Effects Of TNF-α On Production Of NO And Activity Of ENOS From Human Umbilical Vein Endothelial Cells

Object and BackgroundVascular endothelial cell (VEC) is one of the most important cell colonies in the body, which has lots of functions, such as secretion, synthesis, metabolism, immunity and so on. It widely distributes in the body and forms the interface between blood and tissue, not only relates to angiotasis, vasopermeability and blood coagulation, but also has a critical role in the inflammation by producting proinflammatory cytokines interact with the leukocytes in the circulation. The lesion of VEC is an important tissue damage response along with the inflammation. Most of inflammatory factors producted by macrophages, lymphocytes, neutrophils and VECs themselves, such as tumor necrosis factor-a (TNF-a), interferon γ (DFNγ), interleukin-1β (IL-1β) can cause the lesion of VEC, which inevitably result in endothelial functional disorder that the production of nitric oxide (NO) is abnormal. NO is a free radical synthesized by nitricoxide synthase (NOS) from L-Arginine, which serves as a key signalling molecule in physiological processes such as vasodilation, anti-platelet aggregation, inhibits the proliferation of the smooth muscle cells and so on. However, so much NO producted in the inflammatory diseases for the induction of cytokines may promote apoptosis and result in the lesion of VEC .NOS is the rate-limiting enzyme of NO, whose enzymatic activity and expression directly affect the production of NO. There are three subtypes of NOS definited in biological tissue: neuron NOS (nNOS or NOS1), endothelial NOS (eNOS or NOS3) and inducible NOS (iNOS or NOS2). The former two are also called constitution NOS (cNOS). The definitive mechanism how TNF-α affects the apoptosis or necrosis of the endothelial cells is not clear. But it can inhibit eNOS, activate iNOS and then affect the synthesis and release of NO, which may play an important role in its cytotoxic effect to tissue damage. The effect of TNF-α on the production of NO in endothelial cells is still a controversy, because the source of EC or the dosage of TNF-α in different study was not same, the results of NO with TNF-α were not same either. In those studies, human umbilical vein endothelial cells (HUVEC) were rarely used to investigate the association of TNF-α and NO and NOS. In our study, we detected the activity of eNOS and the production of NO in HUVEC and supernatant after treatment with different concentration of TNF-α.for different times, thus discussed the influence of TNF-α on NO and the molecular mechanism of regulation of NOS.Materials and MethodsHuman EC were obtained from umbilical vein (HUVEC) and cultured aspreviously described. Routinely HUVECs were digested by 0.1% type I collagenase from umbilical vein at 37°C for about 30min.Then these cells were plated in 24-well plate at a density of 2X 105/mL in Ml 99 medium containing 15% fetal bovine serum (Hyclone, Australia) supplemented with endothelial growth factor (10 ug/mL, R&D, USA). After cultured with low-serum medium for 24h, EC were incubated with different reagents all day long. The experiment of each group was performed in triplicate. (1) the first group of EC were treated with 2ng/ml, 5ng/ml, lOng/ml, 15ng/ml or 20ng/ml TNF-a respectively for 24h;(2) the second group were treated with lOng/ml TNF-a for 3h, 6h, 12h, 24h or 48h respectivly;(3) the third group were pretreated with lmM L-NMMA (Sigma, USA)for lh before TNF-a;(4) the fourth group were pretreated with lpM DXM (Xianju pharmacy, China)for lh before TNF-a;(5) the last was blank group. After these treatments, NO in cell supernatant were determined by NO kit (Nanjing jiancheng, China). Otherwise, after washed by PBS, adherent EC were digested by 0.25% trypsin for 5min. cell suspension was collected and centrifugated by lOOOrpm for lOmin. Then the precipitates in 300|il normal saline were crushed by ultrasonic crusher for determination of the activity of NOS by NOS avtivity kit (Nanjing jiancheng, China) and the production of NO by NO kit. The procedure of kits was refered to the directions. All results were expressed as means±SD. The data were analyzed by ANOVA (SPSS12.0). A value of P<0.05 was considered to be statistically significant.Results(l)Change of activity of eNOS and production of NO in HUVEC after treatmentwith different concentration of TNF-a. It is found that activity of eNOS was down-regulated by TNF-a in concentration-dependent manner, while production of NO in both cell lysate and supernatant increased, and NO in supernatant increased earlier than that in cell lysate. (2) Change of activity of eNOS and production of NO in HUVEC after treatment with TNF-a for different times. It is found that activity of eNOS attenuated after long-time intervention with TNF-a, but marked augment of NO in both cell lysate and supernatant was detected after 24h, and compared with HUVECs in normal conditions. (3) Effects of L-NMMA and DXM on production of NO in HUVEC. It is found that NO induced by lOng/ml TNF-a can be inhibited by L-NMMA and DXM.ConclusionTNF-a reduce the activity of eNOS, but high-concentration and long-time treatment of TNF-a make the production of NO increase, and this effect can be blocked by glucocorticosteroid. That may be contribute to activation of iNOS by TNF-a, and the mechanism is dependent on further investigation.