Effects Of Propofol On The Expression Of Monocyte Protein From Rat Peripheral Blood With Endotoxemia

Propofol (2,6-diisopropylphenol) is a potent intravenous hypnotic agent widely administered for induction and maintenance of anesthesia and for sedation in the intensive care unit.In recent years,the function of inhibiting the inflammatory reaction on propofol was attented by more and more people.The research and reports are increasing in this area.But With regard to propofol specific anti-inflammatory signal transduction mechanism is not clear and not formed a complete theory.Limitations and hinder the clinical application of specific anti-inflammatory effects of propofol.In the systemic inflammatory response, monocyte are the most important cells. Therefore, from the protein spectrum of mononuclear cells of blood to start, as the study of anti-inflammatory mechanism of propofol important entry point,a lot have been reported about this field. In inflammatory conditions, propofol can inhibit the inflammatory mediators IL-10,IL-6, TNF-a release, and for mechanism of the specific role of propofol inhibited the release of these inflammatory factors are poorly understood.In this study, the central link of inflammation-vascular response to start the establishment of rat model of endotoxemia on rat blood mononuclear cells two-dimensional electrophoresis protein analysis, to look for inflammation-related, subject to regulation and control of propofol protein. on the basis of in the rat with endotoxemia,we extracted the mononuclear cells of peripheral blood and Cleavage cell, Two-dimensional electrophoresis, Proteins identified by mass spectrometry,then obtained proteins of differences in the expression. We choose membrane calcium protein A1 (Annexin A1)relating to inflammatory closely as a further study. The results showed that the results are consistent with the two-dimensional electrophoresis and further verified by membrane calcium-inflammatory proteins played an important role in the process of anti-inflammatory of propofol.At the same time we detected inflammatory cytokines(IL-1β, IL-6 and TNF-α) in serum and found that result showed significant higher than control groups. Membrane calcium-protein A1 plays an important role in the propofol-inflammatory signal transduction mechanism. Research has shown that:AnnexinAl strongly inhibit the expression of IL-6, this effect is activated by impact mRNAd P38MAPK stability.P38MAPK are a class of evolutionarily conserved serine/threonine mitogen-activated protein kinases. The activation of the p38 pathway plays essential roles in the production of proinflammatory cytokines (IL-1β, TNF-a and IL-6);induction of enzymes such as COX-2 which controls connective tissue remodeling in pathological conditions; expression of intracellular enzymes such as iNOS, a regulator of oxidation;induction of VCAM-1 and other adherent proteins along with other inflammatory related molecules.This inhibitory effect of propofol on the activation of p38MAPK may be the reason why propofol decreases the infiltratin of neutrophils in the liver of conscious rats with endotoxic shock; reduces the mortality rate of endotoxaemic rats and attenuates their cytokines responses.Materials and methods:1,Animal preparation and grouping:18 male SD rats weighing 180-220 g, were fasted overnight, with urethane (1.0g/kg) intramuscular anesthesia, the right femoral vein was cannulatied to establish intravenous access.The 18 SD rats were randomly divided into LPS group (L group), propofol+ endotoxin group (L+P group) and control group (C group) and 6 rats in each group.2,Animal model-making and drug infusion:L groups:Received the femoral vein injection of endotoxin from Escherichia coli 0111:B4 10mg/kg, then at a rate of 10mg.kg-1h-1 infusion of 10% Intralipid, continuous 6h.C groups:Received the femoral vein injection of saline With the same volume of endotoxin,then at a rate of 10 mg.kg-lh-1 infusion of 10% Intralipid,then continuous 6hL+P groups:Received the femoral vein injection of endotoxin from Escherichiacoli 0111:B4 10 mg/kg, then at a rate of 10 mg.kg-lh-1 infusion of propofol, then continuous 6h.3,Specimen collection:The right carotid artery were cannulated for collecting3-5ml blood,Anticoagulation with heparin anticoagulant tubes.4,Specimens treatment(1)3ml anticoagulanted rat blood layered onto separation liquid surface.(2) At room temperature,centrifuge at 400×g for 30min, blood layered, aspirate the upper layer to within 2-3 mm of the opaque interface containing the mononuclear cells,discard upper layer, Carefully transfer the opaque interface, containing the mononuclear cell band into a clean 15ml conical centrifuge tube. Add 10ml isotonic PBS Mix tube by gentle inversion several times Centrifuge at 250xg for 10 minutes. Aspirate the supernatant and discard.Add an additional 5.0ml of isotonic PBS,Mix several times by gentle inversion,Centrifuge at 250xg for 10 minutes. Aspirate the supernatant and discard.(4)Repeat Step upper(5)In accordance with the cell volume by adding lysis buffer four times,4℃efrigerator for 30min,4℃.(6) centrifuge at 12000rpm for 15min,supernatant were stored at-80℃.5,Two-dimensional electrophoresis5.1 quantificationEach group will be mixed with cell within the group, With bradford method protein quantification,operate according to kit instructions, Protein concentration in each group in the 10-15ug/ul and Concentration among the three groups close to one another.5.2 first isoelectric focusingUsing Amersham IPGghor the company's first to the isoelectric focusing system Ettan Daltsix Vertical electrophoresis system,operate according to the instructions, and use 24cm, ph value of the tape 3-10, the sample for the 150ug, Soak up 12 hours. Turn first to the isoelectric focusing system,Electrophoresis condition set is:S1 (200v, 1hr,200vhs, step),S2 (500v, 1hr,500vhs, step),S3(1000v, 1hr, 1000vhs, step),S4(5000v, 1hr,5000vhs, step),S5(8000v,0.5hr,4000vhs, Grad), S6(8000v,6.5hr,60000vhs, step),S7 (500v,5hr,500vhs, step)5.3 Balance:Balanced by two-step balance method, each for 15min.5.4 SDS-PAGE electrophoresisElectrophoresis conditions set is:s1 (5w per gel,45min),s2 (20w per gel) 6h When the bromophenol blue migrated to the bottom of glue and electrophoresis end.5.5 Staining:After the two-dimensional electrophoresis, using Coomassie Brilliant Blue R250 dye buying from using Coomassie Brilliant Blue R250 and according to the proposed process to dye,.6,Scanning and AnalysisTest after transfection of the two-dimensional lectrophoresis gel, With Umax PowerLook 1100 scanner to scan projection,store the images in TIF format, Using Bio-Rad two-dimensional pattern analysis software PDQuest 8.0 image to analysis, including background reduction, removal of stripes, spot detection, matching and automatic matching and manual matching software combined, Set 2-fold difference of expression for the differential expression of the above range, the software automatically analyzed,then differentially expressed protein spots of the data were given.7,Mass spectrometry, protein detection and data analysismass spectrometry of protein samples with 4800 MALDI TOF/TOF Analyzer (Applied Biosystems, USA); With peptide mass fingerprinting (Peptide Mass Fingerprint,PMF) and peptide sequence tags through the MASCOT (http://www. matrixscience.com/, MatrixSicence Ltd.,London, UK) search engine and the control of protein database SwissProt protein detection and data analysis. Peptide mass error in the 0.01-0.1% range. Matched peptides over 4, MASCOT protein scores more than 64 points that was statistically significant.8,Annexin A1 verification8.1 Animal preparation and grouping18 male SD rats weighing 180-220 g, were fasted overnight, with urethane (1.0g/kg) intramuscular anesthesia, the right femoral vein was cannulatied to establish intravenous access.The 18 SD rats were randomly divided into LPS group (L group), propofol+ endotoxin group (L+P group),control group (C group) and propofol group(p group) and 6 rats in each group. Animal model-making and drug infusionL groups:Received the femoral vein injection of endotoxin from Escherichia coli 0111:B4 10 mg/kg, then at a rate of 10 mg.kg-lh-1 infusion of 10% Intralipid, continuous 6h.C groups:Received the femoral vein injection of saline With the same volume of endotoxin, then at a rate of 10 mg.kg-lh-1 infusion of 10% Intralipid,then continuous 6hL+P groups:Received the femoral vein injection of endotoxin from Escherichia coli 0111:B410 mg/kg,then at a rate of 10 mg.kg-lh-1 infusion of propofol, then continuous 6h.P groups:Received the femoral vein injection of saline With the same volume of endotoxin, then at a rate of 10 mg.kg-lh-1 infusion of propofol,then continuous 6h8.2 Sample collection and cell protein cleavageAfter 6h collect blood sample, each blood sampling from rats is about 6ml, which 3ml were used to separate mononuclear cells and 3ml for the separation of serum, the isolated mononuclear cell lysis methods, such as 4. Parallel8.3 western blot detectionWe verify the mass spectrometry of protein spots by Western blot. After the extraction of 500μg total protein From each group,with 12% SDS-PAGE gel separation, and transferred onto PVDF membrane,then closed PVDF membrane 1 h use TBST solution Containing 5% skim milk. After closed ending,in the shaking bed,a resistance to anti-Annexin Al rabbit polyclonal antibody(1:1000; Abcam) 4℃was overnight incubation. After incubation, having been combined with a resistance of PVDF membrane wound be rinsed 3 times with TBST solution. Then with horseradish peroxidase conjugated secondary antibodies horseradish peroxides-conjugated anti-rabbit immunoglobulin G(1:2000; Cell Signaling8.4 ELISA assayAnnexin A1 express increased and inhibit the expression of inflammatory factors such as IL-1,IL-6 and TNF-α,we with the method of ELISA to detect serum levels of these inflammatory factors in serum of each group.8.5 Phospho-p38MAPK detectionthe membrane was probed with anti-Phospho-p38MAPK (Thrl80/Tyr182)(3D7) rabbit polyclonal antibody(1:1000; Cell Signaling Technology) and horseradish peroxidase-conjugated anti-rabbit immunoglobulin G(1:2000; Cell Signaling Technology, Danvers MA)9. Data AnalysisMeasurement data were express as mean±tandard(x±s)deviation.All data were analysed with the Statistics Package for Social Sciences (SPSS,Version 13.0 for WINDOWS;SPSS Inc.;Chicago,IL,USA).Differences were considered statistically significant when P was less than 0.05.We use the one-factor one-way classification for measurement data.Results1. Cells lysised, were mixed among the same group and then quantitative with bradford method,according to the instructions. Protein concentrations in each group were 10-15ug/ul, closer to each other. Reproducibility of protein extraction method was good.2. By two-dimensional electrophoresis and mass spectrometry identification, we got 18 differences in protein expression and successfully identified 15,among these:a.The expressing of UMP-CMP kinase and Myosin-9 in the control group are the lowest. b.Cofilin-1,ATP synthase subunit alpha,mitochondrial precursor and Hemoglobin subunit alpha in the LPS group are the lowest.c.In the control group,LPS group and LPS+propofol group,expression of Destrin and Glutathione peroxidase 1 has a trend to gradually increase.d.Expression of Protein S100-A8,L-lactate dehydrogenase A chain,Peptidyl-prolyl cis-trans isomerase B precursor,Peptidyl-prolyl cis-trans isomerase,mitochondrial precursoe and Annexin A1 only in the LPS + propofol group was significantly higher.e. S-phage kinase-associated protein 1 occurs only in the LPS group;f. Ubiquitin expressed only in the control group is the most;The further validation of Annexin A13. Western blot test results showed that Annexin A1 expression in the L group was the lowest,while in C group and P group expressed had a very high volume, while in the L+P group significantly increased.Annexin A1 is a negative regulator of IL-1β,IL-6, and TNF-αand has inhibitory effects on the activation of p38 MAPK signal transduction pathway. Our 2D and Western Blot results showed that in endotoxaemic rats, propofol promotes the expression of Annexin A1 of monocytes in the blood. Therefore, we further studied the effect of propofol on the activation of p38 and release of IL-1β,IL-6,and TNF-αin endotoxaemic rats.Without the treatment of LPS,the phosphorylation of p38 remains in a low basal levels both in intralipid and propofol group. LPS dramatically increased the phosphorylation level of p38 in the monocytes, and this effect could be partially inhibited by propofol.The total p38 andβ-actin loaded on SDS-PAGE was used as internal control.4. ELISA assay of each group serum levels of inflammatory factors in the above-mentioned results showed that LPS group compared with the LPS+propofol group,IL-1,IL-6 and TNF-αof serum were significantly higher,the difference had statistical meaning.Conclusion:1.Using proteomics approach, we first found that propofol increase the expression of proteins Annexin A1 of monocytes in endotoxemic rats significantly;2.In endotoxemia rat mononuclear cells, propofol may via inflammatory protein Annexin A1 upregulation, inhibitit p38MAPK phosphorylation, thereby inhibit the production of inflammatory factor IL-1β,IL-6 and TNF-α.