Relativity Of Amyloid-β And Vascular Endothelial Cell Damage Induced By High Glucose And Intermittent High Glucose

Objective: The serum of patients with type2diabetes mellitus (T2DM) candamage the endothelial cells through oxidative stress, which is related to amyloid-β (Aβ)40and Aβ42. However, the reason of elevated Aβ levels in T2DM patients is still notclear. And it is to be further studied whether the simple elevation of blood sugar levelscan cause endothelial injury by affecting the levels of Aβ40and Aβ42. Therefore, thisstudy aims to interpose the human umbilical vein endothelial cells (HUVECs) throughusing high glucose and intermittent high glucose, and explore the endothelial injurycaused by high glucose and intermittent high glucose and that whether the injury isrelated to Aβ40and Aβ42.Methods:1. HUVECs in the logarithmic growth phase were inoculated in a culture flast andsynchronized respectively, followed by intervene with different concentrations of culturemedium for different time. Normal glucose group (NG group) was intervened with1640culture medium containing5.5mmol/L of glucose for30minutes,3hours,3days. Highglucose group1(HG1group) was intervened with1640culture medium containing11mmol/L of glucose for30minutes,3hours,3days. High glucose group2(HG2group)was intervened with1640culture medium containing20mmol/L of glucose for30minutes,3hours,3days. Mannitol group (MG group) was intervened with1640culture medium containing14.5mmol/L of mannitol for30minutes,3hours,3days.Intermittent glucose group1(IG1group) was intervened with culture mediumcontaining5.5/20mmol/L glucose (firstly with culture medium containing5.5mmol/Lof glucose for24hours, secondly with culture medium containing20mmol/L of glucosefor24hours, and finally with culture medium containing5.5mmol/L of glucose for24hours) for3days. Intermittent glucose group2(IG2group) was intervened with culturemedium containing20/5.5mmol/L of glucose (firstly with culture medium containing20 mmol/L of glucose for24hours, secondly with culture medium containing5.5mmol/Lof glucose for24hours, and finally with culture medium containing20mmol/L ofglucose for24hours) for3days.2. The morphological changes were observed using an inverted microscope. Eachgroup of cell supernatant was taken respectively for examinations after intervention for30minutes,3hours and3days. The superoxide dismutase (SOD) vitality was measuredto assess the cell antioxidant capacity by xanthine oxidase method. The content of maleicdialdehyde (MDA) was measured to assess the lipid peroxidation conditions bythiobarbituric acid method. The content of nitric oxide (NO) was measured to assess theendothelial function by nitrate reductase method. The content of lactate dehydrogenase(LDH) was measured to assess the damage extent of cells or cell membranes bychemical colorimetric analysis. The contents of Aβ40and Aβ42were measured to assessthe effects of high glucose and intermittent high glucose on Aβ40and Aβ42by ELISAassay.Results:1. Morphological changesNG, HG1, HG2and MG group were observed for30min and3h respectively withan inverted microscope. There were no significant morphological changes in each group.The cell outlines were clear in NG, HG1and MG at the3rd day, and were mostlyspindle-shaped, uniform and cobblestone-like closely packed. The cell outline was notclear in HG2group, turned round, shrunk and fragmented in cell body. The changeswere more obvious in IG1and IG2groups, with smaller in volume and fewer inquantity.2. Changes of MDA in the culture supernatants of HUVECsDuring the same time period, after intervention for30min, there was no significantdifference in the content of MDA in each group (P>0.05). After intervention for3h,there was no significant difference in the contents of MDA in HG1and MG groupcompared with the NG group (P>0.05); the content of MDA in HG2group wasincreased significantly (P=0.000), but without significant difference in HG2and MGgroup compared with HG1group (P>0.05); and the contents of MDA in MG and MDAgroup were reduced significantly compared with HG2group (P=0.000). Afterintervention for3d, the contents of MDA in HG1, HG2, IG1and IG2groups weresignificantly increased compared with the NG group (P=0.000); there was no significant difference in MG group (P>0.05); the contents of MDA in HG2, IG1and IG2groupswere significantly increased compared with HG1group (P=0.000,0.001,0.000); thecontent of MDA was significantly reduced in MG group (P=0.000); the contents ofMDA were increased significantly in IG1and IG2groups (P=0.000,0.003), andsignificantly reduced in MG group compared with HG2group (P=0.002). No significantd i f f e r e n c e w a s f o u n d b e t w e e n I G1a n d I G2g r o u p s (P>0.05).There were no significant differences in the contents of MDA among differenttime periods of30min,3h and3d in NG group (P>0.05). In HG1group, compared withthe content of MDA on30min, the content of MDA on3h was not significantlydifferent (P>0.05), and the content of MDA on3d was increased significantly(P=0.000); compared with the content of MDA on3h, the content of MDA on3d wasincreased significantly (P=0.001). In HG2group, compared with the content of MDA on30min, the contents of MDA on3h and3d were significantly increased (P=0.001,0.000);compared with the content of MDA on3h, the content of MDA on3d was increasedsignificantly (P=0.000). No significant difference was found in MGgroup between different time periods (P>0.05).3. Changes of SOD in the culture supernatants of HUVECsDuring the same time period, after intervention for30min, there was nosignificant difference in each group of SOD vitality (P>0.05). After intervention for3h,there was no significant difference in HG1and MG groups (P>0.05), and the SODvitality was reduced significantly in HG2group (P=0.000) compared with NG group;there was no significant difference in HG2and MG groups compared with HG1group(P>0.05); the SOD vitality was increased significantly in MG group compared withHG2group (P=0.000). After intervention for3d, the SOD vitalities were increasedsignificantly in HG1, HG2, IG1, and IG2groups (P=0.000) and no significant differencein MG group (P>0.05) compared with the NG group. The SOD vitalities were reducedsignificantly in HG2, IG1, and IG2groups (P=0.000), and the SOD vitality wasincreased significantly in MG group (P=0.000) compared with HG1group. The SODvitalities were reduced significantly in IG1and IG2groups (P=0.002,0.005), andincreased significantly in MG group (P=0.001) compared with HG2group, withoutsignificant difference between IG1and IG2groups (P>0.05).There were no significant differences in SOD vitalities in NG group amongdifferent time periods of30min,3h, and3d (P>0.05). In HG1group, there was no significant difference in SOD vitality after intervention for3h (P>0.05), but the SODvitality was reduced significantly after intervention for3d (P=0.000) compared with30min; the SOD vitality was reduced significantly after intervention for3d (P=0.001)compared with3h. In HG2group, the SOD vitalities were reduced significantly afterintervention for3h and3d compared with30min (P=0.001,0.000); the SOD vitalitywas reduced significantly after intervention for3d compared with3h (P=0.000). Nosignificant difference was found in MG group among different time periods (P>0.05).4. Changes of NO in the cultured supernatants of HUVECsDuring the same time period, there were no significant differences in the contentsof NO in each group after intervention for30min and3h (P>0.05). After interventionfor3d, the contents of NO were reduced significantly in HG1, HG2, IG1, and IG2groups (P=0.000), and there was no significant difference in MG group compared withNG group (P>0.05); the contents of NO were reduced significantly in HG2, IG1, andIG2groups (P=0.000), and increased significantly in MG group (P=0.000) comparedwith HG1group; the contents of NO were reduced significantly in IG1and IG2groups(P=0.000), and increased significantly in MG group (P=0.004) compared with HG2group.No significant diference was found between IG1and IG2groups (P>0.05).During different time periods, in NG group, the contents of NO were increasedsignificantly after intervention for3h and3d compared with30min (P=0.002,0.000);the content of NO was increased significantly after intervention for3d compared with3h (P=0.003). In HG1group, the contents of NO were reduced significantly afterintervention for3h and3d compared with30min (P=0.001,0.000); the contents of NOwere reduced significantly after intervention for3d compared with3h (P=0.001). InHG2group, c and3d compared with30min (P=0.001,0.000); the contents of NO werereduced significantly after intervention for3d compared with3h (P=0.04). In MGgroup, the content of NO was increased significantly after intervention for3h(P=0.002), the content of NO was increased significantly after intervention for3d(P=0.003) compared with30min; the content of NO was increased significantly afterintervention for3d compared with3h (P=0.008).5. Changes of LDH in the cultured supernatants of HUVECsDuring the same time period, after intervention for30min, there was nosignificant difference in LDH vitalities between HG1and MG groups (P>0.05), and the LDH vitalities was increased significantly in HG2group (P=0.000) compared with NGgroup; the LDH vitalities was increased significantly in HG2group (P=0.005), andreduced significantly in MG group (P=0.003) compared with HG1group. Afterintervention for3h, there was no significant difference in MG group (P>0.05), and theLDH vitality was significantly increased in HG1and HG2groups (P=0.000) comparedwith NG group; the LDH vitality was significantly increased in HG2group (P=0.000),and the LDH vitality was significantly reduced in MG group (P=0.005) compared withHG1group; the LDH vitality was significantly reduced in MG group (P=0.009)compared with HG2group. After intervention for3d, the LDH vitalities weresignificantly increased in HG1, HG2, IG1, and IG2groups (P=0.000), and there was nosignificant difference in MG group (P>0.05) compared with NG group; the LDHvitalities were increased significantly in HG2, IG1, and IG2groups (P=0.000,0.001,0.000), and the LDH vitality was reduced significantly in MG group (P=0.000)compared with HG1group; the LDH vitalities were increased significantly in IG1andIG2groups (P=0.000,0.003), and the LDH vitality was reduced significantly in MGgroup(P=0.002)compared with HG2group.No significantdifference was found between IG1and IG2groups (P>0.05).During different time periods, in NG group, there were no significant differencesin LDH vitalities after intervention for30min,3h and3d (P>0.05). In HG1group, theLDH vitalities were significantly increased after intervention for3h and3d comparedwith30min (P=0.000); the LDH vitalities were significantly increased afterintervention for3d compared with3h (P=0.001). In HG2group, the LDH vitalitieswere significantly increased after intervention for3h and3d (P=0.018,0.000respectively) compared with30min; the LDH vitalities were significantly increasedafter intervention for3d compared with3h (P=0.000). No significant difference wasfound in the MG group during different time periods(P>0.05).6. Changes of Aβ40and Aβ42in the cultured supernatants of HUVECsThere were no significant differences in Aβ40and Aβ42levels in each groupduring the same time period (P>0.05). There were also no significant differences inAβ40and Aβ42levels within a group in different time periods (P>0.05).Conclusions:1. Constant high glucose and intermittent glucose could increase the production ofvascular endothelial MDA, reduce SOD activity, increase LDH activity and reduce the production of NO, and be characterized with certain time dependence and concentrationdependence. Simultaneously, the changes were more significant induced by the highglucose. It was indicated that the increase of glucose level could damage the endothelialcells. The greater it fluctuated, the larger the cells were damaged. It was consistent withthe vascular complications of clinical DM. Therefore, controlling the high bloodglucose in patients with DM and keeping the blood glucose stable would be helpful toreduce the vascular endothelial injuries and delay the onset of DM vascularcomplications.2. Under the condition of constant levels of high glucose and intermittent highglucose, there were no significant changes in Aβ40and Aβ42levels in endothelial cells.It was indicated that the endothelial injuries caused by high glucose and intermittenthigh glucose were not related to Aβ40and Av42. The changes of Aβ40and Aβ42mightbe associated with other factors or the joint action of high glucose and other factors.