Relationship Between Amyloid-β40/42and Type2Diabetes With Macroangiopathy And Vascular Endothelial Injury

Objective: Type2Diabetes mellitus (T2DM) serum can damage Human UmbilicalVein Endothelial Cells (HUVECs), at the same time accompanied by level of Amyloid-β(Aβ)40and Aβ42increase, and Aβ40can induce injury of HUVECs. This study was tofurther to discuss the relationship between Aβ40and Aβ42and T2DM withmacroangiopathy and the role of Aβ42in vascular endothelial as well as the mechanism.Methods:1.78cases of patients with T2DM were selected and divided into isolated T2DMgroup (IDM group, N=38) and T2DM with macroangiopathy group (DMA group,N=40); health control group (HC group, N=40) was selected. All the research subjectswere performed comprehensive physical examination, weight measurement, waistcircumference (WC), systolic blood, pressure (SBP), diastolic blood pressure (DBP)measurement and body mass index (BMI) calculation; elbow venous blood was drawnafter12h overnight fasting to measure the fasting plasma glucose (FPG), glycosylatedhemoglobin A1C(HbA1C), total cholesterol (TC), triglyceride (TG), low densitylipoprotein cholesterol (LDL-C); ELISA method was used to measure the serum ofAβ40and Aβ42.2.30min,3h,3D after injury of human umbilical vein endothelial cells (HUVECs)induced by Aβ42, the microscope was inverted to observe the morphological changes ofthe cells; methyl thiazolyl tetrazolium (MTT) method was used to detect theproliferation of cells when Aβ42was0.005μM,0.05μM,0.5μM,5μM,50μM at3d andthen the most suitable damage concentration was selected.3. HUVECs injury was induced by Aβ42of0.5μM and5μM for30min,3h,3d;cell culture supernatant at different time points was maintained and chemicalcolorimetry was used to measure the activity of lactate dehydrogenase (LDH),thiobarbituric acid method was used to measure the content of maleic dialdehyde (MDA), xanthine oxidase method was used to measure the activity of superoxidedismutase (SOD) and nitrate reductase method was used to measure the content of nitricoxide (NO).4. Selecting endothelial cells at different time points, real time-RT PCR methodwas used to measure the mRNA expression level of receptor for advanced glycationend-products (RAGE) and Western Blot method was used to measure the expressionlevel of RAGE protein.Results:1. Comparison of clinical data of all the groups: the FPG (P=0.000), HbA1C(P=0.002), BMI (P=0.012) and WC (P=0.006) of IDM group were higher than that ofHC group; FPG (P=0.000), HbA1C (P=0.003), BMI (P=0.000), WC (P=0.000), SBP(P=0.000), DBP (P=0.000), TC (P=0.010), TG (P=0.000), LDL-C (P=0.001) of DMAgroup were higher than that of HC group; FPG (P=0.004), WC (P=0.005), SBP(P=0.000), DBP (P=0.001), TG (P=0.000), LDL-C (P=0.016) of DMA group werehigher than that of IDM group.The comparison of serum Aβ40and Aβ42of all the groups: compared with HCgroup, the Aβ40level of IDM group (P=0.002) and DMA group (P=0.000) wasincreased, and the Aβ40level of IDM group was higher than that of IDM group(P=0.000); the difference of Aβ42among all the groups was not statistically significant(P>0.05).2. Changes of cell morphology: for control group, endothelial cells morphologywas normal, with polygonal shape, clear boundary of cells, cells confluent to monolayer,cobblestone-like arrangement. No obvious changes of cell morphology were observed at30min and3h; the visible cells of Aβ4250μM group at3d was decreased, intercellularspace was increased, the cell boundary was fuzzy, cell body was swelled, thearrangement of cells was in disorder, while no obvious changes occurred in othergroups.3. Cell survival rate of groups of all concentrations at3d: compared with controlgroup, the cell survival rate of0.5μM group,5μM and50μM group was decreased,50μM group decreased significantly (P=0.000); there was no statistically significantdifference between0.05μM group and0.005μM group (P>0.05); there was nostatistically significant difference between0.5μM group and5μM group （P>0.05）;compared with other groups, the cell survival rate of50μM group was significantly decreased (P=0.000), but the survival rate was too low. Therefore,0.5μM and5μM wasselected to evaluate the endothelial damage effect in this study.4. The activity of LDH: comparison of all the groups at the same time point: at30min, there was no statistically significant difference among all the groups (P>0.05);at3h, compared with control group, the activity of LDH of0.5μM group (P=0.001) and5μM group (P=0.000) was significantly increased,5μM group increased significantly; at3d, compared with control group, the activity of LDH of0.5μM group (P=0.000) and5μM group (P=0.000), with5μM group increased significantly. At3h and3d, comparedwith0.5μM group, the activity of LDH of5μM group was significantly increased,(P=0.034,0.004), with certain concentration dependence.Comparison among the groups of the same concentration at different time points:in the control group, there was not statistically significant difference at all the timespoints (P>0.05); in0.5μM group, compared the activity of LDH at30min, the activity at3h was increased (P=0.000), and compared with the activity of LDH at30min (P=0.000)and3H (P=0.018), the activity at3d was obviously increased; in5μM group, comparedwith the activity of LDH at30min, the activity at3h was increased （P=0.000）;compared with the activity of LDH at30min (P=0.000) and3H (P=0.002), the activityat3d was obviously increased. In0.5μ M and5μ M group, the increase of LDH activityhad certain time independence with the time prolonged.5. Content of MDA: the comparison among all the groups at the same time point:at30min, there was no statistically significant difference (P>0.05); at3h, compared withthe control group, the content of MDA of0.5μM group (P=0.001) and5μM group(P=0.000) was obviously increased, with5μM group increased significantly. At3h and3d, compared with0.5μM group, the content of MDA of5μM group was increased(P=0.018,0.022), with creation concentration dependence.Comparison among the groups of the same concentration at different time points:in the control group, there was no statistically significant difference at different timepoints (P>0.05); in0.5μM group, compared with the content of MDA at30min, thecontent at3h was increased（P=0.032）and compared with the content of MDA at30minand3h, the content at3d was significantly increased; in5μM group, compared with thecontent of MDA at30min, the activity at3h was increased (P=0.000) and comparedwith the content of MDA at30min (P=0.000) and3h (P=0.000), the content at3d wasobviously increased. In0.5μM and5μM group, the increase of content MDA had certain time dependence with the time prolonged.6. The activity of SOD: comparison among all the groups at the same time point: at30min, there was no statistically significant difference among all the groups (P>0.05); at3h, compared with the control group, the activity of SOD of0.5μM group (P=0.003)and5μM group (P=0.000) was obviously decreased,5μM group decreased significantly;at3d, compared with the control group, the activity of SOD of0.5μM group (P=0.000)and5μM group (P=0.000) was obviously decreased, with5μM group decreasedsignificantly. At3h and3d, compared with0.5μM group, the activity of SOD of5μ Mgroup was decreased and had certain concentration dependence.Comparison within the group of the same concentration at different time points: inthe control group, there was no statistically significant difference at all the time points（P>0.05）；in0.5μM group, compared with the activity of SOD at30min, the activityat3h was decreased (P=0.003), and compared with the activity of SOD at30min(P=0.000) and3H (P=0.001), the activity at3d was obviously decreased. In5μM group,compared with the activity of SOD at30min, the activity at3h was decreased, andcompared with the activity of SOD at30min (P=0.000) and3h (P=0.001), the activityat3d was obviously decreased. In0.5μM and5μM group, the decrease of activity ofSOD had certain time dependence with the intervention time prolonged.7. Content of NO: comparison among all the groups at the same time point: at30min, there was no statistically significant difference among all the groups (P>0.05); at3h, compared with the control group, the content of NO of0.5μM group (P=0.000) and5μM group (P=0.000) was obviously decreased, with5μM group decreasedsignificantly; at3d, compared with control group, the content of NO of0.5μM group(P=0.000) and5μM group (P=0.000) was obviously decreased, with5μM groupdecreased significantly. At3h and3d, compared with0.5μM group, the content of NOof5μM group was obviously decreased (P=0.000,0.047) and had certain concentrationdependence.Comparison among the groups of the same concentration at the different timepoints: in the control group, there was no statistically significant difference at all thetime points（P>0.05）；in0.5μM group, compared with content of NO at30min, thecontent at3h was decreased (P=0.000), and compared with the content of NO at30min(P=0.000) and3h (P=0.000), the content at3d was significantly decreased; in5μMgroup, the compared with the content of NO at30min, the content at3h was decreased (P=0.000), and compared with the content of NO at30min (P=0.000) and3h (P=0.000),the content at3d was obviously decreased. In0.5μM and5μM group, the decrease ofcontent of NO had certain time dependence with the intervention time prolonged.8. The expression of RAGE mRNA: comparison among all the groups at the sametime points: at30min, there was no statistically significant difference among all thegroups (P>0.05); at3h, compared with the control group, the expression of RAGEmRNA of0.5μM group (P=0.038) and5μM group (P=0.000) was enhanced, with5μMgroup enhanced significantly; at3d, compared with the control group, the expression ofRAGE mRNA of0.5μM (P=0.000) and5μM (P=0.000) was obviously enhanced, with5μM group enhanced obviously. At3h and3d, compared with5μM group, theexpression of RAGE mRNA of5μM was increased（P=0.025，0.000）, with certainconcentration dependence.Comparison within the group of the same concentration at the different time points:in the control group, there was no statistically significant difference at all the timepoints (P>0.05); in0.5μM group, there was no statistically significant differencebetween at3h and30min (P>0.05), and compared with the expression of RAGE mRNAat30min (P=0.000) and3h (P=0.000), the expression at3d was obviously enhanced; in5μ M group, compared with the expression of RAGE mRNA at30min, the expressionat3h was enhanced, and compared with the expression of RAGE mRNA at30min(P=0.000) and3h (P=0.000), the expression at3d was obviously enhanced. In0.5μMand5μM group, the enhancement of expression of RAGE mRNA had certain timedependence with the time prolonged.9. The expression of RAGE protein: comparison among all the groups at the sametime point: at30min, there was no statistically significant difference among all thegroups (P>0.05); at3h, compared with control group, the expression of RAGE of0.5μM (P=0.000) and5μM group (P=0.000) was obviously enhanced, with5μM groupenhanced significantly. At3h and3d, compared with0.5μM group, the expression of5μM group was enhanced (P=0.002,0.000), with certain concentration independence.Comparison within the group of the same concentration at the different time points:in control group, there was no statistically significant difference at all the time points(P>0.05); in0.5μM group, compared with the expression of RAGE at30min, theexpression at3h was obviously enhanced, and compared with the expression of RAGEat30min (P=0.000) and3h (P=0.000), the expression at3d was obviously enhanced; in 5μM group, compared with the expression of RAGE at30min, the expression at3h wasenhanced (P=0.000), and compared with the expression of RAGE at30min (P=0.000)and3h (P=0.000), the expression of RAGE at3h was obviously enhanced. In0.5μMand5μM group, the enhancement of expression of RAGE had certain time dependencewith the time prolonged.Conclusions:1．The serum Aβ40level of patients with T2DM is increased, and the increase ofAβ40level of the patients with T2DM combined with macroangiopathy is moresignificant. Aβ40may participate in the occurrence and development of T2DM andlesion of large vascular vessel.2．Aβ42had the injury on endothelial cells, with LDH release increased, MDAgeneration increased, the activity of SOD decreased and NO synthesis decreased, andbesides, with certain concentration and time dependence, whose damage mechanismmay be related to oxidative stress.3．Aβ42induces the injury of endothelial cells, and in addition, the level of RAGEmRNA and protein expression is increased and has creation concentration and timedependence, which indicates that Aβ42inducing oxidative stress damage of endodermisthrough RAGE to block some link to play the role of endothelial protection.