Effects Of Propofol On Apoptosis And The Production Of Inflammatory Cytokines Of Lipopolysaccharide-stimulated Rat Alveolar Macrophages

Objective:This study explored the effects of different concentrations of PPF on apoptosis and the production of inflammatory cytokines of lipopolysaccharide-stimulated rat AMs at different time in vitro.Methods:AMs isolated from situ bronchoalveolar lavage fluid of rats were used for the following experiments after identified and cultured for 6 h,24 h,48 h.Rat AMs were randomly divided into eleven groups:PBS group,DMSO group,LPS group(1?gg/ml),L+P1 group(1?g/ml LPS+1?M PPF),L+P10 group(1?g/ml LPS+10?M PPF),L+P25 group(1?g/ml LPS+25?M PPF),L+P50 group(1?g/ml LPS+50?M PPF),L+P100 group(1?g/ml LPS+100?M PPF),L+P300 group(1?g/ml LPS+300?M propofol),L+DMSO group(1?g/ml LPS+DMSO),L+PBS group(1?g/ml LPS+1?M PBS).Rat AMs morphology were observed by Wright-Giemsa staining method,the viability of rat AMs were determined by MTT,the nuclear morphology of rat AMs was tested by Hoechst33258 staining assay.Furthermore,the secretion of TGF-?,IL-12,IL-10 and TNF-? in rat AMs were detected by enzyme-linked immunosorbent assay.Results:1.Cell viability was analyzed by colorimetric MTT assay.There were no differences between LPS group and L+P1 group,L+PBS group,L+DMSO group(P>0.05).Exposure of rat AMs to 1?g/ml LPS caused a significant increase of viability at 6h,24h,48h(P<0.05).Treatment of rat AMs with combination of LPS and 10?M,25?M,50?M PPF for 6h,24h,48h was not cytotoxic.However,PPF(100?M?300?M)induced significant decrease of cell viability at 6h,24h,48h.2.Wright-Giemsa dye assay:the rat AMs in PBS group or DMSO group were a moderate density,with both fibroblastoid and round cells.Treatment with 1?g/ml LPS led to adopt a fully differentiated amoeboid morphology in rat AMs.In addition,the number of rat AMs was obviously increased compared with the PBS group,and presented a trend of centralized aggregation.Treatment with a combination of LPS and 10,25?M PPF caused rat AMs to have strongly adherent ability and well diopter.However,Treatment with combination of 100,300?M PPF and 1?g/ml LPS led to adopt a condensed morphology with weakly adherent ability and easy suspension.3.Rat AMs were stained with Hoechst33258.We found that the nuclear of rat AMs in PBS group,DMSO group,LPS group,L+P1 group,L+P10 group,L+P25 group,L+P50 group showed normal size and light blue fluorescence,but rat AMs in L+P100 group and L+P300 group started to change their shape and color.The cell nuclei of L+P100 group and L+P300 group were shrunk and deformed,and they displayed trachychromatic and granular fluorescence,as well as that many fluorescent fragments of DNA could be seen in them.4.The results of ELISA indicated that exposure to 1?M propofol did not change LPS-induced augmentation of TNF-a,IL-12,IL-10 and TGF-?.Exposure of rat AMs to 1?g/ml LPS for 6h significantly increased cellular TNF-? levels compared with PBS group(P<0.05).However,treatment of rat AMs with combination of LPS and 25?M,50?M,100?M PPF for 6h significantly decreased the LPS enhanced synthesis of TNF-a(P<0.05).The production of IL-12 of L+P25,L+P50 and L+P100 groups were lower than that of LPS group at 24h(P<0.05).Compared with LPS group,the secretion of IL-10 in L+P25 and L+P50 groups was suppressed at 24h and 48h(P<0.05).After the treatment of the combination of PPF(1?M or 50?M)and LPS for 24h,the production of TGF-? was significantly increased compared with the LPS group.The synthesis of TGF-? in L+P25 group was increased compared with the LPS group at 48h(P<0.05).Conclusion:There results firstly indicate that PPF may protect against LPS-induced ALI in rat.