| Being the main reason for traumatic amputation in war time, limb's explosion injury frequently occurs in modern wars, causing damages to blood vessel, neurotmesis and extensive soft tissue. Immune system is often excessively activated after limb's explosion injury, subsequently resulting in remote organ damage. Amongst, incidence rate of acute lung injury is the highest, which often portends multiple organ dysfunction syndrome, so mitigation and prevention of acute lung injury become the very key step to improve prognosis of limb's explosion injury.Previous investigations indicate that controlled whole-body hypothermia obviously mitigates lung injury caused by sepsis, endotoxin and ischemic-reperfusion injury etc. while for limb's explosion injury accompanying with active bleeding, a long-time hypothermia may lead to fateful hemorrhage. Recent studies found that for the therapy of acute lung injury, early and short-time (2h) application of whole-body hypothermia is better than long-time hypothermia (5h). Transient(30min) whole-body hypothermia can significantly increase ischemic tolerance. Moreover, we also found that transient regional hypothermia can mitigate lung injury and intestinal tract barrier damage by inhibiting the systemic inflammatory reaction and oxidative injury in our early investigations. Therefore, the animal model is made for limb's explosion injury, meanwhile observing effects of transient(30min) whole-body hypothermia or regional hypothermia on lung injury.Endogenous protective system damage has driven considerable interests recently. NF-E2 related factor (Nrf2) is the key transcription factor in endogenous protective system, playing important roles in reducing oxidative stress, inflammatory damage and accumulation of toxic metabolites which are all involved in secondary lung injury after limb's explosion injury. Latest study points out that Nrf2 is activated to regulate antioxidant defenses during hibernation. However, relevant reports about hypothermia protection effects of Nrf2 on lung injury after limb's explosion injury are presently unavailable.In view of that, this paper is divided into four parts:Part 1: Animal model was made by detonating 80mg DDNP fixed on left hind limb of SD rats, meanwhile observing effects of transient (30min) whole-body hypothermia or regional hypothermia on secondary lung injury after limb's explosion injury.Part 2: Effects of transient (30min) whole-body hypothermia or regional hypothermia on Nrf2 of lung after limb's explosion injury was observed.Part 3: Pulmonary micro-vascular endothelial cells (PMVECs) were cultured. In order to investigate changes of Nrf2-transcriptional activity, PMVECs were treated with different doses of hydrogen peroxide (H2O2) or tumor necrosis factorα, expecting to explore the mechanism of Nrf2 transcriptional activity regulated by transient (30min) whole-body hypothermia or regional hypothermia.Part 4: Nrf2 expression in PMVECs was inhibited by RNA interference. Simultaneously, that whether Nrf2 exerts effects on anti-oxidative injury and anti-inflammation was investigated, exploring Nrf2'role in hypothermia protection.The main results and conclusions are as follows:1. Secondary lung injury resulted from limb's explosion injury are characterized of inflammatory reaction, oxidative injury and PMN infiltration. MDA contents and MPO activity in lung tissue are significantly increased (p<0.05), SOD activity decreased, TNFα,IL-6 and IL-10 concentration are remarkably increased (p<0.01).2. Transient whole-body hypothermia or regional hypothermia obviously inhibits inflammatory cells infiltration, mitigates pulmonary edema, decreases MDA content and MPO activity (p<0.01), increases SOD activity and decrease the level of TNFα, IL-6 and IL-10 (p<0.01) in lung tissue. All these show that transient whole-body hypothermia and regional hypothermia can mitigate lung injury after limb's explosion injury by inhibiting oxidative stress and inflammatory reaction.3. Transcriptional activity of Nrf2 in lung tissue is increased after limb's explosion injury, and further increased by early application of transient whole-body or regional hypothermia. However, Nrf2 mRNA levels are not changed, indicating that regulation of Nrf2 transcriptional activity does not occur at gene transcriptional level.4. Nrf2-ARE binding activity is increased after PMVECs exposed to 0.125mM, 0.25mM and 0.5mM hydrogen peroxide (H2O2) or 2.5 ng/ml, 5 ng/ml and 10ng/ml TNFαsolution, showing concentration-dependent. Nrf2-ARE binding activity is decreased after PMVECs exposed to 1mM or 2mM hydrogen peroxide and 20 or 40ng/ml TNF-αsolution compared with 0.5mM hydrogen peroxide or 10ng/ml TNF-αsolution. In consideration that transient whole-body or regional hypothermia can significantly inhibit oxidative injury and inflammatory reaction in lung after limb'explosion injury, it is concluded that Nrf2 transcriptional activity and endogenous protective competent can be increased due to inflammatory cytokines and free radicals are reduced by early application of transient whole-body or regional hypothermia, which is helpful to mitigate lung injury after limb's explosion injury.5. Hydrogen peroxide (H2O2) or tumor necrosis factorαcan lead to more severe injury of PMVECs after Nrf2 gene expression is inhibited by RNA interfere, indicating Nrf2 play important role in the process of anti-oxidative injury and anti-inflammatory reaction. It is suggested transient whole-body or regional hypothermia alleviates lung injury via enhancing transcriptional activity of Nrf2 in the lung tissue.
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