Study Of Hypothermia For The Activation Of Inflammasome Of Hemorrhagic Shock

Part 1 Study of hypothermia for the activation of inflammasome of hemorrhagic shockObjective: To explore the effect of therapeutic hypothermia for the activation of inflammasome in the lung of hemorrhagic shock(HS) rats. Methods: 24 Male Sprague-Dawley rats were randomly divided into three groups: sham group, normothermia resuscitation(NR) group and hypothermia resuscitation(HR) group. Each group was subjected to pressure-controlled(MAP 40 mm Hg) HS for 1 h, then the NR group and the HR group were resuscitated with lactated Ringer and maintained MAP to 90 mm Hg for 1 h. After 4 h, the rats in each group were killed by exsanguination. Hematoxylin Eosin staining was used to observe the injury of lung tissue. The drying wet method was used to detect the edema of lung tissue. q RT-PCR and western blotting were employed to evaluate the m RNA and protein expression of NLRP-3, IL-1β, caspase-1.A 2-tailed t test was used to compare difference between two independent groups. Results:(1) The injury of lung tissue in HR group was significantly lower than that in NR group;(2) Wet/dry(W/D) weight ratio of lung in HR group decrease compared with NR group [HR group 5.85 + 0.102; NR group 6.471 + 0.1658(t=3.14, P < 0.05)];(3) Relative to NLRP-3 and of caspase-1 protein expression were lower in the HR group than NR.(4) The NLRP-3 m RNA expression level of HR group was reduce compared with NR group [(HR group 1.027 + 0.143; NR group 1.3487 + 0.163(t=4.36, P < 0.05)] and IL-1 m RNA expression level of HR group decreased compared with NR group [HR group 162.3 + 0.125; NR group 2.388+0.229(t=7.72, P < 0.05)].Conclusions: Therapeutic hypothermia attenuated HS induced ALI in rats by modulation of signal way of inflammasome. Part 2 Hypothermia preserves mitochondria function of endotheliocytes after ischemia reperfusion injuryObjective: Hemorrhagic shock after traumatic injury carries a high mortality. Therapeutic hypothermia has been widely used in critical illness to improve the outcome in hemorrhagic shock activation signaling pathways. However, the role played by the mitochondria and inflammasomes ’activation in the endotheliocytes protective effects of therapeutic hypothermia remains unclear. We investigated the effects of therapeutic hypothermia on mitochondrial function integrity and inflammasomes’ activation after hemorrhagic shock using an in vitro ischemia reperfusion model. Methods: HUVECs received a simulated ischemia reperfusion injury under normothermic(37 ℃) and hypothermic(31℃) conditions. The were treated with hypoxic condition for 6 h in serum-free, glucose-free culture medium at p H 6.9 and then shifted to re-oxygenation status for 2 h in serum-containing cell culture medium at p H 7.4. Cellular survival, mitochondrial integrity, energy metabolism were studied. Results: Hypothermia treatment lessened cell death [31℃ group 9.23±1.772; 37℃ group 12.63±1.943(t =3.139,P <0.05)] and preserved mitochondrial number against simulated ischemia reperfusion injury. Mitochondrial ATP concentrations were maintained with hypothermia treatment after injury [31 ℃ group 9.65±2.134; 37 ℃ group 14.58±1.316(t=-4.82, P<0.05)]. Mitochondrial membrane potential was preserved by hypothermia treatment [31℃ group 5.85 + 0.102; 37℃ group 6.471 + 0.1658(t=3.14, P< 0.05)] after the injury. Conclusions: Hypothermia preserves mitochondria function of endotheliocytes after ischemia reperfusion injury by attenuating ROS mediated NLRP3 inflammasome activity. Part 3 Hypothermia preserves of endotheliocytes after ischemia reperfusion injury by attenuating NLRP3 inflammasome activityObjective: Hemorrhagic shock activation of inflammatory pathways, produce large amounts of inflammatory mediators, resulting in tissue damage. This experiment will study the therapeutic hypothermia for inflammation body NLRP3 activation of endothelial cells in simulated ischemia and reperfusion. Methods: HUVECs received a simulated ischemia reperfusion injury under normothermic(37 ℃) and hypothermic(31 ℃) conditions. The were treated with hypoxic condition for 6 h in serum-free, glucose-free culture medium at p H 6.9 and then shifted to re-oxygenation status for 2h in serum-containing cell culture medium at p H 7.4. ROS analysis, caspase-1 and NLRP3 protein expression and inflammatory mediator IL-1β and IL-18 expression were studied. Results: 31℃ group inflammasome NLRP3 protein expression was significantly lower than 37℃ group. caspase-1 protein expression was significantly lower than 31 ℃ 37℃ group, but both higher. Applications NLRP3 inhibitors BAY11-7082 display, NLRP3 and caspase-1 protein expression were decreased. Hypothermia for endothelial cells can be reduced ROS concentrations [37℃ group 765.32 ± 20.358; 31℃ group 679.80 ± 17.472,(P<0.05, t=7.808)]. 31 ℃ group of inflammatory mediators IL-1β expression was significantly lower than 37℃ group [37℃ 496.49 ± 52.671; 31℃ group 315.57 ± 23.168,(P<0.05, t=7.701)]. Applications NLRP3 inhibitors BAY11-7082 show, 37℃ and 31℃ resuscitation decreased IL-1β expression were lower, but both compared to the difference was not statistically significant. [Group 31℃ 63.49±18.374; Group 37℃ 70.21 ± 14.52,(P>0.05, t=-0.702)]. IL-18 protein expression was significantly lower than 31 ℃ 37 ℃ group [Group 37℃ 362.62 ± 33.163; Group 31℃ 317.34 ± 28.450,(P<0.05, t=2.538)]. Applications NLRP3 inhibitors BAY11-7082, IL-18 express reduced [Group 31℃ 63.93±13.225; 37℃ Group 73.18±14.479,(P>0.05, t=-1.155)]. Conclusions: Hypothermia preserves endotheliocytes after ischemia reperfusion injury by attenuating ROS mediated NLRP3 inflammasome activity reduce the production of ROS-induced NLRP3 activation, reducing expression of inflammatory mediators IL-1β and IL-18.