The Influences Of TNF-α And IL-1β On Endothelial Cells To Express PAPP-A And The Intervention Of Atorvastatin

PartⅠInfluences of TNF-αand IL-1βon Endothelial Cells to express pregnancy-associated plasma protein– AObjective To investigate the effects of TNF-αand IL-1βon pregnancy-associated plasma protein-A(PAPP-A) expression in primary cultured rat aorta endothelial cells.Methods The rat Aortic endothelial cells(RAEC) were obtained from thoracic aortas and cultured by tissue explant method. The morphology of RAEC was studied by phase contramicroscope. Its molecular markers were observed byⅧfactor immunocytochemistry. Passage 3～4 cells were used in experiment, and they were randomly divided into 5 groups: (1) Blank control group: treated without intervention ;(2) Groups of different TNF-αconcentrations: RAEC were coincubated with different concentrations of TNF-α(10,20,40,60,100ng/mL) for 24h.(3) Groups of different IL-1βconcentrations: RAEC were coincubated with different concentrations of IL-1β(1,5,10,20,50ng/mL) for 24h. (4) TNF-αgroups of different intervention time: RAEC were coincubated with 60μg/mL TNF-αfor different periods(2,4,8,16,24,48h). (5) IL-1βgroups of different intervention time: RAEC were coincubated with 20ng/ml IL-1βfor different periods(2,4,8,16,24,48h).After intervention, the culture cells and supernatant were collected. LDH activity in supernatant was detected by LDH test box. PAPP-A mRNA was detected by RT-PCR. The levels of PAPP-A in supernatant of culture cells were detected by ELISA.Results1. Normal cells were flat polygonal and nucleus were round or oval at the center of cell. With the increase of cell density, cultured cells were "paving stone-like" arrangement . Most cells display green fluorescence under fluorescent microscopy, which accords with the characteristics of EC.2. The activity of LDH in groups of TNF-α100ng/ml and IL-1β50ng/ml were higher than blank control group(p<0.05).These showed that TNF-αand IL-1βhave direct cytotoxic effects.(These two groups should be rule out during the experiments. )3. After the intervention of TNF-α(10,20,40,60ng/ml)and IL-1β(5,10,20ng/ml) for 24 hours, the levels of PAPP-A expression increased initially with the concentration raise(p<0.05). Cytokines intervention and the endothelial cells PAPP-A expression have a dose - response relationship.4. After cells were coincubated with 60ng/mL TNF-α, the expression of PAPP-A increased beginning from 4h,which raised as the stimulation extension in a time-dependent manner(p<0.05). Also the expression of PAPP-A increased in IL-1βgroups of different intervention time (p<0.05), but beginning from 8 h.PartⅡEffects of atorvastatin on TNF-αand IL-1βinduced expression of PAPP-AObjective To investigate the effects of atorvastatin on TNF-αand IL-1βinduced endothelial cells expression of pregnancy-associated plasma protein– AMethods There were five groups:(1) Blank control group: treated without intervention ;(2) TNF-αgroups: cells were coincubated with 60ng/mL TNF-αfor 24h. (3) IL-1βgroups: cells were coincubated with 20ng/mL IL-1βfor 24h. (4)Atorvastatin group: cells were coincubated with 10μmol/L atorvastatin for 24h.(5) Atorvastatin intervent group:Cells were coincubated with 0.05μmol/L, 0.1μmol/L, 1.0μmol/L, 10μmol/L atorvastatin respectively as well as 40ng/mL TNF-αor 20ng/mL IL-1βfor 24h. MTT reduction assay was used to observe the atorvastatin drug effect on cell proliferation. PAPP-A mRNA was detected by RT-PCR. The levels of PAPP-A in supernatant of culture cells were detected by ELISA.Results1. After the intervention of different concentrations of atorvastatin for 3,6,12,24,48h,compared with the blank control group, there was no significant changes in cell proliferation (P>0.05).It showed that drug intervention itself had no toxic effects on the cells.2. There was no significant difference of PAPP-A expression between atorvastatin group and blank control group(P>0.05).3. PAPP-A expressions of atorvastatin intervent groups(10μmol/L, 1.0μmol/L, 0.1μmol/L, 0.05μmol/L ) decreased significantly VS. TNF-αgroup or IL-1βgroups (P<0.05). The levels of PAPP-A expression decreased initially with the atorvastatin concentration raise(p<0.05), in a concentration -dependent manner.Conclusions1. TNF-αand IL-1βupregulate the expression of PAPP-A in a concentration-dependent and time-dependent manner .2. Atorvastatin itself doesn't influence the PAPP-A expression, but inhibits the expression of PAPP-A activated by inflammatory factors in a concentration-dependent manner in primary cultured rat aorta endothelial cells.