| Dementia is a general term for the decline or loss of memory and other cognitive abilities caused by various diseases.The second leading cause of dementia after Alzheimer’s disease(AD)is Vascular dementia(VD),accounting for 10-20% of dementia cases.According to epidemiological statistics,there are approximately 7.2 million people with VD in the world,and this number is expected to reach 13.2 million by 2030 and 23 million by 2050.This undoubtedly poses a huge burden for both society and individuals,and at present,there is a lack of effective treatments and drugs for VD.VD is a cognitive impairment caused by cerebrovascular disease risk factors or cerebrovascular lesions.Insufficient cerebral perfusion drives the proliferation of central nervous system immune cells such as microglia and astrocytes,which release inflammatory factors and cause a neuroinflammatory response,which in turn drives physiological responses such as apoptosis and autophagy in neurons,ultimately leading to neuronal death and damage to hippocampal structures.Damage to the hippocampus,an important tissue area for brain memory and spatial cognition,can lead to cognitive decline.Therefore,reducing gliosis in the hippocampus and mitigating neuronal apoptosis and autophagy is a way of neuroprotection.The PI3K/Akt/m TOR signaling pathway is one of the important pathways regulating apoptosis and autophagy,and there are many papers confirming the involvement of this pathway in various neurological diseases,but more experiments are still needed to further investigate it because the complexity of its associated signaling molecules.Dulaglutide is a novel hypoglycemic agent that agonizes the glucagon-like peptide-1 receptor(GLP-1RA).From previous studies,it can be concluded that Dulaglutide can improve insulin resistance,is a good hypoglycemic agent,and can reduce apoptosis and regulate autophagy in the nervous system.However,studies related to Dulaglutide in VD have not been reported.Therefore,in this experiment,we explore whether Dulaglutide can improve VD cognitive impairment.The clinical part explores whether the Ty G index is associated with an increased risk of cognitive dysfunction,that is,to clarify whether insulin resistance is associated with cognitive impairment.In the basic experiment,a rat model of VD will be constructed by establishing a bilateral common carotid artery occlusion(BCCAO),and three different doses of Dulaglutide,high,medium,and low,will be applied to the rats,and their blood glucose and body weight will be monitored.After 4 weeks of drug administration,the Morris water maze and the open field test were applied for behavioral testing.The neuronal and glial cell changes,apoptosis,autophagy,and PI3K/Akt/m TOR signaling pathway protein and gene expression levels were detected by immunofluorescence,western blotting,and RNA-Sequencing techniques in each group of the hippocampal area.Thus,the possibility of Dulaglutide as a therapeutic candidate for VD was explored.Part One Clinical impact of triglyceride glucose product index associated with cognitive impairment in the middle-aged and elderly populationObjective: To investigate whether the Ty G index is associated with an increased risk of cognitive impairment.Methods: This study retrospectively collected a total of 134 non-diabetic patients who attended the Department of Neurology at the People’s Hospital of Hebei Province from January 2018 to December 2021 and had completed cognitive function tests.After patients were enrolled,general conditions and patient laboratory blood test indexes such as triglycerides(TG),fasting blood glucose(FPG),high-density lipoprotein,low-density lipoprotein,total cholesterol,uric acid,and total serum homocysteine were collected.Ty G index was calculated by fasting TG and FPG,and its Montreal cognitive assessment(Mo CA)scale assessment was collected.SPSS 23.0 statistical software was used to analyze the data,and the measurement data were expressed as mean ±standard deviation,and independent samples t-test was used to compare the differences between groups;the categorical data were expressed as percentages,and chi-square test was used to compare the differences between groups.Multifactor binary logistic regression analysis was used to determine the factors influencing the risk of cognitive dysfunction.Results:1.134 eligible patients with a mean age of 62.4 ± 10.7 years and 56% male(n = 75)were included in this retrospective study.All patients were divided into a non-cognitively impaired group(n = 45)and a cognitively impaired group(n= 89)according to the Mo CA score.Compared with the non-cognitively impaired group,patients in the cognitively impaired group were older,had a higher prevalence of hypertension,and were more likely to have intracranial plaques(P < 0.05).2.In the univariate binary logistic regression analysis,elevated Ty G index was associated with cognitive pairing(OR: 6.87;95% CI: 2.30-20.55;P < 0.05).In the multifactorial binary logistic regression analysis,the correlation remained significant,and after adjusting for age,history of hypertension,intracranial atherosclerosis,and FPG,increased Ty G index was still associated with the risk of cognitive impairment in patients(OR: 6.04;95% CI: 1.49-24.46;P < 0.01).And age also showed a strong correlation both in the univariate regression analysis(OR: 1.05;95% CI: 1.01-1.10;P < 0.05)and in the multivariate regression analysis(OR: 6.87;95% CI: 2.30-20.55;P < 0.05).3.To further clarify whether Ty G index was an independent risk factor for cognitive impairment,a multivariate binary logistic regression analysis was performed with cognitive impairment as the dependent variable,including past history and general and laboratory index data,respectively,and the results showed that Ty G index was an independent risk factor for the development of cognitive impairment(P < 0.05).Conclusions: It was positively that Ty G index levels associated with the risk of cognitive impairment in middle-old aged adults.Age is also a risk factor for the development of cognitive impairment.Part Two Effects of Dulaglutide on blood glucose,body weight,and cognitive function in rats with vascular dementiaObjective: To establish a rat VD model by BCCAO and observe the effects of Dulaglutide on blood glucose,body weight,and cognitive function in rats 4weeks after surgery.Methods: 220-250 g clean-grade male Sprague-Dawley rats were selected and randomly divided into five groups: Sham,Model,Low,Middle,and High.The intraperitoneal injection was performed for 4 weeks.The groups were as follows: Sham group(sham surgery + saline injection);Model group(BCCAO surgery + saline injection);Low group(BCCAO surgery + 0.15 mg/kg/week Dulaglutide);Middle group(BCCAO surgery + 0.3 mg/kg/week Dulaglutide);High group(BCCAO surgery + 0.6 mg/kg/week Dulaglutide).The rats’ blood glucose and body weight were monitored during Dulaglutide injection,and behavioral tests were performed in a Morris water maze and open field experiments after 4 weeks of Dulaglutide injection to clarify whether Dulaglutide could reduce cognitive dysfunction in VD rats.Results:1.All rats had their body weight recorded every 3 days after mimicry.In all groups,the mean body weight tended to increase over time.In contrast,Dulaglutide did not show a weight-lowering effect during the measurement period.2.We recorded fasting blood glucose at the beginning of surgery,2 weeks,and 4 weeks after surgery in all groups of rats.The mean levels of fasting blood glucose during the experimental recording period did not differ between the groups.3.In the open field experiment,there were no significant differences between the groups in total distance,number of central area traversals,resting time ratio,number of standing behaviors,number of wash events,and central area distance ratio.There was no significant change in the behavior of rats in the Dulaglutide group compared to rats in the Sham group in the open-field experimental test,and Dulaglutide drug treatment did not cause more anxiety or depression-like behavior in rats.4.In the Morris water maze positioning navigation experiment,the escape latency was significantly longer in the Model group than in the Sham group on days 2 to 5(day 2: P<0.05;days 3 to 5: P<0.01),whereas the escape latency was significantly better in the Dulaglutide treated group than in the Model group(day 3: P<0.05,Middle group vs.Model group;day 4.P<0.01,High group vs.Model group;day 5: P<0.01,all doses of Dulaglutide treatment group vs.Model group).No significant difference was expressed among the Dulaglutide treatment groups in escape latency.Overall,escape latency decreased progressively with increasing training time in all groups.5.In the Morris water maze in the spatial exploration experiment,rats in the Sham and Dulaglutide treated groups both spent a longer time in the target quadrant than those in the Model group(P < 0.05,Sham,Low,and Middle groups vs.Model group;P < 0.01,High group vs.Model group),and there was no difference in the frequency of platform crossing between the five groups.Conclusions:1.In this experiment,no hypoglycemia and significant weight loss were observed in VD rats during the treatment time of Dulaglutide.2.Dulaglutide can improve the cognitive impairment of VD rats and has a neuroprotective function.3.In this experiment,Dulaglutide did not induce anxiety or depressionlike mood in VD rats.4.The three dose groups of Dulaglutide,high,medium,and low,did not show any significant difference in the water maze and open field experiments.Part Three Effects of Dulaglutide on neurons and glial cells in the hippocampal region of rats with vascular dementiaObjective: To clarify the effects of Dulaglutide on microglia,astrocytes,and neurons in VD rats.Methods: One part of the rats treated in the second part of the experiment was perfused and frozen section,and the other part of the rats was severed and hippocampal tissues were left.The structural changes of CA1 and CA3 regions in the hippocampus of each group of rats were observed by HE staining,and the number of neuroglia and neurons was quantified by immunofluorescence of microglia marker Iba1,astrocyte marker GFAP and neuron-specific nuclear protein Neu N,and the protein expression of Iba1,GFAP,and GAD67 in the hippocampus of VD rats was detected by western blotting technique.GAD67 protein expression in the hippocampus of VD rats.Results:1.In HE staining,neurons in the hippocampal CA1 and CA3 regions of the Sham group rats were regularly arranged with normal morphology,moderate size,clear nuclei,and densely arranged cytoplasm.In contrast,neurons in the Model group were lost,disordered in arrangement,irregular in morphology and size,with indistinct boundaries between the nucleus and cytoplasm and lose cytoplasm.The neuropathological changes and neuronal damage were significantly improved in all three groups of Dulaglutide-treated rats,indicating that Dulaglutide has a protective effect on neuronal damage in VD2.In the experiment,we quantified the number of microglia in the hippocampus by Iba1 immunofluorescence labeling.The results showed that the number of Iba1+ cells in the hippocampal CA1,CA3,and DG regions were significantly higher in the Model group than in the Sham group(three regions:P<0.01),indicating that microglia were extensively activated in all three regions of the hippocampus in VD rats.In contrast,Iba1+ cells in the CA1,CA3,and DG regions of the hippocampus were significantly reduced in the Dulaglutidetreated group compared to the Model(three regions: P<0.01,all Dulaglutidetreated groups vs.Model).Among the three different dose groups treated with Dulaglutide,the number of Iba1+ cells in the CA3 region of the hippocampus was significantly reduced in the High dose group compared to the Middle dose group(CA3 region: P<0.01,High group vs.Middle group).In addition,we applied western blot technique to determine the protein levels of hippocampal Iba1 in each group of rats.the protein levels of Iba1 in the Model group were significantly higher than those in the Sham group(protein levels of Iba1: P <0.01),and relatively low levels of Iba1 were also observed in each Dulaglutide treatment group(protein levels of Iba1: P < 0.01,Low group vs.Low group vs.Model group,P < 0.05,Middle group and High group vs.Model group).3.We performed quantitative immunofluorescence counting and western blotting protein quantification to detect astrocyte expression in the hippocampus of rats in each group of the experiment.The number of GFAP+ cells in the CA1,CA3,and DG regions of the hippocampus was significantly increased in untreated VD rats compared to the Sham group(three regions: P < 0.01).Treatment with Dulaglutide reduced the number of GFAP+ cells(three regions.P < 0.01,all Dulaglutide treated groups vs.Model group),and astrocytes were larger and more branched in the Model group.In the three dose groups treated with Dulaglutide,astrocytes were slightly higher in the hippocampus of rats in the Middle group than in the Low and High groups(CA1 and CA3 regions.P <0.01,Low and High groups vs.Middle group).western blotting quantification results also confirmed these findings(protein levels of Iba1: P < 0.01,Model group vs.Sham group;P < 0.01,all Dulaglutide treated groups vs.Model group;P < 0.01,Low and High groups vs.Model group).4.In quantitative immunofluorescence counts of Neu N,the number of Neu N+ cells in the hippocampal CA1 and CA3 regions of the rats in the Model group was significantly reduced,compared with the Sham group(CA1 and CA3regions: P < 0.01).In the CA1 area,Neu N+ cells were significantly more in the Dulaglutide-treated group than in the Model group(CA1 area: P < 0.01,Low and High groups vs.Model group;P < 0.05,Middle group vs.Model group),while in CA3 area,the number of Neu N+ cells in the Dulaglutide-treated group was significantly higher than in the Model group only in the High group(CA3region: P<0.01,High group vs.Model group).5.We applied western blot to determine the GAD67 content in the hippocampus of rats in each group.The GAD67 content was significantly decreased in the Model group compared with the Sham group(P < 0.01),indicating that there were fewer GABAergic neurons in the hippocampus of VD rats.This GABAergic neuronal damage was alleviated by Dulaglutide treatment(P < 0.05,Low group vs.Model group;P < 0.01,Middle,High group vs.Model group).Conclusions:1.hippocampal tissue neurons of VD rats showed significant histopathological changes,which were attenuated by Dulaglutide.2.hippocampal microglia and astrocytes in the VD rat model induced by BCCAO showed significant proliferation,and Dulaglutide inhibited the proliferation of glial cells and decreased the levels of Iba-1 and GFAP protein.3.The hippocampus of VD rats showed significant neuronal damage,and the hippocampal neuronal damage was reduced after Dulaglutide intervention,in which the effect of the high-dose group was better than the other two dose treatment groups.4.The hippocampal GAD67 protein level was decreased in VD rats,and Dulaglutide could improve this phenomenon to some extent.Part Four Effect of Dulaglutide on apoptosis and autophagy in hippocampal tissues of rats with vascular dementiaObjective: Both HE and immunofluorescence staining in the third part of the experiment showed that neurons in the hippocampal region of VD rats showed damage and reduction.Since apoptosis and autophagy are two major processes of neuronal injury and death,we examined the changes in the expression of various proteins associated with apoptosis and autophagy to explain why Dulaglutide treatment could reduce neuronal death.Methods: Western blot was applied to detect the expression of Bcl-2,Bax,c-caspase-3,LC3 B,Beclin 1,and p62 proteins in the hippocampal tissues of rats in each group.Results:1.both Bcl-2/Bax ratio and sheared caspase-3(c-caspase-3)were markers of apoptosis.The Bcl-2/Bax ratio was significantly decreased(P<0.01)and ccaspase-3 expression was increased(P<0.01)in the Model group compared with the Sham group.In addition,all three Dulaglutide treatment groups exhibited a significant increase in Bcl-2/Bax ratio(P<0.01,all Dulaglutide treatment groups vs.Model group)and a decrease in c-caspase-3(P<0.05,Low and High groups vs.Model group;P<0.01,Middle group vs.Model group).In addition,among the three Dulaglutide treatment groups,the Bcl-2/Bax ratio was higher in the Low group than in the other two groups(P<0.05,Middle group vs.Low group;P<0.01,High group vs.Low group),while the Middle group had better results than the other two groups in reducing the protein expression of ccaspase-3(P<0.01.Low group and High group vs.Middle group).The LC3B-II/LC3B-I ratio,P62,and Beclin-1 were all indicators involved in the regulation of autophagy.the LC3B-II/LC3B-I ratio was significantly higher in the Model group than in the other four groups(P<0.05,High vs.Group).In addition,the LC3B-II/LC3B-I ratio was similar in the three Dulaglutide treatment groups.There was no significant difference in Beclin-1between the five groups.Rats in the Sham group showed elevated expression of P62 compared to the other four groups(P<0.01,other four groups vs.Sham group).P62 expression was also significantly higher in the Dulaglutide-treated group than in the Model group(P<0.01,all Dulaglutide-treated groups vs.Model group compared).Conclusions:1.chronic cerebral ischemia and hypoxia-induced apoptosis and excessive activation of autophagy,resulting in neuronal degeneration and death in the hippocampus of VD rats.2.Dulaglutide may exert neuroprotective effects by reducing neuronal apoptosis and regulating autophagy.Part Five Effect of Dulaglutide on PI3K/Akt/m TOR signaling pathway in rats with vascular dementiaObjective: To investigate whether PI3K/Akt/m TOR signaling pathway is involved in the neuroprotective effect of Dulaglutide.Methods: Western blotting was applied to detect PI3 K,Akt,and m TOR protein levels and their phosphorylation levels,and RNA-Sequencing was used to detect the gene differences between the High,Sham,and Model groups,and according to the differentially Expressed Genes between groups(Differentially Expressed Genes,DEGs)between groups were statistically analyzed,and the results of differentially expressed gene analysis,differentially expressed gene GO enrichment analysis,and differentially expressed gene KEGG pathway enrichment analysis were obtained.Results:1.To investigate whether PI3K/Akt/m TOR signaling pathway was involved in the process of the neuroprotective effect of Dulaglutide,western blotting was applied to detect the protein levels of PI3 K,Akt,and m TOR and their phosphorylation levels.In the Dulaglutide-treated group,the protein expression levels of p-PI3 K,p-Akt,and p-m TOR were relatively increased compared with other groups,while PI3 K,Akt,and m TOR did not differ significantly between the groups.p-Akt and p-m TOR showed an increasing trend in VD rats(treated and untreated groups)relative to Sham rats(p-Akt and p-m TOR: P < 0.05,Model group vs.Sham group;P < 0.01,all Dulaglutide treated groups vs.Sham group).In the detection of p-PI3 K protein,p-PI3 K protein was higher in the Model group than in the Sham group,but the difference was not significant,whereas there was a significant increase in the Dulaglutide treatment group compared with the Sham group(P < 0.05,Low group vs.Sham group;P < 0.01,Middle group and High group vs.Sham group).In the three groups treated with Dulaglutide,p-PI3 K and p-Akt were not significantly different between the three dose groups,while p-m TOR was more significantly increased in the Middle group compared to the other two groups(P<0.05,Low and High groups vs.Middle group).2.RNA was extracted from hippocampal tissues of the Sham,Model,and High groups for RNA-seq sequencing.The Model group had 17 up-regulated genes and 23 down-regulated genes compared to the Sham group,while the Dulaglutide High dose treatment group had 19 up-regulated genes and 78 downregulated genes compared to the Model group.DEGs were classified into three main GO groups: molecular functions,cellular components,and biological processes.According to the results,DEGs in the High and Model groups were mainly enriched in the cellular component and molecular function classes of genes.KEGG pathway analysis showed that DEGs in the Model and Sham groups were significantly enriched in neuroactive ligand-receptor interactions,and the High and Model groups were significantly enriched in four pathways,including the m TOR signaling pathway.According to differential gene statistics,Deptor was significantly downregulated in the High group compared with the Model group,while Pdpk1 was downregulated in the High group compared with the Model group,but there was no significant difference after statistical analysis.ribosome-associated Rpl17 was significantly upregulated in both the High and Sham groups compared with the Model group(P<0.05,Sham group vs.Model group;P<0.01,High group vs.Model group).LOC100362149 was also significantly upregulated in the High group compared to the Model group(P<0.01,High group vs.Model group).Conclusions:1.The results of this study demonstrate for the first time that dulaglutide drives an increase in PI3K/Akt/m TOR phosphorylation during VD treatment,a phenomenon that may be related to its neuroprotective effect.2.The increase of RPL17 in the Duroc glucotide treatment group suggests that the application of Duroc glucotide may require attention to emotion-related problems. |
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