| Objective: At present the main chronic complications of diabetes, kidney,heart, eye to diabetic foot and neuropathy are associated with vascular injury.The main pathogenesis of diabetic vascular lesions have activated proteinkinase (PKC), accumulation of advanced glycation end products, polyolpathway abnormalities, the doctrine of oxidative stress, inflammation,cytokines participate in and so on. The oxidative stress theory is currentlyrecognized as the pathogenesis, and it is also accredited that this fact couldplay a center role of the above-mentioned mechanisms.High concentration ofglucose could make the ability to clear oxygen free radica decreased, resultingin the accumulation of oxidation products. Oxidative stress may cause theactivation of PKC, accumulation of glycation products, abnormal polyol andother metabolic pathways. Vascular lesions involve the correlation result ofmany factors, but the injury of vascular endothelial cell is the key target cells.In the anaerobic and high sugar state, blood vessels of Patients with diabetescan promote vascular endothelial growth factor (vascular endothelial growthfactor, VEGF) produce, thus cause endothelial cell proliferation andangiogenesis. Therefore, in the pathogenesis of diabetic vascular lesionsprocess, VEGF plays a very important role. Soluble endothelial formationfactor receptor1(soluble fms-like tyrosine kinase-1, sFlt-1) is a correspondingreceptors combined with VEGF, expressed by umbilical vein endothelialcell.Through competitive binding VEGF with the receptors on the surface ofvascular endothelial cell, blocking its biological activity and inhibitingendothelial cell proliferation and the formation of new blood vessels.CaiHaiRui, confirmed that human umbilical vein endothelial cells (humanumbilical vein endothelial cell, HUVEC) can also express sFlt-1, but atpresent, the research about sFlt-1mainly focus on diabetic retinopathy, whether it plays a similar role in diabetic peripheral vascular lesions, theresearch is few. In addition, the injury of endothelial cells, can also make theplasma low-density lipoprotein（LDL）,through the injured endothelial cells,concentration in subcutaneous clearance,and oxidation.The oxidized low-density lipoprotein(ox-LDL)cause endothelial dysfunction, foam cellformation and atherosclerosis. Sawamura, etc found that new endothelial cellox-LDL specific receptor, namely Lectin-like oxidized low-density lipoproteinreceptor1(LOX-1) in1997, mediated ox-LDL biological activity, play animportant role in AS, and then cause the vascular lesions.But in high glucoseenvironment whether it can cause endothelial cells express LOX-1increase,reported at home and abroad is less. Tea polyphenols, as a wide range ofantioxidant, is no exception for umbilical vein endothelial cells.The researchof ChenLuLu, has confirmed that tea polyphenols can effectively restrain theoxidative damage of endothelial cells caused by high glucose. Mitogenactivated protein kinase (mitogen activated protein kinases, MAPK) familyis very conservative serine/threonine protein kinase, is the main signalmolecules in the signal transduction process, and plays an important role inthe process of development and disease. The family has four members, areextracellular signal conditioning kinase (extracellular regulated proteinhnase l/2, ERK1/2), c-jun N end kinase (c-jun N-terminal kinase, JNK)/emergencyactivated protein kinase (stre activated protein kinase, SAPK), p38MAPK,ERK5/BMK1(big mitogen-activated protein kinase, BMKl). p38proteinkinase is by Han etc endotoxin stimulus mammalian cells, from which theseparation and purification of phosphoric acid tyrosine protein kinase. p38isthe most important members in MAPK family to control the inflammatoryresponse, it may be activated，due to physiological stress, lipopolysaccharide,osmotic stress and UV irradiation.This experiment is aimed to observe sFlt-1,LOX-1expression activity， through the different concentrations of teapolyphenols and p38MAPK inhibitor SB203580act on HUVEC cells culturedin high glucose in vitro forty-eight hours，and discuss the mechanism of highglucose result in the change of sFlt-1, LOX-1expression and protection of tea polyphenols, and whether the expression of the index through p38MAPKsignal path, to find a new possible way for diabetes vascular lesionsprevention and treatment.Methods: To extract neonatal umbilical cord, after pancreatic enzymedigestion obtained umbilical vein endothelial cells to primary culture, over80%of the fusion to subculture, using3to5generations of cells for the nextstep. Select Logarithmic phase of cell growth in good condition into the group.Trials are divided into15groups: normal control group (glucose5.5mmol/L,DMEM nutrient solution), high glucose group (glucose30mmol/L, DMEMnutrient solution), tea polyphenols intervention group: concentration were10mg/L, and20mg/L and30mg/L, p38MAPK inhibitor group：SB203580concentration were10μmol/L,15μmol/L, and20μmol/L, respectively tonormal control group and high glucose group, hypertonic group (30mmol/Lmannitol concentration). In addition to the the hypertonic control group, eachdrug group30minutes prior to the processing of high-glucose were added tothe drug. Each group had four hole cells. After medicaments, continue tocultivate for48hours, to observe the shape of the HUVEC cells, MTT methodto detect survival vitality, RT-PCR and Elisa were testing sFlt-1and LOX-1mRNA expression level and quantity of protein expression.Results:1Compared with normal control group, although mannitol group cellactivity decreases, but without statistical difference (P>0.05); Normal+teapolyphenols group compared with normal control group, the cell activity of10mg/L concentration group decreased,30mg/L group was obviously higher,the difference had statistical significance (P<0.05), cell activity of theconcentration of20mg/L group had no significant difference (P>0.05); cellactivity of High glucose group was suppressed obviously, the difference wasstatistically significant (P<0.05); Intervention with different concentrations oftea polyphenols, found that each concentration group were statisticallysignificant (P<0.05). and compared with the high glucose group, cell vitalityof HG+10mg/L TP decreased (P<0.05), HG+20mg/L TP group increased but still lower than the normal control group (P<0.05), HG+30mg/L TP wasincreased significantly (P<0.05), so selected20mg/L of TP concentration foroptimal experimental dose，as the experimental function of concentration.HUVECs vitality of NG+p38resistance (10μmol/L,15μmol/L,20μmol/L)groups were lower than normal control group, but there was no statisticallysignificant difference (P>0.05), and in the three groups,The cell vitality ofNG+15p38resistance is the strongest.In HG+p38resistance (10μmol/L,15μmol/L,20μmol/L) groups，HUVECs vigor increased than pure highglucose，but still lower than the normal control group (P<0.05), and the cellvitality,is no difference among three groups, so choose15μmol/L p38MAPKinhibitor concentration as the best dose, as the role of the concentration infollowing experiments.2Compared with normal control group, the sFlt-1mRNA and proteinlevels of high permeabilityare reduced, but there was no statisticallysignificant difference (P>0.05); High glucose group, sFlt-1mRNA and proteinexpression significantly reduced, the difference was statistically significant(P<0.05). After the intervention of tea polyphenols, the expression in highsugar group and normal control group were increased, and respectively werestatistically compared with before the intervention (P<0.05).3Compared with normal control group, the LOX-1mRNA expressionof high glucose group increased, the difference was statistically significant(P<0.05). After TPs intervention, LOX-1mRNA expression decreased, butthere was no statistically significant difference between the normal controlgroup with tea polyphenols and before intervention。And after joining teapolyphenols high glucose group, the difference was statistically significant(P<0.05).4Compared with normal control group, p38MAPK protein concentrationof the high glucose group increased, the difference was statistically significant,after intervention of the tea polyphenols, its expression decreased, thedifference has statistical significance.Respectively, compared with normalcontrol group and the high glucose group, after joining p38MAPK inhibitor SB203580, sFlt-1mRNA, protein and LOX-1mRNA almost no expression.Conclusion:1High glucose environment can cause HUVEC the expression of sFlt-1activity increased, LOX-1content decreased, may play an important role inthe process of diabetic peripheral vascular lesions.2Tea polyphenol can observably reduce HUVEC the LOX-1highexpression and raise sFlt-1expression, mitigate the damage effects of highglucose on human umbilical vein endothelial cells, reduce the occurrence ofarterial sclerosis and the formation of new blood vessels.3p38MAPK can be activated in the high-glucose environment, and itsinhibitor can significantly affect the LOX-1and sFlt-1expression quantity，and the expression of LOX-1and sFlt-1may through the p38MAPK path. |
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