| Background and Objection:Endotoxemia which induced by trauma, infection, decreased immune function, gastrointestinal mucosal ischemia, and even mechanical ventilation, is the most common form of systemic inflammatory response in the clinical. In surgery perioperative, endotoxemia occurred is particularly common, if not timely intervention, caused by postoperative acute kidney injury, lung injury, or even multiple organ dysfunction syndrome (multiple organ dysfunct ionsyndrome, MODS), which is often an important factor to cause deaths. Therefor, how can effectively reduce and inhibit excessive inflammation, postoperative rehabilitation in order to facilitate the patient’s clinical research has been a hot issue.Propofol, is a new intravenous anesthetic discovered in recent years and a chemical called2,6-double-isopropyl phenol (2,6-di-isopropyl phenol). It can be as an anesthetic induction agent can be used as intravenous anesthesia agent. Propofol has a fast-acting and short duration, no accumulation in vivo toxicity and some analgesic effect has been widely used in clinical and ICU sedation. Recent studies have found that propofol can reduce pulmonary artery pressure and inhibit the response of ischemic pulmonary vasoconstriction, reducing the right heart load, but can also inhibit calcium influx and release, so is expected to have a protective effect of hypoxic organ. In addition, most attention is propofol anti-inflammatory protective effect. From1992, O’Donnell et al reported the clinical dose range of propofol can inhibit in vitro neutrophil polarization (neutrophil polarization), and showed a dose-dependent propofol and inflammatory response The study has been a focus of the research direction of the propofol. Propofol can inhibit the inflammatory response with unified understanding, however, the specific molecular mechanism for this function is not clear, but can not form a complete theoretical foundation to guide clinical practice, which greatly limits the specific application of propofol in the inflammatory response in this area.Recent studies show that propofol by inhibiting the signal transduction process in a variety of molecules can inhibit the inflammatory response, and enhance the reduction of TLR-4protein, inhibit the expression of the CD14gene and the phosphorylation of Iκ B to reduce NF-κB transferring to the nucleus, thereby reducing the plasma and cell cytokines such as, IL-8, IL-1, TNF-a and so on. Propofol can also serve to protect the tissue cells by inhibiting the adhesion molecules involved in inflammation and reduce the damage of microvascular endothelial cells. Corcoran, TB showed that propofol inhibited the high expressiong of vascular cell adhesion molecule-1and Intercellular adhesion molecule-1induced by oxidative damage. They prove that propofol can inhibit the expression of P-selectin on endothelial cells and impede the adhesion of leukocytes and endothelial cells fot the first in vitro experiments, which is important in heart transplants and coronary bypass surgery on myocardial protective effects in myocardial reperfusion.Study found that endotoxemia, propofol can inhibit mitogen-activated protein kinase activation, inflammatory cytokine release, NO generation and the change of the permeability of the cytoskeleton. However, the specific molecular mechanisms of propofol inhibition of MAPK activation and inflammatory cytokine release is not clear. In order to further the anti-inflammatory molecular mechanism of action of propofol in endotoxin blood, we pre-build a rat model of endotoxemia, the use of proteomics analysis of propofol on serum and mononuclear cell protein The expression profile. Propofol control group of normal rats, rats in the endotoxemia group and endotoxemia treated rat mononuclear cell protein expression in-depth study of spectral changes and found that propofol can contribute to mononuclear cells in the Annexin The expression of the A1. But the question now is:1. Propofol inhibition of p38phosphorylation increased expression of Annexin A1directly caused? If propofol directly inhibits p38phosphorylation by Annexin Al, then the release of IL-1, IL-6and TNF-alpha and other inflammatory factors in this model is whether it is directly affected by the regulation of p38phosphorylation? In addition to p38outside, in this model of inflammation propofol MAPK in inflammatory signaling pathways ERK, JNK signaling pathway also have some impact, whether this effect is also related with Annexin A1?Methods and materialsSection1:The influence of propofol on Annexin Al expression and p38phosphorylation in the blood serum mononuclear cells of endotoxin rats from the general level1.1The influence of propofol to expression of Annexin A1and p38in the blood serum mononuclear cells of endotoxin ratsThe experiment rats are randomly divided into6groups:control group (group C); LPS group (group L); propofol group (group P); LPS+propofol group (group L+P); intralipid group (group I); LPS+intralipid group (group L+I). Every group contains6rats. Take the rat serum6h later after the treatment, separate the mononuclear cells, extract the cell protein, and detect the expression of Annexin A1and p-p38by the western blot.1.2The release of inflammation factors (TNF-α、IL-1β、IL-6) in the endotoxin rat influenced by propofolThe experiment rats are randomly divided into6groups:control group (group C); LPS group (group L); propofol group (group P); LPS+propofol group (group L+P); intralipid group (group I); LPS+intralipid group (group L+I). Every group contains6rats. Take the rat serum6h later after the treatment, and detect the concentration of TNF-α、IL-1β、IL-6by ELASA.Section2:The influence of propofol to expression of Annexin A1and MAPK family in the THP-1cell stimulated by LPS from in vitro cellular level1.1The effect of different propofol concentration on the expression of Annexin A1:The experiment rats were divided into6groups:group C:normal control group; L group:stimulated6h by LPS (10μg/ml); L+P (5) group:stimulated6h by LPS (10μg/ml) and propofol (5μg/ml):L+P (10) Group:stimulated6h by LPS (10μg/ml) and propofol (10μg/ml); L+P (20) group:stimulated6h by LPS (10μg/ml) and propofol (20μg/ml); L+P (30) Group:stimulated6h by LPS (10μg/ml) and propofol (30μg/ml); L+P (50) Group:stimulated6h by LPS (10μg/ml) and propofol (50μg/ml). After6h of stimulation, cells were collected and lysised to extract protein, then detection the Annexin A1expression by Western blot1.2Influences of propofol on the time effects of Annexin A1expression in the THP-1cells stimulated by LPS. The experiment rats were divided into4groups: group C:normal control group; L group:stimulated by LPS (10μg/ml); L+P group: stimulated by LPS (10μg/ml) and propofol (20μg/ml); group P:stimulated by propofol (20μg/ml). Every group were separately stimulated for1h.2h,6h and then cells were collected. Western blot was used to detect the expression of Annexin Al.1.3Influences of propofol on the time effect of MAPK family activation in the THP-1cells stimulated by LPS. The experiment rats were divided into4groups: group C:normal control group; group L:stimulated by LPS (10μg/ml); group L+P: stimulated by LPS (10μg/ml) and propofol (20μg/ml); group P:stimulated by propofol (20μg/ml). Every group was separately stimulated for0.5h,1h,2h.6h and then cells were collected. Western blot was used to detect the expression of p-p38, p-ERK1/2, p-JNK1/2.1.4Influences of propofol on the release of inflammation factors in the THP-1cells stimulated by LPS. The experiment rats were divided into4groups:group C: normal control group; group L:stimulated by LPS (10μg/ml); group L+P: stimulated by LPS (10μg/ml) and propofol (20μg/ml); group P:stimulated by propofol (20μg/ml). EL AS A was used to detect the concentration of TNF-α、IL-1β and IL-6. 1.5Influences of intralipid on the expression of Annexin A1and p-p38in the THP-1cells stimulated by LPS. The experiment rats were divided into4groups: group C:normal control group; group L:stimulated by LPS (10μg/ml); group L+I: stimulated by LPS (10μg/ml) and intralipid (20μg/ml); group I:stimulated by intralipid (20μg/ml). Cells were collected after6h stimulation and detect the expression of Annexin A1and p-p38by Western blot.1.6The THP-1cells were transienly transfected by siRNA-Annexin A1. The synthesis siRNA-Annexin A1was transfected into THP-1cells by electroporation method.1.7Detection of transfection and interfere efficiency of siRNA-Annexin Al: Transfection efficiency was observed by fluorescence microscopy; RT-PCR was used to detect the expression of Annexin A1to confirm the interference efficiency.1.8The influence of propofol on the expression of p-p38after Anneinx A1silence. The experiment rats were divided into5groups:group L. stimulated by LPS (10μg/ml) for6h; group L+P:stimulated by LPS (10μg/ml) and propofol (20μg/ml) for6h; group P, stimulated by propofol (20μg/ml) for6h; group L’:stimulated by LPS (10μg/ml) for6h after siRNA-Annexin A1transfection; group L’+P’, stimulated by LPS (10μg/ml) and propofol (20μg/ml) for6h after siRNA-Annexin A1transfection. Collect the cells and detect the expression of Annexin A1and p-p38by the way of Western blot.1.9The influence of propofol on the release of inflammation factors after Anneinx A1silence. The experiment rats were divided into5groups:group L, stimulated by LPS (10μg/ml) for6h; group L+P:stimulated by LPS (10μg/ml) and propofol (20μg/ml) for6h; group P, stimulated by propofol (20μg/ml) for6h; group L":stimulated by LPS (10μg/ml) for6h after siRNA-Annexin Al transfection; group L’+P,stimulated by LPS (10μg/ml) and propofol (20μg/ml) for6h after siRNA-Annexin A1transfection. Collect the cells and detect the mRNA level of TNF-α、 IL-1β and IL-6by the way of RT-PCR.Results:Section1:1.1The influence of propofol on the expression of Annexin Al and p38in the blood serum mononuclear cells of endotoxin rats:The results show that, Compared with the L group, the expression of Annexin A1in the group L+P and group P were significantly higher (P=0.000), while no difference between groups I and I+L. Compared with group C, the level of p38phosphorylation significantly increased (P<0.01) in the group L, L+P and L+I. Compared with group L, the level of p38phosphorylation significantly decreased (P<0.01) in the group L+P, While no significant difference between L and group L+I (P>0.05)1.2The release of inflammation factors (TNF-α、IL-1β、IL-6) in the endotoxin rat influenced by propofol. Compared with the C group, the content of TNF-α, IL-1β and IL-6were significantly higher (P<0.01) in the group L, L+P, and L+I. Compared with the group P, the content of TNF-a, IL-1β and IL-6were significantly higher (P<0.01) in the group L+P. Compared with the group I, the content of TNF-a, IL-1β and IL-6were significantly higher (P<0.01) in the group L+I. Compared with the group L, the content of TNF-a, IL-1(3and IL-6significantly decreased (P<0.01) in the group L+P, While no significant difference between group L+P and group L+I (P>0.05).Section2:1.1The effect of different propofol concentration on the expression of Annexin Al:With the propofol concentration increasing, the expression of Annexin A1increased. When the propofol concentration was30μg/ml, Annexin A1activation achieved the maximum effect. When the propofol concentration was50μg/ml, the expression of Annexin A1was decreased. There was no significant difference when the propofol concentration was20μg/ml or30μg/ml.1.2Influences of propofol on the time effects of Annexin A1expression in the THP-1cells stimulated by LPS. Compared with the group L, the expression of Annexin A1was no difference between group L+P and P stimulated for1h and2h by propofol. Compared with the group L, the expression of Annexin A1was significantly increased (P<0.01) in the group L+P and P stimulated for6h propofol.1.3Influences of propofol on the MAPK family in the THP-1cells stimulated by LPS. Compared with the group C, the level of p38phosphorylation was significantly higher in the group L (P<0.01), while no difference between group C and P. Compared with the group L. the level of p38phosphorylation at0.5h had no significant difference in the group L+P, decreased from1h, and significantly decreased (P<0.01) at2h and6h, and show a decreasing trend. When stimulated for0.5h and1h, compared with group C, the level of ERK1/2phosphorylation of the group L was significantly higher (P<0.01), while no significant difference between the two groups at2h and6h. When the propofol stimulated for1h, the level of ERK1/2phosphorylation in group L+P was significantly lower than the group L(P<0.01). Compared with the group C, the level of JNK1/2phosphorylation was significantly higher in the group L (P<0.01). Compared with the group L. JNK1/2phosphorylation of group L+P was no significantly difference (P>0.05).1.4Influences of propofol on the release of inflammation factors in the THP-1cells stimulated by LPS. Compared with the group C, the content of TNF-a, IL-1β and IL-6were significantly higher (P<0.01) in the group L, L+P. Compared with the group P. the content of TNF-a. IL-1β and IL-6were significantly higher (P<0.01) in the group L+P. Compared with the group L, the content of TNF-a. IL-1β and IL-6were significantly lower (P<0.01) in the group L+P.1.5Influences of intralipid on the expression of Annexin A1and p-p38in the THP-1cells stimulated by LPS. The expression of Annexin A1in group C, L, I and L+I was no significant difference (P>0.05; For the expression of p-p38, compared with group C, p38phosphorylation of group L and L+I were significantly increased (P<0.01), while no significant difference in group I; compared with group L, p38phosphorylation of group L+I was no significant difference (P>0.05).1.6Transfection and interfere efficiency of Annexin Al. The transfection efficiency could be up to about60%. Compared with the control group, the mRNA of Annexin A1was significantly lower (P<0.05) in the cells transfected siRNA-Annexin A1, and and its interference efficiency is up to75%.1.7The influence of propofol on the expression of p-p38after Anneinx A1 silence. In the wild type, compared with the group L, the p38phosphorylation of the group L+P was significantly decreased (P<0.05). In the Annexin A1-/-cells, the p38phosphorylation was no significant difference between group L and L+P (P>0.05).1.8The influence of propofol on the release of inflammation factors after Anneinx A1silence. Compared with the group L. IL-1β, IL-6and TNF-a of group L+P released significantly reduced (P<0.01). Compared with the L’group, inflammatory factors IL-1β, IL-6and TNF-a released no significant difference in theGroup L’+P’(P>0.05).Conclusion1. the overall level of proof of propofol can upregulate the expression of Annexin A1, inhibition of p38MAPK phosphorylation, the final suppression of inflammatory cytokines IL-1β, IL-6and TNF-a release of anti-inflammatory effects.2. The experiments prove that the effects of propofol on the activation of Annexin A1is a dose-dependent. The maximal effect is the propofol concentration of30μg/ml. when the propofol concentration is50μg/ml, the expression of Annexin Al decreased.3. The influence of propofol on the MAPK family:LPS stimulation can promote of p38MAPK, ERK1/2and JNKl/2phosphorylation of THP-1cells. Propofol can play anti-inflammatory effect by the inhibition of p38MAPK and ERK1/2phosphorylation, but no significant correlation with the JNK1/2phosphorylation.4. Propofol may inhibit the activation of p38through up-regulating the expression of Annexin A1to inhibit the inflammatory cytokines TNF-a, IL-1β, IL-6to exert its anti-inflammation effect in THP-1cells. |
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