Effect Of Propofol On The Secretion Of Cytokines From Human Monocytes Stimulated With LPS

ObjectiveCytokines realeased after stimulating human monocytes with LPS.This study explored the effect of propofol on the secretion of proinflammatory cytokines TNF-a,IL-6 and anti-inflammatory cytokine IL-10 induced by LPS,and explored the influence of propofol on TLR4 expression induced by LPS.Methods1.Human peripheral blood mononuclear cells were isolated from healthy donors by density gradient centrifugation.After adhering to the plastic flat plate,the cells were collected to be verified through Wright-Giemsa stain,nonspecific esterase stain and anti-human CD14 antibody label.The survival rate of monocytes was also determined.2.The monocytes were resuspended in RPMI1640 medium and incubated in 48-well flat-bottomed microplates at a concentration of 5×10⁵/500μl/well.We explored the concentration-effect relationship and time-effect relationship between LPS and cytokines.1) Concentration-effect relationship of LPS and cytokines:after stimulating monocytes with different concentration of LPS(blank control,1ng/ml,10ng/ml,100ng/ml and 1000ng/ml) for 24h,the supernatant was harvested for the measurement of TNF-a,IL-6 and IL-10 by ELISA.2)Time-effect relationship of LPS and cytokines:after stimulating monocytes with LPS at the concentration of 10ng/ml for 2h,6h,12h,24h and 48h respectively in 37℃,5%CO₂ incubator,the supernatant was harvested for the measurement of TNF-a,IL-6 and IL-10.3.The effect of propofol on the production of cytokines and the expression of TLR4 induced by stimulating monocytes with LPS.1) The effect of propofol on the production of cytokines induced by stimulating monocytes with LPS:monocytes were divided into following groups:blank group,LPS group,0.1%DMSO group,propofol(100μg/ml) group and differernt dose of propofol(1μg /ml,5μg/ml,25μg/ml and 100μg/ml)+LPS groups.After incubating the cells with LPS and different dose of propofol for 24h,the supernatant was harvested for the detection of cytokines including TNF-a,IL-6 and IL-10.2) The effect of propofol on the expression of TLR4 after stimulating monocytes with LPS:monocytes were divided into following groups:blank group,LPS group,0.1%DMSO group,propofol(100μg/ml) group and differernt dose of propofol(1μg/ml,5μg/ml,25μg/ml and 100μg/ml) + LPS groups.After the monocytes were incubated for 24h,the monocytes were labeled with fluorescently labeled anti-human CD14 and anti-human TLR4.We analysed the percentage of dual-positive monocytes by flow cytometry.Results1.Through density gradient centrifugation and adherence method,we could get high purity of monocytes which account for 80%of all cells;the survival rate was equal or more than 95%through trypan blue exclusion test.2.1)The concentration of cytokines increased gradually with the increase of LPS. When we stimulated monocytes with LPS at a concentration of 10ng/ml,from the time-effect relationship curve we could see TNF-a,IL-6 and IL-10 were sensitive and measurable.2)TNF-a,IL-6 and 1L-10 increased gradually after stimulating monocytes with LPS, and got their peak concentration at 6h,24h and 24h respectively.3.1)TNF-a,IL-6 and IL-10 were very low in the medium of propofol and 0.1%DMSO groups,which was the same as blank control.After stimulating monocytes with LPS,the cytokines increased significantly(P＜0.01).Propofol reduced the production of cytokines induced by LPS in a dose dependent manner,that is,with the increase of propofol from 1μg/ml,5μg/ml,25μg/ml to 100μg/ml,the inhibition effect was augmented.2)Compared with blank control group,the expression of TLR4 showed no difference in 0.1%DMSO group and propofol group.The expression of TLR4 decreased after monocytes stimulated with LPS,and became less after applying propofol.Conclusions1.Cytokine could release by stimulating Human monocytes with LPS at the concentration of 10ng/ml.2.Propofol showed inhibitory effect on the production of cytokines induced by LPS in a dose-dependent manner.3.Propofol could decrease the expression of TLR4 on the cell surface after monocytes stimulated with LPS.The mechanism for the inhibitory effect on the secretion of cytokines might be partly attributed to the TLR4 expression decrease.