| PrefaceAristolochic acid nephropathy(AAN)is a rapidly progressive interstitial nephropathy.The underlying mechanism is not fully understood.In recent years,lots research of tubulointerstitial ischemia and hypoxia,tubular atrophy and interstitial fibrosis caused by AAN peritubular capillary loss have been clone.Wall mounting vascular endothelial cells,vascular injury is the key target tissue.It directly poisoned the blood of harmful substances.And it subject to all kinds of stress.Endothelial cells have a variety of cytokine secretion functions,messaging,anticoagulation,such as complex regulatory function relaxant directly involved in blood vessel formation and remodeling. So research of aristolochic acid(AA)effects on vein endothelial cells(VEC) damage and antagonistic way could lead to new treatment of chronic aristolochic acid nephropathy(CAAN).We use cultured human umbilical vein endothelial cells(HUVEC),Microscope and transmission electron microscopy were used to observe changes of cell morphology and ultrastructure,ELISA method of cell culture supernatant thrombomodulin(TM), fluorescent indicator FLuo-3/AM were applied to examine intracellular calcium concentration([Ca2+]i.).This article will explore the impact of AA on the VEC([Ca2+]i), influence of different concentrations of calcium dobesilate in VEC calcium homeostasis and protective effects of calcium dobesilate on VEC.Material and methodsMaterialsChief reagents:Human umbilical vein endothelial cells line(HUVEC),Dulbecco modified Eagle medium(DMEM) low sugar culture,Excellence in fetal calf serum (FBS),trypsin,4-Hydroxyethyl Piperazine acid(HEPES),Dimethyl sulfoxide(DMSO), aristolochic sodium salt(AA-Na)(the work of the concentration of 10mg/l),Calcium dobesilate original powder,FLuo-3/AM fluorescent indicator,Thrombomodulin(TM) ELISA Kit pre-coated.Chief equipment:incubation box,Olympus light microscope, autoclave sterilizer,refrigerator,a microscope with automatic photomicrography device, intracellular calcium fluorescence imaging measurement system.Methods(1) Cell cultureCells were inoculated in DMEM supplemented with 10%FBS,10μg/ml streptomycin,10U/ml penicillin.Cells were incubated at 37℃in a humidified chamber with 5%CO2.(2) Experimental grouping and processingcultured HUVEC were randomly divided into three groups:①normal control group(control group);②AA-Na group(AA group);③calcium dobesilate treatment group(intervention group).AA-Na powder was joined into DMEM medium, configured to 10mg/l solution.While the calcium dobesilate original powder adding DMEM medium,configured concentration of 10μM,25μM,50μM solution.The control group were cultured with Dulbecco modified Eagle medium for 24h.AA group were cultured with 10mg/l AA-Na for 24h,48h.The intervention group were cultured with 10mg/l AA-Na for 24h,then adding different concentrations of calcium dobesilate (10μM,25μM,50μM),and further cultured 24h,48h.(3) Cells in each group([Ca2+]i) measurementThe cells in each group were inoculated in plastic culture dishes.Cells of each group to be treated according to the above,abandoning the original culture medium, each dish with calcium-free phosphate buffer(PBS solution)(pH7.2) washing 2 times volume to 1ml.Then cells were loaded with FLuo-3,working fluid 5μM(50μl),37℃, 5%CO2 incubator incubated 40min.After loading,the cells were washed twice with calcium-free PBS.Will load FLuo-3,cells of each group placed in intracellular calcium measured fluorescence imaging system platform,37℃measured calcium fluorescence intensity of single cell.Average fluorescence intensity on behalf of the group average cell([Ca2+]i).(4) Each group cell culture supernatants TM determinationthe collection of the control group and AA group trained 24h,the intervention group developed 24h,48h of cell culture supernatant of the 100μl,in accordance with the TM as shown in ELISA detection kit methods and procedures for detection,each repeated 6 times,testing each group TM content.(5) Each group of morphology examinationCell were subjected to phasecontrast microscope analysis and,to observe the overall morphology and single-cell changes.Meanwhile cell were subjected to transmission electron microscopy analysis.Statistical analysisAll measurement measurement material were present as mean±SEM,SPSS16.0 for windows was adopted to undertake variance analysis,multi-group samples were compared with the number of analysis of variance.A probability value of＜0.05 was considered significant.ResultsCells in each group([Ca2+]i) changesEach group of([Ca2+]i) was shown as average fluorescence intensity.After applying AA-Na,HUVEC([Ca2+]i) increased rapidly,which reached the maximum within 5 minutes and declined to the baseline by 70 minutes.AA group average ([Ca2+]i)was significantly higher than control group,P＜0.05.Intervention group and AA group compared,average(Ca2+]i) had a certain degree of decline.Escalating single doses of calcium dobesilate(10,25,and 50μM) caused a dose-dependent decreased in ([Ca2+]i).Incubation for 48h did not yield results significantly different from those obtained with the 24h treatment.Each group cell culture supernatant TM test resultsAfter applying AA-Na,TM was significantly higher than control group,P＜0.05.TM values compared with the control group,p＞0.05,compared with AA group, P＜0.05,prompted dobesilate calcium concentration of 25μM,50μM,the cell culture 24h or 48h,TM values compared with AA group have a significantly decreasedCells were observed morphologically(1) Under phasecontrast microscope control group:cultured cells had a cobblestone appearance with a strict monolayer growth,cell outline clear,nucleus for round or oval in shape,cell growth is exuberant,almost without loss;AA group:cell adhesion ability weakening,partly suspended in culture medium,the volume of adherent cells shrink round;the intervention group:under different concentrations of adherent cells grew well,rarely falling.(2) Under transmission electron microscopyUnder electron microscope,each group of cell nuclear change situation control group:normal cell morphology,nuclear integrity;AA group:irregular-shaped nucleus, depression;intervention group:the nuclear-shaped cells are relatively rules.control group:ER and mitochondria morphological are integrity and structural clarity;AA groupRER is pool expansion shaped,and a small number of mitochondrial cristae defect,vacuolar degeneration;intervention group:mitochondfial defects appear fewer cristae,rough endoplasmic reticulum relatively complete.ConclusionAA induced VEC calcium overloading,TM secretion and injury of endothelial cells,ER and mitochondria destruction;Dobesilate calcium could protect ER and mitochondria,reduce the AA induced VEC calcium overloading and These could protect VEC.
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