| Amniotic fluid embolism (AFE) is a most serious obstetric complication that the incidence is low and the death rate is very high but can not anticipate. Owing to the Onset of AFE is very urgent,the advancement of course of disease is quick and the mortality is very high , it is difficult to investigate the pathogeneny of it. The conventional idea considered the cause is that the amniotic fluid ingredients got into parent to result in mechanicalness embarrass.Recent years late, many scholars proposed it related with hypersensitive-ness . However, up to now, there is a lack of enough experiment evidences a-bout it, furthermore it had a dispute about the pathogenesis of it. Main death reason of AFE is that the AFE induced acute respiratory failure bright heart function failure,exchange function disorder of capillary blood gas, ultimately resulted in tissue ischemia and hypoxia , peripheral circulation failure and MODS. Once happened MODS, the case fatality was almost 100% , this was main reason that the death rate of AFE is very high. The specific mechanism of AFE patients happened the MODS is unknown. Therefore, it is a important task of modern obstetric emergency medicine to discuss deeply the genesis mechanism of MODS . In the last ten years studies indicated the SIRS which was induced by many etiological factors was a capital pathology foundation and formative basical reason of MODS. SIRS induced extensive damage of host autologous tissue structure and physiologic function, finally caused MODS even death. NF - κB is an transcription factor that it mainly participated in the expression regulation of organism inflammation molecule and participated in organize injury caused by many reasons. The aim of our study is that to manufacture a amniotic fluid entering blood model by input the different character self amniotic fluid into the late trimester of pregnancy rats and research the morbidity cause, further genesis , development.Our study plan to investigate the origin , the mechanism of more happen ,development and prevention measures of AFE through three aspect. The primary part: explore the change and the function mechanism of Tryptase (the indicator of de-granulation of mast cells) and complement C3,C4 in AFE. The second part; explore the function of NF - κB in more happen, development in AFE. The third part: explore the inhibitory action and mechanism of action of dexamethasone on NF - κB.Materials and Methods一. Experimental animals and grouping50 late trimester of pregnancy Wistar rats ( gestation age 20 days) , body weight 282 -388g( offered by The Second Clinical Institute Experimental Animal Center of China Medical University, supply animal according to experiment condition). The animals were divided into five groups randomly: control group (normal saline treatment group,NS) ,amniotic fluid group(AFG) , meconium fluid group(MFG) , intervention group A(amniotic fluid + Dex0.1mg/100g) ,intervention group B( meconium + Dex).二. Experimental methods1. Preparation amniotic fluid and meconium fluid for experiment.2. Preparation the animal model: made a subtotal hysterectomy for rat and closed abdomen, separated the right external jugular vein and left common carotid artery, made a left common carotid arterial cannula and switched the three -waytube to link the two qigong methods electrophysiolograph, continuous monitoring mean aortic pressure( MAP) ; the right external jugular vein switched three-waytube, from here infused the injection(0. 25ml/100g). drew 1.0ml blood from left common carotid artery at pro - experiment and post - experiment 60 min respectively, disposed the preparation blood, deepfreezed at - 70℃. at 6omin killed the animals with 10% KCL 5ml and sheared the chest and abdominal cavity, took the right lung,the right kidney,heart,liver and quantity sufficient intestinal tract tissues to test, took the left lung for test.3. The complement C3 and C4 were checked by immunotuebidimetry.4. The Tryptase activity was tested with colormetrically using special sub-strate, BAPNA.5. Immunohistochemistry of NF - κB P65: Semiquantitativecount method was used to count the number of P65 positive cells in 10 visual fields of every smear under 400 micro - scope.6. Expirical procedure of hybridization in situ of NF - κB( brief).7. HE stain: Observe the pathologic changes of tissue.8. Measurement of MPO in pulmonary tissue: MPO activity(u/g) = △A × 13.5/tissue wet weight.三. Reagent and equipment:1. Mostly reagent: Immunohistochemistry and hybridization in situ of NF -kB P65 kit, complement C3,C4,MPO reaction kits.2. Equipment; Blood pressure monitor, Supercentrifuge, Electrophoresis apparatus, Homogenizer, Figure image analysis system.四. Statistical analysis:Data were analyzed by using SPSS statistical software and were expressed as median standard error. T test were used to analysize all data.Experimental Results1. Pathology examinationIn HE stain, experimental groups Lung tissues have different degree dropsy and abundant blood vessel, part has small amouts bleeding in the lung parenchyma, around the blood vessel and bronchus have abundant inflammatory cells soakage including leukocyte(main neutrophil) , macrophage, and little lymphocyte. Kidney: bleeding can be seen everywhere in interstitium, particularly in glomerulus, and interstitium edema. Intestines: inflammation cells increase a-round glandular organ, chief is lymphocyte , eosinophile granulocyte, dropsy is obvious, liver; hepatic cells swell, hepatic lobules have a little bleeding, inflammation cells increase, dropsy is light. Heart: it has a little congestion spot and inflammation cell soakage . But we do not find the pathology change above -mentioned in control group. Principal injury organs are lung and kidney in the amniotic fluid group; In the meconium fluid group principal injury organs are lung , kidney and intestines, but the heart and liver injury are light. The degreeof organ injury in the amniotic fluid group are light than the those in the meconium fluid group, contrast to the control group, the degree of organ injury is light in the treatment group.2. The level of complement C3,C4 at pro - infusion and post - infusion in every groupThere are no obvious difference between before experiment and after experiment in NS group(P >0. 05) ; There are obvious difference between before experiment and after experiment in the amniotic fluid group (P <0. 01). 3. The level of serum tryptase at pro - infusion and post - infusion in every groupThere are no obvious difference between before experiment and after experiment in NS group(P >0. 05) ; Although contrast to pro -experiment, in the experimental groups the level of serum tryptase increase in the amniotic fluid group and meconium fluid group , there are no significant differences( P >0.05) .4. Expression of NF - κB P65 in the lung tissue Immunohistochemical results: The part cytoplasm of lung tissue are pale -staining in the control groups, the cell nucleus are almost no color stain; Contrast to the comparison group, in the amniotic fluid group, the cytoplasm of lung tissue can see buffy granules, the nucleus staining increase significantly(P <0. 01); The nucleus staining in the meconium fluid are very stronger than those in the comparison group( P <0.01) ; In the lung tissue of amniotic fluid group the cytoplasm can see buffy granules, Contrast to the amniotic fluid intervention group the nucleus staining increase significantly (P <0.01) ; The nucleus staining in the meconium fluid group are significantly increased than those in the meconium fluid intervention group( P <0.01) .5. The results of hybridization in site of NF - κB P65The part cytoplasm are pale - staining in the lung tissue of comparison group, the nucleus are almost no stain; Contrast to the comparison group, in the amniotic fluid group, the cytoplasm can see buffy granules, the nucleus staining increase significantly(P <0. 01) ; The nucleus staining in the meconium fluid are very stronger than those in the comparison group (P <0. 01) ; The expression of NF - κB in the Dex intervention group are significant decreased than those in the animal model group, and there are significant difference in each group(P <0.01).6. The contents of MPO in the lung tissueThe contents of MPO in the lung tissue in every experimental group are higher than those in the comparison group, and there are significant difference in each group( P <0. 01) . The contents of MPO in the lung tissue of Dex intervention group significantly decrease than those in the animal model group, and there are significant difference in each group (P <0. 01).DiscussionAmniotic fluid embolism is a rare but calamitous obstetric event, the maternal mortality rate reached up to 60 ~ 80%. The pathophysiology of AFE is a MODS syndrome including pulmonary vasospasm, respiration and circulation failure, and DIC. In the past, embolism was considered a primary pathogeny , but nowadays, the key of AFE is thought that the endogenous medium were released when the amniotic fluid entrance the mother body. When the AFE happened , the hypersensitiveness medium in the blood, such as: interleukin, oros-tin, and leukotriene, were significantly increased. Therefore, many scholars considered that it related with hypersensitiveness. Benson et al considered it was a I type anaphylaxis depending on IgE. But Machael et al considered the mechanism of AFE might be a anaphylactoid reaction without antibody participation, and called it a "Anaphylactoid syndrome relation with pregnancy" .Tryptase, which is a neutral protease of mast cells, is most abundant protease in the mast cell granules, which it releases paralleling to histamine at mast cell degranulation. It is a marker of mast cell degranulation. Therefore, determining the change of level of the serum tryptase, it would help us to undersand whether or not there has a degranulation when the AFE happens. Our study find, after the difference property amniotic fluid are infused into the blood circulation of rats, although the levels of tryptase have difference degree increase, contrast to the comparison group, there are no significant difference, it show that degranulation is not significantly increasely, it only hint us that hypersensitiveness phenomenon ever appeared in our body, it is still necessary to study the mechanism of AFE.The complement system, which is composed of about 30 species membra-nine protein and plasma protein, is a perplexing fintity protein hydrolysis system, consisting of C2, C3, C4 et al. In normal physiologic function station, it exert important function to clean pathogenic microorganism and dispose immuno-complex et al. But the abnormity excess activation of it is a genesic foundation of many clinical diseases.Our study find, the average content of serum complement C3, C4 are significantly decreased after infused the amniotic fluid and meconium fluid of rats, it show that the complement system are activated and consumed abundantly in the AFE, it may act as importance action in the pathogenesis of AFE; The study also find, In experimental group Lung tissues have different degree dropsy, a-bundant blood vessel, have a few bleeding in the lung parenchyma, have abundant inflammatory cell soakage around the blood vessel and bronchia, such as leukocyte ( main neutrophil) , macrophage, and lymphocyte. Myeloperoxidase (MPO) exists in the azurophil of neutrophil (PMN) , MPO were released at the activation of PMN, so it is an indicator which sensitively reflects the degree of PMN infiltration. The contents of MPO in the lung tissues in every experimental group are higher than those in the comparison group, especially in the meconium fluid group. The possible mechanism of pathology changes of these as follows: besides the formed elements in meconium fluid bring the machine emblolism leading the more soakage of neutrophilic leukocyte, the complement system was activated and consumed abundantly in the AFE, its activation and spallation would produce the segment C5a which is a stronger leukocyte chemotactic factor ( LCF) , it can recruit and activate leucocyte, resulting in soakage aggregation of leucocyte to injury the lung tissue. Meantime, the complement segment C3a, C5a are effective anaphylatoxin , they make the blood vessel endothelium to contract, and blood plasma to exosmose causing the tissue dropsy and transition of leucocyte. The complement C3b also can couple with majority of phagocytes act as opsonization, accelerate the injury cells were phagocytose. Inaddition, MAC, C5b -9 which is produced by the injury of complement, can directly insert the surface of cells , form a hydrophilic channel, and cause the inside and outside substances of cells to communicate resulting in the cells death. The acti-vation of complement and the production of segment make the cells more activate and release much toxicity oxygen free radical, and cell factors, many cell factors such as leukotriene , arachidonic acid and metabolites et al had been found in the animal model of AFE and acted as a determinate effect. Ultimately, as a result of excess activation of complement, organism autologous tissues were inju-ried by direct and mediately way. The study show that the mass activation of complement may be a main reason of AFE, on the foundation of it, appear the MODS syndrome including pulmonary vasospasm, respiration and circulation failure, and DIC.AFE often accompany MODS, this is also main reason that the AFE has higher mortatily. A great deal of study in the past showed: many media, such as activation trypsogen, cell factor, endotoxin, peroxide and metabolic product of arachidonic acid might articipate morbidity. But these researches and treatments were all studied the isolated and numerous cell factors, couldnt make for us further comprehend the the pathogenesy of the AFE from the molecular level.It is Know to all that the essence of inflammation is a process that the body immunity system clean the foreign body antigens or itself quassation and necrotic histiocyte. The moderate inflammation reaction has protection to the body, but when the body was stimulated excessively, it could induce excessiveness, loss of control or unbridled systemic inflammatory response, resulted in the extensive damage of the autologous tissue structure and physiologic function of parasitifer, ultimately caused MODS even death.Along with the development at full speed of molecular biology, the people have farther knows about the mechanism of excess inflammatory response which was induced by many factors. The recent research results showed that when the inflammation cell of body ( such as neutrophilic leukocyte, monomacrophage, and lymphocyte) were stimulated by the outside stimulation, through the propot-ional receptors, it initiated the signal transduction process inside the cells, activate many kinds of nuclear transcription factors, further caused the replication and transcription of inflammation medium genes, finally caused the super quantity release of many inflammation media.NF - κB , which is a ubiquitous transcription factor, is a convergence pointof many kinds of signal transduction approach, and almost exist in all cells . Not only participated the expression regulation of various gene of immune response , the virus replication, the cell apoptosis and proliferation, but also acted as a key effect in gene regulation inflammation reaction.With the help of the rat model, we observe the expression change of NF -kB , experimental results show: After infuse into the amniotic fluid and meconium fluid, protein expression of NF - κB inside the lung tissue obviously strengthen, it transfers from the cytoplasm to cell nucleus, thus activate the transcription and synthesis of various kinds of media. Many media are activated but it again promote the expression of a great deal of cell factor, produce more cell factors and media, such form vicious cycle, finally caused SIRS with the characteristic of cell autodestruction, and ultimately developed MODS.Our research results show that when the amniotic fluid enters the blood circulation of rat, the complement system is activated to complete the clearance process of the foreign body and foreign antigen, in the clearance process the complement would produce a great deal of segment with inflammation activity media, such as C3a, C4a and C5a et al, they are called the anaphylatoxin, which can combine with the propotinal acceptor of the surface of the mast cells and the basophile granulocyte , promote release of the blood vessel active media such as histamine and tryptase, increase vasopermeability, aggravate the local dysaemia , meantime, C5a is a effective chemotatic factor, it can stimulate the neutrophilic leukocyte to move following the concentration grads of C5a. Many inflammation media which are released by neutrophilic leukocyte cause dropsy and injury of endotheliocyte, if they were excessive, they would made the endotheliocyte injury or even death, and severity restrict the responsiveness of vasculature to vaso - excitor material such as endothelin( ET) , vasotonin A and catecholamine ( CA) et al. Resulting in the extensive or excessive distension of vas-cellum, periphery circulatory disorder, decrease of blood pressure, finally the microcirculation get along with paralysis condition. A great deal of inflammation media not only cause the above - mentioned pathological change, and but also can activate the NF - κB, when the NF - κB is activated, mediate the cell interaction of the endotheliocyte and the leukocyte , make the leukocyte infiltratethe parenchyma cells of each organs, meantime, in the local part by the way of respiratory burst it would produce oxygen free radical, and release many inflammation media, such as lysosomal enzyme, prostaglandin enzyme, leukotriene and PAF, injuryed the capillary vascular endotheliocyte of the every vital organs, increased the vasopermeability, cause the parenchyma cells damage and MODS.This experiment result show that after given a intervention with Dex, the activity of nuclear factor significantly decrease , infiltrateion of neutrophil relieve . Comparing with before intervention , pathology check show that each organ damage obviously relieve . It show the Dex has a regulation function to nuclear factor. Ever since a long time ago, the glucocorticoids was used to cure the amniotic fluid embolism , only thought that it had antiinflammatory and anti - anaphylaxis effect, but its precisely mechanism was not quite understand. The recent research discovered that the educe of glucocorticoids treatment effect was that it intercept NF - κB effect through two pathway, first , the glucocorticoids receptor combined with P65 directly, thus influence the combination of NF - κB and DNA; Second, glucocorticoids promoted production of I kB a.This research result show that after the organism suffered the outside information such as wound, can activate the NF - κB , mediate cell factor release excessively and cause loss of control inflammation reaction and MODS. The Dex can obviously inhibit the activity of NF - κB, influence genetic transcription of many kinds of cell factors in the cell factor network, thus bring extensive influence on the network,thus relieve inflammation process.With the quickly development of the molecular biology technique, many mankind diseases occurrence and development can incriminate the abnormal expression of some genes, thus cause diseases occurrence . So we should enter the current molecular level from whole level and cellular level, further research the concrete pathogenesis, but the NF - κB is a hot spot to study the pathogensis of MODS in recent years, it is a kind of ubiquitous transcription factor, is a convergence point of many signal conduction pathway, through combine with kB locus of target gene , cause target gene to transcription, cause a series of pathologic physiology process. Our research result show that NF - κB may act as a...
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