Clinical And Basic Research Of Atherosclerosis And Diabetic Encephalopathy

Atherosclerosis(AS)is the most common vascular complication in elderly patients with type 2 diabetes mellitus(T2DM).Cognitive dysfunction is one of complication in nervous system in T2 DM,which is of great importance.Whether is there any association between AS and cognitive function in patients with T2DM? Previous studies have found a common regulatory mechanism in AS and diabetesmacrophages autophagy.We then come up with two hypotheses:(1)There is a relationship between AS and non-cerebrovascular accident induced cognitive impairment in elderly diabetic patients.(2)There is a common pathway to regulate macrophage autophagy that influence the occurrence and development in AS and diabetic encephalopathy(DE).To further validate our hypothesis,we design the following clinical and basic research.PART 1 Cognitive status in atherosclerosis and type 2 diabetic elderly patientsObjective: This study was designed to investigate the relationship between AS and cognitive status in T2 DM patients with non-cerebrovascular accident by noninvasive AS testing,including ba PWV,ABI,AIP,AASI and c IMT.Furthermore,to explore the value of those noninvasive AS test method in DM.Methods: 120 elderly DM patients were selected as the experimental group(DM group)and 100 non-DM elderly people as the control group(NDM group).Non-invasive AS detection methods(ba PWV,ABI,AIP,AASI and c IMT)were used on those two groups.Meanwhile,to assess total cognitive status in each group with MMSE,Mo CA,ADL,and cognitive domain(memory function,executive function evaluation,visual spatial ability,speech function evaluation,attention function)with other assessment scales.Then,in the DM group,to assess the relationship between AS and overall cognitive status and each cognitive domain,respectively,by Spearman correlation analysis.Results: 1.there was no significant difference in each index of ba PWV,AASI and AIP between the two groups(P> 0.05).The ABI of DM group was lower than that of non-DM group,while the c IMT of DM group was higher than that of non-DM group with significant difference(P <0.05).2.Compared with non-DM patients,DM was showed a decrease trend in overall cognitive status,MOCA score was decreased with the difference of statistically significant(p <0.05),ADL between the two groups have no significant difference.In the cognitive domain,compared with non-DM patients,DM patients showed a worse memory and language status with significant difference(p <0.05).3.there was a positive correlation between ABI and MMSE,MOCA,respectively,(r = 0.626,r = 0.628,P <0.05)in DM group and negative correlation with ADL(r =-0.540,P < 0.05).There was a negative correlation between c IMT and MMSE,MOCA,respectively,(r =-0.387,r =-0.455,P <0.05)and a positive correlation with ADL(r = 0.238,P <0.05).4.There was a positive correlation among ABI in memory function,executive function and language function in DM group(r = 0.547,r = 0.627,r = 0.482,P <0.05);c IMT was associated with memory function,executive function and language function with a negative correlation(R =-0.365,r =-0.280,r =-0.361,P <0.05).For the visual space and attention,there were no correlation.Conclusion: The most two valuable non-invasive AS detection methods are ABI and c IMT for detection the severity of AS in T2 DM.The overall cognitive function of the DM group was worse than that of the non-DM group,with the memory function and language function decreased.The WHO-BCAI VFT scale is more sensitive in language function testing.There was a significant correlation between AS and general cognitive status in DM group,especially in memory,executive,language function.The correlation between ABI was better than c IMT.PART 2 Regulating effects of miR-384-5p on macrophage autophagy in the development and progression of atherosclerosisObjective: To investigate the regulation of miR-384-5p on macrophage autophagy in the development and progression of AS.Methods: 20 male ApoE(-/-)mice of 10 weeks old were divided into normal diet group(HF)and high fat diet group(HFD)randomly and aqually,HFD group were fed with HFD 12 weeks,which successfully induced AS model;the animals were sacrificed to extract the aortic stem tissue,then,making HE staining,immunohistochemistry;The macrophages were sorted by flow cytometry.The levels of m RNA in extracellular matrix were detected by RT-PCR and LC3-II / I,ATG7,p62 and Beclin-1 were detected by Western blot.Macrophages were transfected with miR-384-5p mimics,as-miR-384-5p,null controls,and miR-384-5p.For the expression of miR-384-5p,bioinformatics analysis and dual luciferase assay were used to detect.Results: Compared with NOR group,macrophage autophagy in HFD group was inhibited compared with that in NOR group;the levels of Beclin-1 in NOR group was significantly higher than HFD group(p <0.05)by RT-q PCR.Compared with the control group,the expression of miR-384-5p in macrophages in HFD group was significantly increased(p <0.05).Luciferase Reported showed that the fluorescence activity of Beclin-1 3’UTR fluorescent plasmid transfected miR-384-5p macrophages was significantly decreased compared with the null group(p <0.05).Conclusion: HFD induced miR-384-5p expression in cells increased,inhibiting Beclin-1 protein translation and translation,decreasing the protein synthesis of Beclin-1 and decreasing the autophagic activity of macrophages,Apoptosis of macrophages promotes the formation of foam cells,leading to the development of AS.PART 3 Regulating effects of miR-384-5p on macrophage autophagy in the development and progression of diabetic encephalopathyObjective: To investigate the regulating effects of miR-384-5p on macrophage autophagy in the development and progression of diabetic encephalopathy.Methods: 40 male C57 BL / 6 mice aged 10 weeks old were randomly divided into normal diet group(n = 10)and diabetic encephalopathy group(STZ group)(n = 30).STZ group received 1% streptozotocin intraperitoneal injection for inducing DM model.Meanwhile,10 mice in the STZ group were injected with adeno-associated virus via tail vein as AAV-null group and STZ + AAV-null group,10 mice received AAV-miR-384-5p injection as STZ + AAV-miR-384-5p group.Water maze test was used to test learning and memory function in mice.Animals were sacrificed to extract brain tissue,and macrophages were sorted by flow cytometry.m RNAof Beclin-1 in extracellular matrix were detected by RT-PCR,LC3-II / I,ATG7,p62 were detected by Western blot.The brain tissue and pancreatic tissue of mice were detected by immunohistochemistry for measuring the number of β cells,the contents of MDA,nitrite / nitrate,peroxidase and superoxide anion.Luciferase reporter assay was used for detection of miR-384-5p expression.Results: 1.Compared with CTL group,the autophagy-related protein Beclin-1 expression was increased in STZ group(p <0.05),macrophage autophagy was enhanced in STZ group.2.miR-384-5p was Beclin-1 targeted m RNA,which was capable of inhibiting the expression of Beclin-1.3.The blood glucose and insulin levels in the experimental group were only related to the STZ injection,however,with no significant correlation with the AAV transfection.AAV transfection,miR-384-5p overexpression did not affect the modeling of STZ diabetic mice;4.Overexpression of miR-384-5p significantly reduced macrophage in mice brain;5.miR-384-5p through the inhibition of macrophage activity,leading to brain tissue degradation product of which malondialdehyde,catalase and superoxide dismutase were increased and nitric oxide was decreased.Conclusion: The increase of Beclin-1 in STZ mice was mediated by the inhibition of miR-384-5p transcription.Beclin-1 expression increased the macrophage autophagy activity,which was significantly increased brain damage.After transfection of miR-384-5p mediated by adenovirus injection in STZ mice,macrophage activity in mouse brain was significantly inhibited,which benefited for brain recovery to some extent.