Il-1¦Â And Tnf-¦Á Expression Levels Of Atorvastatin The Intervention Effect On Rat Smooth Muscle Cells In Endothelial Lipase

PartⅠThe influence of IL-1βand TNF-αon endothelial lipase in rat aortic smooth muscle cellsObjective: To investigate the effects of IL-1βand TNF-αon endothelial lipase(EL) in rat aortic smooth muscle cell.Methods: The rat Aortic smooth muscle cells (RASMC) were obtained from thoracic aortas and cultured by tissue explant method. The morphology of RASMC was studied by phase contramicroscope. Its molecular markers were observed by alpha-actin factor mmunocytochemis -stry. Passage 3～5 cells were used in experiment, and They were randomly divided into 3 groups. GroupⅠ: treated without intervention as the control; GroupⅡ: the rat Aortic smooth muscle cells were treated in serum-free culture medium containing IL-1β(1 to 30ng/mL) for 24 hours or with IL-1β(30ng/mL) for up to 72 hours; GroupⅢ: TNF-α(5 to 50ng/mL) for 24 hours or with TNF-α(50ng/mL) for up to 72 hours. Thirty minutes before harvest, heparin stock solution was added to bring the final heparin concentration to 0.4mg/mL. The culture medium and cells were collected, centrifuged at 3000g for 10 minutes to remove cell debris, and stored in 0.5-mL aliquots at -70°C until use. The levels of EL in supernatant of culture cells were detected by the method of double mono-cloned antibody ELISA. we tested the levels of ELmRNA by the method of RT-PCR, which were adjusted by GAPDH. Then we compared the values of OD among the groups.Results:⑴The indentification of smooth muscle cells: most cells displayed green fluorescence under fluorescent microscopy, which accords with the characteristics of SMC.⑵The activity of LDH in groups of IL-1β50ng/mL and TNF-α100ng/mL were higher than blank control group(p<0.05).These showed that IL-1βand TNF-αhave direct cytotoxic effects.(These two groups should be rule out during the experiments. )⑶SMCs were treated with increasing concentrations of IL-1β, ranging from 1ng/mL to 30g/mL. Dose-dependent increases in secreted EL protein and EL mRNA were noted. The relative increases in EL protein and mRNA were greater at the highest concentrations (30ng/mL) of IL-1β. There was a significant difference between the intervention group and the control group (P<0.05). There were significant differences among the intervention groups(P<0.05). The relative increases in EL protein and mRNA were greater at 72 hour. There was a significant difference between the different time group and the control group (P<0.05). There were significant differences among the different time groups(P<0.05).⑷SMCs were treated with increasing concentrations of TNF-α, ranging from 5ng/mL to 50 ng/mL. Dose-dependent increases in secreted EL protein and EL mRNA were noted. The relative increases in EL protein and mRNA were greater at the highest concentrations (50ng/mL) of TNF-α. There was a significant difference between the intervention group and the control group (P<0.05). There were significant differences among the intervention groups(P<0.05). The relative increases in EL protein and mRNA were greater at 72 hour. There was a significant difference between the different time group and the control group (P<0.05). There were significant differences among the different time groups(P<0.05).Conclusion: SMCs were treated with increasing concentrations of IL-1βand TNF-α, then a time-dose-dependent increases in secreted EL protein and EL mRNA were noted.PartⅡEffects of Atorvastatin on endothelial lipase secreting by rat aortic smooth muscle cellsObjective: To investigate the effects of atorvastatin on endothelial lipase(EL) secreting by rat aortic smooth muscle cells.Methods: The rat Aortic smooth muscle cells(RASMC) were cultured and divided into groups when cultured in the passage 3. They were randomly divided into 3 groups. GroupⅠ: treated without intervention as the control; GroupⅡ: IL-1β(30ng/mL) or TNF-α(50ng/mL) GroupⅢ: atorvastatins(10-5 mmol/L); GroupⅣ: atorvastatins+IL-1β(30ng/mL) or TNF-α(50ng/mL). Rat aortic smooth muscle cells were first incubated in serum-free medium with 3 ng/mL IL-1βand 5 ng/mL TNF-α;1 hour later,atorvastatins with different concentration(1×10-7 mmol/L ,1×10-6mmol /L,1×10-5 mmol/L) were added to the medium. The culture medium and cells were collected after 24 hours.Results:⑴After 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.⑵ELISA results suggest that: Atorvastatin inhibited the secretion of EL in a dose-dependent manner. There was a statistically significant difference between the drug intervention group and the control group (P<0.05). The effect of atorvastatin on the IL-1β(TNF-α)-mediated vascular smooth muscle cells EL expression was a dose-dependent inhibition.The RT-PCR had the same results.Conclusion: Atorvastatin can inhibit EL secreting by IL-1βand TNF-αstimulated rat aortic smooth muscle cells.