| Hepatic injury secondary to warm ischemia and reperfusion (I/R) is an important clinical issue. It has been implicated in the pathogenesis of a variety of clinical conditions including trauma, thermal injury, hypovolemic and endotoxin shock, reconstructive vascular surgery, liver transplantation, and liver resectional surgery. A considerable number of experimental studies have indicated that I/R induced liver injury occurs in a biphasic manner. In the early stages of reperfusion, endothelial cell swelling, vasoconstriction, leukocyte entrapment, and platelet aggregation within the sinusoids result in failure of the microcirculation. Endothelial and Kupffer cell swelling is the result of intracellular edema, following the failure of active transmembrane transport secondary to the ischemia-induced energy deficiency. Vasoconstriction is a result of a deterioration in the delicate balance between nitric oxide and endothelin. These result in narrowing of the sinusoidal lumen with consequent decreased leukocyte velocity. The frequency of leukocyte-endothelial cell contacts is thus increased, promoting leukostasis. Stagnant leukocytes, although unable completely to occlude the sinusoidal lumen, further contribute to the flow hindrance in the sinusoidal network of the hepatic microcirculation. This prolongs the period of hypoxia, with areas of the liver remaining ischemic after the onset of reperfusion. This in turn isfollowed by the activation of Kupffer cells and neutrophils, which produce inflammatory cytokines and oxygenderived free radicals (ODFR), further aggravating hepatic injury.Leukotriene (LT)s are important preinflammatory arachidonic acid-derived metabolites, which are metabolized and secreted to bile in a ATP-dependent manner in liver. LTd synthesis enzymes, including leukotriene d synthase (LTdS), microsomal glutathione S-transferase (mGST) 2 and mGST3, conjugates LTA4 with glutathione (GSH) to form LTd, the parent compound of the cysteinyl leukotrienes. LTd synthesis enzymes (LTdS, mGST2 and mGST3) were found in human and rat liver.It has been reported that LTs were involved in cholestasis, hepatic inflammation, portal hypertension, hepatorenal syndrome, fulminant hepatic failure and primary graft nonfunction following liver transplantation. There were only few studies about the relationship between hepatic I /R injury and LTs. Recent study had observed that the cysteiny LTs content in the hepatic tissue after 12 and 24 h reperfusion was increased compared to controls and accompanied by the enhancement of hepatic edema and plasma ALT elevation. But the alterations in the protein level of mGST2 and mGST3, the cell types responsible for the expression of LTdS, mGST2 and mGST3 and the activities of LTd synthesis enzyme, including LTdS, mGST2 and mGST3 during hepatic I /R injury are not previously studied;the role of LTd synthesis enzymes and LTCi as well as the mechanisms that influence LTd generation in the early phase of hepatic I/R injury are far from being elucidated.Larfars et al first demonstrated that the cysteinyl leukotrienes LTd and LTD4, as well as LTB4, activate NO release from human PMN by surface receptor, G-protein and [Ca2+]i dependent mechanisms. The addition of NO via the infusion of sodium nitroprusside (SNP, 0.05 mM, 1 mM) reduced the effect of leukotriene D4 on portal flow, bile flow and bile acid secretion whereas the leukotriene D4 effects on hepatic glucose output remainedunaffected. Correlation coefficient between decrease in portal flow and reduction of bile flow by infusing leukotriene D., was higher than that while in the presence of SNP. LTB4 decreased hepatocyte NO synthesis in a concentration-dependent manner when the cells were stimulated with a combination of cytokines or IL—1 alone. Reduced synthesis of N02- was associated with reduced iNOS mRNA levels suggesting that the induction of iNOS was inhibited. These findings demonstrate that eicosanoids can regulate hepatocyte NO synthesis in vitro. Research over the past 20 years has identified endogenous nitric oxide (NO) as a key messenger molecule in the cardiovascular, nervous and immune systems. NO may be released from the hepatic vascular endothelium, platelets, nerve endings, mast cells, and Kupffer cells as a response to various stimuli such as endotoxemia, ischemia-reperfusion injury, and circulatory shock.Its release from the endothelium can be elicited by numerous autacoids such as histamine, vasoactive intestinal peptide, adenosine, ATP, 5-HT, substance P, bradykinin, and calcitonin gene - related peptide. At this present time, the effects of NO on hepatic I /R injury are still paradoxical. In addition, it hasn' t been found that whether and how NO influences the expressions and activities of LTC4 synthesis enzymes (LTC4, mGST2 and mGST3) and LTC4 content up to now. Moreover, the mechanisms of NO actin on these gene expressions remain to be elucidated.In his study, we employed hepatic partial I/R (70%) rat model by Pringle' s pattern to study the mRNA and protein expressions of LTC4 synthesis enzymes, the activities of LTC4 synthesis enzymes, and the content of LTCi after I/R in rat liver tissue;we also investigated the effects of sodium nitroprusside(SNP), a NO donor on the mRNA and protein expressions of LTd synthesis enzymes, the activities of LTC4 synthesis enzymes, and the content of LTC4 after hepatic I/R in rat liver tissue;in addition, themechanisms of a NO donor SNP action on the gene expressions of LTC4 synthesis enzymes were also explored in hepatic I/R injury rats.This paper was divided into 3 parts.Parti. Alterations of the mRNA and protein expressions of LTd synthesis enzymes, LTd synthesis enzymes activities and LTd production during hepatic I/R in ratsObjective: To investigate the changes of the mRNA and protein expressions of LTd synthesis enzymes, LTd synthesis enzymes activities and LTd production during hepatic I/R in rats.Methods: Adult male Spr ague-Daw ley rats were divided into 2 groups: sham group (Control) and ischemic-reperfusion group (I/R) with 6 rats in each group. Liver were subjected to 60 min of partial hepatic ischemia followed by 5 h of reperfusion, saline was administered intravenously. We examined LTd content, the activities and expressions of LTd synthesis enzymes (LTdS, mGST2 and mGST3) with RP-HPLC, immunoblot and immunohistochemistry. Liver damage was assessed by serum ALT, AST measurements and histological observation. SOD activity and MDA level were employed to evaluate lipid peroxidation in the pathological process.Results: Compared with control, the mRNA expression of LTdS was significantly increased during 5h reperfusion while the mRNA expression of mGST2 and mGST3 were lower after hepatic 3h reperfusion in rat liver (FKO. 05), the protein expression of LTdS, LTd synthesis enzymes activities and LTd content were significantly increased during I/R injury of rat liver (fKO. 05). This was accompanied by serum ALT and AST elevation (PCO. 05), liver tissue MDA increase (FKQ. 05), as well as histological damage. Immunohistochemistry staining showed that most hepatocytes andsinusoidal endothelial cells expressed strongly LTCiS in a I/R sensitive manner. There were no differences of the protein expression of mGST3 and SOD activity between control and I/R rats.Conclusions: 1.) Hepatic I/R up-regulated obviously the mRNA and protein expressions of LTC4S;2.) Hepatic I/R down-regulated significantly the mRNA expressions of mGST2 and mGST3 while presents no effects on mGST3 protein expression;3.) Our results suggest that LTC4 increase during hepatic I/R in rats could be partialy resulted from the enhancements of LTCiS expression and LTC4 synthesis enzymes activities which mainly contributed to LTCiS rather than mGST2 (in terms of gene level) or mGST3;4.) The increased LTC4 in I/R rat liver tissue may be related to lipid peroxide, redox state imbalance along with liver functional and histological damage.Part2. Effects of SNP on the protein expressions and activities of LTC4 synthesis enzymes and LTCt accumulation in hepatic I /R injury ratsObjective: To study the effects of SNP on the protein expressions and activities of LTC, synthesis enzymes and LTC4 accumulation in hepatic I /R injury rats.Methods: Adult male Sprague-Dawley rats were divided into 5 groups: sham group (Control), ischemic-reperfusion group (I/R) and SNP (2.5, 5 and 10|ag/kg/min) +I/R groups with 6 rats in each group. Liver were subjected to 60 min of partial hepatic ischemia followed by 5 h of reperfusion. Saline or SNP (2. 5, 5 and 10u.g/kg/min) were administered intravenously. The mRNA expressions of inducible nitric oxide synthase (iNOS) and endogenous nitric oxide synthase (eNOS) in the liver tissues, were measured by RT-PCR;the protein expressions of LTC4 synthesis enzymes were detected with western blotting, LTC4 synthesis enzymes activities in microsomes and LTC4 contentin liver tissue were measured by RP-HPLC. Tissue injuries were assessed by serum ALT and AST and histological changes. In addition, serum N02- and liver tissue GSH was also detected by biochemical methods.Results: Compared with the control group, LTdS protein expression, LTCi synthesis enzymes activities in microsomes and LTd content in liver tissue were markedly increased after 5 h reperfusion (RO. 05), the mRNA expression of eNOS were significantly attenuated (RO. 05) while iNOS mRNA was obviously enhanced, and this was accompanied by plasma ALT, AST and NO*-elevation (R0.05) together with hepatic GSH decline (RO. 05) in I/R group. SNP (5 and 10u.g/kg/min) administered intravenously reversed the effects of hepatic I /R on the protein expression of LTdS, the activities of LTCi synthesis enzymes, LTd content, serum N02-, the gene expressions of eNOS and iNOS, and hepatic tissue GSH (RO. 05). Moreover, serum ALT and AST were also weakened in SNP group (RO. 05). Immunohistochemistry staining showed that the expression of LTdS in hepatocytes and sinusoidal endothelial cells in SNP rat liver tissue were lower than that in I/R groups. But no difference of the protein expression of mGST3 among control, I/R and SNP rats was observed (P>0. 05).Conclusions: 1.) SNP, a NO donor, down-regulated the protein expression of LTdS, but did not affect the protein expression of mGST3 during I/R injury in rat liver;2.) SNP repressed LTC, synthesis enzymes activities and decreased LTd production in rat liver after I/R;3.) Our results suggest that the decline of LTd production in SNP rat liver could be partialy resulted from SNP inhibiting LTC4S protein expression and LTd synthesis enzymes activities which mainly contributed to the inhibition of SNP on LTC,,S rather than on mGST3;4.) The effects of SNP may be partially related to the facts that it increased eNOS but reduced iNOS gene expression and serum N02- level, stabilized redox state and protected liver from functional and histological damage during hepatic I/R in rats.Part3. SNP regulated the mRNA expression of LTd synthase via NF-kB signaling pathway in hepatic I/R injury ratsObjective: To explore the mechanisms of SNP action on the mRNA expressions of LTC4 synthesis enzymes in hepatic I/R injury rats.Methods: On the basis of above experiments , we examined the mRNA level of iNOS, eNOS and LTd synthesis enzymes in rat liver tissue, and the protein expression of NF-k B p65, p50 and I k Boc in liver cell lysates and nuclear extracts by RT-PCR and immunoblot respectively. Immunohistochemistry staining examined the expression of NF~k. B p65 in liver tissue sections. In addition, serum N02-,the levels of GSH and MDA together with SOD activity in liver tissue were also detected.Results: Compared with the control group, the mRNA expressions of LTdS and iNOS were significantly increased while the mRNA expressions of mGST2, mGST3 and eNOS were lower after 5h reperfusion in rat liver (P<0. 05), serum N02- elevation and hepatic GSH decline in I/R group were also observed(P<0. 05), the protein expressions of NF-k B p65 and p50 in nucleus extract were obviously enhanced in I/R group. Immunohistochemistry staining revealed that I/R rat liver exhibited strongly cytoplasmic and nuclear staining, control rat liver displayed negative staining and SNP rat liver with slightly cytoplasmic and nuclear staining. SNP(5 and 10u,g/kg/min) reversed completely the effects of hepatic I /R injury on the gene expressions of LTdS, iNOS and eNOS, the protein expressions of NF- k B p65 and p50 in nucleus extract, as well as the levels of serum N02- and hepatic GSH and MDA. Interestingly, SNP (2.5u.g/kg/min) increased the mRNA expressions of mGST2 and mGST3 while SNP (10u,g/kg/min) increased mGST2 mRNA level but decreased mGST3 mRNA level compared to I/R group. Compared with sham group, the mRNA expressions of mGST2 and mGST3 elevated in SNP (2.5 (P<0. 05), mGST3 mRNA decreased and the mGST2 mRNA exhibited nochanges in SNP (5 and 10u,g/kg/min) groups (P>0.05). But I k Ba protein expression and SOD activity in liver tissue remains unchanged in all groups. Conclusions: 1.) The mRNA expressions of LTC4synthesis enzymes were partially regulated via NF- k. B activation pathway in a I k Ba -independent manner during hepatic I/R injury in rats;2.) SNP, a NO donor, down-regulated obviously the mRNA expression of LTC4S by inhibiting NF- k. B activation independent of Ik Ba degradation, but appeared to be a dual effects on the regulation of the mRNA expressions of mGST2 and mGST3 by other signaling pathway different with NF-kB activation;3) NF-kB activation may be also associated with ROS,lipid peroxidation,GSH depletion.Conclusions1. Up-regulation of the mRNA expression of LTC4S presented continuously and down-regulation of the mRNA expressions of mGST2 and mGST3 developed afterwards in the early phase of hepatic I/R in rats, which are partially caused by NF-k B activation independent of IkBa degradation;2. Hepatic I/R injury enhanced the protein expression of LTC4, but did not affect the protein expression of mGST3;3. LTC4 synthesis enzymes activities and LTC4 content were all increased significantly in I/R liver tissue, suggesting that LTC4 accumulation in I/R rat liver could be partialy resulted from the enhancements of LTC4S expression and LTC, synthesis enzymes activities which mainly contributed to LTC,|S rather than mGST2 (in terms of gene level) or mGST3;4. The increased LTC, in I /R rat liver tissue may be related to lipid peroxide, redox state imbalance along with liver functional and histological damage.5. SNP, a NO donor, down-regulated obviously the mRNA expression of LTC4S by inhibiting NF- k B activation in I k Ba-independent manner, but appearedto be a dual effects on the regulation of the mRNA expressions of mGST2 and mGST3 by other signaling pathway different with NF-kB activation;6. SNP attenuated the protein expression of LTC4S, but not affected the protein expressions of mGST3 during I /R in rat liver;7. SNP repressed LTd synthesis enzymes activities and reduced LTC4 production during I /R in rat liver, suggesting that the decline of LTd production in SNP rat liver could be partially resulted from SNP inhibiting LTCiS protein expression and LTC4 synthesis enzymes activities which may be mainly contributed to SNP repressing LTCS rather than mGST2 (in terms of gene level) or mGST3;8. The above effects of SNP may be partially related to the facts that it activated eNOS but inhibited iNOS and reduced serum N(k levels, stabilized redox state and protected liver from functional and histological damage during hepatic I/R in rats.
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