PPolyHb Resuscitated Severe Rat Hemorrhagic Shock And Pretreatment Effects On Oxidative Stress And Inflammation Of HUVECs

Hemorrhagic shock was the leading cause of death among posttraumatic patient worldwide. Bleeding control, intravascular volume expansion, restoration of vital organ perfusion and tissue oxygenation with fluid resuscitation, as well as maintenance of mean arterial pressure and normothermia remain mainstays of therapy for patients with hemorrhagic shock. The most efficient solution for use in resuscitation is still under debate. Isotonic fluids including isotonic saline solution (NS) and Lactated Ringer's (LR), colloid solutions including hydroxyethyl starch (HES) and dextran, whole blood, blood fraction and balance-ratio component therapy have been approved for resuscitation. However, experimental studies have revealed that resuscitation with NS or LR in the setting of massive hemorrhage contributes significantly to oxyradical formation and systemic inflammation. Specifically, LR increases tissue neutrophil activation, which in turn potentiates superoxide formation and inflammation cascade, which cause systemic inflammatory response syndrome (SIRS).50% patients eventually evolve Acute Respiratory Distress Syndrome (ARDS) following hemorrhagic shock/resuscitation (HS/R) and even may end in multiple organ dysfunction syndrome (MODs). Resuscitation may concern on modulating the immune response and reactive oxygen species (ROS) generation. To improve the oxygen delivery and alleviate ischemia/reperfusion (I/R) injuries, blood is required for repletion of oxygen-carrying capacity on setting of severe hemorrhagic shock and resuscitation except volume expansion and maintenance of organ perfusion with fluid resuscitation. Blood shortage worldwide has been aggravated for potential infectious complications, immune suppression, short-term storage, needs for crossmatching and proper preservation. The design of an alternate therapeutic oxygen carrier for use as an alternative to whole blood or packed red blood cells has become increasingly urgent. Hemoglobin-based oxygen carriers (HBOCs) as an oxygen carrier compounds are promising substitutes for packed red blood cells (PRBCs) in hemorrhagic shock and resuscitation therapy. Our research group designed an HBOCs, Polymerized porcine hemoglobin (pPolyHb), which has been used in 100% blood exchange transfusion and verified to be safety and capable of oxgen carrier and release.Herein, the objective of this work is to investigate the application of pPolyHb in severe rat hemorrhagic shock and resuscitation and the effects on oxidative stress and inflammation of endothelial cells as well as the underlying molecular signal pathways.Firstly, the effects on severe rat hemorrhagic shock and resuscitation with pPolyHb were assessed from the aspects of mean arterial pressure (MAP) and arterial blood gas, oxidative stress and inflammation, as well as histopathology results. All experiments were conducted on pentobarbital-anesthetized SD rat. A controlled hemorrhage was performed to lower mean arterial pressure (MAP) to 35±5mmHg and maintained for 90min and total blood loss about 60 ± 5%(total blood 7%body wet). Different resuscitation fluids plus twice volume loss of NS were perfused at a ratio of 0.5 mL/min from left femoral vein over 30 minutes. The animals randomized into 5 groups (n=6):? Sham;?Normal Saline (NS),?2g/kg pPolyHb (pPolyHb),? 0.6%HES (HES),?self-blood transfusion (Blood). MAP and arterial blood gas parameters were compared. Blood samples were withdrawn to evaluate TNF-a, IL-1?, MPO and MDA in the progress of shock and resuscitation. At 6 h reperfusion, the heart, liver, lung and kidney were biopsied for HE histopathology and TUNEL apoptosis or flash-frozen for analyses of lactic acid (LAC), MPO, MDA and HMGB1. Cys C, NGAL ? KIM-1 were measured to assess renal injury.During the phase of resuscitation, MAP (mmHg) increased and maintained in pPolyHb (84 ± 11) vs. NS (55±10); In addition, pPolyHb more effectively augmented circulating total base excess BE (-6.3±5.7 vs-13.7±4.2) and reduced Lac (3.22±1.5 vs.6.0±2.2) level. pPolyHb as well as Blood resuscitation significantly rectify the metabolic acidosis and hyperventilation. Inflammation cytokines TNF-a and IL-1? level are lower in pPolyHb group than in NS and HES groups (P< 0.05) in plasma. HMGB1, a late proinflammation cytokine was significantly increased in NS group but no difference was observed in the other groups compared to Sham. Taking together, inflammmtion cytokines were suppressed in pPolyHb resuscitated hemorrhagic shock. MPO indicated the neutrophil infiltration. pPolyHb prevented the increase in circulating neutrophils that occurred following NS resuscitation (P < 0.05) and showed no difference with HES and Blood groups (P> 0.05). pPolyHb increased lipid peroxidation marker MDA and showed higher level than HES and Blood (P< 0.05) but no difference with NS group. In a certain degree, pPolyHb suppress the oxidative stress following resuscitation.During hemorrhagic shock and resuscitation, peripheral organs such as kidney, intestine and lung are usually insulted by Ischemia/Reperfusion (I/R) injury. Lac content indicated that pPolyHb significantly reduced anaerobic metabolites compared to NS group (P< 0.05). Lung MPO and MDA amounts increased in all groups (P< 0.05 vs. Sham). The highest MPO and MDA was observed in NS group while there was no difference among the other groups (P>0.05). pPolyHb attenuated MPO level but not MDA. pPolyHb also inhibited HMGB1 increasing observed in NS group and suggested no difference with Blood group, especially in Lung tissue. In the aspect of histopathology, pPolyHb as well as Blood resuscitation was found to reduce the increased tissue injury observed in NS group.Kidney tends to be injury for severe ischemia during hemorrhagic shock and resuscitation. HMGB1 level in kidney was no difference in all resuscitated group at 6h perfusion. Although kidney injury marker (KIM-1) suggested no difference, Cystain C (Cys C) and Neutrophil Gelatinase Associated Lipocalin (NGAL) fell greatly in pPolyHb, NS and Blood group than that in HES groups. In the aspect of histopathology stained and analyzed by HE stain, renal injury degree in pPolyHb was agree with NS group but inferior to Blood group. The same results were suggested by TUNEL apoptosis.Collectively, after 6 h resuscitation, NS causes augment of circulatory and organic inflammation and oxidative level, tissue hypoxia remained, increase of pulmonary neutrophil infiltration, and occurrence of renal hydropic degeneration. pPolyHb could attenuate most of these effects in addition with the same lipid peroxidate and renal injury probablely due to pseudo-peroxidase activity. As an 'oxygen bridge', pPolyHb resuscitated severe hemorrhagic shock will benefits patient.In addition to oxygen carrier capability, pPolyHb can decrease the oxidative stress and inflammation indicated that pretreatment with pPolyHb will be useful in reduce I/R injury. Heme oxygenase-1 (HO-1) was reported play a role in preventing oxidative stress caused by I/R injury for HBOCs. I/R injury have been demonstrated to largely arise from the oxidative stress. During the insult, endothelial cells is one of the determinants initiated PMNs infiltration and activation. The protective effects of polymerized porcine hemoglobin (pPolyHb) pretreatment and the underlying mechanisms were explored against hydrogen peroxide (H2O2)-induced cell viability, apoptosis, and ROS production in human umbilical vein endothelial cells (HUVECs), which were elucidated by analyzed the signaling protein related to apoptosis, survival, and redox. pPolyHb protected cells against H2O2-induced cell death and apoptosis without any cytotoxicity, as evidenced by significant improvement in cell viability and attenuation of LDH leakage, the decrease of apoptotic gene expression (Bax) and activation of anti-apoptotic gene, Bcl2 as well as reducing Cyt C and cleave-Caspase-3 release in HUVECs. Moreover, pretreatment with pPolyHb augmented HO-1 production in a time- and concentration-dependent manner and inhibiting HO-1 expression by Snpp abolished the protective effect of pPolyHb on cell viability and LDH leakage, which demonstrated that HO-1 play a pivotal role underlying these cytoprotective effects. Furthermore, inhibiting HO-1 expression by SnPP increased the activation of JNK/p38 MAPK pathway and intracellular ROS generation suggesting that the cytoprotective effects of HO-1 induced by pPolyHb was involved in regulation of JNK and p38 MAPK pathway and ROS generation.pPolyHb was demonstrated to decrease the inflammation response in hemorrhagic shock resuscitation and the endothelial cell activated in neutrophil infiltration pathogenesis. Moreover, HO-1 was verified to suppress inflammation response. Therefore, the effects of pPolyHb pretreatment on TNF-a induced HUVECs inflammation and the underlying signal transduction were investigated. Cytokine levels, signal pathway activation, as the primary experimental outcomes, was detected by ELISA, Quantitative Real-time PCR, Western blot and immunofluorescence technique respectively after HUVECs exposure to lOng/mL TNF-a. pPolyHb pretreatment inhibited TNF-a-induced expression of MCP-1, ICAM-1, VCAM-1 and MMP-9. These responses were abolished by HO-1 inhibitor SnPP, indicating the role of HO-1 in pPolyHb protective effects. Furthermore, pPolyHb pretreatment was proved to activate p38 MAPK/Nrf2 signal pathway and induce HO-1 gene expression. Typically, TNF-a markedly stimulate expression of inflammation cytokines including MCP-1, ICAM-1, VCAM-1 and MMP-9 through NF-?B pathway. pPolyHb can inhibit NF-?B pathway and crosstalk with HO-1 pathway. During the phase of pPolyHb pretreatment, intracellular glutathione (GSH) was decreased and pPolyHb Fe3+ content increased, which suggested that ROS may play a role in pPolyHb protective effects. Taken together, theses results demonstrated that protective effects of pPolyHb pretreatment on TNF-a-induced HUVECs was mediated by ROS/p38 MAPK/Nrf2 dependent HO-1 expression and NF-?B pathway suppression.In conclusion, these studies suggested that pPolyHb can attenuate oxidative stress, inflammation and tissue injury during hemorrhagic shock and resuscitation and the benefits outweigh the risks when administration in severe hemorrhage patients. pPolyHb pretreatment can induce HO-1 whereby activation of ROS/p38 MAPK/Nrf2 axis and play a role in counteracting with oxidative stress and inflammation. pPolyHb is a promising therapy strategy in organ preservation, elective cardiac surgery, anemia and acute cardiovascular Diseases.