Involvement Of Androgen Receptor In Human Umbilical Vein Endothelial Cells Injure Induced By Intermittent High Glucose And Its Possible Mechanisms

The incidence of cardiovascular disease in men compared with premenopausal age women is significantly high, even taking into account other risk factors. Male sex is an independent risk factor for coronary artery disease. But some studies showed that male cardiovascular disease patients'androgen levels were lower than men without cardiovascular disease. In vitro and in vivo studies had shown that physiological doses of androgen can reduce the occurrence of atherosclerosis, so whether androgens are truly causative of CVD is not clear.The androgen receptor (AR) is a type of nuclear receptor that is activated by binding of either of the androgenic hormones testosterone or dihydrotestosterone. Sex hormone receptors, including AR and estrogen receptor (ER) a and β, are important in vascular tissue. AR expressed in vascular endothelial cells and vascular smooth muscle cells. But the the importance of AR in these cells is still inconclusive.Glycemic variability is an important factor used to resolve potential clinical problems in diabetic patients. It is known that glycemic variability contributes to the development of macro-and microvascular complications in diabetes. The role of glycemic variability is an independent risk factor for diabetic complications; it can generate excess reactive oxygen species (ROS) because hyperglycemia triggers activation of oxidative stress through the mitochondrial electron-transport chain. Recently, many in vitro and in vivo studies have shown the importance of ROS involvement in the core mechanism of glycemic variability-induced vascular complications.The PI3K/Akt/mTOR pathway is an intracellular signalling pathway important in cell survival and apoptosis. PI3K activation activates Akt which activates mTOR. In many stressful situations, as diabetes, cancers and hypoxia, this pathway is overactive to reduce apoptosis and allow cell proliferation. When this pathway is blocked or knocked down, cell damage or apoptosis will increase. Previous studies have shown that there is cross-talk between AR and PI3K/Akt/mTOR pathway.Whether glucose fluctuation will affect the expression of AR in HUVEC? Whether androgen can improve the damage induced by glucose fluctuation in HUVEC via AR? Whether the improvement requires the PI3K/Akt/mTOR signal pathway? To solve the above problems, we cultured HUVEC in different glucose concentration to detect cell injury, AR expression and PI3K/Akt/mTOR signaling pathway. Then we used drugs and siRNA blocking or knock down the pathway, observed whether AR can improve the damage induced by glucose fluctuation in HUVEC through the PI3K/Akt/mTOR signaling pathway. Part one:The models of human umbilical vein endothelial cells induced by constant and intermittent high glucose in vitroObjective:To establish constant and intermittent high glucose injure human umbilical vein endothelial cells (HUVEC) modles and detect oxidative/antioxidative system in the cell culture supernatant.Methods:HUVEC was cultured in different Endothelial Cell Medium (concentration of glucose:5.5mM, lOmM,30mM,50mM, and5.5mM+mannitol44.5mM) different times (Oh,24h,48h and72h). Inverted phase contrast microscopy and MTS assay were used to evaluate cell growth. Then HUVEC was randomly divided into control group (concentration of glucose:5.5mM, group N), constant high glucose group (concentration of glucose:30mM, group C) and intermittent high glucose group (concentration of glucose:5.5mM and30mM alternately every12hours, group F). After different time, inverted phase contrast microscopy and MTS assay were used to evaluate cell growth. The activity of GSH-Px and8-iso content in supernatant were measured too.Results:1.From inverted phase contrast microscope, group5.5mM and lOmM were in good condition with normal morphology. Group30mM and50mM became irregular in shape and some cells detach from bottom of wells. Group50mM was distorted more apparently. MTS array showed that cell proliferative activity of group5.5mM, lOmM and30mM were increased gradually within72h. But the growth rate of group30mM was significantly slower than group5.5mM. Cell proliferative activity of group50mM was decreased gradually between48-72h. Compared with group5.5mM, group5.5mM+mannitol44.5mM had no significantly distinction.2. Comparing with group N, from inverted phase contrast microscope, group C and group F cultured Oh and24h were without significant distinction. The group C cultured48h was without significant distinction comparing with group N. The cells of group F cultured48h changed to fusiform or irregular in shape, some cells changed to unclear in profile, and the nuclei were enlarged. Group N cultured72h was grown in good condition with normal morphology. Group C became fusiform or irregular in shape and some cells detach from bottom of wells. Group F cultured72h was distorted more apparently.3. Cell proliferative capacity (OD value) of three groups cultured Oh and24h showed no significant difference (P>0.05). The group N and C cultured48h showed no significant difference (P>0.05). Comparing with group N and C, group F cultured48h OD value was decreased significantly (P<0.01). Comparing with group N, group C and F cultured72h, OD value were significantly reduced (P<0.01); group F was significantly lower than C(P<0.01).4.8-iso and GSH-Px contents of three groups cultured Oh and24h had no significant difference (P>0.05). GSH-Px contents of group F cultured48h was significantly lower than N (P<0.05), but group C and N showed no significant difference (P>0.05).8-iso content of group F cultured48h compared with group N and C was significantly increased (P<0.01; P<0.05), but group C and N showed no significant difference (P>0.05).8-iso contents of group C and F cultured72h compared with group N were significantly increased (.P<0.05), while GSH-Px contents were significantly lower (P<0.01); comparing with group C, group F had higher8-iso contents (P<0.05) and lower GSH-Px contents (P<0.01).Conclusion:Constant and intermittent high glucose induced the cell proliferative capacity descending, promoted lipid peroxidation. decreased anti-oxidation capacity, and caused HUVEC injury. The effect of intermittent high glucose was earlier and stronger than constant high glucose. Part two:Involvement of androgen receptor in human umbilical vein endothelial cells injure induced by intermittent high glucoseObjective:To elucidate the effect of intermittent high glucose on androgen receptor (AR) expression and to clarify whether testosterone can improve the injury induced by glucose fluctuation via AR in human umbilical vein endothelial cells (HUVECs).Methods:HUVEC was cultured in different Endothelial Cell Medium (ECM) different times (Oh,24h,48h and72h). The experiment was randomly divided into control group (concentration of glucose:5.5mM, group N), constant high glucose group (concentration of glucose:30mM, group C) and intermittent high glucose group (concentration of glucose:5.5mM and30mM alternately every12hours, group F). After different time, expression of AR was determined by quantitative real-time PCR, western blot and immunofluorescence. Different concentrations (1nM,10nM,100nM and1000nM) of testosterone (T1, T10, T100, and T1000) and flutamide (FLU) were added in the group N and F to observe the changes in the expression of AR and the cells damages caused by intermittent high glucose.Results:1. Oh and24h, AR mRNA and protein expression of three groups showed no significant difference (P>0.05). Group N and C showed no significant difference at48h (P>0.05).48h and72h, comparing with group N and C, AR mRNA and protein were decreased significantly in group F (P<0.01). Comparing with group N cultured72h, AR mRNA and protein were significantly reduced in group C (P<0.01). 2. Comparing with group F, from inverted phase contrast microscope, group F+T1and F+T10were grown in better condition and group F+T10was improved more apparent. When testosterone concentration increased to100nM, cells began to appear damage. When the concentration increased to1000nM, cell was distorted more apparent. Comparing with group N, other groups'cell proliferative capacity were decreased significantly (P<0.01). Comparing with group F, cell proliferation was significantly increased in group F+T1and F+T10(P<0.05; P<0.01) but decreased in group F+T1000(P<0.01). Group F+T100showed no significant difference comparing with F group (P>0.05). Comparing with group N, other groups8-iso contents were increased significantly and GSH-Px contents were decreased significantly (P<0.01). Comparing with group F,8-iso contents were significantly decreased in group F+T10and F+T100(P<0.05; P<0.01), group F+T1and F+T1000showed no significant difference comparing with F group (P>0.05). Comparing with F group, GSH-Px contents were increased significantly in F+T1, F+T10and F+T100but decreased significantly in group F+T1000(P<0.05). Comparing with N group, apoptosis rates were increased significantly in other groups (P<0.01or P<0.05). Comparing with F group, apoptosis rates decreased significantly in F+T1, F+T10and F+T100but increased in group F+1000(P<0.01or P<0.05). Each group compared with N group, cleaved-caspaseS expression was increased significantly (P<0.01or P<0.05). Compared with F group, cleaved-caspase3expression were declined significantly in group F+T10and F+T100(P<0.01or P<0.05), group F+T1and F+T1000showed no significant difference.3. Comparing with N group, AR mRNA and protein expression were higher in F+T1000group (P<0.05), and other groups were lower (p<0.01). Comparing with F group, AR mRNA and protein expression had no significant difference in F+T1group (P>0.05) and increased significantly in F+T10, F+T100and F+T1000(P<0.01).4. Comparing with group F, cell proliferation activity,8-iso, GSH-Px, apoptosis rates and cleaved caspase3protein expression showed no significant difference in group F+FLU and F+FLU+T10(P>0.05).Conclusion:Both constant and intermittent high glucose can decrease AR expression in HUVEC. Intermittent high glucose reduced AR expression earlier and more apparent than constant high glucose. Testosterone can increase the expression of AR in HUVEC. With testosterone concentration increasing, AR expression was gradually up-regulated. Various testosterone concentrations improve the damage induced by intermittent high glucose in HUVEC are different. The concentration of10nM has greater benefit, and the benefit is realized through AR. Part three:Involvement of androgen receptor in human umbilical vein endothelial cells injure induced by intermittent high glucose and its possible mechanismsObjective:To detect the effect of intermittent high glucose on PI3k/Akt/mTOR signaling pathway in human umbilical vein endothelial cells (HUVECs) and to explore whether androgen receptor (AR) influences the cell injury induced by glucose fluctuation via PI3K/Akt/mTOR signaling pathway.Methods:HUVEC was cultured in different Endothelial Cell Medium (ECM) different times (Oh,24h,48h and72h). The experiment was randomly divided into control group (concentration of glucose:5.5mM, group N), constant high glucose group (concentration of glucose:30mM, group C) and intermittent high glucose group (concentration of glucose:5.5mM and30mM alternately every12hours, group F). After different time, expression of Akt, p-Akt, mTOR and p-mTOR were determined by western blot. Using LY294002, Rapamycin, Akt siRNA and mTOR siRNA to block or knock down PI3k/Akt/mTOR signaling pathway. According to the results of the second part, HUVEC were divided into control group (group N) and intermittent high glucose group (group F), group F is further divided into intermittent high glucose without intervention (group F), intermittent high glucose with10nM testosterone (F+T10), intermittent high glucose with10nM testosterone and LY294002group (F+T10+LY), intermittent high glucose with10nM testosterone and rapamycin group (F+T10+RAPA), intermittent high glucose with10nM testosterone, LY294002and rapamycin group (F+T10+LY+RAPA), intermittent high glucose with10nM testosterone with Akt siRNA (F+T10+Akt siRNA), intermittent high glucose with lOnM testosterone with mTOR siRNA (F+T10+mTOR siRNA), intermittent high glucose with lOnM testosterone with Akt siRNA and mTOR siRNA (F+T10+Akt siRNA+mTOR siRNA) to observe the cells'damage and apoptosis caused by intermittent high glucose.Results:1. At Oh, p-Akt and p-mTOR protein expression of three groups showed no significant difference (p>0.05). At24h, p-Akt of group F was increased than group N and C (P>0.05). At48h, p-Akt of F and C were increased than N group (P>0.05); p-mTOR of F was increased than N and C (P>0.05). At72h, p-Akt and p-mTOR of F and C were increased than N (P<0.05). But there was no significant difference between group C and F (P>0.05).2. MTS, ELISA, flow cytometry and western blot's results showed that:When drug blocking or siRNA knocking down the PI3K/Akt/mTOR signaling pathway, T10failed to improve HUVEC's injure induced by intermittent high glucose via AR.Conclusion:Constant and intermittent high glucose can activate the PI3K/Akt/mTOR signaling pathway in HUVEC. When drug blocking or siRNA knocking down the PI3K/Akt/mTOR signaling pathway, testosterone can't improve HUVEC's injure induced by intermittent high glucose via AR.