| ObjectiveIn patients with type 2 diabetes mellitus(T2DM) the incidence and mortality rate of cardiocerebrovascμlar events are significantly increasing, T2DM is as dangerous as cardiocerebrovascμlar diseases. T2DM patients with hyperglycemia, hypertension and dislipidemia are prone to suffering from cardiocerebrovascμlar diseases, however, it still happened when blood glucose, pressure and lipid were well controlled. It is imbalanced between system of oxidation and anti-oxidation in patients with T2DM, increasing oxidative stress accelerates developing of macrovascμlar complications by endothelium dysfunction, vessel inflammation, atherosclerotic plaque formation and fragilicity, promotion of pre-thrombotic state.Advanced oxidation protein products (AOPP) is presented first by Witko-Sarsat from plasma of patients with uremia in 1996, it is cross- linking protein products mainly from oxidative modification of albumin by hypochlorite from active neutrophils. AOPP is a good marker of oxidative stress and also as inflammation mediator which can promote oxidative stress. It is confirmed that AOPP is associated with oxidative stress, but the exact mechanisms remain unclear.Stromal cell-derived factor-1α(SDF-1α) is one efficient chemoattrac- tant factor which can chamoattract monocyte, lymphocyte, it is expressed on vessel endothelial cell, vessel smooth muscle cell and so on. It is manifested that it is highly expressed in atherosclerotic plaques and can chemoattract monocyte to infiltrate through the vessel wall. so we conclude that SDF-1αis associated with atherosclerosis.Mitogen active protein kinase (MAPK) signal transduction catalase system is the major system that transduct signals which is important for the cell from outer of the cell into the nuclear. It include mainly three pathways in human beings which are ERK, JNK/SAPK, p38.In this study we investigated the effects of AOPP on adhesion between ECV304 cells and THP-1 cells, whether AOPP can promote SDF-1αchemoattracting THP-1. We explored the effects of AOPP on expression of SDF-1αmRNA and protein in ECV304, and also searched for probable transduction pathway involved in AOPP stimulating SDF-1αexpression. At last, we studied the serum AOPP concentration in patients with T2DM and the association between AOPP and cadiovascular risk factors.Methods1. To culture ECV304 cell line and THP-1 cell lineTwo types of cells were cultured in RPMI1640 with 10% fetal bovine serum under the condition of 37℃, 5%CO. the 3rd to 5th generations of the cells were used to perform the experiment.2. Preparation of AOPP-BSA200mg/ml BSA was mixtured with the same dose of 0.2mol/L NaOCl, cultured under room temperature for 30 minitues, then dialysed in PBS(0.01mol/L, pH7.4)for 24 hour at 4℃, filtrated and sterilized with nitrocellulose membrane,stored under -70℃.BSA without NaOCl under same treatment was as control. AOPP-BSA content was determined by measuring absorbance at 340nm in acidic condition and was calibrated with Chloramines-T in the presence of potassium iodine.3. THP-1/ECV304 cells adhesion testECV304 cells were cultured in six-well plates with AOPP-BSA of different concentrations, then THP-1 cells were put in the plates for 30 minutes at 37℃, at last counted the number of THP-1 cells adhering on the ECV304 cells under microscope。4. SDF-1αchemoattract THP-1 testTranswell permeable system(8μm) were used to examine the THP-1 cells chemotaxis. THP-1 cells pretreated with AOPP-BSA of different concentrations for 24 hours were put in upper chambers, SDF-1αof different concentrations were put in lower chambers, after 10 hours at room temperature upper chambers were taken out, counted the cell number in 4 vision continuously under microscope(300×), the average cells were as the results.5. SDF-1αmRNA expression were examined by RT-PCRECV304 cells were pretreated with medium,200μmol/l BSA, 50, 100, 200μmol/l AOPP-BSA respectively for 6 hours, or pretreated with 200μmol/l AOPP-BSA for 0, 2, 6, 12, 24hours respectively, then collected the cells, extract the total RNA of the cells by Trizol reagent, determined the expression of SDF-1αmRNA by RT-PCR.6. SDF-1αprotein levels were examined by western blot and ELISAECV304 cells were pretreated with medium, 200μmol/l BSA, 50, 100, 200μmol/l AOPP-BSA respectivly for 6 hours, or pretreated with 200μmol/l AOPP-BSA for 0, 2, 6, 12, 24hours respectively, then collected the cells, extract the total proteins of the cells, measured the SDF-1αprotein levels by western blot. Inhibition test were performed as that ECV304 cells were precultured with SB203580, a special inhibitor of p38MAPK or U0126, a special inhibitor of ERK1/2 of different concentrations for 1 hour, then cultured with 200μmol/l AOPP-BSA for 12 hours, measured the SDF-1αprotein concentrations by ELISA7. p-p38MAPK and p-ERK1/2 protein were measured by western blotECV304 cells were pretreated with medium, 200μmol/l BSA, 50, 100, 200μmol/l AOPP-BSA for 15 minutes, or pretreated with 200μmol/l AOPP- BSA for 0, 15, 30, 60, 120 minutes respectively, then collected the cells, extract the total proteins of the cells, measured the expression of p-p38MAPK and p-ERK1/2 protein by western blot.8. To measure the serum AOPP and SDF-1αconcentrations in patients with T2DMFasting blood were drawed and put into tube with 10% EDTA, centrifuged for the serum. Serum SDF-1αlevels were determined with ELISA by measuring the absorbance at 450nm. Serum AOPP concentrations were measured by spectrophotometer,serum and PBS were mixtured by 1:5, Chloramines-T were diluted as the same, then 1.16 mmol/L KI and 20μl acetic acid were put in, immediatly measured the absorbance at 340nm.Results1. AOPP promote adhesion between THP-1 and ECV304 cells by dose- dependent mannerNumber of THP-1 cells adhering on ECV304 cells was a few without AOPP-BSA stimulating, after cultured with 50μmol/l AOPP-BSA, the adhesion cell number increased significantly, pretreated with 200μmol/l AOPP-BSA made the most adhesion cells number as compared with those of controls, it increased by 26 times.2. AOPP promote SDF-1αchemoattracting THP-1 cells by dose-dependent manner10ng/ml SDF-1αchemoattracted THP-1 cells which were petreated with AOPP-BSA of different concentrations for 24 hours, the results were that chemoattracted THP-1 cells increased while AOPP-BSA concentration increased. Number of chemoattracted THP-1 cells pretreated with 200μmol/l AOPP-BSA was 11 times compared with those of the control.3. AOPP stimulates SDF-1αmRNA expression in ECV304 cells by time- and dose-dependent mannerCompared with the control after cultured with 200μmol/L AOPP-BSA for 2 hours, the expression of SDF-1αmRNA increased significantly, the expression was at peak after 6 hours, after that the expression decreased, at 24 hours there was no difference with the control. The expression of SDF-1αmRNA increased while the precultured AOPP-BSA concentration increased.There was a little expression with no stimμlant or BSA only, and no difference were seen between them.4. AOPP stimulates SDF-1αprotein expression in ECV304 cells by time- and dose-dependent mannerThe expression of SDF-1αprotein increased significantly after pretreated with AOPP-BSA for 2 hours, the maximum was at 24 hours.Compared with that of the control the expression of SDF-1αprotein increased while the AOPP-BSA concentration increased.5. AOPP stimulates p-p38MAPK and p-ERK1/2 protein synthesis in ECV304 cells by time- and dose-dependent mannerAfter 15 minutes precultured with AOPP-BSA p-p38MAPK and p-ERK1/2 protein increased significantly, then decreased gradually,it still higher at 2 hours than the control.p-p38MAPK and p-ERK1/2 protein synthesis increased while the AOPP-BSA concentration increased.6. Stimulating effect of AOPP-BSA on SDF-1αprotein expression in ECV304 were blocked by p38MAPK special inhibitor SB203580 or ERK1/2 special inhibitor U0126 by dose-dependent manner0.1uM SB203580 or U0126 can block the stimulating effect of AOPP-BSA on SDF-1 protein expression. The inhibition rate increased with the inhibitor concentration increasing.7. Serum level of AOPP and SDF-1αwas higher in patients with T2DM, serum levels of AOPP were correlated with cardiovascμlar risk factors of T2DM patientsCompared with the control, serum levels of AOPP and SDF-1αwere significantly increased in patients with T2DM, AOPP levels were signi- ficantly higher in good glycemic control group than those of poor glycemic control group. Compared with insulin resistance group, serum AOPP levels were higher in non-insulin resistance group. Increased AOPP levels were found in patients with macrovascular complications than those without complications. Of all the patients with T2DM, higher serum SDF-1αlevels, body mass index(BMI), fasting plasma glucose(FPG), glycosylated hemo- globin A1c( HbA1c) and triglyceride(TG)were found in high AOPP group than those of normal AOPP group.Serum levels of AOPP were positively correlated with FPG, HbA1c, TG, BMI, HOMA-IR and SDF-1α. Logistic multivariable regression analysis found that age, mean blood pressure (MBP) and AOPP were the independent risk factors which have influence on macrovascular complications of T2DM patients.Conclusions1. AOPP promoted adhesion between ECV304 and THP-1 cells and enhanced chemoattracting effects of SDF-1αon THP-1 cells by dose- dependent manner.2. AOPP stimlated SDF-1αmRNA expression and protein synthesis in ECV304 cells by dose-and time-dependent manner.3. The stimulating effect of AOPP on SDF-1αprotein synthesis was mediated by p38MAPK and ERK1/2 signal transduction pathway.4. Serum AOPP and SDF-1αlevels increased in patients with T2DM, and AOPP serum levels were correlated with many cardiolvascular risk factors, it was an independent risk factor contribute to the pathogenesis of macrovascular complications in T2DM.
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