| IntroductionThe intestinal ischemia-reperfusion(IIR) can take place not only in a series of intestinal diseases, such as the mesenteric arterial embolism, volvulus and bowel obstruction, but also in the course of hemorrhagic shock, serious trauma and infection, abdominal aortic aneurysm surgical operation etc.. The intestinal ischemia can destroy the intestinal function as a barrier, while the reperfusion can cause the serious damage of local tissue, and do harm to the remote organs when supplying energy and clearing toxic metabolites. If the damage followingⅡR is so severe that SIRS comes into being, multiple organ dysfunction will happen.The mechanism of MODS is not entirely clear now, and it has been a focus to find a way to control MODS duringⅡR. Carbon monoxide (CO), despite extreme toxicity at high concentrations, can confer sytoprotection during ischemia/reperfusion or inflammation-induced tissue injury. CO has been shown to affect several intracellular signaling pathways, and such pathways mediate, in part, the known vasoregulatory, anti-inflammatory, anti-apoptotic and anti-proliferative effects of this gas. Exogenous CO delivered at low concentrations is showing therapeutic potential as an anti-inflammatory agent and as such can modulate numerous pathophysiological states. This research focus on that whether CO can provide protection against multiple organ injuries duringⅡR and what its mechanism is. It consists of the following three parts: first, the effects of exogenous CO on carboxyhemoglobin blood concentration duringⅡR in rats; second, the effects of exogenous CO on multiple organ injuries duringⅡR in rats; third, the mechanisms of the protection of exogenous CO against multiple organ injuries duringⅡR in rats.Materials1. Animals: 64 male Wistar rats, weighing 220-260g, were provided by experimental animal center of China Medical University.2. Main Reagents and kits: standard CO, one step TUNEL apoptosis assay kits, kits of TNF-αand IL-10, p38 MAPK antibody, polyacrylamide and so on.3. Main Apparatus: CIBA-CORNING238 blood gas analyzer, Detax pressure monitor, Hitachi H-600-4 transmission electron microscope, N1KON 801FL-FUW-PF fluorescence microscope, GC-911-γ-counter, Olympus ML2000 microscope, BIO-RAD-PAC300 electrophoresis apparatus, GIS-2020 gel imaging analytical system, Heraus-Biofuge-PrimoR hypothermic high speed centrifuge.Methods1. Animal modelThe animals were housed in a licensed, climate-controlled animal-care facility, with 12-hour light/dark cycle and flee access to standard chow and water. Before the experiment the animal were deprived of food for 12 hours but had flee access to water. Rats were anesthesized with 20%urethane(1g·kg-1 intraperitoneally). Right carotid artery and right femoral vein were cannulated for BP and fluid and blood samples being taken. The animals were tracheotomized and breathed through a T-tube. A midline laparotomy was performed, and the superior mesenteric artery (SMA) was clamped with a microvascular clip. The incision was closed, and 60 minutes later it was reopened with the clip removed. Reperfusion was confirmed by the return of pulsation to the mesenteric arcade. 120 minutes after reperfusion, the animals were killed and samples were taken for advanced examination.2. Animal grouping64 rats were randomly allocated into 8 groups with 8 animals in each.Group A: sham operation, no SMA clamped;Group B: SMA clamped 60 minutes and reperfusion 120 minutes;Group C1: inhaled 100ppm CO at the time 10 minutes before SMA clampedGroup C2: inhaled 250ppm CO at the time 10 minutes before SMA clampedGroup D1: inhaled 100ppm CO at the time 60 minutes after SMA clampedGroup D2: inhaled 250ppm CO at the time 60 minutes after SMA clampedGroup E1: inhaled 100ppm CO at the time 60 minutes after reperfusionGroup E2: inhaled 250ppm CO at the time 60 minutes after reperfusion3. Observation of parameters and method(1) MAP duringⅡR (by pressure monitor)(2) COHb concentrations before and after CO inhaled (by blood gas analyzer)(3) Tissue ultrastructures (by transmission electron microscope)(4) number of PMN in different tissues (by light microscopy)(5) number of apoptotic cells in different tissues (by fluorescence microscope)(6) plasma TNF-αconcentrations (radio-immunifaction)(7) plasma IL-10 concentrations (radio-immunifaction)(8) expressions of p38 MAPK in different tissues (western blotting)Results1. The effect of exogenous CO on carboxyhemoglobin blood concentration duringⅡR in rats1.1 The COHb blood concentrations were not different at each time point in Group A and B.1.2 The COHb blood concentrations increased more significantly at T3 and T4 points in Group C1 and C2 than those in Group B (P＜0.05). 2. The effects of exogenous CO on multiple organ injury induced byⅡR in rats2.1 the degree of ultrastructures damaged in different tissues(1) in Group B, intestinal＞lung＞liver, and no significant change in kidney.(2) in different groups, Group C＜Group D＜Group E＜Group B (P＜0.05);(3) no significant different changes between the 250ppm group and the 100ppm group.2.2 number ofapoptotic cells in different tissues(1) few apoptotic cells can be seen in liver and kidney, but lots of in intestinal and lung;(2) in different groups, Group C＜Group D＜Group E＜Group B (P＜0.05);(3) more in the 100ppm group than in the 250ppm group.3. The mechanisms of the protection of exogenous CO against multiple organ injury induced byⅡR in rats3.1 number of PMN in different tissues(1) compared with in Group A, no more PMN in kidney in other groups;(2) in different groups, Group C2＜Group C1＜Group D2＜Group D1＜Group E2＜Group E1＜Group B (P＜0.05);(3) more in the 100ppm group than in the 250ppm group (P＜0.05).3.2 plasma TNF-αconcentrations(1) compared with in Group A, it increased significantly in Group B (P＜0.05).(2) in different groups, Group C2＜Group C1＜Group D2＜Group D1＜Group E2＜Group E1＜Group B (P＜0.05);(3) more in the 100ppm group than in the 250ppm group (P＜0.05).3.3 plasma IL-10 concentrations(1) compared with in Group A, it increased in Group B (P＜0.05).(2) in different groups, Group C2＞Group C1＞Group D2＞Group D1＞ Group E2＞Group E1＞Group B (P＜0.05);(3) less in the 100ppm group than in the 250ppm group (P＜0.05).3.4 expressions of p38 MAPK in different tissues(1) compared with in Group A, it increased in Group B (P＜0.05).(2) in different groups, Group C2＞Group C1＞Group D2＞Group D1＞Group E2＞Group E1＞Group B (P＜0.05);(3) less in the 100ppm group than in the 250ppm group (P＜0.05).Conclusions1. It is safe to inhale 100ppm and 250ppm CO duringⅡR in rats according to the changes of COHb blood concentration.2. Exogenous CO can provide protection against multiple organ injury induced byⅡR in rats. The effect of 250ppm CO is more significant than that of 100ppm CO. The effect of CO is more significant to be used before intestinal ischemia than after.3. Exogenous CO provides protection against multiple organ injury induced byⅡR through modulating cells apoptosis, inhibiting PMN aggregation in tissues, inhibiting the elevation of TNF-αand promoting the release of IL-10 in part.4. Exogenous CO can modulate the expression of p38 MAPK in cells, which is one of the biomolecular elements of its protection against multiple organ injury.
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