| Diabetes mellitus(DM)is a chronic metabolic disorder characterized by hyperglycemia and a wide spectrum of complications including cardiovascular,ocular,renal,and immunological disturbances.Currently,more than five hundred million people worldwide are affected by diabetes and this number is expected to increase dramatically by the next 30 years.In recent years,more and more attention has been focused on diabetes-associated neurological deterioration,represented by a progressive impairment of cognitive abilities,that is,diabetes-associated cognitive dysfunction.Diabetes-associated cognitive dysfunction is mainly characterized by the decline of memory,learning ability and psychomotor efficiency in patients with type2 diabetes mellitus(T2DM).Diabetes-associated cognitive dysfunction not only leads to a severe dysfunction in self-care ability and quality of life in T2 DM patients,but also increases the morbidity and mortality of related diseases.Therefore,the research on the pathogenesis of diabetes-associated cognitive dysfunction and the prevention and treatment of drugs has great socio-economic value.Unfortunately,the underlying pathogenesis of diabetes-associated cognitive dysfunction and related interventions or treatment measures are still unclear.Resveratrol(RSV)is a natural non-flavonoid polyphenolic compound extracted from plants,RSV has various pharmacological activities such as anti-DM,anti-endoplasmic reticulum stress(ERS),anti-inflammatory,antioxidant,and anti-apoptosis.RSV has a variety of potential therapeutic targets for neurodegenerative diseases,DM,cardiovascular and cerebrovascular diseases and many other diseases.Although more and more studies have begun to focus on the improvement of RSV on cognitive function in recent years,the effect of RSV on improving diabetes-associated cognitive dysfunction and related mechanisms are still worthy of further exploration.Therefore,this study intends to observe the improvement effect of RSV on cognitive dysfunction in T2 DM rats,and to explore the molecular mechanism of the effect of RSV on diabetes-associated cognitive dysfunction from the perspectives of anti-neuronal apoptosis,anti-inflammatory and anti-ERS.This study is divided into two parts.The first part observes the improvement effect of RSV on cognitive dysfunction in T2 DM rats,and explores whether its mechanism is related to the improvement of rat hippocampus neuronal damage,the reduction of neuronal apoptosis,inflammatory damage,and ERS,and up-regulating mi R-146a-5p,down-regulating Thioredoxin-interacting protein(TXNIP).The second part further verifies the molecular mechanism of RSV improving diabetes-associated cognitive dysfunction is through regulating mi R-146a-5p/TXNIP axis to play an anti-inflammatory and anti-ERS role in vitro cell experiments.This study is of great significance in exploring the relevant mechanisms of diabetes-associated cognitive dysfunction and effective control measures.Part Ⅰ Preliminary study on the effect and mechanism of resveratrol on cognitive dysfunction in type 2 diabetic ratsObjective: To observe the effects of RSV on T2DM-associated cognitive dysfunction.To observe the effects of RSV on the pathological changes of T2 DM rats hippocampal neurons,inflammatory factors,ERS markers,mi R-146a-5p and TXNIP expression levels.To explore the possible mechanism of RSV improving cognitive dysfunction in T2 DM rats.Methods: 36 male SD rats were divided into normal control group(Normal group,n=12)and T2 DM model group(n=24)by random number table method.After the T2 DM model group was fed with high-sugar and high-fat diet for 4 weeks,0.45%streptozotocin was injected into the left lower abdomen at a dose of 35 mg/kg,and continued to be fed with high-sugar and high-fat diet for 2 weeks.The rat blood glucose was greater than 16.7mmol/L,which was regarded as successful in T2 DM rats.24 T2 DM rats were divided into 2 groups by random number table method:T2DM group(n=12),T2DM+RSV group(n=12).The T2DM+RSV group was given RSV 50 mg/kg/d by gavage,and the Normal and T2 DM groups were given the same volume of sodium carboxymethyl cellulose by gavage every day for 4 weeks.The body weight and blood sugar of the rats in each group were measured weekly.Novel object recognition test and Morris water maze test were used to evaluate the cognitive function of the rats.HE staining was used to observe the morphological changes of neurons in the hippocampal CA1 area of the rats.TUNEL staining was used to detect the apoptosis of hippocampal neurons of the rats.The content of IL-1β and TNF-α in the hippocampus of the rats were detected by ELISA,the expression of ERS markers in the hippocampus of rats were detected by Western blot,and the expression of IL-1β,TNF-α,mi R-146a-5p and TXNIP m RNA was detected by q RT-PCR.Results:(1)Novel object recognition experiment showed that: compared with the Normal group,the discrimination index of the new objects in the T2 DM group was significantly decreased(P ﹤ 0.05),compared with the T2 DM group,the discrimination index of the T2DM+RSV group was significantly improved(P﹤0.05).(2)Morris water maze test: The results of the positioning navigation test showed that from day 2 to day 4,the escape latency of rats in T2 DM group was significantly longer than that in Normal group(P﹤0.05).On days 3 and 4,the escape latency in T2DM+RSV group was significantly shorter than that in T2 DM group(P﹤0.05).The results of the space exploration test showed that the number of platform crossings and the time spent in the target quadrant were significantly less than those in the Normal group(P﹤0.05).The number of platform crossings and the time spent in the target quadrant were significantly higher in the T2DM+RSV group than those in the T2 DM group(P﹤0.05).(3)HE staining: The neurons in the hippocampal CA1 area of the rats in the Normal group were arranged neatly and tightly,and the cytoplasm and nuclei were plump and clearly visible.The neurons in the hippocampal CA1 area of the T2 DM group were disordered and loose,and the cells became smaller,with nuclear pyknosis,chromatin aggregation,and decreased cytoplasm.The morphology of neurons in the hippocampal CA1 region of the T2DM+RSV group was significantly improved compared with the T2 DM group.(4)TUNEL staining:Compared with the Normal group,the number of apoptotic neurons in the T2 DM group was significantly increased(P﹤0.05).Compared with the T2 DM group,the number of apoptotic neurons in the T2DM+RSV group was significantly reduced(P﹤0.05).(5)Compared with the Normal group,the content and m RNA expression of IL-1β and TNF-α in the hippocampus of the T2 DM group were significantly increased(P ﹤ 0.05).Compared with the T2 DM group,the content and m RNA expression of IL-1β and TNF-α in the T2DM+RSV group were significantly decreased(P﹤0.05).(6)Compared with the Normal group,the protein expressions of ERS markers GRP78,CHOP and IRE1 in the T2 DM group were significantly increased(P ﹤ 0.05).Compared with the T2 DM group,the protein expressions of ERS markers GRP78,CHOP and IRE1 in the T2DM+RSV group were significantly decreased(P ﹤ 0.05).(7)Compared with the Normal group,the expression of mi R-146a-5p in the T2 DM group was significantly decreased(P ﹤ 0.05),and the expression of TXNIP m RNA was significantly increased(P﹤0.05).Compared with T2 DM group,the expression of mi R-146a-5p in T2DM+RSV group was significantly increased(P ﹤ 0.05),and the expression of TXNIP m RNA was significantly decreased(P﹤0.05).Conclusion: RSV can ameliorate cognitive dysfunction in T2 DM rats,and its mechanism may be related to ameliorating neuronal damage in the hippocampus,reducing neuronal apoptosis,inflammatory damage,and ERS,and up-regulating mi R-146a-5p,down-regulating TXNIP.Part Ⅱ Resveratrol attenuates high glucose-induced inflammation and endoplasmic reticulum stress in rat primary hippocampal neurons via the mi R-146a-5p/TXNIP axisObjective: To observe the anti-inflammatory and anti-ERS effects of RSV on high glucose-induced primary hippocampal neurons,as well as the effects on the expression of mi R-146a-5p and TXNIP.And to further verify whether RSV exerts anti-inflammatory and anti-ERS neuroprotective effects by regulating the mi R-146a-5p/TXNIP axis.Methods:(1)Rat primary hippocampal neurons were isolated and cultured in vitro,and a model of high-glucose-injured hippocampal neurons was established.They were divided into control(Con)group,high-glucose(HG)group,and HG+RSV group.The high glucose concentration was 45 m M,the RSV intervention concentration was 45 μM,and the intervention time was 48 h.ELISA and q RT-PCR were used to detect the content and m RNA expression of IL-1β and TNF-α in hippocampal neurons.Western blot was used to detect the expression of ERS markers GRP78,CHOP and IRE1 in hippocampal neurons.And q RT-PCR was used to detect the expression of mi R-146a-5p and TXNIP m RNA in hippocampal neurons.(2)Mi R-146a-5p was inhibited by transfecting mi R-146a-5p inhibitor in hippocampal neurons,and divided into HG group,HG+RSV+mi R-146a-5p inhibitor negative control(HG+RSV+inhibitor NC)group,and HG+RSV+mi R-146a-5p inhibitor(HG+RSV+mi R-146a-5p inhibitor)group.ELISA and q RT-PCR were used to detect the content and m RNA expression of IL-1β and TNF-α in hippocampal neurons of each group.Western blot was used to detect the expression of ERS markers GRP78,CHOP and IRE1 in hippocampal neurons in each group.(3)The dual-luciferase reporter gene assay verified that TXNIP was a target gene regulated by mi R-146a-5p.(4)Mi R-146a-5p mimics,mi R-146a-5p mimics negative control(mimics NC),mi R-146a-5p inhibitor and inhibitor NC were transfected to construct mi R-146a-5p overexpression and expression inhibition model.Then,q RT-PCR and Western blot were used to detect the expression of TXNIP m RNA and protein,and to further verify the regulation effect of mi R-146a-5p on TXNIP.Results:(1)Compared with the Con group,the content and m RNA expression of IL-1β and TNF-α in hippocampal neurons in the HG group were significantly increased(P ﹤ 0.05).Compared with the HG group,the content and m RNA expression of IL-1β and TNF-α in hippocampal neurons of the HG+RSV group were significantly decreased(P ﹤ 0.05).(2)Compared with the Con group,the protein expressions of ERS markers GRP78,CHOP and IRE1 in hippocampal neurons of the HG group were significantly increased(P﹤0.05).Compared with the HG group,the protein expressions of ERS markers GRP78,CHOP and IRE1 in the HG+RSV group were significantly decreased(P ﹤ 0.05).(3)Compared with the Con group,the expression of mi R-146a-5p in the hippocampal neurons of the HG group was significantly decreased(P ﹤ 0.05),and the expression of TXNIP m RNA was significantly increased(P﹤0.05).Compared with the HG group,the expression of mi R-146a-5p in hippocampal neurons in the HG+RSV group was significantly increased(P ﹤ 0.05),while the expression of TXNIP m RNA was significantly decreased(P﹤0.05).(4)The results of transfection with mi R-146a-5p inhibitor or its negative control showed that compared with the HG group,the IL-1β and TNF-αcontent,m RNA expression of hippocampal neurons in the HG+RSV+inhibitor NC group were significantly decreased(P ﹤ 0.05),the protein expressions of ERS markers GRP78,CHOP and IRE1 were significantly decreased(P﹤0.05).Compared with the HG+RSV+inhibitor NC group,the IL-1β and TNF-α content and m RNA expression of hippocampal neurons in the HG+RSV+mi R-146a-5p inhibitor group were significantly increased(P ﹤ 0.05).The protein expression of ERS markers GRP78,CHOP and IRE1 were significantly increased(P﹤0.05).(5)The results of dual-luciferase reporter gene assay showed that the relative luciferase activity was significantly decreased in the transfected mi R-146a-5p mimics and TXNIP 3’UTR(wild-type)luciferase plasmid groups(P ﹤ 0.05).(6)In the mi R-146a-5p overexpression and expression inhibition model,the results showed that compared with the transfected mimics NC group,the TXNIP m RNA and protein expression in the mi R-146a-5p mimics transfection group were significantly decreased(P﹤0.05).Compared with the transfected inhibitor NC group,the TXNIP m RNA and protein expression in the mi R-146a-5p inhibitor transfection group were significantly increased(P﹤0.05).Conclusion: RSV may play a protective role in high glucose-induced inflammatory injury and ERS injury in rat hippocampal neurons by up-regulating the expression of mi R-146a-5p and then down-regulating the expression of TXNIP. |
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