Effect Of Propofol On Apoptosis And The Production Of IL-10and TNF-α In Rat Alveolar Macrophages

Objective:To evaluate the potential effect of different concentrations of propofol on apoptosis and the production of interleukin-10(IL-10) and tumor-necrosis factor-α(TNF-α) in rat alveolar macrophages(AMs). Methods:AMs separated from bronchoalveolar lavage fluid(BALF) of rats were randomly divided into ten groups:PBS group, DMSO group, P1group to P6group(the concentrations of propofol were1μM,10μM,25μM,50μM,100μM,300μM), lipopolysaccharide(LPS) group(1μg/ml), dexamethasone(DEX) group(10-6M). A series of method is adopted to detected the effect of propofol on apoptosis of rat AMs:the biological morphology of AMs was observed by Wright-Giemsa dye assay, cell viability was determined by the colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, nuclear morphology was detected using Hoechst33258staining, the apoptosis rate of rat AMs were detected by PI staining and annexin V-FITC/PI double staining, the activity of caspase-3was tested by caspase-3kit, and the phagocytotic activities were determined by fluorescence microscope. In addition, the production of TNF-α and IL-10in rat AMs were determined by enzyme-linked immunosorbent assays(ELISAs) and immunocytochemistry staining. Results:1. Cells were stained with Wright-Giemsa stain. The AMs in PBS group or DMSO group were a mixed morphology, with both fibroblastoid and round cells. Treatment with propofol(1μM,10μM,25μM,50μM,100μM) caused rats AMs to adopt a fully differentiated amoeboid morphology with strongly adherent ability and well diopter, whereas LPS-treated AMs was significantly increased compared with the blank group, and presents a trend of centralized aggregation. Propofol(300μM)-treated AMs adopted a condensed morphology with weakly adherent ability and easy suspension, while treatment with DEX caused a more obvious decrease of rat AMs compared with P6group.2. The viability of rat AMs in P1group to P5group and DMSO group were not statistically significant compared with PBS group(P>0.05), whereas the viability of rat AMs in P6group and DEX group were significantly decreased after24h(P<0.05), and the viability of rat AMs in LPS group were significantly increased after6h by MTT(P<0.05).3. Using Hoechst33258staining, we observed that the cell nuclear of AMs in PBS group, DMSO group, P1to P5groups and LPS group appeared normal nuclear size and blue fluorescence, whereas AMs in P6group and DEX group start to change their shape(they shrunk and round up).4. PI staining showed that in P6group and DEX group, we detected an apoptotic peak and the number of AMs in G0/G1phase increased, while the number of AMs in S and G2/M phases reduced. The apoptosis rate of rat AMs in PI to P5groups, DMSO group and LPS group were not statistically significant compared with PBS group(P>0.05).5. Analysis by annexin V-FITC/PI double staining indicated that the apoptosis rate of rat AMs in P1to P5groups, DMSO group and LPS group were not statistically significant compared with PBS group(P>0.05). However, the apoptosis rate of rat AMs in P6group and DEX group were significantly increased(P<0.01).6. The P6group and DEX group led to a significant increase in caspase-3activity as compared to PBS group after24h(P<0.05), while the caspase-3activity in P1to P5groups, DMSO group, LPS group were not statistically significant(P>0.05).7. Propofol at1μM or DMSO do not affect the phagocytotic activity of rat AMs, while the phagocytotic activity of rat AMs in P3to P4group and DEX group were significantly reduced(P<0.01).8. The ELISAs and immunocytochemistry staining showed that the production of TNF-a and IL-10were not affected in P1to P6groups(P>0.05), whereas the production of IL-10in DEX group was significantly increased, and the production of TNF-a was significantly increased in LPS group. Conclusion:1. Exposure of rat AMs to lμM,10μM,25μM,50μM,100μM propofol did not affect AMs viability. When the administered concentration reached300μM, propofol would cause AMs apoptosis.2. Propofol at300μM and DEX(10-6M) induces apoptosis of rat AMs which is probably caused by the activated caspase-3.3. A therapeutic concentration of propofol inhibits the phagocytotic activity of rat AMs, and the mechanism of the inhibiting effect have no relationship with the apoptosis of rat AMs. DEX inhibits the phagocytotic activity and induces apoptosis of rat AMs, while LPS increases the phagocytotic activity and the viability of rat AMs.4. The production of TNF-a and IL-10were not affected in rat AMs after stimulated with different concentrations of propofol respectively, whereas the production of IL-10in DEX group was significantly increased, and the production of TNF-a was significantly increased in LPS group.