Investigation On The Mechanism Of Secretion Of ET-1, EMMPRIN By Huamn Umbilical Vein Endothelial Cell Caused By High Glucose And Intervention Of TPs

Objective:Diabetic vasculopathy is the leading cause of death in patientswith diabetes, because of the complexity, the etiological mechanism is still notclear. Recently, more and more researches showed that vascular endothelialcell injury and the pathological process of type2diabetes vascular lesions areclosely related. Endothelin-1is the stronger vaso-excitor material found in thehuman body, is an important indicator of endothelial dysfunction. Theincreasing of ET-1plays an important role in diabetic vasculopathy.Extracellular matrix metallop roteinase inducer can reduce the degradation ofExtracellular matrix, through reducing the expression of matrixmetalloproteinases, leading to pathological reconstruction of Extravascularmatrix. Finally, it can lead to diabetic vasculopathy. Mitogen-activated proteinkinase pathway is the main signal transduction pathway of cellular stress anddamage responses, p38MAPK has the closest relationship with the endothelialcell damage.With the high glucose, vascular endothelial cells can activatep38MAPK, leading to VECs damage and obstacle of function. It can not onlyreduce more ET-1synthesized, leading to vasoconstriction and proliferatingviability of VSMCs. But also can reduce the expression of EMMPRIN, whichcan reduce MMPs through paracrine, reducing the degradation of Extracellularmatrix, consequently, too much Extracellular matrix accumulate inextravascular. ET-1and EMMPRIN jointly participate Occurrence anddevelopment of diabetic vasculopathy. SB203580can play an inhibitory partthrouth competitiving advantage ATP of p38. Researches showed that a certainconcentration of Tea Polyphenols can resist the damage of VECs and thenprevent morphogeny of diabetic vasculopathy.We design this experiment to observe the activity of Human umbilical vein endothelial cells and secretion of ET-1and EMMPRIN in culturedHuman umbilical vein endothelial cells with high glucose, and TeaPolyphenols and inhibitor SB203580, to observe the effect of Tea Polyphenolson actvity of HUVECs and explore the possible mechanism, provideexperimental basis for clinical application of diabetic vasculopathy.Methods: Cultivate human umbilical vein endothelial cell line in virto,take those in logarithmic stage in good growth condition in experiment.Divide the cels into15groups:1normal control group (NG, glucose concentration:5.5mmol/L)2high concentration glucose group (HG, glucose concentration:30.0mmol/L)3mannitol control group(5.5mmol/L glucose+24.5mmol/L mannitol)4Tea Polyphenols treatment group:⑴NG+TPs with various concentration (10mg/L,20mg/L,30mg/L)group(NT1, NT2, NT3)⑵HG+TPs with various concentration (10mg/L,20mg/L,30mg/L)group(HT1, HT2, HT3)5SB203580treatment group:⑴NG+SB203580(10μmol/L,15μmol/L,20μmol/L) group(NP1, NP2,NP3)⑵HG+SB203580(10μmol/L,15μmol/L,20μmol/L) group(HP1, HP2,HP3).Cultivate the cells in each group in different nutrient solutions for48h,keep the samples to measure. Use MTT method to measure the activity of cells,observe the expression of EMMPRIN mRNA, ET-1mRNA using reversetranscriptase polymerase chain reaction, measure the expression of p38MAPK and EMMPRIN protein phosphorylated using Western blot method.Results:1Compared with normal control group, the activity of HUVECs isreduced in high concentration glucose group (P＜0.05); No differencebetween control group and mannitol control group (P＞0.05); NG+SB203580 (10μmol/L,15μmol/L,20μmol/L) groups have lower HUVECs activity thannormal control group, but no statistical significance (P＞0.05). Meanwhile theactivity of cells in NP2is the strongest in these three groups. All the activitiesof HUVECs of HG+SB203580(10μmol/L,15μmol/L,20μmol/L)groups arehigher than pure high glucose group but lower than normal control group (P＜0.05) and the degree of increase of cell activities are similar to each other,which can prove that the optime p38MAPK inhibitor concentration should be15μmmol/L and the later experiments will use this concentration.2Compared with the normal control group, the activity of HUVECs inNP1group declined dramatically (P＜0.05), there is no obvious difference inHUVEC activities between the NP2and normal control group (P＞0.05). TheHUVECs activity in NP3is much higher than the normal control group (P＜0.05), HP1has a lower HUVECs activity than pure high glucose group (P＜0.05), while HP2group has a much higher HUVECs activity but still lowerthan the control group (P＜0.05), the activity of HUVECs in HP3is muchhigher than pure high glucose group (P＜0.05), therefore the optimeconcentration of TPs is20mg/L, which will be used in later experiments.3Compared with the normal control group, the p38MAPK, ET-1ofHUVECs in high glucose group takes high expression (P＜0.05), whileEMMPRIN shows low expression (P＜0.05); There are no huge differencesbetween mannitol control group and normal control group (P＞0.05).Theexpression of p38MAPK of HUVECs in NP2group is decreased compared tothe normal control group (P＜0.05)，there is also no difference in expression ofEMMPRIN, ET-1of HUVECs in NP2group compared to the control group (P＞0.05), the expression of p38MAPK, ET-1of HUVECs in HP2group isdecreased (P＜0.05) and the expression of EMMPRIN goes up but still lowerthan the normal control group(P＜0.05).4The expression of p38MAPK of HUVECs in NT2group is decreasedcompared to the normal control group(P＜0.05)，there is no difference inexpression of EMMPRIN, ET-1of HUVECs in NT2group compared to thecontrol group (P＞0.05), the expression of p38MAPK, ET-1of HUVECs in HP2group is decreased compared to the pure high glucose (P＜0.05) and theexpression of EMMPRIN goes up but still lower than the normal controlgroup (P＜0.05).Conclusions：1High concentration of glucose can inhibit the activity of HUVECsthrouth p38MAPK signal pathway, and20mg/L TPs can restrain this affect.2High concentration of glucose can induce HUEVCs increasingexpression of ET-1, but decreasing expression of EMMPRIN throuthactivating p38MAPK signal pathway.320mg/L TPs can promote the expression of EMMPRIN, but inhibit theexpression of ET-1throuth inhibiting activation of p38MAPK signal pathwayinduced by high glucose. It is suggested that20mg/L TPs can protectHUVECs through this ways.