Research On The Mechanisms Of Glucose Metabolism-related Proteins(GluT1,TPI1) Promoting The Formation Of AD

Aortic dissection(AD)is a group of acute cardiovascular diseases,due to the structural remodeling and functional degradation of the aortic wall,the impact of the high-pressure blood flow led to intima rupture and medial tear,accompanied by inflammatory infiltration.Which characterized by dangerous onset,rapid progress,and high mortality.Lacking the effective cure drugs and preventive measures,with an increasing incidence.Finding the etiology and incentives,and studying the pathogenic mechanism has important clinical significance and positive social significance for the prevention and treatment of AD.This research investigates the relationship between glucose metabolism-related proteins(Glu T1,TPI1)and the occurrence of aortic dissection.The content is divided into two parts,Part I: To explore the relationship between Glu T1-mediated insulin resistance(IR)and AD.The essence of IR is the abnormal function and quantity of Gluts on the cell membrane,resulting in decreased the ability of cells to uptake glucose,which is a high-risk factor for cardiovascular disease.However,there are few studies about the relationship between AD and IR.Using glycosylated hemoglobin(Hb A1c)as an indicator for evaluating IR,we retrospectively analyzed clinical epidemiological survey data and found that IR was closely related to AD,and the m RNA and protein expression of Glu T1 that were analyzed by RT-q PCR,Western Blot and IF which were significantly down-regulated in AD aorta,p < 0.05;Establishing IR and AD mouse models,it was found that Glu T1 mediated IR to promote AD occurrence,vascular smooth muscle cell phenotype switch and MMP2/9expression,p < 0.05;The insulin resistance model of HA-VSMCs was established by hyper-insulin induction method,and it was found that IR obviously reduced the m RNA and protein expression of Glu T1 and SM22 in HA-VSMCs,P < 0.05,while these expression of OPN were opposite,P < 0.05.The expression of Glu T1 were downregulated in HA-VSMCs by transfected with Si-Glu T1 liposome,which led to the m RNA and protein expression of the phenotype-specific molecule SM22 were deceased(P < 0.05)and that of OPN were increased(P < 0.05).The above experimental results show that Glu T1-mediated IR induces the phenotype of vascular smooth muscle cells to switch from contractile to synthetic,which promotes the formation of aortic dissection.Part II: In order to explore the relationship between TPI1 and aortic dissection.Glycolysis/Gluconeogenesis is the basis of the energy source for cell metabolism.investigate the mechanisms of the TPI1 contribute to the formation of AD by research the effect of key enzyme in metabolic pathways(TPI1)on the function and phenotype of cells.Differentially expressed proteins were screened by proteomic analysis,and GO and KEGG analysis were performed by Fun-Rich software.founding that the protein expression of TPI1 in the glycolysis/gluconeogenesis pathway was the most elevated,and the protein expression tendency of TPI1 in AD aortic smooth muscle were verified by Western Blot and immunohistochemistry,P < 0.05.Then,the protein expression of TPI1 was upregulated in the aortic smooth muscle of AD model mouse induced by Ang-II,P < 0.05.Finally,HA-VSMCs were transfected with liposome TPI1-m RNA,the over expression of TPI1 promote the phenotype switch of HA-VSMCs from contractile to synthetic and promote the expression of MMP2/9,(P < 0.05).The result of this research indicates that TPI1 contribute to the formation of AD by affecting the phenotype of VSMCs and the expression of MMP2/9,in addition to affecting intracellular metabolism.Part I: The Research on the Mechanisms of Glu T1-Mediated Insulin Resistance Contribute to the Formation of ADChapter One:A Study on the Relationship Between Insulin Resistance and Aortic DissectionObjective: To explore the fact that Glu T1-mediated insulin resistance is a high-risk factor that promotes the occurrence of AD.Methods: 1)Excluding these cases that did not meet the conditions,and taking Hb A1 c ≥ 5.7 that were detected at the time of admission as IR,we collected data on patients with acute thoracic aortic dissection(ATAD)in cardiovascular surgery from July 2017 to July 2020 in Fujian Medical University Union Hospital and calculated the proportion of patients with insulin resistance;2)Real time-q PCR(RT-q PCR)and Western Blotting(WB)were used to study the m RNA and protein expression of Glu T1 in thoracic aorta from normal people and AD;3)using the techniques of immunohistochemistry and immunofluorescence to detect the protein expression of Glu T1.Results: 1)The patients with ATAD were divided into four groups according to the value of Hb A1c: Hb A1 c < 5.7: 129 cases,35.64%;6.0 > Hb A1 c ≥ 5.7: 85 cases,23.48%;6.5 > Hb A1 c ≥ 6.0: 99 cases,27.3%;Hb A1 c ≥ 6.5: 49 cases,13.54%;2)the m RNA expression of Glu T1 in the artery of patients with TAD was significantly reduced(P < 0.05);3)the protein expression of Glu T1 was decreased in the aorta of AD,with statistically significant(p < 0.05).Conclusion: Glu T1-mediated insulin resistance is a high-risk factor which associated with aortic dissection.Chapter Two: Induces IR by D12492 in Apo E-/-mice and establishes AD model with BAPN combined with Ang-IIObjective: To investigate the mechanism that IR promoting AD by establishing a mouse disease model of IR and AD.Methods: Apo E-/-mice were induced to develop IR by fed with D12492,and AD model was established by subcutaneously implanting 24 h Ang-II(1ug/Kg/min)osmotic mini-pumps after 28 days of free drinking water containing 0.25% BAPN.3-week-old male healthy Apo E-/-mice were used for our research,all mice were divided into two groups based on the similar average body weight per mouse(control group n=7,model group n=15).collecting venous blood(centrifuged at 1500 g at 4°C for 15 min,extracting plasma,storing in-80°C refrigerator for detect insulin concentration by ELISA)and measuring fasting glucose concentration(FGC)by tail-cuff,after 12 hours of fasting,and using HOMA-IR to assess the sensitivity of insulin.The mice in the model group were fed with D12492,while the control group was fed with normal chow,and all drinking water contained 0.25% BAPN.collecting venous blood and detecting FGC after 4 weeks,then the Ang-II mini-pumps were implanted subcutaneously,and the mice were euthanized 24 h after implantation,Fresh aortic tissue was obtained for protein verification,and 4% paraformaldehyde was used to fix specimens before paraffin embedding.Results: 1)D12492 diet induces IR in mice;2)The incidence of AD in the model group was 13/14,which higher than the control group that 3/7;3)The protein expression of Glu T1 in the aorta of the mice from the model group was significantly decreased,p < 0.05;4)In the aorta of the model group mice,the protein expressions of phenotype-specific proteins α-SMA,SM22 were decreased and OPN was increased,and the protein expression of MMP2 and MMP9 were increased,all the results have statistical significance(p < 0.05);5)Pathological examination: H&E staining showed that the VSMCs in the aorta middle layer of the model group were hyperplasia,disordered,extracellular matrix increased,and double-lumen sign was seen;EVG staining showed the elastic fibers in the aorta of the model group became smaller and thinner,broken.and the semi-quantitative statistics show a significant decrease,p < 0.05;Masson staining showed the collagen fibers increased and thickened,the result of semi-quantitative statistical is significance up-regulate,p < 0.05.Conclusion: D12492 lead to IR by reducing the protein expression of Glu T1,promoted phenotypic switch of VSMCs,secretion of MMP2/9,and the occurrence of AD.Chapter Three: Glu T1 mediates IR induces phenotypic switch of HA-VSMCsObjective: To investigate the molecular mechanism of Glu T1-mediated IR to promote AD by establishing insulin-resistant HA-VSMCs model and reducing the expression of Glu T1 by liposome transfection of Si-Glu T1.Methods:1)Establishing the insulin-resistant HA-VSMCs model by hyperinsulinemia induction;2)Reducing the protein expression of Glu T1 in HAVSMCs by transfection of Si-Glu T1 with liposomes;3)RT-q PCR and WB were used to study the m RNA and protein expression of Glu T1,SM22 and OPN in HAVSMCs of IR;4)the protein Expression of Glu T1,SM22 and OPN in HA-VSMCs were shown using immunofluorescence methods.Results: 1)When HA-VSMCs were cultured in DMEM containing10-7mmol/L insulin for 36 h,the glucose consumption of the same unit counted cells were decreased most significantly(p < 0.01);2)The m RNA and protein expression of Glu T1 was decreased in HA-VSMCs with insulin resistance(p < 0.001);3)The m RNA and protein expression of SM22 was decreased and OPN was increased in insulin-resistant HA-VSMCs,p < 0.01;4)The m RNA and protein expressions of SM22 were downregulated,p < 0.01,while the OPN was the opposite tendency,p < 0.01,after HA-VSMCs were transfected with Si-Glu T1.Conclusion: Glu T1-mediated IR induces the switch of HA-VSMCs phenotype from contractile to synthetic.Final conclusion: Glu T1-mediated IR induces phenotypic switch of VSMCs promotes the occurrence of AD.Part II: TPI1 induces phenotypic switch of VSMCs that contributing to the formation of ADObjective: To expose the mechanism of Triosephosphate isomerase-1(TPI1)promoting the occurrence of aortic dissection.Methods: 1)Fun-Rich analysis was performed based on differential proteins screened by TMT proteomics analysis;2)Western Blot and immunohistochemistry were used to verify the protein expression changes of TPI1;3)Ang-II micropumps were implanted subcutaneously for 28 days to induce the formation of AD in mice,and the protein expression of TPI1 in aortic smooth muscle was detected;4)TPI1-m RNA was transfected with liposomes to make TPI1 was overexpressed in HAVSMCs;5)RT-q PCR and Western Blot were used to detect the transfection efficiency of TPI1-m RNA,the expression of SM22,α-SMA,OPN and MMP2/9.Results: 1)the protein expression of triosephosphate isomerase-1(TPI1)in the glycolysis/gluconeogenesis pathway was the most elevated;2)Western Blot and immunohistochemistry verified the change trend of TPI1 in AD,which was statistically significant(p < 0.01);3)Continuous pumping of Ang-II can induce AD in mice;4)The protein expression of TPI1 in the aortic smooth muscle of mice treated with Ang-II was increased,with statistical significance(p < 0.05);5)After HA-VSMCs were transfected with TPI1-m RNA,the expressions of SM22 and α-SMA were downregulated,and the expressions of OPN and MMP2/9 were upregulated,all of these changes were statistically significant(p < 0.01).Conclusion: TPI1 induces the phenotype switch of VSMCs and the expression of MMP2/9 to promote AD formation.