The Mechanism Of Propfol-induced Depression Of Myocardial Contraction And Reduction Of Macrophages Production Of TNF-α And IL-6

BackgroundPropofol (2,6-diisopropylphenol) is a potent sedative and hypnotic agent which is known to depress cardiac contraction, but the molecular mechanisms involved are not known. The central mechanism seems to involve altered cardiac excitation-contraction coupling. But the exact steps in cardiac excitation-contraction coupling which propofol affect have been a matter of debate. Here we hypothesized that decreasing myofilament Ca2+responsiveness is central to the depression of contraction. We first demonstrated that propofol depressed contraction without affecting intracellular Ca2+availability at low and moderate doses, and the depression of force development was a result of direct myofilament effect.Macrophages respond to invading microbes at multiple levels, promoting the production of reactive oxygen species (ROS) in macrophages. Despite their well-established cytotoxic activities, recent studies have shown that ROS are immunomodulatory agents that can enhance the immune response to an infection. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are major sources of ROS in immune cells. Propofol also exhibits anti-inflammatory and antioxidant activity on macrophages. The effect of propofol treatment on NADPH oxidases and ROS production in macrophages has not been studied. Here, we investigate the effect of propofol pretreatment on LPS-induced changes to ROS levels, NF-κB phosphorylation, and NADPH oxidase activity in murine macrophage cell line RAW 264.7.Part 1 The Mechanism of Myofilament Ca2+Sensitivity in Propofol-induced Depression of Myocardial Contraction ObjectiveIn this study, we test the decreasing myofilament Ca2+responsiveness is central to the depression of myocardial contraction. We first demonstrated that propofol depressed contraction without affecting intracellular Ca2+availability at low and moderate doses, and then the depression of force development was a result of direct myofilament effect.Methods1. Trabeculae PreparationThe rats were anesthetized via intra-abdominal injection with pentobarbital (100 mg/kg) and the heart was dissected out. Trabecular muscle from the right ventricle of the heart was dissected and mounted between a force transducer and a motor arm, superfused with K-H solution at a rate of～10ml/min, and stimulated at 0.5 Hz. Force was measured by a force transducer system (KG7, Scientific Instruments GmbH, Heidelberg, Germany) and expressed in millinewtons per square millimeter of cross-sectional area.2. Measurement of Intracellular Ca2+([Ca2+]i) [Ca+]i was measured by using the free acid form of fura-2. Fura-2 epifluorescence was measured by excitation at 380 and 340 nm. Fluorescent light was collected at 510 nm by a photomultiplier tube. The output of the photomultiplier was collected and digitized.3. Steady-state Activation of TrabeculaeRyanodine (1.0μM) was used to enable steady-state activation. After the muscles were exposed to ryanodine for 15 min, different levels of tetanizations were induced briefly (-4-8 s) by stimulating the muscles at 10 Hz at various concentrations of extracellular Ca2+([Ca2+]。; 0.5-20 mM).4. Skinned TrabeculaeAfter the steady-state experiments, the same trabeculae were immediately skinned by 15-20 min of exposure to Triton X-100 (1%). Different Ca2+concentrations were achieved by mixing the activating solution and relaxing solution in different ratios. The readiness of the skinned preparation was assured by the loss of pink color of the muscle and clear visibility of the sarcomeres, and the instantaneous force development upon exposure to activating solutions.5. Determination of myofibrillar ATPase activityMyofibrils were prepared from cardiac ventricle as previously described with careful use of protease inhibitors. Assays were performed at pH 7.0 with 50 mM imidazole,50 mM KCl and 2 mM MgATP. In the final assay conditions, myofibrillar protein concentration was diluted in buffer to have a concentration of 0.2 mg ml-1 in the assay, and protein concentration determined once again for final calculations. Mg-ATPase activity was calculated in nanomoles of inorganic phosphate liberated per milligram of myofibrillar protein per minute.Results1. Propofol Depressed Twitch Force without Affecting [Ca2+]i TransientsThe effects of propofol on force development and [Ca2+]i transients was first studied in isolated intact trabecular muscles. The typical raw recordings of force and [Ca2+]i transients at different propofol concentrations and the pooled data of all muscles tested. Propofol depressed force development in a dose-dependent manner without significantly altering amplitudes of [Ca2+]i transients until the concentration was 100μM.2. Propofol reduced maximal Ca2+-activated force (Fmax) and increased the [Ca2+]i required for 50% of activation (Ca50) in the intact musclesWe determined the steady-state force-[Ca2+]i relations before and during anesthetic exposure. Propofol (50μM) reduced maximal Ca2+-activated force (Fmax) (48.21±2.15 vs.76.87±3.32 mN/mm2 control, P<0.01) and increased the [Ca2+]i required for 50% of activation (Ca50) from 0.51±0.11 (control) to 0.71±0.12μM (P<0.05). Propofol didn’t affect the Hill coefficient.3. Propofol depressed maximal Ca2+-activated force (Fmax) and increased the [Ca2+]i required for 50% of activation (Ca50) in the skinned musclesIn the intact muscle, the myofilaments are bathed in a cellular milieu. To rule out all potential confounding factors in the milieu that contains many factors that could be potentially modified by the anesthetics and modulate the steady-state force-[Ca2+]i relationship, we skinned the same trabecular muscle in which the steady-state force-[Ca2+]i relations had been obtained. This enabled us to obtain force-[Ca2+] relations devoid of potential soluble confounding factors in these muscles. Under propofol (50 μM), Fmax was significantly depressed (from 87.87±6.63 to 40.93±2.13 mN/mm2, P<0.01) and Ca50 was significantly increased (from 1.01±0.18 to 2.02±0.14 μM, P<0.01).4. Propofol decreased myofibrillar Mg-ATPase activityTo further evaluate whether the direct myofilament effect of propofol and isoflurane involves thick/thin myofilament proteins or regulatory proteins, we determined the Mg-ATPase activity in isolated myofibrils treated with propofol or isoflurane. Both propofol and isoflurane decreased maximal myofibrillar Mg-ATPase activity and shifted the Mg-ATPase-pCa relationship towards the left.ConclusionsIn summary, we demonstrated that, at clinically relevant doses, propofol directly depress myofilament contractility by decreasing Ca2+ responsiveness. The findings have provided fundamental insights about anesthetic effects in the heart.Part 2 Propofol Reduces Lipopolysaccharide-Induced, NADPH Oxidase (NOX2) Mediated TNF-a and IL-6 Production in MacrophagesObjectivePropofol (2,6-diisopropylphenol) is a potent sedative and hypnotic agent that exhibits anti-inflammatory and antioxidant activity. In particular, propofol reduces the release of inflammatorymediators such as IL-6 and TNF-a and inhibits ROS in phagocytic cells. However, the effect of propofol treatment on NADPH oxidases and ROS production, in macrophages has not been studied. Here, we investigate the effect of propofol pretreatment on LPS-induced changes to ROS levels, NF-κB phosphorylation, and NADPH oxidase activity in murine macrophage cell line RAW 264.7.Methods1. Detection of cytokine productionA total of 2×105 macrophages were seeded into 24-well plates and incubated overnight. Cells were treated with DMSO or propofol for 40 min, and then stimulated with LPS for 8 h. TNF-a and IL-6 concentration in culture was measured by an ELISA kit.2. Analysis of Cytokine mRNA levels using RT-PCRRAW264.7 macrophages were pretreated with DMSO or propofol for 40 min and then stimulated with LPS for 2 h. Total RNA was extracted with TRIzol reagent, Data are normalized to β-actin expression in each sample. Amplification, detection.3. Detection of Akt and nuclear NF-κB ActivationRAW264.7 macrophages were pretreated with DMSO or propofol for 40 min and then stimulated with LPS for 60 min. Cells (5×106) were harvested and resuspended. Equal amounts of protein were resolved using 10% SDS-PAGE and electro-transferred onto nitrocellulose membrane.. The primary antibodies were added and the membrane was then incubated with a secondary antibody. Protein bands were detected with an enhanced chemiluminescence kit and captured by Image-Pro Plus 6.0.4. Measurement of superoxide productionThe oxidative fluorescent dye hydroethidine (DHE) was used to evaluate levels of superoxide as described previously. After a 40 min incubation with propofol or DMSO, cells were stimulated with LPS for 40 min, then stained with 2μM DHE for 30 min at 37℃ in the dark. Nuclei were stained with DAPI. Finally, cell samples were analyzed using an Olympus Ⅸ71 fluorescence microscope.5. NADPH oxidase activity assayNADPH oxidase activity was measured using an assay kit. After a 40 min treatment with propofol or DMSO, cells were stimulated with LPS for 6h, and then harvested. The supernatants were transferred to a tube, to which specific substrate conjugates for oxidase were added. NADPH oxidase activity was determined by spectrophotometry at 340 nm.6. Determination of NADPH oxidase expressionMacrophages were pretreated with DMSO or propofol for 40 min and then stimulated with LPS for 8h. The primary antibodies were added. Protein bands were detected with an enhanced chemiluminescence kit and captured by Image-Pro Plus 6.0.Results1. Effect of propofol on LPS-induced TNF-α and IL-6 expressionLPS treatment induced a significant increase in cellular TNF-α and IL-6 levels (P<0.05). However, propofol pretreatment reduced LPS-induced TNF-α and IL-6 expression by 20.01±5.4%(P<0.05),46.15±6.8%(P<0.05),61.53±10.2%(P<0.05) in response to 10μM,50μM and 100μM propofol, respectively (Fig.1). Treatment of macrophages with a therapeutic concentration of propofol (50μM) decreased LPS-induced production of TNF-α and IL-6 mRNA..2. Effect of propofol on LPS-induced nuclear NF-κB and Akt phosphorylationAfter treatment with 100 ng/ml LPS for 1 h, a significant increase in NF-κB and Akt phosphorylation was observed. However,50μM propofol pretreatment reduced the level of NF-κB and Akt phosphorylation. Similar to its effect on TNF-α and IL-6, propofol treatment alone did not alter NF-κB and Akt phosphorylation.3. Effect of NF-κB Inhibitor on LPS-induced TNF-α and IL-6 expressionA significant increase in TNF-a and IL-6 expression was observed after treatment with 100 ng/ml LPS for 1 h. However,20μM pyrrolidine dithiocarbamate (PDTC), a selective chemical NF-κB Inhibitor pretreatment 1h reduced the expression of TNF-a and IL-6.4. Effect of propofol on LPS-induced ROS generationAs expected, LPS (100 ng/ml) treatment significantly increased the intracellular ROS in macrophages. Remarkably, pre-treatment with 50μM propofol significantly reduced LPS-induced increases in intracellular ROS. Again, propofol pretreatment alone did not alter levels of intracellular ROS.5. Effect of propofol on LPS-stimulated NADPH oxidase activityLPS treatment stimulated NADPH oxidase activity (298±13.69%), while propofol pretreatment significantly lowered the degree of NOX stimulation (136±13.69%). Treatment with 50μM propofol alone had no effect on NADPH oxidase activity.6. Effect of propofol on LPS-induced NADPH oxidase expressionLPS (100 ng/ml) treatment for 8 h led to a significant increase in protein expression of NOX subunits (p47phox, gp91phox and p22phox). Pretreatment with propofol effectively reduced the expression of p47phoxand gp91phox in LPS-stimulated cells, but had no effect on p22phox expression. Propofol alone did not elicit effects on protein expression of oxidase subunits.ConclusionsThe current study has demonstrated the immunomodulatory effects of propofol on macrophage cytokine and ROS production. Specifically, these include the inhibitory effects of propofol on LPS-induced TNF-a and IL-6 production and expression, as well as on NADPH oxidase expression and activity.