Hepatic cytoprotection: from fibrosis to ischemia and reperfusion

Abstract

Our research group has been studying the hepatic modulation of kinin system for decades. The kinin system is represented mainly by bradykinin (BK), a B2 receptor agonist (B2R), constitutively expressed. Kininase I acts on BK and produces des-Arg9-bradykinin (DABK), which binds to the B1 receptor (B1R), induced by inflammation. In the liver, BK induces portal hypertensive response mediated by B2R. B1R modulates neither hypertensive nor hypotensive hepatic response, but we found that it is expressed during various stages of liver injury: inflammation, cirrhosis and ischemia-reperfusion injury (IRI). In this case, B1R activation is related to apoptosis and B2R to necrosis.In experimental models of fibrogenesis, we verify that B1R knockout mice have different response to CCl4 or cholestatic (bile duct ligation, BDL). The results suggest that activation of B1R protects the liver from fibrosis in the CCl4 model, but not in the BDL model. We also found the hepatoprotective effect of condroitin sulfate in fibrogenesis induced by BDL. The main goal is to study the liver cytoprotection with different interventions in various experimental models of liver injury. Regarding the experimental model of IRI, the aim is to study the relation between the activation of B2R or B1R and cell death by analyzing the intracellular signaling pathways of kinins and cell death. In addition, we will examine the role of the pre-treatment with omega-3 or 6 enriched diet in the cell death induced by kinins in IRI. In relation to the fibrogenesis, the objectives are to evaluate the inflammation, cell death and regeneration after different interventions: chondroitin administration or laser therapy.To develop the experimental model of IRI, the liver will be isolated, exsanguinated and preservation solution of the University of Wisconsin at 4ºC will be infused. The liver will be kept in this solution for 24 hours at 4° C (ischemia). After this period, the organ is reperfused with Krebs solution (37º C) and saline, BK or DABK will be injected in bolus in the portal cannula and portal pressure monitored. To analyse the role of pre-treatment with omega-3 or -6, male Wistar rats will be fed with control. É-3 or É-6-enriched diet for 8 weeks, and after this period submitted to the model of IRI. Alanine- and aspartate-aminotransferase serum activity will be performed to assess the prior condition of the animal. B1R will be detected by Western blotting and cytokines and chemokines by Magpix system (Millipore). Trypan blue will be used to assess necrotic cell death. Apoptosis will be studied by immunodetection of cleaved caspase-3, Bcl-2, Bcl-X, Caspase-3, mcl-1 and TUNEL assay.For the experimental model of BDL, Wistar rats will be anesthetized with mixture of isoflurane 1.5% and nitrous oxide at 0.8 L/min and medicinal oxygen at 0.4 L/min for anesthesia induction. Body temperature will be maintained at 37° C using a heated operating table. After trichotomy and asepsis with iodized alcohol, laparotomy will be performed and the common bile duct isolated, doubly ligated with 5-0 silk and divided between these points. Next the peritonium muscle layers and skin will be sutured. In the control group (Sham), similar procedure will be performed, but without the common bile duct ligation. The animals are euthanized 7, 14, 21 and 28 days after surgery. In the treated-animals, C4S or C6S (120 mg / kg of animal) will be administered intraperitoneally one day before, the day of surgery and at 48 hours after the surgery, once a day in the morning. After this period the animals will be treated every 48 hours. In untreated-animals, vehicle (0.9% NaCl) will be administered at the same intervals described for condroitin administration.Thus, this project aims to understanding the pathophysiology of IRI or fibrogenesis and also verifies the role of therapeutic interventions in these situations to improve the quality of the organ and consequently the patient. (AU)