| BackgroundApoptosis of vascular endothelial cells is a key component of vascular disease. Since Ross raised "inflammatory response" theory, the endothelial injury has been gradually recognized as crucial component element of atherosclerosis (AS). In the early of AS, vascular endothelial cells of AS prone-region have apoptosis phenomena. Apoptosis of endothelial cells can accelerate the process of AS, releases various kinds of factors which can promote AS. These factors can further aggravate AS, and thereby forming a vicious circle. A growing number of experiments in vitro and vivo have confirmed that the endothelial cells dysfunction and apoptosis can be mediated by high glucose. Apoptosis in vascular endothelial cells induced by high glucose not only could increase the permeability of blood vessel walls but also lose the regulation of normal vascular endothelial relaxation and anti-adhesion function. It is considered to be an important factor for vascular diseases which accelerated atherosclerosis of diabetes. However, the mechanisms of endothelial cell apoptosis in high glucose have not yet been fully clarified.In the recent study, it has been found that Tribble3 can not only promote apoptosis but cause significantly higher fasting blood glucose through inhibition of Akt activity. It suggested that TRIB3 might be the target between diabetes and AS.TRIB gene was found in Drosophila, also known as neural cell death induced protein kinase, which inhibits mitosis in the early development of Drosophila. Tribbles is a family of Drosophila protein including TRIB1, TRIB2, TRIB3, which expresses in a variety of cells, such as vascular smooth muscle cells, macrophages, Hela cells. Its expression in cells has a specific regulation. Because TRIB3 is the homologue of mammalian, the research of TRIB gene focuses on the TRIB3. TRIB3 may have a wide range of biological activity. In the fasting state of db-/db- mice (a genetic defect in diabetic mice), TRIB3 mRNA and protein synthesis of liver were significantly increased, leading to increased glycogen output and blood glucose levels in a state of fasting. TRIB3 is possible to be not only the reason for impaired glucose tolerance of diabetes, but also the result of high blood glucose.Up to now, whether high glucose could promote TRIBs expression on the vascular endothelial cells, as well as role of TRIB3 in high glucose-induced endothelial cell apoptosis has not been reported. The above come into being the present study design and objectives.Objectives1. To investigate the relations of high glucose-stimulated time and endothelial cells survival through detecting HUVECs cell survival by MTT and apoptosis by AnnexinV-FITC/PI;2. To investigate the relations of TRIBs mRNA expression and glucose concentration, stimulating time by RT-PCR.3. To investigate the relations of TRIB3 expression and glucose-stimulated time by RT-PCR, immunofluorescence and Western-Blot;4. To explore the relations of TRIB3 and the atherosclerosis of diabetes by siRNA transfection inhibiting the expression of TRIB3.Subjects and methodsIn this study, we studied the third to fifth HUVECs and adopted MTT, AnnexinV-FITC/PI, cells immunohistochemistry, real time RT-PCR, Western blot respectively to observe the corresponding results.1. Methyl thiazolyl tetrazolium (MTT) determined cell survival rates of HUVECs by being treated with different concentrations glucose (5.5, 10, 20, 30, 40mmol/L) for 24h, 48h, 72h, respectively.2. In vitro diabetic states, after HUVECs were dealt with 30mmol/L glucose for 4h, 8h, 12h, 24h, 48h, 72h, we used SYRB Green I real-time PCR to detect TRIBs mRNA expression; after different concentrations glucose (10, 20, 30, 40mmol/L) and normal conditions stimulating HUVECs for 24h, TRIBs mRNA expression was detected by real-time PCR to test the expression of TRIBs mRNA; when HUVECs were treated by 30mmol/L glucose, 25mmol/L mannitol, and normal concentration for 4h, 8h, 12h, 24h, 48h, 72h, TRIB3 mRNA expression were detected.3. When HUVECs were respectively treated by 30mmol/L glucose for 24h and 48h, we observed cells changes by immunofluorescence compared with the control group.4. After HUVECs were dealt with 30mmol/L glucose for 24h, 48h, 72h, TRIB3 protein was assayed by Western-Blot.5. After TRIB3 were inhibited by siRNA transfection, cell apoptosis dealt with 30mmol/L glucose for 48h was assayed by AnnexinV-FITC/PI.Results1. MTT tested the effects of different glucose concentrations on cell survival rate of HUVECs: Compared with the control group, HUVECs stimulated by 5.5, 10, 20, 30, 40mmol/L glucose for 24 hours have no significant difference in survival rate (all P>0.05); the endothelial cells treated with 20, 30, 40 mmol/L glucose for 48h and72h have lower survival rate, and there are statistically significant differences concerned with concentrations (all P<0.05); Dealt with different glucose concentrations for 24h, 48h and 72h, cell survival rate decreased gradually. Survival rates of 20, 30, 40 mmol/L glucose between the groups was statistically significant, demonstrating the time-dependence. While glucose stimulating time extended, cell survival rates went down. The cell survival rate at 40mmol/L glucose is less than 50%, therefore we chose 30mmol/L for conventional glucose stimulation group.2 When 30mmol/L glucose treating HUVECs for 4h, 8h, 12h, 24h, 48h and 72h, real-time PCR detected TRIBs mRNA (TRIB1, TRIB2, TRIB3). The results showed that TRIB1, TRIB2, TRIB3 were all expressed in HUVECs. TRIB3 mRNA expression was markedly increased at 12h, peaked at 24h, but decreased at 48h, indicating that TRIB3 mRNA expression depends on time. TRIB1 mRNA, TRIB2 mRNA expressions are less and have no regularity.3 HUVECs were treated with different glucose concentrations (normal, 10, 20, 30, 40mmol/L) for 24h. TRIB3 mRNA expression increased significantly (P<0.05), while TRIB1 and TRIB2 mRNA expressions are less and have no significant differences. TRIB3 mRNA expression depends on concentrations, 30mmol/L glucose had the highest expression which have the statistical significance (P<0.01).4 HUVECs were treated with 30mmol/L glucose, hypertonia and normal conditions for 4h, 8h, 12h, 24h, 48h and 72h, real-time PCR detected TRIB3mRNA expression. The results showed that TRIB3mRNA was time-dependent, increased from 12h, peaked at 24h, then gradually decreased. There is statistically significant difference (P<0.05). In the hypertonic group TRIB3 mRNA increased slightly, suggesting that TRIB3 mRNA had no relation with hypertonic.5 With TRIB3 IgG immunofluorescence staining we can see nuclear staining of control group, 24h glucose group, 48h glucose group, and no obvious staining cytoplasm, which indicated that TRIB3 exists in the nucleus of HUVECs, little or none in cytoplasmic; among the three groups we can see the strongest immunofluorescence at 24h, weaker at 48h, the weakest in control group. This suggested that TRIB3 expresses the highest when HUVECs were stimulated by high glucose for 24h.6 With 30mmol/L glucose stimulating HUVECs for 24h, 48h and 72h, Western Blot detected TRIB3 protein expression. The results showed that TRIB3 protein expression was not significant at 72h and TRIB3 protein expression increased at 24h and 48h compared with the control group. TRIB3 protein expression at 24h is 5.44-fold to normal (P<0.01).7 After siRNA inhibiting the expression of TRIB3 and 30mmol/L glucose stimulating HUVECs for 48h, AnnexinV-FITC/PI detected apoptosis. The results showed that the apoptosis of siRNA group significantly decreased compared with the high glucose group.Conclusions1. TRIBs exist in HUVECs; 2. High glucose is one stimulating factor of TRIB3;3. After HUVECs stimulated by high glucose, the expression of TRIBs, mainly TRIB3, depend on concentration and time;4. After TRIB3 gene silencing, the apoptosis caused by high glucose significantly decreased. This suggested that TRIB3 might partly participate in the process of high glucose-induced apoptosis in endothelial cells.
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