Inhibitory Effect And Possible Mechanisms Of Naringin On Adhesion Of High Glucose-induced Human Umbilical Vein Endothelial Cell To Monocytes

Diabetes mellitus refers to a number of disorders characterized by chronic hyperglycemia and alterations of cellular homeostasis, which lead to diffuse vascular damage and multiorgan dysfunction. Vascular complications in diabetes mellitus are chronic diseases which are commonly occurred in heart, brain, kidney and retina, contributing to the general morbidity and mortality.Adhesion of leukocyte to vascular endothelial cells is one of the earliest responses to hyperglycemia in the development of diabetic complications. Hyperglycemia induced vascular endothelial cells to express numerous inflammatory factors, chemotactic factors and adhesion molecules, which promote circling leukocytes platelets to adhere to endothelium, and transmigrate to under-endothelium and aggravate vascular inflammation. These factors are tightly associated with cell adhesion including monocyte chemotactic protein 1(MCP-1), P-selectin, E-selectin, vascular endothelial cell adhesion molecule-1(VCAM-1) and intercellular adhesion molecule (ICAM). MCP-1 mainly mediates movement of monocyte and lymphocyte to vascular wall. P-selectin, a member of selectin family in adhesion molecules mainly mediates rolling of leukocyte on endothelial cells, which serve as an important role in the initial stage of inflammation. In the later stage of inflammation, it functions with other selectin molecules. E-selectin is mainly expressed on endothelial cells in the inflammatory location. During inflammation, it mediates recruitment and rolling of leukocyte to endothelial cells. VCAM-1 is a main member of immunoglobulin super family. The VCAM-1 protein is an endothelial ligand for VLA-4 (Very Late Antigen-4 orα4β1) of theβ1 subfamily of integrins, and for integrinα4β7. The VCAM-1 protein mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. It also functions in leukocyte-endothelial cell signal transduction, and it may play a role in the development of atherosclerosis and rheumatoid arthritis. ICAM is an endothelial-and leukocyte-associated transmembrane protein long known for its importance in stabilizing cell-cell interactions and facilitating leukocyte endothelial transmigration.Mechanism of diabetic complications is complicated and involved in five metabolic pathways which are related to activation of polyol pathway, increased production of advanced glycation end products (AGEs), activation of protein kinase C (PKC), nuclear factor-κB and hexosamine biosynthetic pathways. Hyperglycemia-induced overproduction of reactive oxygen species (ROS) such as superoxide, the hydroxyl radical and hydrogen peroxide is recognized as the causal factor responsible for diabetic vascular injury. It has been shown that ROS increases the adhesiveness of endothelial cells via activation of nuclear factor-κB (NF-κB), a transcriptional factor for regulating gene expression including CAMs. Under normal condition, NF-κB is sequestrated in cytoplasm bound to inhibitory factor of NF-κB(IκB). Once IκB is degraded, NF-κB is released from the NF-κB/IκB complex and translocated from cytoplasm to nucleus, followed by increasing gene expression of CAMs such as VCAM-1 and E-selectin. Therefore, the inhibition of ROS overproduction and NF-κB signal pathway activation may be useful in preventing the diabetic vascular injury.Nitric oxide (NO) is a key biological messenger, playing a role in a variety of biological processes. It is known as the 'endothelium-derived relaxing factor' and biosynthesized endogenously from arginine and oxygen by various nitric oxide synthase (NOS) enzymes and by reduction of inorganic nitrate. In endothelial cells it is synthesized by endothelial nitric oxide synthase (eNOS). The endothelium (inner lining) of blood vessels uses nitric oxide to signal the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow. Nitric oxide contributes to vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Humans with atherosclerosis, diabetes, or hypertension often show impaired NO pathways. Nitric oxide is one of important sign of endothelial integrity.Naringin (4,5,7-trihydroxyflavanone 7-rhamnoglucoside) is a well-known flavanone glycoside exacted from grape fruits, Citrus paradise, Citrus sinensis, Citrus unshiu, Artemisia selengensis, roots of Cudrania cochinchinensis and fruits of Pon cirus. Naringin has been reported to possess potent anti-oxidant, superoxide scavenging, anti-apoptotic, anti-atherogenic and metal chelating activity. However, effect of naringin on adhesion of endothelial cell to leukocytes is not investigated. Thus, we performed experiments to examine inhibitory effect of naringin on cell adhesion in HUVECs induced by high glucose and possible mechanisms of anti-vascular injury activity by this compound.Methods and results1. Naringin inhibits THP-1 cells adhesion to HUVECs induced by high glucose.To explore whether naringin inhibits high glucose-induced endothelial cell adhesion to leukocytes, the HUVECs were pretreated with 10,25,50μg/ml naringin for 2 h and then with 33 mmol/L glucose for 48 h. Fluorescently labeled THP-1 cells were added to high glucose-treated HUVECs and co-cultured for 1 h. After removal of the non-adhesion cells, the remaining adhesion cells were photographed with fluorescence microscopy. The cells were lysed and measured using a spectrofluorometer. The results showed that after treatment with high glucose for 48 h, adhesion of monocytes to HUVECs significantly increased (P<0.01 compared to normal glucose treatment).When HUVECs were pretreated with 10,25, and 50μg/mL naringin for 2 h, this compound was found to markedly decrease monocyte adhesion to HUVECs induced by high glucose concentration. (P<0.01 compared to high glucose treatment alone).2. Naringin inhibits the expression of CAMs and MCP-1 of HUVECs induced by high glucose concentration.Since naringin can inhibit high glucose-induced cell adhesion, we investigated whether naringin inhibited expression of CAMs and MCP-1 in the HUVECs under high glucose condition. HUVECs were pretreated with increasing concentrations of naringin for various lengths of time and stimulated with high glucose for 48 h. ELISA is using to mearsure MCP-1 secreted by HUVCs induced by high glucose and surface ELISA is using to measure P-selectin, E-selectin,VCAM-1 and ICAM on the surface of HUVECs induced by high glucose. Results showed that treatment with high glucose (33 mmol/L) significantly increased the surface expressions on surface of HUVECs of P-selectin, E-selectin,VCAM-1 and ICAM, as well as secretion of MCP-1 of the HUVECs (P<0.01 compared to normal glucose treatment). However, pretreatment with 10,25,50μg/ml naringin inhibited high glucose-induced the increase of P-selectin, E-selectin,VCAM-1, ICAM and MCP-1 in dose-dependent manner, with a maximal inhibition effect achieved at 50μg/mL. The significant inhibition caused by naringin was seen within 1 h after the start of pretreatment and increased in a time-dependent manner. In addition, we used Western Blot to determine the expression of VCAM-1 and ICAM in whole cells. The results showed that pretreatment with 10,25,50μg/ml naringin inhibited the expression of VCAM-1 and ICAM in whole cells induced by high glucose in dose-dependent manner. Moreover, we used RT-PCR to investigate whether naringin inhibit the mRNA expression of VCAM-1 and ICAM in HUVECs induced by high glucose. The results showed that naringin inhibited the mRNA expression of VCAM-1 and ICAM in whole cells induced by high glucose in dose-dependent manner.3. Naringin inhibits high glucose-induced ROS overproductionTo determine whether the effect of naringin on CAM expression is related to its ability to inhibit ROS production, intracellular ROS generation was measured with fluorescent probe. The result showed that, exposure to high glucose resulted in more than 5-fold increase compared to normal glucose treatment. In contrast, pretreatment with the various concentration of naringin significantly decreased high glucose-induced ROS production compared to high glucose treatment alone.4. Naringin inhibits high-glucose induced NF-κB signaling pathway activationWe explored the effect of naringin on high glucose-induced NF-κB signaling pathway activation using western blotting and immunocytochemistry. The results showed that high glucose significantly increased NF-κB p65 expression by about 3-folds in the nuclear extracts compared to normal glucose concentration. In contrast, pretreatment with 10,25,50μg/mL naringin for 1 h markedly inhibited high glucose-induced increase of nuclear NF-κB p65 in dose-dependent manner. Furthermore, high glucose significantly decreased the IκB expression levels in cytoplasm. However, pretreatment with 10,25,50μg/mL naringin for 1 h markedly inhibited high glucose-induced decrease of IκB expression levels in dose-dependent manner.To confirm the consistency to the result of western blotting, we observed the translocation of NF-κB p65 by using immunocytochemistry. The result showed that high glucose caused NF-κB p65 translocation into nucleus compared to normal glucose concentration. However, pretreatment with 25μg/mL naringin inhibited the translocation of NF-κB p65 from cytoplasm to nucleus. These results were consistent with the result of western blotting.5.Naringin reverses the decrease of phosphorylated eNOS and NO production in HUVECs induced by high glucoseWe used fluorescent probe to determine the concentration of NO in HUVECs. The level of phosphorylated eNOS was detected by Western Blot. The resulted showed that the decreased NO production induced by high glucose was reversed by pretreatment with 10,25,50μg/ml naringin. Coincidently, the decreased level of phosphorylated eNOS induced by high glucose is also reversed by pretreatment with 10,25,50μg/ml naringin.6. Effect of high glucose and naringin on HUVEC viabilityPrimary human umbilical vein endothelial cell (HUVEC) was isolated from human umbilical cord and cultured. The cell viability assessment was performed using Cell Counting Kit-8. HUVECs were treated with various concentration of glucose for 24, 48 h. The results showed that treatment with various concentration of glucose had no effect on HUVEC viability. HUVECs were treated with various concentration of naringin for 24,48 and 72 h. The results showed that treatment with various concentration of naringin had no effect on HUVEC viability.Taken together, the conclusions of present study are as follow:1. Naringin inhibits THP-1 cells adhesion to HUVECs induced by high glucose concentration.2. Naringin inhibits the expression of CAMs and MCP-1 of HUVECs induced by high glucose concentration.3. Naringin inhibits high glucose-induced ROS overproduction.4. Naringin inhibits high-glucose induced NF-κB signaling pathway activation.5. Naringin reverses the decrease of phosphorylated eNOS and NO production in HUVECs induced by high glucose.