Xiaofa Qin

Apolipoprotein AIV: a potent endogenous inhibitor of lipid oxidation

Overexpression of apolipoprotein (apo) AIV in transgenic mice confers significant protection against atherosclerosis in apoE knockout animals even in the presence of a more severe atherogenic lipid profile. Because lipoprotein oxidation has been recognized to be pivotal in development of atherosclerosis, the antioxidative activity of apoAIV was investigated. Fasting intestinal lymph was used to mimic conditions in the interstitial fluid, the potential site for lipoprotein oxidation in vivo. ApoAIV (10 micrograms/ml) significantly inhibited copper-mediated oxidation of lymph. This inhibitory effect was further evaluated using purified low-density lipoprotein. Addition of apoAIV (2.5 micrograms/ml) increased the time of 50% conjugated diene formation by 2.4-fold, whereas apoE or BSA did not show such a protection even at 20 micrograms/ml. Addition of apoAIV during the propagation phase also resulted in a dose-dependent inhibition. ApoAIV also protected macrophage-induced oxidation of fasting lymph. These results provide the first evidence that apoAIV is a potent endogenous antioxidant.

The mucus layer is critical in protecting against ischemia-reperfusion-mediated gut injury and in the restitution of gut barrier function.

It is well documented that the gut injury plays a critical role in the development of systemic inflammation and distant organ injury in conditions associated with splanchnic ischemia. Consequently, understanding the mechanisms leading to gut injury is important. In this context, recent work suggests a protective role for the intestinal mucus layer and an injury-inducing role for luminal pancreatic proteases. Thus, we explored the role of the mucus layer in gut barrier function by observing how the removal of the mucus layer affects ischemia-reperfusion-mediated gut injury in rats as well as the potential role of luminal pancreatic proteases in the pathogenesis of gut injury. Ischemia was induced by the ligation of blood vessels to segments of the ileum for 45 min, followed by up to 3 h of reperfusion. The ileal segments were divided into five groups. These included a nonischemic control, ischemic segments exposed to saline, the mucolytic N-acetylcysteine (NAC), pancreatic proteases, or NAC + pancreatic proteases. Changes in gut barrier function were assessed by the permeation of fluorescein isothiocyanate dextran (molecular weight, 4,000 d) in ileal everted sacs. Gut injury was measured morphologically and by the luminal content of protein, DNA, and hemoglobin. The mucus layer was assessed functionally by measuring its hydrophobicity and morphologically. Gut barrier function was promptly and effectively reestablished during reperfusion, which was accompanied by the restoration of the mucus layer. In contrast, treatment of the gut with the mucolytic NAC for 10 min during ischemia resulted in a failure of mucus restitution and further increases in gut permeability and injury. The presence of digestive proteases by themselves did not exacerbate gut injury, but in combination with NAC, they caused an even greater increase in gut injury and permeability. These results suggest that the mucus layer not only serves as a barrier between the luminal contents and gut surface epithelia, but also plays a critical role in the maintenance and restitution of gut barrier function.

The role of apolipoprotein AIV on the control of food intake.

Apolipoprotein AIV (apo AIV) is a protein synthesized by the human intestine. The synthesis and secretion of apo AIV are stimulated by fat absorption. In 1992, Fujimoto et al. [1] first demonstrated that apo AIV is a satiety signal secreted by the small intestine following the ingestion of a lipid meal. This initial observation was followed by a number of studies supporting apo AIVs role as a satiety signal. This review article discusses the regulation of synthesis of apo AIV in the small intestine as well as the hypothalamus. In addition, the evidence that apo AIV is a satiety factor and its role of apo AIV in diet induced obesity will be discussed. We hope this review will serve as a catalyst to promote apo AIV research in the future. With most of the required reagents available, e.g., the apo AIV knockout and transgenic animals and apo AIV antibodies, the next few years should bring considerable new information on the function of apo AIV.

LOSS OF THE INTESTINAL MUCUS LAYER IN THE NORMAL RAT CAUSES GUT INJURY, BUT NOT TOXIC MESENTERIC LYMPH NOR LUNG INJURY

There is substantial evidence that gut barrier failure is associated with distant organ injury and systemic inflammation. After major trauma or stress, the factors and mechanisms involved in gut injury are unknown. Our primary hypothesis is that loss of the intestinal mucus layer will result in injury of the normal gut that is exacerbated by the presence of luminal pancreatic proteases. Our secondary hypothesis is that the injury produced in the gut will result in the production of biologically active mesenteric lymph and consequently distant organ (i.e., lung) injury. To test this hypothesis, five groups of rats were studied: 1) uninstrumented naive rats; 2) control rats in which a ligated segment of distal ileum was filled with saline; 3) rats with pancreatic proteases placed in their distal ileal segments; 4) rats with the mucolytic N-acetylcysteine (NAC) placed in their distal ileal segments; and 5) rats exposed to NAC and pancreatic proteases in their ileal segments. The potential systemic consequences of gut injury induced by NAC and proteases were assessed by measuring the biological activity of mesenteric lymph as well as gut-induced lung injury. Exposure of the normal intestine to NAC, but not saline or proteases, led to increased gut permeability, loss of mucus hydrophobicity, a decrease in the mucus layer, as well as morphological evidence of villous injury. Although proteases themselves did not cause gut injury, the combination of pancreatic proteases with NAC caused more severe injury than NAC alone, suggesting that once the mucus barrier is impaired, luminal proteases can injure the now vulnerable gut. Because comparable levels of gut injury caused by systemic insults are associated with gut-induced lung injury, which is mediated by biologically active factors in mesenteric lymph, we next tested whether this local model of gut injury would produce active mesenteric lymph or lead to lung injury. It did not, suggesting that gut injury by itself may not be sufficient to induce distant organ dysfunction. Therefore, loss of the intestinal mucus layer, especially in the presence of intraluminal pancreatic proteases, is sufficient to lead to injury and barrier dysfunction of the otherwise normal intestine but not to produce gut-induced distant organ dysfunction.

Role of lipase-generated free fatty acids in converting mesenteric lymph from a noncytotoxic to a cytotoxic fluid.

Recent studies have shown that mesenteric lymph plays a very important role in the development of multiple-organ dysfunction syndrome under critical conditions. Great efforts have been made to identify the biologically active molecules in the lymph. We used a trauma-hemorrhagic shock (T/HS) model and the superior mesenteric artery occlusion (SMAO) model, representing a global and a localized intestinal ischemia-reperfusion insult, respectively, to investigate the role of free fatty acids (FFAs) in the cytotoxicity of mesenteric lymph in rats. Lymph was collected before, during, and after (post) shock or SMAO. The post-T/HS and SMAO lymph, but not the sham lymph, manifested cytotoxicity for human umbilical vein endothelial cells (HUVECs). HUVEC cytotoxicity was associated with increased FFAs, especially the FFA-to-protein ratio. Addition of albumin, especially delipidated albumin, reduced this cytotoxicity. Lipase treatment of trauma-sham shock (T/SS) lymph converted it from a noncytotoxic to a cytotoxic fluid, and its toxicity correlated with the FFA-to-protein ratio in a fashion similar to that of the T/HS lymph, further suggesting that FFAs were the key components leading to HUVEC cytotoxicity. Analysis of lymph by gas chromatography revealed that the main FFAs in the post-T/HS or lipase-treated T/SS lymph were palmitic, stearic, oleic, and linoleic acids. When added to the cell culture at levels comparable to those in T/HS lymph, all these FFAs were cytotoxic, with linoleic acid being the most potent. In conclusion, this study suggests that lipase-generated FFAs are the key components resulting in the cytotoxicity of T/HS and SMAO mesenteric lymph.

Intraluminal nonbacterial intestinal components control gut and lung injury after trauma hemorrhagic shock.

Objective:To test whether the mucus layer, luminal digestive enzymes, and intestinal mast cells are critical components in the pathogenesis of trauma shock–induced gut and lung injury. Background:Gut origin sepsis studies have highlighted the importance of the systemic component (ischemia-reperfusion) of gut injury, whereas the intraluminal component is less well studied. Methods:In rats subjected to trauma hemorrhagic shock (T/HS) or sham shock, the role of pancreatic enzymes in gut injury was tested by diversion of pancreatic enzymes via pancreatic duct exteriorization whereas the role of the mucus layer was tested via the enteral administration of a mucus surrogate. In addition, the role of mast cells was assessed by measuring mast cell activation and the ability of pharmacologic inhibition of mast cells to abrogate gut and lung injury. Gut and mucus injury was characterized functionally, morphologically, and chemically. Results:Pancreatic duct exteriorization abrogated T/HS-induced gut barrier loss and limited chemical mucus changes. The mucus surrogate prevented T/HS-induced gut and lung injury. Finally, pancreatic enzyme–induced gut and lung injury seems to involve mast cell activation because T/HS activates mast cells and pharmacologic inhibition of intestinal mast cells prevented T/HS-induced gut and lung injury. Conclusions:These results indicate that gut and gut-induced lung injury after T/HS involves a complex process consisting of intraluminal digestive enzymes, the unstirred mucus layer, and a systemic ischemic-reperfusion injury. This suggests the possibility of intraluminal therapeutic strategies.

Intestinal mucus layer preservation in female rats attenuates gut injury after trauma-hemorrhagic shock.

BACKGROUND We tested the hypothesis that females are more resistant to trauma-hemorrhagic shock (T/HS)-induced gut injury than males, and this is related to better preservation of their intestinal mucus layer, which is influenced in turn by the estrus cycle stage at the time of injury. METHODS Male, proestrus and diestrus female rats underwent a laparotomy (trauma) and 90 minutes of shock ( approximately 35 mm Hg). At 3 hours after reperfusion, terminal ileum was harvested and stained with Carnoys Alcian Blue for mucus assessment, hematoxylin and eosin, and periodic acid schiff for villous and goblet cell morphology and injury. Ileal permeability was measured in separate intestinal segments using the ex vivo everted gut sac technique. RESULTS When compared with males, proestrus female rats were significantly more resistant to T/HS-induced morphologic gut injury, as reflected in both a lower incidence of villous injury (14% vs. 22%; p < 0.05) and a lesser grade of injury (1.0 vs. 2.8; p < 0.05) as well as preservation of gut barrier function (17.9 vs. 32.2; p < 0.05). This resistance to gut injury was associated with significant preservation of the mucus layer (87% vs. 62%; p < 0.05) and was influenced by the estrus cycle stage of the female rats. There was a significant inverse correlation between mucus layer coverage and the incidence (r = 0.9; p < 0.0001) and magnitude (r = 0.89; p < 0.0001) of villous injury and gut permeability (r = 0.74; p < 0.001). CONCLUSIONS The resistance of female rats to T/HS-induced intestinal injury and dysfunction was associated with better preservation of the intestinal mucus barrier and was to some extent estrus cycle-dependent. Preservation of the mucus barrier may protect against shock-induced gut injury and subsequent distant organ injury by limiting the ability of luminal contents such as bacteria and digestive enzymes from coming into direct contact with the epithelium.

Testosterone depletion or blockade in male rats protects against trauma hemorrhagic shock-induced distant organ injury by limiting gut injury and subsequent production of biologically active mesenteric lymph.

BACKGROUND We tested the hypothesis that testosterone depletion or blockade in male rats protects against trauma hemorrhagic shock-induced distant organ injury by limiting gut injury and subsequent production of biologically active mesenteric lymph. METHODS Male, castrated male, or flutamide-treated rats (25 mg/kg subcutaneously after resuscitation) were subjected to a laparotomy (trauma), mesenteric lymph duct cannulation, and 90 minutes of shock (35 mm Hg) or trauma sham-shock. Mesenteric lymph was collected preshock, during shock, and postshock. Gut injury was determined at 6 hours postshock using ex vivo ileal permeability with fluorescein dextran. Postshock mesenteric lymph was assayed for biological activity in vivo by injection into mice and measuring lung permeability, neutrophil activation, and red blood cell deformability. In vitro neutrophil priming capacity of the lymph was also tested. RESULTS Castrated and flutamide-treated male rats were significantly protected against trauma hemorrhagic shock (T/HS)-induced gut injury when compared with hormonally intact males. Postshock mesenteric lymph from male rats had a higher capacity to induce lung injury, Neutrophil (PMN) activation, and loss of red blood cell deformability when injected into naïve mice when compared with castrated and flutamide-treated males. The increase in gut injury after T/HS in males directly correlated with the in vitro biological activity of mesenteric lymph to prime neutrophils for an increased respiratory burst. CONCLUSIONS After T/HS, gut protective effects can be observed in males after testosterone blockade or depletion. This reduced gut injury contributes to decreased biological activity of mesenteric lymph leading to attenuated systemic inflammation and distant organ injury.

Anticoagulants influence the in vitro activity and composition of shock lymph but not its in vivo activity

Many models of trauma-hemorrhagic shock (T/HS) involve the reinfusion of anticoagulated shed blood. Our recent observation that the anticoagulant heparin induces increased mesenteric lymph lipase activity and consequent in vitro endothelial cell cytotoxicity prompted us to investigate the effect of heparin-induced lipase activity on organ injury in vivo as well as the effects of other anticoagulants on mesenteric lymph bioactivity in vitro and in vivo. To investigate this issue, rats subjected to trauma-hemorrhage had their shed blood anticoagulated with heparin, the synthetic anticoagulant arixtra (fondaparinux sodium), or citrate. Arixtra, in contrast to heparin, did not increase lymph lipase activity or result in high levels of endothelial cytotoxicity. Yet, the arixtra-treated rats subjected to T/HS still manifested lung injury, neutrophil priming, and red blood cell dysfunction, which was totally abrogated by lymph duct ligation. Furthermore, the injection of T/HS mesenteric lymph, but not sham-shock lymph, collected from the arixtra rats into control mice recreated the pattern of lung injury, polymorphonucleocyte (PMN) priming, and red blood cell dysfunction observed after actual shock. Consistent with these observations, citrate-anticoagulated rats subjected to T/HS developed lung injury, and the injection of mesenteric lymph from the citrate-anticoagulated T/HS rats into control mice also resulted in lung injury. Based on these results, several conclusions can be drawn. First, heparin-induced increased mesenteric lymph lipase activity is not responsible for the in vivo effects of T/HS mesenteric lymph. Second, heparin should be avoided as an anticoagulant when studying the biology or composition of mesenteric lymph because of its ability to cause increases in lymph lipase activity that increase the in vitro cytotoxicity of these lymph samples.