The Effects Of Different Synthetic Colloids On Hemorheology And Inflammatory Response In A Rat Hemorrhagic Shock Model And The Anti-inflammatory Mechanisms Of CNP

Hemorrhagic shock (HS) is the leading cause of death in civilian and militarytrauma. Fluid resuscitation is a common intervention for the management of HSvictims to maintain the organ perfusion and reduce tissue injury. However, afterresuscitation, the initial survivors of HS are still susceptible to systemic inflammatoryresponse syndrome (SIRS), which triggers multiple organ dysfunction syndrome(MODS) and post-traumatic death. Previous research have indicated that oxidativestress, inflammation respone, and microcirculation disturbances contribute to theoccurrence of MODS. Fluid resuscitation may contribute to oxidative stress andinflammation due to reperfusion injury. Oxidative stress and inflammation directlypromotes the development of gut barrier dysfunction; this, in turn, causes endotoxintranslocating to the systemic circulation, leading to the occurrence of SIRS andMODS. Moreover, Hemorrhagic shock/resuscitation (HS/R) can affect thehemorheological parameters, futher correlate with the microcirculation, which isdirectly related to the occurrence of MODS. Therefore, ideal resuscitation strategiesshould suppress oxidative stress and the systemic inflammatory response and improvehemorheological parameters and microcirculation after HS in addition to maintainingeffective organ perfusion. Previous studies have indicated that the choice ofresuscitation fluid correlates with the hemorheological parameters, oxidative stress,and inflammation response, which are directly relate to the treatment of hemorrhagicshock. In our previous investigation, there are still lack of unified standards for thechoice of resuscitation fluid in treatment of hemorrhagic shck. Due to the differencesin animal model, infusion dose, examination index, systemic comparisons ofoxidative stress, inflammatory response, and hemorheological parameters aftercommonly used synthetic colloid infusions in hemorrhagic shock are lacking. In part1and2, we evaluate the effects of synthetic colloids on hemorheology, oxidativestress, and the inflammatory response in rat HS model, to provide evidence for thechoice of fluids for the early treatment of hemorrhagic shock. With the enhanced understanding of of pathophysiology of hemorrhagic shock,the present study has upgraded fromthe whole organ level into to cellular andmolecular levels. The discovery of new, safe, and effective anti-shock drugs withclear mechanism shows broad prospects for the treatment of hemorrhagic shock.C-type natriuretic peptide (CNP), one member of the natriuretic peptide family, playsan important role in the regulation of vascular homeostasis, not only as regulators ofblood volume but also by directly altering vascular reactivity. In the treatment ofhemorrhagic shock, CNP play a role by reducing tissue edema, improvingmicrocirculation, and protecting renal function, and is a potential new anti-shock drug.Previous study have reported local expression of CNP inhibits the expression ofadhesion molecules suppresses in injured carotid arteries in rabbit, indicated that CNPmay exert a protective anti-inflammatory effect. However, the effect of CNP onLPS-induced inflammation in endothelial cells still requires further verification. Inparticular, the mechanisms underlying the anti-inflammatory effects of CNP on thehuman vasculature, specifically on endothelial cells, are still largely unknown. In part3, we investigated whether CNP exerts anti-inflammatory effects via the attenuationof LPS-induced endothelial activation in vitro model of the HUVECs. In addition, theunderlying mechanisms and intracellular signaling pathways affected by CNP inLPS-stimulated endothelial cells were investigated.Part1: Effects of synthetic colloids on hemorheology in vitro and in hemorrhagicshock modelMethods: For the in vitro experiments, the Wistar rats were anesthetised using sodiumpentobarbital, which were injected intraperitoneally. The right femoral artery and veinwere isolated with minimal dissection and catheterised using polyethylene catheters(PE-50) containing heparinised saline. Fresh blood samples were obtained from theright femoral arterial catheter using heparin-treated tubes. The rat blood wascentrifuged and adjusted to a hematocrit of40%by adding or removing autologousplasma. The well-distributed erythrocyte samples were subsequently diluted with6%HES130/0.4(HES130),6%HES200/0.5(HES200), or4%succinylated gelatin(GEL) at volume ratios of5:1and3:1, respectively. The control group consisted ofthe erythrocyte suspension without dilution. All samples were incubated for15min at37°C and then blood samples were adjusted to a hematocrit of40%before measuringerythrocyte deformability (elongation index, EI), erythrocyte aggregation index (AI),and aggregation amplitude (AMP). The plasma was obtained using centrifugation to detect the viscosity.For the in vivo experiments, the rats were anesthetised using sodiumpentobarbital, which were injected intraperitoneally. The right femoral artery and veinwere isolated with minimal dissection and catheterised using polyethylene catheters(PE-50). After surgical preparation and10min of stabilisation, a volume-controlledhemorrhage of18ml/kg (approximately30%of total blood volume) was performedusing pumps at a rate of0.26ml/min for30min through the right femoral arterialcatheter. Rats were then subjected to a slower hemorrhage of12–15ml/kg(approximately20–25%of total blood volume) a rate of0.13ml/min for35min. Theanimals with a base excess of-9to-12mmol/L were resuscitated after bloodwithdrawal via the femoral venous catheter. After the volume-controlled hemorrhagewas induced, the animals were randomly assigned to one of four groups. The shamgroup underwent all instrumentation procedures without blood collection. The otherthree groups each received a different colloid solution (HES130, HES200, GEL).The volume of resuscitation for the colloid solutions was equal to that of the bloodwithdrawal. After2hrs had elapsed following resuscitation, blood samples werecollected from the femoral artery with heparin-treated tubes for hemorheologicalevaluation. Hemorheology was assessed through the plasma viscosity, erythrocytedeformability (elongation index, EI), erythrocyte aggregation index (AI), andaggregation amplitude (AMP).Results: In the in vitro study, the plasma viscosity was significantly increased in thepresence of GEL, HES130, and HES200compared to that of the control group. Theplasma viscosity in the HES200group was lower than that in the GEL group.Moreover, there was no significant difference between the values in the HES130andHES200groups. In the in vivo study, plasma viscosity increased significantly in GELgroup and decreased significantly after the infusion of HES130and HES200compared to the sham group. There was no significant difference between the valuesin the HES130and HES200groups.In the in vitro study, for the ratio of3:1, the erythrocyte deformability, asmeasured using EI, significantly decreased in the presence of the three plasmaexpanders at a shear rate of100s-1. However, the EI values at high rates (>100s-1) didnot differ significantly among the groups. For the ratio of5:1, there were nosignificantly differences among the groups at the various shear rates. In the in vivostudy, the EI values at various shear rates for the study groups were not significantly different from those of the sham group.For the ratio of3:1, GEL induced hyperaggregation by significantly increasingthe AI and AMP compared to those of the control group. AI was significantly lower inthe HES130and HES200groups compared to the GEL group; AMP wassignificantly reduced in the HES200group as compared to that of the GEL group.There were no significant differences among HES130, HES200, and the controlgroups with respect to AI and AMP. For the ratio of5:1, GEL induced a dramaticelevation in AI and AMP compared to the control group. The AI and AMP weresignificantly reduced in the HES200group compared to those in the GEL group.There were no significant differences among HES130, HES200, and the controlgroups with respect to AI and AMP. In addition, there were no differences for AIamong the various ratios. The AMP values in the HES130and HES200groups forthe ratio of3:1were significantly increased compared to those for the ratio of5:1. Inthe in vivo study, there was no significant difference among the groups with respect tothe AI values in vivo. GEL induced a significant elevation in AMP compared to that ofthe sham group. The AMP values in the HES130and HES200groups weresignificantly lower than that of the GEL group. There were no significant differencesamong HES130, HES200, and the sham groups with respect to AMP.Conclusions: Hydroxyethyl starch didn’t change erythrocyte aggregation comparedto the control in a rodent hemorrhagic shock model. GEL significantly accelerates theerythrocyte aggregation and elevates the plasma viscosity compared to hydroxyethylstarch.Part2: Effects of synthetic colloids on oxidative stress and inflammatoryresponse in hemorrhagic shockMethods: The rats were anesthetised using sodium pentobarbital, which were injectedintraperitoneally. The right femoral artery and vein were isolated with minimaldissection and catheterised using polyethylene catheters (PE-50). After surgicalpreparation and10min of stabilisation, a volume-controlled hemorrhage of18ml/kg(approximately30%of total blood volume) was performed using pumps at a rate of0.26ml/min for30min through the right femoral arterial catheter. Rats were thensubjected to a slower hemorrhage of12–15ml/kg (approximately20–25%of totalblood volume) a rate of0.13ml/min for35min. The animals with a base excess of-9to-12mmol/L were resuscitated after blood withdrawal via the femoral venous catheter. After the volume-controlled hemorrhage was induced, the animals wererandomly assigned to one of four groups. The sham group underwent allinstrumentation procedures without blood collection. The other three each received adifferent colloid solution (HES130, HES200, GEL). Synthetic colloid solutions wereresuscitated using the same volume as blood withdrawal. Blood gas analysis wasperformed at baseline, after blood withdrawal, and2hrs after resuscitation. All of theanimals were euthanized under anesthesia2hrs after resuscitation. MDA and MPOativity levels in tssue samples were determined. In addition, the intestinal levels ofTNF-α and IL-6were also assayed.Results: The pH, pCO2, pO2, and BE values were not different between groups atbaseline. No significant differences in pH, pCO2, pO2, and BE values were observedin the groups that underwent hemorrhagic shock at the end of hemorrhage. MDAconcentrations in the liver, lungs, intestine, and brain of rats that were resuscitatedwith HES130were all significantly lower compared to GEL group. HES130significantly suppressed the elevation of MDA levels in the liver, intestine, and braincompared to HES200, but similar MDA levels were observed in the lungs. Nosignificant difference was observed between the HES200and GEL groups in alltissues. MPO activity in the liver, lungs, intestine, and brain in the HES130groupwere significantly reduced compared to the HES200group. The infusion of HES130also decreased MPO activity in all measured tissues compared to the GEL group. Nosignificant difference between the HES200and GEL groups were observed in all fourtissues. The HES130group significantly suppressed intestinal TNF-α and IL-6elevation compared to the HES200group. Only intestinal TNF-α was lower in theHES130group than the GEL group. However, no statistically significant differencesin TNF-α and IL-6levels were observed between the HES200and GEL groups.Conclusions: Hydroxyethyl starch130/0.4, but not hydroxyethyl starch200/0.5andsuccinylated gelatin, treatment after hemorrhagic shock ameliorated oxidative stressand the inflammatory response in this rat model. No significant differences wereobserved after hydroxyethyl starch200/0.5and succinylated gelatin administration atdoses of approximately33ml/kg.Part3: C-type natriuretic peptide attenuates LPS-induced endothelial activationand its mechanismMethods: The effect of CNP on cell viability was determined by MTT assay. Theeffect of CNP on adhesion molecule expression was assessed using quantitative real-time RT-PCR and western blotting analyses. The nuclear factor-κB (NF-κB),MAPK, and PI3K/Akt signaling pathways in LPS-stimulated HUVECs wereinvestigated using western blotting analyses, and the production of intracellularreactive oxygen species (ROS) was measured using a fluorescence method.Results: The viability of HUVECs significantly decreased the after24h incubationwith1μg/ml LPS. CNP did not decrease the viability of HUVECs when they wereincubated with LPS in the presence of CNP (0.01,0.1,1μM). Pretreated with1μMCNP significantly inhibited the expression of VCAM-1, ICAM-1, P-selectin, andE-selectin. LPS significantly increased phosphorylation of the NF-κB P65subunit andNF-κB DNA binding activity in HUVECs after a1-h incubation. CNP markedlyreduced the phosphorylation of the NF-κB p65subunit and NF-κB DNA bindingactivity. LPS stimulation resulted in a rapid activation of ERK1/2, p38, and JNK.CNP suppressed LPS-induced phosphorylation of ERK1/2and p38MAPK at15and30min after incubation with LPS. However, CNP did not affect the LPS-inducedphosphorylation of JNK. The mRNA and protein expression of VCAM-1andICAM-1were significantly inhibited when p38MAPK inhibitor was used alone or incombination with ERK1/2inhibitor. However, ERK1/2inhibitor did not inhibit themRNA and protein expression of ICAM-1. LPS stimulation resulted in a rapidphosphorylation of Akt, with peak levels occurring15min after incubation with LPS.Pretreatment with CNP prior to LPS stimulation initially elevated Aktphosphorylation and further increased steadily at30min after incubation with LPS.The addition of PI3K/Akt pathway inhibitor reversed the inhibitory activity of CNPon LPS-induced mRNA and protein expression of ICAM-1and VCAM-1. In addition,CNP increased HO-1expression in LPS-stimulated cells in a concentration-dependentmanner. Treatment with LPS significantly elevated the production of intracellularROS after1and3h incubation. Moreover, CNP significantly reduced LPS-inducedintracellular ROS production.Conclusions: CNP effectively attenuated LPS-induced endothelial activation byinhibiting the NF-κB and p38signaling pathways, eliminating LPS-inducedintracellular ROS production, and activating the PI3K/Akt/HO-1pathway.In the treatment of hemorrhagic shock, appropriate fluid therapy is critical in themanagement of HS victims. Due to the decreased plasma and whole blood viscosity,GEL can be used for correction of abnormal blood rheology in early treatment ofhemorrhagic shock. HES130/0.4ameliorates the oxidative stress and inflammatory response, may have an advantage in preventing SIRS after hemorrhagicshock/resuscitation. In addition, CNP exerts anti-inflammation effects via severalsignaling pathways. It is suggested that CNP has the potential to be a multifunctionalanti-shock drug. However, these results are needed to be verified in the clinical trialsand animal experiment. This study provides an experimental basis for the fluid choicein the treatment of hemorrhagic shock and lays the foundation for the development ofnew anti-shock drug, has certain clinical importance and scientific significance.