M+NC),model+Mimics(M+Mimics),normal+negative control(C+NC),normal+Mimics(C+Mimics),model+Inhibitor(M+Inhibitor)and normal+Inhibitor group(C+Inhibitor),respectively,to explore the regulatory effects of elevated or reduced mi R-449a-5p expression levels on HT-22 cell injury induced by PA stimulation.LDH was used to detect the effects of HT-22 cell injury,Western Blotting with flow cytometry to detect apoptosis levels,and Western Blotting was used to detect changes in key proteins regulating synaptic plasticity and the Akt/GSK-3β signaling pathway,chemiluminescence and ELISA were used to detect levels of MDA,LPO,GSH/GSSG and 4-HNE,respectively,and immunofluorescence was used to detect levels of ROS,an indicator of oxidative stress.The bioinformatics software predicted the possible binding m RNA of mi R-449a-5p,followed by q RT-PCR for analysis and validation.Results1.Compared with the control group,141 up-regulated Lnc RNAs and 136down-regulated Lnc RNAs were detected in the hippocampus of T2 DM mice.q RT-PCR results were consistent with the microarray screening results.GO and KEGG enrichment analysis showed that the differentially expressed Lnc RNAs were mainly focused on metabolism,growth and development processes,cell signaling and other aspects.Compared to controls,Lnc RNA TUG1 expression was significantly enhanced in both hippocampal tissue and high-fat-induced HT-22 cells in type 2 diabetic mice,and palmitic acid(100u M)induced an increased LDH release rate in HT-22 cells,and showed decreased expression of apoptosis-related protein Bcl-2,increased expression of Cleaved-Caspase-3,synaptic plasticity in The expression of the key regulatory proteins Synapsin-1 and Synaptotagmin-1,as well as the brain-derived neurotrophic factor BDNF were reduced,and the p-Akt/ Akt and p-GSK-3β/ GSK-3β ratios of proteins related to the Akt/GSK-3β signaling pathway were decreased.2.Silencing Lnc RNA TUG1 expression reduces PA-induced elevated levels of apoptosis and lipid peroxidation in HT-22 cells in vitro and exhibits alleviating effects on altered Akt/GSK-3β signaling pathway and synaptic plasticity damage.3.mi R-449a-5p,a direct target of Lnc RNA TUG1,was downregulated in PA-induced HT-22 cells.Moreover,mi R-449a-5p was able to directly reduce Caspase-3 levels and exhibited protective effects against high lipid-induced HT-22 cells in terms of apoptosis,oxidative stress,lipid peroxidation levels and synaptic plasticity damage.ConclusionThe hippocampal tissues of mice in the T2 DM group induced by high-fat diet combined with STZ intraperitoneal injection had a spectrum of Lnc RNAs differentially expressed from normal mice,with a significant increase in the expression of Lnc RNA TUG1.In vitro PA stimulation induced HT-22 cell injury and upregulation of Lnc RNA TUG1 expression,accompanied by imbalance in the expression of apoptosis,synaptic plasticity-related proteins and key molecules of the Akt/GSK-3β signaling pathway,effectively mimicking hippocampal injury in T2 DM mice.The abnormally elevated TUG-1 was positively associated with Cleaved Caspase-3 expression and inversely associated with Bcl-2,p-AKT/AKT,and p-GSK-3β/ GSK-3β expression.Silencing of Lnc RNA TUG1 was effective in ameliorating PA-induced HT-22 cell injury,and the mechanism involved the regulation of HT-22 cells in terms of apoptosis,oxidative stress,lipid peroxidation levels and synaptic plasticity after binding to mi R-449a-5p. |
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