The Protective Effect Of Polydatin On Lung Injury After Hemorrhagic Shock In Postpartum Rabbit

Postpartum hemorrhage(PPH) is considered the leading cause of pregnancy-related deaths worldwide, with an estimated 140,000 women dying annually from this complication. Especially in developing coutries, mortality from PPH has remained high despite international efforts to decrease maternal mortality since the launch of the Safe Motherhoold Initiative in 1987. In our country, postpartum hemorrhage is the major reason of maternal death and morbidity.After hemorrhagic shock induced by postpartum hemorrhage, the systemic inflammatory response was activated and the first organ injured was lung. In the last five years it has gained recognition that transfusion of blood products, a treatment method to postpartum hemorrhage could induce acute lung injury by itself, which is called Transfusion-related acute lung injury. One word, acute lung injury (ALI) is the major reason for peripartum women's entering in Intensive Care Unit. For peripartum women, ALI is readily develop into acute respiratory distress syndrome (ARDS), multiple organ dysfunction syndrome (MODS), which results in death finally.Even until now, the mechanism of lung injury after shock is still unclear. It is widely accepted that polymorphonuclear is the major cell mediating this process. The unbalanced between over expression of pro-inflammatory mediators and the relatively less expression of anti-inflammatory mediators, over-activated polymorphonuclears, ischemia-reperfusion injury and cellular apoptosis is major mechanism leading to the ALI/ARDS after hemorrhagic shock. The over-activation of nuclear factor-kappa B (NF-κB) could lead to the synthesis and release of inflammatory mediators, promote the development of ALI and ARDS.Polydatin is extracted from a traditional Chinese herbal medicine, Polygonum cuspidatum,3,4',5 trihydroxystibene-3-monoglucoside. and has a special effect in shock treatment in terms of improvement of heart function and microcirculatory insufficiency.OBJECTIVEBecause of the beneficial effects of Polydatin in improving microcirculation and prolongation of survival time of pregnant rabbits in hemorrhagic shock. We speculated that it could also improve lung injury after hemorrhagic shock in postpartum rabbit. So, in the present study, we tested the efficacy of Polydatin in improving lung injury in a controlled hemorrhagic shock in postpartum rabbit.METHODS40 anesthetized New Zealand white rabbits with 6 hours within postpartum were underwent controlled hemorrhagic shock by bleeding via femoral artery to mean arterial pressure (MAP) of 40-45mmHg. The experiment consisted of two phases, shock phase (T0-T60min) and resuscitation phase (T60-T360min). Animals were randomly divided into four groups (n=10 per group); The first group, SS ("sham shock"), underwent a sham operation in which they were subjected to all the surgical procedures experienced by the hemorrhaged animals but were not hemorrhaged. The second group, SH ("shock"), underwent hemorrhage but without resuscitation. The next two groups, NS, PD, underwent hemorrhage and received a single volume infusion of 4ml/Kg normal saline or Polydatin(1mg/ml) at T60 followed by transfued with half of the heparinized shed blood. At T360, three animals from group SS, NS, PD were euthanized for lung tissue harvest for further test(all the animals in group SH were died during 2-3 hours after hemorrhagic shock and were immediately harvested the lung for further examination); while to the remain animals, the cannulas were then removed after ligation of the femoral artery, femoral vein. The animals were placed in a caged with food and water and observed for survival. Mean arterial pressure (MAP) were continuously monitored, Blood samples for hematocrit(Hct), alanine aminotransferase (ALT), Creatinine (Cr), PaO2, PaCO2, PaO2/FiO2, lactate, pH, Tumor necrosis factor-alpha (TNF-α), interleukin-10(IL-10) were taken at baseline, 60min post bleeding (Postbleeding 60 min),20min post drug (Postdrug 20 min), 280min post transfusion of blood products (Posttransfusion 280min). Lung tissue examination included pathological exmaination, wet weight/dry weight ratio, myeloperoxidase (MPO) assay,Malondialdehyde (MDA), the expression of Intercellular adhesion molecule-1(ICAM-1) mRNA, Heat shock protein-70(HSP-70) and NF-κB. Liver and Kidney underwent pathology examination with HE stained. Statistical Analysis:results are presented as mean±standard deviation (SD) unless otherwise noted, and analyzed by two-way analysis of variance with repeated measures over time. Post hoc analyses were performed with Student-Newman-Keuls multiple comparisons test. The survival time was analyzed by Kaplan-Meier plots and compared by the Breslow test. Analysis was completed with a statistical software package for desktop computers (SPSS13.0). P≤0.05 (two-sided) was considered significant.RESULTSBasic status and Blood lossAll groups were comparable in baseline measurements (such as age or body weight) and the volume of blood withdrawl via the femoral artery for induction and maintenance of shock (P>0.05)Hemodynamic changesMAP of the rabbits before hemorrhage was between 94.2±97.3 mmHg. After blood withdrawl via femoray artery, MAP decreased markedly over a 15 min period (T0-T15) to a comparable values of 40～45 mmHg in all of the hemorrhaged groups. In the sham shock group SS, MAP remained stable at normotensive levels throughout the experiment. In the group SH, in which the hemorrhagic shock was not resuscitated, MAP remained stable at hypotensive levels and died 2-3 hours after hemorrhage. Immediately after polydatin infusion, the MAP of PD group recovered to (70.5±0.9) mmHg, which was significantly higher than the NS group (50.9±0.9) mmHg and remained at that level for about 10 minutes. After that, MAP was gradually decreased to (58.4±0.8) mmHg, which is also higher than the relative time point of NS group, which was (46.5±0.8) mmHg. After twenty min of drug or normal saline infusion, half of the heparinized blood was reinfused to group NS and PD. In the following time, MAP of group PD increased to (78.2±0.7～81.7±1.0) mmHg, which was significantly higher than that in group NS(68.5±1.0～73.2±0.7) mmHg (P<0.05).HCTAll groups showed a significant reduction of Hct after hemorrhagic shock and resuscitation, no significant difference was found between group NS and group PD.Serum TNF-αand IL-10 assayThe blood concentration of TNF-αand IL-10 was increased after hemorrhage and peak at 20min after drug (equal to 280min after hemorrhage) and decreased at 6 hour after hemorrhage. At 6 hour after hemorrhage, the blood concentration of TNF-a was significantly lower in PD group compared with NS group(P<0.05), while no significant differences was found between these two groups.PaO2, Pa CO2 and PaO2/FiO2No significant differences was found between PaO2, PaCO2 and PaO2/FiO2 in the baseline between four groups.60 minutes after bleeding, animals respiratory rate increases, PaO2 and PaO2/FiO2 was increased while PaCO2 was decreased.280 minutes after transfusion, PaO2 and PaO2/FiO2 was higher in PD group than NS group, no significant differences was found in PaCO2 between PD, NS and SS group.Lactate and pH level There was significant metabolic acidosis as shown by the decrease in pH from 7.41～7.43 to 7.25～7.27 and the increase in lactate increased from 1.4～1.7 to 8.6～9.7 mmol/L. Although, lactate tended to decrease, and pH value tended to increase at 280min after transfusion of blood product in group NS and group PD compared with shock period(P<0.05), all of which had not recovered. However, in PD group, lactate was significantly lower, while pH value was significantly higher than corresponding values in the group NS.Lung tissue Malondialdehyde (MDA) contentAt the end of experiment, the lung tissue MDA content was increased in all the shock groups compared to SS group. The animals in PD group had significantly lower MDA content than those in SH or NS group (P<0.05).Lung tissue HSP-70 expressionCompared with SS group, HSP-70 expression in lung tissue is up-regulated in the other three groups, and the up-regulation of HSP-70 expression is much evident in PD group than that in other three groups(P<0.05).Lung tissue NF-κB expressionThe down-regulation of NF-κB expression in lung tissue is much evident in PD group than that in SH or NS group (P<0.05). The expression of NF-κB is similar between PD and SS group (P>0.05)Lung tissue NF-κB activityLung tissue NF-κB activity was significantly decreased in PD group than that in SH or NS group (P<0.05). The activity of NF-κB was similar between PD and SS group (P>0.05)Lung tissue mRNA expression of ICAM-1Compared with SS group, the mRNA expression of ICAM-1 was significantly higher in other three groups. Compared with NS group, PD inhibited the mRNA expression of ICAM-1 (P<0.05).Lung MPO activity, wet weight/dry weight ratio, and lung injury score The lung MPO activity, wet weight/Dry weight ratio and lung injury score was significantly lower in PD group than those in NS or SS group, but was significantly higher than those in SS group (P<0.05).Serum ALT content & Serum creatinine contentNo significant differences was found in serum ALT or creatinine content in the baseline between four groups.60 minutes after bleeding, both ALT and creatinine were increased in SH, NS, PD group.20 minutes after Polydatin administration, ALT was decreased, while AST was still increased. Until blood transfusion, both creatinine and ALT was decreased, while they were still not recovered to the baseline level.ALT in the PD group was significantly lower than that in NS group, while no significant difference was found in creatinine between the two groups.Liver and Kidney pathology examinationLiver and kidney in the SS group were almost normal. Hemorrhagic shock resulted in severe liver hemorrhagic edema with inflammatory cell inflitration and vacuolar changes of renal tubular epithelial cell. Compared with NS group, Polydatin reduced inflammatory cell infiltration in the hepatic gate-duct area. No significnat differences was found between NS and PD group in the kidney pathological examination, both were shown cell enlargement and vacuolar changes in the renal tubular epithelial cells.Survival timeMedian survival time in PD group (22.0±1.3 hours) was significantly longer than that in NS group(10.0±0.6 hours) (P<0.05).CONCLUSIONPolydatin has protective effect on hemorrhagic shock induced lung injury in postpartum rabbit, which maybe mediated by alleviates ischemia-reperfusion injury, induces HSP-70 expression, inhibits overactivation of NF-κB and mRNA ICAM-1 expression in lung tissue, finaly decrease lung tissue PMN infiltration and ameliorate lung injury. Polydatin administration could improve liver function while has no obvious effect on kidney function after hemorrhagic shock in postpartum rabbit.