The Effect And Mechanism Of L-arginine On High Glucose-Induced Human Umbilical Vein Endothelial Cell Senescence

Today, diabetes has become a global epidemic disease to damage human health. Chronic complications are the major cause of disability and death in patients with diabetes. Compared with non-diabetic people, morbidity of atherosclerosis disease is higher, age of incidence is younger and development of illness is fast in patients with diabetes. Some studies have shown that high glucose accelerates cell senescence and vascular aging, thereby accelerates the process of atherosclerosis. Therefore, it is therapeutic agent for prevention and treatment of diabetic vascular complications that study mechanism of high glucose-induced cell senescence and thereby development of effective blocking drug.Endothelial cell is the key target tissue to diabetic vascular disease. It not only plays a protective function in the formation of anti-coagulation factor, but also it can secret some cellular factor to regulate blood vessel lumen and maintain internal environment. In normal physiological state, the update rate of endothelial cells is very low, and constant cells cleavage induced by injury (For example high glucose) will lead to telomere shortening. So the cells enter growth arrest period, which is aging period. In senescent state, the cells showed some characteristics, for example, increased SA-β-gal activity, the cells stuck in the cell cycle G0/G1 phase, aging-related gene and protein overexpression, as well as telomere shortening, decreased telomerase activity and so on. Simultaneously, in vascular endothelial cell senescence process, a variety of secretion biologically active factors are also changing, such as decreased the formation of endothelium-dependent vascular relaxing factor, increased synthesis of inflammatory factors and adhesion molecule expression. These changes will result in endothelial cell dysfunction, and eventually lead to the occurrence of atherosclerosis. Therefore, there was a close relationship between aging and diabetes-associated atherosclerosis, namely diabetic macrovascular disease.Telomere clock theory is one of the doctrine mechanisms of aging. Telomere is the DNA sequence of chromosome ends in eukaryotic cells. Telomere shortens with each cell cleavage. But chromosome stability can't be maintained when telomere reduced to a threshold value. So it will lead to cells lose their proliferative ability and cells enter into the aging phase. Thus, telomere shortening is considered as biomarkers of cell senescence and it enable the molecular clock to start. Telomerase is a ribonucleoprotein reverse transcriptase, can add new repeat sequences to shortened telomere ends so that maintain telomere length. It plays an important role in the regulation of life and cell proliferation, is a multifactor, PI3K/Akt pathway is one of the multifactors in regulating telomerase activity.Study found that Akt can enhance telomerase activity. On the melanoma cell line SK-MEL28 study found that Akt activity in tumor increased after incubation with the absence of serum for 24 h, and telomerase activity increased 2-fold. Two sequences XXRXRXXSXX of human telomerase reverse transcriptase (hTERT) correspondingly emerged phosphorylation. Application of PI3K inhibitor reduced telomerase activity in tumor cells and inhibited the hTERT phosphorylation. Thus, Akt is become a very important upstream regulator of telomerase.Increased oxidative stress and reduced NO bioavailability play an important role in cell senescence. Studies showed that high glucose can increase ROS and latter may shorten telomere length through inhibited Src Family kinase activity to accelerate cell senescence process. NO and excess ROS generated response so that attenuated oxidative stress in cell. NO is considered as the most important endothelium-derived aotocrine factor which L-arginine (L-Arg) is catalyzed by nitric oxide synthase (NOS).The effect of NO is extremely broad, which can adjust vascular tube cavity size, inhibit platelet aggregation, anti-apoptosis, inhibit smooth muscle cell proliferation and leukocyte adhesion, which plays an important role in the anti-atherosclerosis. Many studies found that endothelial NOS (eNOS) expression and NO production decrease with age, although the mechanism is not yet clear, studies have shown that phosphatidylinositol 3-kinase(PI3K)/Akt may mediate the phosphorylation of eNOS. Some studies have shown that entothelial dysfunction is related to inhibit PI3K/Akt/eNOS pathway in patients with type 2 diabetes. Thus, upregulation PI3K/Akt/eNOS signaling system should be an important target for prevention and treatment of diabetic vascular disease in endothelial cells.L-Arg is the precursor of NO synthesis. In 1992, Creager and Dubois-Rarde, for the first time, concluded that complementary role of L-Arg which is the substrate of eNOS, is to increase biological activity of endothelium-generated NO. L-Arg to improve the mechanism of endothelial cell function is relatively diversified. This effect may be relate to increase NO synthesis and eNOS protein expression, reducing reactive oxygen species (ROS) formation.In summary, this experiment, under high glucose in vitro, aimed to study whether high glucose may accelerate human umbilical vein endothelial cell senescence and its effect on NO, eNOS protein expression and transcription level, as well as the relationship between it and the PI3K/Akt signaling pathway. To study whether L-Arg may delay high glucose-induced cell senescence process and its mechanism is related to inhibit PI3K/Akt/eNOS pathway due to provide experimental evidence for pathogenesis mechanism, prevention and treatment for diabetic macroangiopathy.Materials and methods1. We cultured human umbilical vein endothelial cell with different concentration of glucose solution, and high glucose (33mmol/L) and/or different concentration of L-arginine co-incubated cells, as well as the same concentration incubated in different time point, then we collected cells and cell culture medium. Respectively with PI3K/Akt inhibitor LY294002 or eNOS inhibitor L-NAME pretreated for 2 h, then incubated cells with the above-mentioned factors, and collected cells and cell culture medium to used for target detection.2. Senescence-associated (3-galactosidase (SA P-gal) activity was determined by the percentage of SAβ-gal staining positive cells. The latter was determined by counting, the number of blue-green cells within a sample of 1000 cells.3. We used PCR-ELISA assay to detect the telomerase activity.4. ROS and analysis of the cell cycle were determined by flow cytometry assay.5. Applied RT-PCR to detect the mRNA expression of PI3K, Akt and eNOS.6. Used Westernblot to detect the p-Akt (Ser-473), Akt, p-eNOS (Ser-1177) and eNOS protein expression in HUVEC.7. Restored the nitrate to nitrite by nitrate reductase, and then detected the nitrite through the classic Griess reagent, which measured a total nitric oxide.Results1. With the glucose concentration increasing, that is 5.5,11,22,33 mmol/L, SA P-gal activity gradually increased (P<0.01), telomerase activity gradually decreased (P<0.01), the percentage of G0/G1 phase cells increased (<0.01), S phase cells decreased (P<0.01) and cell proliferation ability gradually weakened.2. With the glucose concentration increasing, intracellular ROS gradually increased (P＜0.001), whereas NO production gradually reduced (P<0.001).3. Compared with normal control group, the mRNA expression of PI3K, Akt and eNOS was significantly decreased in 33 mmol/L glucose concentration group at 72 h (P＜0.05).4. Compared with normal control group, the ratio of p-Akt (Ser-473) to Akt and p-eNOS (Ser-1177) to eNOS protein expression were significantly reduced (P<0 .01).5. Co-incubation with different concentration of L-arginine (0.4 mmol/L,0.8 mmol/L, 1.6mmol/L) and high glucose (33 mmol/L) in HUVEC, SAβ-gal activity weakened in varying degrees (compared with high glucose group, P＜0.01), and telomerase activity obviously increased (compared with high glucose group, P<0.01), and G0/G1 phase cells significantly reduced, however, S phase cells increased (compared with high glucose group, P＜0.01), so cell proliferation was improved. But increasing L-arginine to 3.2 mmol/L exerted the opposite effect, it was no statistical difference compared with high glucose group.6. L-arginine (0.4 mmol/L,0.8 mmol/L,1.6mmol/L) reduced intracellular ROS (P＜0.001), and increased NO formation (P＜0.001).7. Compared with high glucose group, L-arginine (0.8 mmol/L) increased the mRNA expression of PI3K, Akt and eNOS (P＜0.01).8. Compared with high glucose group, L-arginine (0.4 mmol/L,0.8 mmol/L, 1.6mmol/L) significantly increased the ratio of p-Akt (Ser-473) to Akt and p-eNOS (Ser-1177) to eNOS protein expression (P＜0.01).9. PI3K/Akt inhibitor LY294002 or eNOS inhibitor L-NAME can eliminate the anti-senescence effect of L-arginine.Conclusion1. High glucose can significantly accelerate the cell senescent process, which presents the role of glucose concentration-dependent and time-dependent.2. L-arginine can significantly delay high glucose-accelerated cell senescent process.3. L-arginine maybe exerts anti-senescence effect through upregulation PI3K/Akt pathway to increase telomerase and eNOS activity.