High Glucose Activates TNRF1 Signaling And Further Promotes Endothelial Progenitor Cells Apoptosis Through Inducing Oxidative Stress Reaction

Objectived: Diabetes mellitus(DM) is a chronic disease caused by genetic and environmental factors leading to high blood glucose and imbalance of carbohydrate,fat and protein metabolism.It not only causes glucose metabolic disorder, but also cause vascular complications.Endothelial progenitor cells(EPCs) play an important role in maintaining healthy blood vessels and promoting reconstruction of the damaged blood vessels. Currently, research on the mechanism of angiogenesis in diabetes mellitus impaired, EPCs dysfunction related factors have been widely attention, among them, the theory of oxidative stress playing an important role.It has been shows that: hyperglycemia environment of diabetic can make the EPCs apoptosis increased, decreased, and increased apoptosis may be the main mechanism of EPCs reduction in Diabetic vascular disease. Cell apoptosis is a autonomy, programmed death process controlled by genes, caused by a variety of incentives and mainly through death receptor mediated apoptosis pathway. Tumor necrosis factor receptor(TNFR) is the most representative one. In death receptor family,especially TNFR1,which mainly mediates the apoptosis signal transmission process. It has been reported that high blood glucose can induce TNFR1 expression,which can be inhibited by antioxidant.The main aim of this experiment is to investigate whether high glucose in vitro activates TNFR1 and further promotes rat bone marrow-derived endothelial progenitor cells(EPCs)apoptosis. Methods : SD rats were executed by taking off the neck, be put in 75% alcohol for 15 mintutes.Then we separated rat femur and tibia with animal anatomy instrument,then washing marrow cavity with PBS containing 1% heparin until rinses became colorless.After high speed centrigugal of flushing fluid, we collected the cells in the bottom of the tubes. The cells were seperated by using Ficoll density gradient centrifugation.After the separation, the Monocytes were plated into 6-well plates, changing cell medium for the first time after three days.For each 3 or 4 days,we renewed the cell medium.Morphological changes of the cells were observed through inverted microscope and EPCs were identified by the method of the double-labeled of Di I-ac-LDL/ FITC-UEA-1.Then EPCs were treated with high glucose(5.5, 15,30,60 mmol / L), high glucose(30 mmol / L) + Tempol and high glucose(30mmol/L)+ MAB430. Apoptosis rate of the cells were detected by flow cytometry;Molecular probe(DCFH- DA) were used to detect reactive oxygen species(ROS) content changes after treatment of different glucose concentration;the expression level of TNFR1 and related protein(TRADD, TRAF2, RIP, NF-k Bp65, Caspase3) were detected by Western blotting.Resulted: After culturing with M199 for three days, most of EPCs were spherical and began to adhere.Seven days later, cell grew rapidly and became colony-like. After fourteen days, "paving stone" cells can be found. By laser confocal microscopy, dual-positive cells were considered the endothelial progenitor cells after dealing with the Di I-ac-LDL/ FITC-UEA-1. When in the early stage(24-48h) of high glucose stimulating, the number of apoptosis EPCs were approximately same in HG group with in Con group. When in the later stage(72 h-96 h) of high glucose stimulating, the number of apoptosis EPCs increased apparently in HG group compared with Con group.The high glucose group(15 mmol/L, 30 mmol/L, 60 m mol/L) group showed statistical significance compared with control group(P < 0.01).In the early stage(24-48h), the number of apoptosis EPCs in high glucose group(30 mol/L, 60 mol/L), respectively, comparing with high glucose group(15mmol/L, were with no difference. However in the later stage(72 h-96 h),we found that the number of apoptosis EPCs in HG group(60mmol/L)increased apparently compared with HG group(15mmol/L),and it showed statistical significance(P < 0.01). Compared HG(30mmol/L) group with using antioxidants Tempol and TNFR1 receptor antagonist MAB430 treatment, apoptosis cells were less in the early stage(24h),while were more in the later stage(72 h), statistically significant(P < 0.01).While high glucose stimulate EPCs in the early stage(24h),,In the early stage, there is no significant difference with HG and Control group, with the extension of time,In the later stage(72 h),ROS were more in HG group(P<0.01).Tempol and MAB430 treatment, Compared with HG(30mmol/L) group, ROS were less in the early stage(24h),while were more in the later stage(72 h), statistically significant(P < 0.01), High glucose(30mmol/L) stimulate EPCs in the early stage(24h), TNFR1 protein and TNF apoptosis signal pathway related protein(TRAF2、TRADD、RIP、Caspase3) expression level not increase in the earlier stage( before 24 h) but increase significantly in the later stage(after 72 h)(P<0.01),with Tempol and MAB430 treatment, TNFR1 protein and TNF apoptosis signal pathway related protein(TRAF2、TRADD、RIP、Caspase3) expression level were decreased(P<0.01).And with the High glucose(30mmol/l), the NF-k Bp65 protein relative expression is gradually reduced, the amount of protein expression in 72 h and 24 h have a statistically significant difference(P < 0.01).After processed by TEMPOL, MAB430, whether early or late, compared to HG group(30mmol/l), the NF-k Bp65 protein expression was elevated(P < 0.01).Conclusion : High glucose can induce oxidative stress activated TNFR1 receptor protein and TRADD protein,which to recruit TRAF2, RIP protein, TRAF2 and RIP protein of which can be further to activate the NF-k Bp65 transcription factors, which is directly related to apoptosis of Caspase3 protein expression increased, and further induce cell apoptosis.