The Central Role Of FIAF In Multiple Organ Injury Induced By Intestinal Ischemia/Reperfusion

Intestinal I/R is a common pathophysiology process occurred in patients in surgery and intensive care unit. Intestinal I/R disrupt intestinal barrier and even lead to MODS which have a high mortality and morbidity. The mortality caused by acute mesenteric thrombosis is high up to 60-80%.Intestinal barrier dysfunction is critical in the pathogenesis of MODS induced by intestinal I/R. Intestinal mucosal barrier is a selective permeability barrier consists of mechanical barrier, immune barrier and microorganism barrier. The mechanical barrier is the most important part and is composed of intestinal epithelial cells, tight junctions and cell adhesion molecules. A normal mucosal barrier can prevent the invasion of pathogens and maintain the homeostasis effectively. Previous studies demonstrated that the inflammatory and oxidative cascaded can disrupt the intestinal barrier, recent studies suggest that excessive activation of apoptosis and autophagy can impaire barrier directly. Further studies indicate myosin light-chain kinase (MLCK) pathway, which regulate the specific protein ZO-1 and claudin-2, is crucial in the maintenance of intestinal barrier function.Intestinal I/R not only induces intestinal barrier dysfunction but also triggers remote organs injury sequentially including liver, lung and kidney and even leads to MODS. Therefore, treatment on intestine per se is vital to the intestine-originated multiple organ injury. Studies have shown that apoptosis is the primary form of intestinal epithelial cell death after I/R, further studies found that excessive activation of autophagy can also damage the intestinal barrier directly. However, inhibition of apoptosis and autophagy can not completely reverse the multiple organ injury induced by intestinal I/R. Almost recently, increasing studies have focused on a novel mode of cell death-necroptosis, which also named programmed necrosis with unclear regulative mechanism. The receptor-associated protein-3 (RIP3) escapes from inhibition of caspase-8 is demonstrated indispensable in necroptosis and RIP3 has been believed as the crucial mediator of necroptosis. In addition, mixed lineage kinase domain like (MLKL), a target and interaction protein with RIP3, is necessary to the regulation of necroptosis. Regulation of necroptosis pathway is neither traditional caspase-3 mediated apoptosis nor the classical necrosis pathway. Other studies demonstrated that it is not only involved in intestinal injury, but also lead to the lethal-systemic inflammatory response and increased mortality of mice. Necroptosis also occurred in caerulein-induced acute pancreatitis. Thus, we speculate that necroptosis pathway may play the critical role in the intestinal I/R-induced multiple organ dysfunction syndrome.Fasting-induced adipose factor (FIAF) is involved in homeostasis maintenance, wound repair, and apoptosis. It may play the central role in MODS induced by intestinal I/R. Firstly, FIAF maintain the integrity of blood-brain barrier and regulate the expression of ZO-1. This provides a potential role of FIAF on the integrity of the intestinal barrier. Secondly, FIAF has involved in macrophage-mediated inflammatory reaction of intestine and is essential during the development of the lymphatic system. FIAF also protect against LPS-induced inflammatory injured pulmonary micro-vascular endothelial cells. Studies have shown that FIAF deficient mice are more vulnerable to suffer severe chronic inflammation induced by high-fat diet in macrophages of mesenteric-lymph nodes. FIAF deficiency induces intestinal lymphatic system development incompletely and death in 3 weeks after birth due to immunodeficiency in mice. This suggests that FIAF may have important value in intestinal I/R induced-lung injury which mediated by lymphatic pathway and dependent on macrophage activation. Finally, FIAF inhibits the release of TNF-a and further blocks binding of tumor necrosis factor to TNF receptor, thereby inhibits RIP1 and RIP3 activation and restores caspase-8, while reduces activation of MLKL, eventually blocks the occurring of necroptosis. FIAF may play a central role in multiple organ injury induced by intestinal I/R because of its diverse biological function.Fish oil (FO), containing major ingredients as ω-3 PUFAs, has been widely used as a therapeutic intervention in critical care settings. The potential health benefits of ω-fatty acids supplementation are controversial. However, growing evidence indicates that co-3 PUFAs have potential benefits in disease models featuring I/R. In addition, ω-3PUFAs is a strong inducer of FIAF which may inhibit lung macrophage inflammatory response induced by intestinal I/R injury. Other studies suggest co-3PUFAs antagonize macrophage inflammatory in an AMPK/S1RT1 dependent manner. Therefore, ω-3PUFAs may relieve lung injury induced by intestinal I/R via AMPK/SIRT1 pathway and inducing FIAF.Natural herb biochanin A (BCA) and curcumin belong to phenolic compounds and BCA is potent PPAR activator. BCA is widely used to protect inflammation, burns and other critical circumstance. PPAR activator can induce FIAF expression strongly. And curcumin inhibits necroptosis of neurons has been reported. Therefore, Phenolic compounds BCA, also known as a PPAR activator, may induce FIAF and inhibit necroptosis to alleviate multiple organ injury induced by intestinal 1/R.Based on these theories, intestinal I/R models were created using mice and rats. The hypoxia/reoxygenation (H/R) models were created to imitate I/R in vivo using intestinal epithelium Caco-2 cells. Following questions will be clarified:i). effects of FIAF in intestinal barrier dysfunction induced by 1/R. ii). whether fish oil (ω-3PUFAs) attenuates lung injury induced by intestinal I/R via FIAF. iii). whether BCA can induce FIAF and protect multiple organ injury against intestinal I/R through necroptosis. Our research will clarify the central role of F1AF in intestinal I/R induced multiple organ dysfunction in future.PART I:Rh FIAF alleviates intestinal barrier dysfunction induced by I/R via regulating the structure and function.Objective:To evaluate the role of FIAF on intestinal barrier dysfunction induced by I/R.Methods:24 healthy male Wistar rats were randomly divided into 3 groups:sham group (sham group), intestinal I/R group (I/R+vehicle group), Rh FIAF group (I/R+Rh FIAF). Intestine I/R models were created by the superior mesenteric artery occlusion for 1 hour and reperfusion for 4 hours. Rh FIAF group were given (28mg/kg, intravenous injection) in the beginning of reperfusion. Histopathological changes of intestine were observed and TNF-a, IL-6, IL-1β and MDA were determined; intestinal autophagy (Beclin-1, LC3T, LC3II and P62) and apoptosis (Cleaved caspase-3/caspase-3) levels; intestinal barrier structure (MLCK, p-MLC/t-MLC, ZO-1 and Claudin-2) and function (FD4 clearance rate and vascular permeability indicator E-cadherin); intestinal tissue FIAF protein and mRNA changes were determined.Results:i) Compared with the sham group, I/R group:intestinal villus were disorganized and the tips were broken, hemorrhage and edema were observed in mucosal and submucosal, subepithelial gap were widened; serum and intestinal TNF-a, IL-6, IL-1β (P<0.01), MPO and MDA levels were increased significantly (P<0.05); while autophagy (Beclin-1, LC3I and LC3II were elevated, P62 were lowered) and apoptosis (numbers of apoptotic cells and Cleaved caspase-3/caspase-3) were increased significantly (P<0.05). Intestinal barrier structure (MLCK, p-MLC/t-MLC were increased; ZO-1 and Claudin-2 were decreased) and function (FD4 clearance rate and E-cadherin) are weakened (P<0.01). FIAF protein and mRNA increased significantly in intestine (P<0.05).ii) Compared with I/R group, Rh FIAF groups:intestinal villus disarranged and tips fracture are relieved, hemorrhage and submucosal edema are soothing, the subepithelial gaps is not widened obviously. Serum and intestinal TNF-α, IL-6, IL-1β, MPO and MDA levels were decreased (P<0.05). Intestinal autophagy (Beclin-1, LC3I, LC3II were decreased, P62 was increased) and apoptosis (number of apoptotic cells and Cleaved Caspase-3) were decreased (.P<0.05). Intestinal barrier structure (MLCK, p-MLC/t-MLC were decreased; ZO-1 and Claudin-2 were increased) and function (FD4 clearance rate and E-cadherin) were recovered (P<0.01).Conclusion:i). Intestinal 1/R can induce intestinal mucosal barrier and vascular endothelial barrier structure and function associated with exacerbated inflammatory cascade and oxidative stress. In addition, excessive activation of autophagy and apoptosis accompanied with reduced survival rate are observed,ii). Rh FIAF protects intestinal barrier dysfunction against 1/R injury and improve survival through following potential mechanism:(a) inhibition of excessive autophagy and apoptosis in intestinal epithelial cells, (b) Reduce inflammatory and oxidative cascade, (c) Inhibits MLCK pathway, FD4 clearance and the loss of ZO-1 and Claudin-2.PART.II. Fish-oil emulsion (omega-3 polyunsaturated fatty acids) attenuates acute lung injury induced by intestinal ischemia/reperfusion through inducing FIAF and Adenosine 5’-monophosphate-activated protein kinase-sirtuinl pathway.Objective:Activated macrophage infiltration into the lungs is paramount in the pathogenesis of acute lung injury (ALI) induced by intestinal ischemia/reperfusion (I/R). Omega-3 polyunsaturated fatty acids (co-3 PUFAs) is a potent activator of the Adenosine 5’-monophosphate-activated protein kinase/sirtuinl (AMPK/S1RT1) pathway against macrophage inflammation. Other studies demonstrated FIAF also induced by ω>3 PUFAs and protects against severe pro-inflammatory effects of dietary saturated fat in mesenteric lymph node macrophages. We aimed to evaluate whether co-3 PUFAs may protect against ALI induced by intestinal I/R via the AMPK/SIRT1 pathway and the role of FIAF in this pathophysiology.Methods:Ischemia in male Wistar rats was induced by superior mesenteric artery occlusion for 60 min and reperfusion for 240 min. One milliliter per day of fish-oil emulsion (FO emulsion, containing major ingredients as co-3 PUFAs) or normal saline (control) was administered by intraperitoneal injection for three consecutive days to each animal. All animals were sacrificed at the end of reperfusion. Blood and tissue samples were collected for analysis.Results:Intestinal I/R caused intestinal and lung injury, evidenced by severe lung tissue edema and macrophage infiltration. Pretreatment with FO emulsion improved the integrity of microscopic structures in the intestine and lungs. Intestinal I/R induced the expression of macrophage-derived mediators (macrophage migration inhibitory factor and macrophage chemoattractant protein-1), inflammatory factors (nuclear factor κ B, tumor necrosis factor a, interleukin 6, and interleukin 10), and pro-apoptosis factor p66shc (P<0.01, P＜0.01; P<0.01, P＜0.05, P＜0.05, P<0.05; P＜0.01). There was a decrease in the expression of AMPK, SIRT1, claudin 5 and increase in the expression of FIAF (P<0.01). FO emulsion significantly inhibited macrophage infiltration into the lungs, inflammatory factors expression, and p66shc phosphorylation (P<0.01; P<0.05; P<0.05). Importantly, FO emulsion restored AMPK, SIRT1, and claudin 5 and further induced FIAF increase in lungs (P<0.01, P<0.01, P<0.05; P<0.05).Conclusions:Pretreatment with ω-3 PUFAs effectively protects intestinal and lung injury induced by intestinal I/R, reduces macrophage infiltration, suppresses inflammation, inhibits lung apoptosis, and improves the lung endothelial barrier after intestinal I/R in a manner dependent on AMPK/SIRT1 and inducing of FIAF. Thus, there is a potential for developing AMPK/SIRT1 and FIAF as novel targets for patients with intestinal I/R-induced ALI.PART Ⅲ:Biochanin A inhibits necroptosis and lessons intestinal I/R induced multiple organ injury via FIAF.Objective:To evaluate the role of necroptosis pathway in intestinal I/R induced multiple organ injury and whether Biochanin A inhibits necroptosis and protect multiple organ injury against intestinal I/R via FIAF.Methods:i) Protective effect and mechanism of biochanin A on multiple organ injury induced by intestinal I/R.24 healthy male C57BL/6J mice were randomly divided into the following 3 groups:sham, I/R and BCA pretreatment group. Intestinal I/R model are constructed as described previously. BCA were given at a dose of 50mg/kg/d intraperitoneal injection with consecutive 5 days before operation. Intestine, liver, lung and kidney histopathological changes were observed. ALT, AST levels and BALFP content; blood urea nitrogen (BUN) and creatinine (Cr) levels; TNF-α and IL-6 levels; were all determined. Intestinal necroptosis indicators (RIP3, MLKL and caspase-8); FIAF, PPAR α, PPAR β and PPAR γ protein; FIAF mRNA levels were determined.ⅱ) To determine the mechanism of BCA on intestinal I/R injury, H/R were performed using Caco-2 cells. BCA, RNA interference, different subtypes of PPAR inhibitors were employed, protein or mRNA levels of the related moleculars including FIAF、RIP3、caspase-8 were determined using western blot or real-time PCR. BCA concentration and reaction time were defined after preliminary experiment. The following scientific questions were explored:(a) Which PPAR subtypes were involved in the induction of FIAF by BCA in Caco-2 cells.(b) FIAF protein and mRNA expression behavior in Caco-2 cells challenged after different times of H/R. (c) Inhibition of Caco-2 cells necroptosis after H/R by BCA. (d) Effects of BCA on FIAF Protein and mRNA expression in Caco-2 cells after H/R. (e) Effect of FIAF in the inhibition of Caco-2 cells necroptosis by BCA after H/R.Results:I. Protective effect and mechanism of BCA in multiple organ injury induced by intestinal I/R.ⅰ) Compared with sham group,I/R group:Inflammatory cells infiltration and villus derangement associated with mucosal and submucosal edema, hemorrhage were observed in intestine. Derangement of the liver cell cord and bleeding were observed in central venous and periportal venules accompanied with inflammatory cell infiltration and hepatic sinusoid dilatation and congestion. Severely congestion and inflammatory cell infiltration in alveolar were observed. In addition, thickened alveolar-wall and hyaline membrane was observed. Renal tubules showed pathological changes, including edema, necrosis and vacuolization. Intestine and liver, lung and kidney histopathology scores were increased significantly (P<0.01, P<0.01, P<0.05,P<0.05). Similarly, ALT, AST and BALFP level; BUN and Cr were increased significantly (P<0.05). Serum TNF-a, TL-6; intestinal necroptosis (RTP3, MLKL increased and Caspase-8 decreased) increased (P<0.05, P<0.05; P<0.05, P<0.05, P<0.01). Intestinal FIAF mRNA and protein expression was significantly increased as well as PPAR a and PPAR y but not PPAR β protein levels (P<0.05, P<0.01; P< 0.05).ii) Compared with I/R group, BCA group:Inflammatory cells infiltration decreased and submucosal edema and hemorrhage were relieved in intestine. Derangement of the liver cell cord and bleeding were relieved. Inflammatory cell infiltration and hepatic sinusoid dilatation and congestion were smoothly. Congestion and infiltration of inflammatory cells in alveolar were slightly. Alveolar wall was not thicker and no hyaline membrane observed. In addition, significant amelioration of renal pathological changes including edema, necrosis and vacuolization was observed. Intestine and liver, lung and kidney histopathology scores were decreased significantly (P<0.05). Similarly, ALT, AST and BALFP level; BUN and Cr were decreased significantly (P<0.05). Serum TNF-a, IL-6; intestinal necroptosis (RIP3, MLKL decreased and Caspase-8 increased) was decreased (P<0.05). Intestinal FIAF mRNA and protein expression was further increased significantly as well as PPAR a and PPAR y but not PPAR β protein levels (P<0.05, P<0.01; P<0.05).Ⅱ. Effect and mechanism of BCA protect Caco-2 cells against H/R injuryi) BCA induce FIAF mRNA expression in Caco-2 cells by activation of PPAR a and PPAR y (P<0.05). ii) FIAF protein and mRNA increase in a time-dependent manner in Caco-2 cells after hypoxia 1h then reoxygenation within 16hs (P<0.05). iii) BCA inhibit Caco-2 cells inflammation and necroptosis after H/R in a dose-dependent manner (P<0.01). iv) BCA further induces FIAF protein and mRNA increased compared with cells after H/R (P<0.05). v) FIAF is required for BCA protect Caco-2 cells against H/R (P<0.01).Conclusion: i. BCA protects multiple organ injury induced by intestinal I/R. ii. BCA activate PPAR a and PPAR y and further induce expression of FIAF. iii. BCA attenuates multiple organ injury and intestinal epithelial cells necroptosis after I/R via FIAF.