| Hemorrhagic shock is a kind of common clinical diseases. It can bring about failure of gut barrier function and intestinal bacteria/endotoxin translocation (BET) during the developing process of hemorrhagic shock, so as to induce gut inflammation and distant multiple organs dysfunction, such as lung, liver, heart, kidney and so on. The result is that multiple organs dysfunction syndrome (MODS) happens, which has done harm to human health and often leads to death. Its mechanism may be associated with systemic inflammatory response syndrome (SIRS) caused by shock and intestinal endotoxemia. Lymphology is a weak field in circulation system study, and for a long time, it has been considered that lymph circulation is an assistant part for blood circulation. Our previous study showed that lymphatic microcirculation had close relations with the occurrence, development and outcome of shock;mesenteric lymph duct ligation could block reflux of intestinal lymphatic circulation, lighten multiple organs injury and inflammation reaction in two-hit rats. Meanwhile, mesenteric lymph can activate neutral granulocytes and promote occurrence of acute lung injury. If mesenteric lymph duct is ligated in advance, acute lung injury can be prevented. Hence, attentions are paid to changes of lymphatic microcirculation in shock and organ injury caused by shock.Objective:To further investigate the effects and mechanisms of mesenteric lymph in shock pathogenesis;to study the effects of mesenteric lymph duct ligation on the changes of structure and function of organs and level of inflammatory mediators in rats with hemorrhagic shock, such as free radical (FR), nitric oxide (NO), tumor necrosis factor-alpha (TNFα), and interleukin (IL) in lungs, heart, kidney and liver;to illustrate the effect of shock lymph on metabolic vitality, proliferation cycle, mRNA expressions of apoptosis related genes and inflammatory mediators in pulmonary micro-vascular endothelial cells (PMVEC) in rats;to explore molecular mechanism of shocklymph in the vital organs injury, such as lung, heart, kidney and liver, so as to provide experimental evidence for pathogenesis mechanism of multiple organs injury in shock .In this study, the damaging effectof mesenteric lymph duct ligation to block lymph on multiple organs of hemorrhagic shock rats was observed in vivo, and the pathogenesis was explored as well;meanwhile, the damaging effect of shock lymph on PMVEC was investigated in vitro in order to illustrate cell molecular machamism of shock lymph on the injry of multiple organs.Methods:1) Hemorrhagic shock modelMale rats were anesthetized with sodium pentobarbital (50mg/kg) injected intramuscularly and the right carotid artery and left jugular vein were isolated by minimal dissection and aseptically cannulated respectively with a catheter containing heparinized saline. In the shock group and the ligation group, blood was withdrawn from the right carotid artery to mean arterial pressure of 40mmHg and were maintained at this level for 90 minutes by adjusting the liquid level of Lamson's bottle containing aseptic Ringers bottle to the fixed height (volume of blood loss should be one fifth of whole blood volume, and the whole blood volume should be calculated as one thirteenth of the body weight). At the end of the shock period, rats were resuscitated by infusing all shed blood and Ringers fluid (infusing volume was equal to whole blood volume) from left jugular vein with micro-volume infusion pump, and the infusing time should last more than 20 minutes, and rats in the sham group were anesthetized and cannulated as described above, but no blood was withdrawn or infused.2) Assessment of survival rate in ratsThirty Wistar male rats were randomly divided into three groups: the mesenteric lymph duct ligation group, the sham group and shock group (n=10). In the shock group and the ligation group, hemorrhagic shock model was established according to method 1, following which were prepared sterilely and received laparotomy of 5cm, then the mesenteric lymph duct was exposed, but the mesenteric lymph duct was ligated in the ligation group and in the shock group the mesenteric lymph duct was threaded without ligation. In sham group, rats were only anesthetized and operated on jugular area and abdomen without any blood loss and resuscitation. The survival rates of all rats for 24 h were recorded.3) Structure and functions for organsEighteen Wistar male rats were randomly divided into three groups: the ligation group, the sham group and the shock group (n=6) in order to observe the structures and functions of the organs. The hemorrhagic shock models were established according to method 1 and 2. All the rats in three groups were respectively anesthetized once more after 6h of shock resuscitation, made blood loss so as to make serum sample. The indexes of pH, PaO2, and PaCO2 in artery blood, which can show the lung function, were determined by the automatic blood gas and acid-alkali analyzer. Automatic biochemical analyzer tested the biochemical indexes of AST, ALT, BUN, Cre, LDH-1 and CK-MB, which can illustrate the function of liver, kidney and heart. Then the rats were killed, selected fixed position on lung, liver, heart and kidney so as to prepare pathological tissue section to observe structural changes of lung, liver, heart and kidney.4) Measurement of inflammatory mediatorSeventy-eight rats were randomly divided into three groups: the ligation group, the sham group and the shock group, which were used to prepare homogenate and determine inflammatory mediators at different time. Hemorrhagic shock models were made according to method 1. Rats were killed respectively according to following different times: for the rats in the shock group, at the end of shock period and Oh, lh, 3h, 6h, 12h, 24h after resuscitation (each time point n=6), for the rats in the ligation group, at lh, 3h, 6h, 12h and 24h after resuscitation (each time point n=6), for the rats in the sham group, at 24h which was equal to the time for the ligated rats of resuscitation (n=6). The lungs, livers, kidneys and cardiac muscles that had been stored at -80° C were weighed, homogenized in aseptic normal saline, and then centrifused at 2500 r/min for 10 mins. The SOD, NOS activity, MDA, NO2 /NO3 contents in the supernatant were assayed by a standard spectrophotometric technique. The TNFot and IL-6 contents were determined with ELISA. To assess neutrophil accumulation in the above all organs, myeloperoxidase (MPO) activity in supernatant was measured with spectrophotometric technique. Protein in organs was estimated by coomassie brilliant blue method. The iNOS mRNA expression in lung was assayed by RT-PCR.5) PMVEC cultureThe Wistar male rats weighing 70-100g were disinfected with 75% alcohol after anesthetized with pentobarbital sodium. Their chest cavity was opened and the lungs were taken out and then were put into the cold Hank's liquid. After taking away the residual blood, carefully cut the lungtissue into lmm3 sections, which would be put into the labeled sterile culture bottle and were left for solidification for 2h in culture apparatus. After this process, add DMEM was added into culture bottle. When the cells converged into mono-layer, continued the passage culture after digested by trypsin. Third passage of PMVEC was used in this study.6) Mesenteric lymph and blood collectionIn the shock group, blood was withdrawn from the right carotid artery to mean arterial pressure of 40mmHg and was maintained at this level for 90 minutes. At the end of the shock period, rats were resuscitated by infusing all shed blood and Ringers fluid from left jugular vein, mesenteric lymph duct and portal vein were cannulated with a catheter containing heparinized saline in different rats. The sham rats were anesthetized and canulated as described above and lymph and portal vein blood were collected, but no blood loss and infusion. The mesenteric lymph and portal vein blood was continuously collected into sterile heparin-wetted iced tuberculin syringes for 2h and 2 minutes, respectively. The collected lymph and portal vein blood was centrifuged at 400g for 15 minutes at 4°C to remove all cellular elements, the humoral cell-free mesenteric lymph and plasma samples were frozen at -80° C for following study.7) PMVEC injury and viability assaysThe experiment was done in six groups: Group A (2% fetal bovine serum group), the culture solution is DMEM+2% fetal bovine serum;Group B (Normal lymph group) the culture fluid is DMEM + normal lymph;Group C (shock lymph group) the culture solution is DMEM + shock lymph. Group D (normal plasma group) the culture solution is DMEM + normal plasma. Group E (shock plasma group) the culture solution is DMEM + shock plasma. Group F (control group), the culture solution is only DMEM. Then the influence of normal lymph, shock lymph, normal plasma and shock plasma on PMVEC with light microscope, electron microscope and scanning electron microscope was observed at the following different concentrations and different cultured time: Concentrations (v/v, final concentration): 2%, 4%, 6%, 8%, 10%;time points: 4h, 8h and 12h. The PMVEC viability was measured by the mitochondrial tetrazolium (MTT). The DNA was dyed with propidium iodide (PI), the proliferation cycle and the apoptosis of the cultured PMVEC was observed with flow cytometry. At the same time, perform electrophoresis analyze on DNA of cell nucleus to observe the effect of shock lymph on PMVEC apoptosis.8) Inflammatory mediator and apoptosis related gene mRNA expressionPMVEC were treated for 6h by 4% collected lymph and plasma samples in method 6, digested by trypsin and the cell suspension was prepared. The total RNA was extracted by UNIQ-10 Trizol total RNA extract kit, then the cDNA was reverse transcripted and stored in -20 °C. According to the protocol of the AMV RT-PCR kit, /?-actin was used as an interior control and the mRNA expression of both the apoptosis related gene such as bcl-2, bax, Fas and FasL and the inflammatory mediator such as TNFa, IL-6, iNOS and VEGF were determined quantitatively by RT-PCR. The PCR product was analyzed by gel electrophoresis with 1.5% agarose, and the density of the strap were scanned and analyzed after taking photo by UV light. The ratio of gray scale between target gene and /3-actin gene was calculated, which indicated the related expression of the target gene.9) Measurement of the toxic substance in samples of blood and lymph24 Wistar male rats were used in this experiment, the samples of the shock lymph and plasma, the normal lymph and plasma were collected as described above in method 6 (n=6). All samples were collected in the heparinized pyrogen-free glass tubes, separated by centrifugation, frozened and strored immediately at -80 °C until the time of assay. The endotoxin level of the above samples was determined by the improved LAL quantitative method. The contents of TNFa and IL-6 were determined according to the above method 4.Results:1) The mesenteric lymph reflux in the hemorrhagic shock rats had injury effects on the function and structure of lung, liver, heart, kidney. After mesenteric lymph duct ligation and blocking the reflux of lymph, the damage on the function and morphology of these important organs could be relieved so that the rats' death rates could be lowered as well.2) Contents of the cytokine and proinflammatory mediators in organs were influenced by blocking the reflux of mesenteric lymph. The MDA and NO contents, NOS and MPO activity, TNFa and IL-6 level of lung, liver, heart, kidney of hemorrhagic shock rats were higher than that of sham group at the different time after resuscitation, and SOD activity was significantly lower than that of sham group (P<0.0\, P<0.05). For the ligation group after resuscitation and blocking the reflux of mesenteric lymph, the MDA and NO contents, NOS and MPO activity, TNFa andIL-6 level of lung, liver, heart, kidney were higher than that of sham group at different time, and SOD activity was significantly lower than that of sham group (P<0.0\, P<0.05). After mesenteric lymph duct ligation for 6h and 12h, the MDA and NO contents, NOS and MPO activity and TNFa and IL-6 level of the organs began to decrease at different degree, SOD activity increased and had no significant difference with that of sham group (/>>0.05). The MDA and NO contents, NOS and MPO activity, TNFa and IL-6 level of each organ homogenate in ligation group were markedly less than that of shock group at 6h, 12h, 24h and also the SOD activity began to increase more than that of shock group (PO.01, PO.05). The results of iNOS mRNA expression in lung showed that iNOS mRNA expressions in shock group after resuscitation at 3h, 6h, 12h, 24h and that of ligation group at 3h, 6h were all higher than that of sham group. And iNOS mRNA expression in ligation group after resuscitation at 6h, 12h, and 24h were all significantly lower than that of shock group (PO.01).3) The primary cultured technique of PMVEC was established and the results showed that mesenteric lymph from shocked rats affected the morphology and proliferation cycle of PMVEC. After PMVEC was treated with mesenteric lymph and plasma from shocked rats, normal lymph and plasma with different concentrations, the observation showed that shock lymph had damaged the morphology of PMVEC with the concentration increasing of shock lymph and prolonging of function time. Shock lymph with 4% concentration could cause some cells shrinked and turned circular gradually after action for 4h. After 6h cells got apoptosis, and apoptotic bodies could be observed under electron microscope. After shock lymph with 10% final concentration affected on PMVEC for 12h, all the cells were broken into pieces. The shock plasma had the similar effect but weaker than that of shock lymph. However, normal lymph and normal plasma with same concentration showed no effect. The PMVEC viability determined by MTT displayed that: with the concentration increasing of shock lymph, the proliferation and viability of PMVEC were gradually inhibited, and dose-effect relationship appeared within definite concentrations. The viability of PMVEC had been remarkably lower than that of control group, after shock lymph with 4% final concentration functioned on it for 12h (PO.01). The results assayed by flow cytometre demonstrated that with the concentration of shock lymph increasing and when the dosage for inducing apoptosis was reached (final concentration 4%), the Go/Gi ratio increased, S and G2/M ratio decreased;with the concentration of shock lymph increasing and the action time prolonged,apoptosis rate of PMVEC increased correspondingly. And this result suggested shock lymph had the effect on inhibiting proliferation of PMVEC and promoting apoptosis and necrosis. The results of DNA electrophoresis analysis for cell nucleus showed that: after shock lymph with 4% final concentration functioned on PMVEC for 6h, DNA of cell nucleus schizolysised into 180-200bp and the oligoucleotide fragments which were integer times of them, so the typical DNA ladder pattern for apoptosis formed. After shock lymph with 10% final concentration affected on PMVEC for 4h, cell nucleus DNA schizolysised into irregular oligoucleotide fragments so as to form disperse electrophoresis. The other groups had no effect on the formation of DNA ladder pattern of apoptosis after functioning on PMVEC.4) The injury effect of shock lymph on PMVEC had relation with expression for inflammatory mediators, apoptotic-related gene and toxic substances that were higher than that of shock plasma. Shock lymph with 4% final concentration and shock plasma with same concentration treated PMVEC for 6h, then compared with the results of normal lymph, normal plasma, fetal bovine serum and DMEM, the mRNA expression of bax, Fas and Fas L and level of TNFa, IL-6 and iNOS significantly increased, and the mRNA expression of bcl-2 and VEGF obviously decreased (/><0.01, P<0.05);and in the shock lymph group, the mRNA expressions of bax, Fas, Fas L, TNFa, IL-6 and iNOS were remarkably higher than that of shock plasma group, and the mRNA expressions of bcl-2, VEGF were significantly lower than that of shock plasma group (P<0.01). The measurement of toxic substance in shock lymph, shock plasma, normal lymph and normal plasma showed that: the contents of endotoxin, TNFa and IL-6 in shock lymph obviously are higher than that of shock plasma, normal lymph and normal plasma (.PO.01).Conclusions:1) Blocking the reflux of mesenteric lymph can lower the death rate with shock rats;improve organ function and histological injury. Its mechanism is related to reduction of intestinal endotoxin translocation, reducing free radical injury, degrading of releasing and expression for inflammatory mediators such as NO, TNFa, IL-6 and so on, decreasing of neutrophils sequestration.2) Shock lymph could lead to the damage of PMVEC, and that is stronger than that of shock plasma. Its mechanism is related to that shock lymph increases the TNFa, IL-6, and iNOS mRNA expressions, promotes the bax, Fas, Fas L mRNA expressions, inhibits bcl-2 and VEGF mRNAexpressions, Simultaneously, it's also relevant with the high concentration of endotoxin, TNFa, IL-6 in shock lymph.3) The results suggest that the mesenteric lymph plays an important role in pathogenesis of shock.. Taking blocking the reflux of lymph as an aim can provide new method for preventing from shock and protection for multiple organs after shock. |
