The Study Of The Effect Of Hypertonic Saline On Hepatic Ischemia Reperfusion Injury In Rats

Part ⅠThe effect of hypertonic saline on neutrophil mediated hepatocellular injury after ischemia reperfusionIntroductionInterruption of blood flow to an organ or tissue (ischemia) and subsequent reperfusion lead to an acute inflammatory response that may cause significant cellular damage and organ dysfunction. Warm ischemia/reperfusion injury of the liver has been shown in a number of clinical settings, including hepatic resectional surgery, liver transplantation, and hemorrhagic shock with fluid resuscitation. Consequences of ischemia/reperfusion injury include liver failure in association with remote organ failure in more severe cases, both of which have significant rates of morbidity and mortality. Work done by Jaeschke et al established that there are two distinct phases of liver injury after warm ischemia and reperfusion. The initial phase of injury is characterized by Kupffer cell-induced oxidant stress. Kupffer cell production and release ofreactive oxygen species, including superoxide anion and hydrogen peroxide, result in acute hepatocellular injury. Despite the contribution of Kupffer cell-derived oxidants, the extent of injury during this initial phase is far less than that observed at later time points.Events occurring during the initial phase of liver injury, including activation of Kupffer cells, initiates a complex inflammatory pathway that culminates with the hepatic accumulation of neutrophils. Recruited neutrophils directly damage hepatocytes by releasing oxidants and proteases and are responsible for the later phase of liver injury induced by ischemia/reperfusion.Recently, more and more studies have demonstrated that hypertonic saline (HTS) had the anti-inflammatory effect and can inhibit a varity of neutrophil functions, which used as resuscitation fluids in many critical illnesses, such as shock, trauma and severe infection illness. Therefore we hypothesized that HTS resuscitation might be effective in the hepatic I/R injury. But now there are still few studies about the HTS in the treatment of hepatic I/R injury. In the present study, we will explore the effect of pretreatment of HTS in the hepatic I/R injury.Materials and MethodsIn vivo model of partial warm hepatic I/R injuryRats were anesthetized with intraperitoneal 4% chloral hydrate (lml/lOOg). After laparotomy, the portal venous and hepatic arterial branches to the left and median hepatic lobes were isolated and occluded with a microvascular clamp. Following 30-minute ischemic period, the clamp was removed, allowing reperfusion. Experimental groupsThe rats were divided into three groups: sham operation group (Sham group), hepatic I/R group (IR group) and pretreatment of HTS group (HTS group). In the Sham group, the rats only underwent laparotomy and isolation of the vessels, without the occlusion of the portal venous and hepatic arterial branches to the left and median hepatic lobes. In the IR group, the animals received 4ml/kg volume of saline intravenous 1 hour before the clamp of the vessels. In the HTSgroup, the rat received 4ml/kg volume of HTS (7.5%) intravenous 1 hour before the clamp of the vessels. The rats were sacrificed at the time of lh, 3h, 6h, 12h and 24h after reperfusion in each group, respectively. Each time point had 5 rats. Assessment of serum aspartate aminotransferase (ALT)Blood samples were obtained from subhepatic inferior vena cava to mesure ALT using the clinical biochemistry machine (HITACHI, Japan). Myeloperoxidase (MPO) assayThe MPO activity of the liver was analyzed by chromatometry using the kit according to the instructions. Flow cytometry analysis of CDllb/CD18(Mac-l) expression on peripheral neutrophilsValues after I/R are expressed as a ratio (%) to the baseline values (100%) obtained from controls. Reverse transcription polymerase chain reaction (RT-PCR) analysisRT-PCR was used to assess ICAM-1 mRNA expression in the liver. Western-blot analysisTo study the protein expression of ICAM-1 in liver. HistologyFormalin-fixed portions of the liver were embedded in paraffin, and 5-um-thick sections were cut and stained with hematoxylin and eosin. The morphology of hepatocytes, infiltration of neutrophils and the structure of hepatic cords were examined. The structures of sinusoid were examined by TEM. Statistical analysis.All values were expressed as mean±standard deviation of the mean (SD). Analysis of variance (ANOVA) and / test of independent means were used for statistical analysis. Data were considered significant at a level of P<0.05.Results Effect of HTS on serum ALT levelsThe serum ALT levels in the IR group began to increase after I/R, peaked at 6h. The pretreatment of HTS resulted in protection against ALT release, which was most significantly at 3h. 6h and 12h (PO.05). The ALT levels remained normal at the Sham group. MPO activityTo determine if HTS pretreatment was accompanied by decreased neutrophils sequestration, we measured liver MPO, a neutrophil marker enzyme.The activity of MPO increased significantly at 6h after reperfusion in IR group. However, HTS protected against I/R-induced hepatic neutrophils infiltration at 6h, showing MPO levels that were comparable with IR group. Mac-1 Expression After Hepatic I/RFlow cytometric analysis revealed that I/R induced a mild but linearly increasing upregulation of Mac-1(CDI Ib/CD18) on the surface of circulating neutrophils in both groups of rats. Mac-1 expression peaked at 6 h and remained almost stable until 24 h. After pretreatment of HTS, the expression was inhibited, which was most significant at 3h and 12h. (PO.05). ICAM-1 expression after hepatic I/RAfter hepatic 1/R, ICAM-1 mRNA expression increased significantly when compared with sham group, peaked at 6h after reperfusion. After the pretreatment of HTS. there showed only mild increase expression of ICAM-1 mRNA after hepatic I/R, which was most significant at 6hu 12h and24h when compared with IR group(/><0.05). Western blot analysis showed the similar results. Histological assessmentSwelling of hepatocytes, structural derangement, necrosis and inflammatory cell infiltration could be found in the hepatic lobes after I/R. Compared with IR group, HTS pretreatment showed moderate hepatocytes swelling and few neutrophils infiltration.ConclusionsPretreatment of HTS had the effect of the prevention of hepatic ischemia reperfusion injury by inhibiting the expression of Mac-1 on circulating neutrophils and the expression of ICAM-1 in the liver.Part IIThe effect of hypertonic saline on cytokines expression during hepatic ischemia injuryIntroductionsMany experimental studies have defined that the hepatic I/R injury includes two phases. The initial phase involves activation of Kupffer cells with the production of the release of reactive oxygen species, molecules that themselves exert hepatocellular injury. The second phase is characterized by further activation of Kupffer cells with the consequent release of chemokines (TNP'-a and IL-I P ) that promote and amplify the development of hepatocellular injury. TNF-a is the most important inflammatory molecules. It will up-regulation a lot of chemokines, such as intercellular adhesion molecule 1 (ICAM-i), liver C-X-C chemokine and ENA-78 generation, which will further promote neutrophil recruitment and directed migration into the liver. The infiltration of neutrophils into liver will cause hepatic parenchymatitis, which is a key element in the hepatic I/R injury. On the other hand, during the I/R injury process, the protective elements are also activated, including the activation of the endogenous IL-IO, which has a great important role in the reversible injury in the I/R process.HTS now is widely used as a resuscitation fluid during critical illness, which has been shown to exert potent anti-inflammatory effects in vivo. After the usage of HTS, cytokines expression profiles would also be changed. From the first part, we found out that HTS pretreatment could inhibit the activation of neutrophils during hepatic ischemia reperfusion. In this part, we will explore the effect of pretreatment of HTS on cytokines expression during hepatic I/R injury.Materials and MethodsIn vivo model of partial warm hepatic I/R injuryRats partial hepatic ischemia reperfusion injury model.Experimental groupsThe rats were divided into three groups: sham operation group (Sham group), hepatic I/R group (IR group) and pretreatment of HTS group (HTS group). In the Sham group, the rats only underwent laparotomy and isolation of the vessels, without the occlusion of the portal venous and hepatic arterial branches to the left and median hepatic lobes. In the IR group, the animals received 4ml/kg volume of saline intravenous 1 hour before the clamp of the vessels. In the HTS group, the rat received 4ml/kg volume of HTS (7.5%) intravenous 1 hour before the clamp of the vessels. The rats were sacrificed at the time of Ih, 3h, 6h, I2h and 24h after reperfusion in each group, respectively. Each time point had 5 rats. Assessment of serum aspartate aminotransferase (ALT)Blood samples were obtained from subhepatic inferior vena cava to mesure ALT using the clinical biochemistry machine. Chemokine determinationsRat TNF-a and IL-10 chemokines were determined by enzyme linked immunosorbent assay (ELISA) using commercially available reagents (R&D systems, USA). Chemokine levels were reported in pg/ml. Myeloperoxidase (MPO) assayThe MPO activity of the liver was analyzed by chromatometry using the kit according to the instructions. Reverse transcription polymerase chain reaction (RT-PCR) analysisRT-PCR was used to assess TNF-a and IL-10 mRNA expression in the liver. Western-blot analysisTo study the protein expression of TNF-a, IL-10, STAT3 and phosphorylated STAT3 (P-STAT3). HistologyFormalin-fixed portions of the liver were embedded in paraffin, and 5-um-thick sections were cut and stained with hematoxylin and eosin. The morphology of hepatocytes, infiltration ofneutrophils and the structure of hepatic cords were examined. Statistical analysis.All values were expressed as mean±standard deviation of the mean (SD). Analysis of variance (ANOVA) and t test of independent means were used for statistical analysis. Data were considered significant at a level of P<0.05.ResultsEffect of HTS on serum ALT levelsThe serum ALT levels in the IR group began to increase after 1/R, peaked at 6h. The pretreatment of HTS resulted in protection against ALT release, which was most significantly at 3h. 6h and 12h (P<0.05). The ALT levels remained normal at the Sham group. Chemokine determinationsPlasma TNF-a levels were determined by ELISA on serum samples from all three group. Reperfusion of the ischemic liver resulted in a significant increase in the plasma TNF-a level in the IR group, peaked at 6h. Pretreatment of the HTS showed a smaller increase in the plasma TNF-a level, which was most significantly at 3h, 6h and 12h compared with IR group (P<0.05). The Sham group showed a low and stable of TNF-a level. In the present study, we observed the I/R caused a substantial increase of the plasma IL-10 levels. The plasma IL-10 levels peaked at 6h and tended to return toward baseline at 24h after reperfusion. By contrast, HTS pretreatment caused a marked rise in IL-10 levels, especially at Ih, 3h and 6h (VO.05), suggesting the HTS could induce the endogenous IL-10 release. The plasma IL-10 levels were low in the Sham group. MPO activityTo determine if HTS pretreatment was accompanied by decreased neutrophils sequestration, we measured liver MPO, a neutrophil marker enzyme.The activity of MPO increased significantly at 6h after reperfusion in IR group. However, HTS protected against I/R-induced hepatic neutrophils infiltration at 6h, showing MPO levels that were comparable with IR group.RT-PCR analysisFrom RT-PCR analysis, we found that the TNF-a mRNA expression was inhibited, but the IL-10 mRNA expression was increase significantly in the HTS pretreatment group compared with the IR group, which suggested HTS could suppress the expression of inflammatory molecules such as TNF-a through inducing the increase release of endogenous IL-10. Western-blot analysisFrom Western-blot results, the expression of TNF-a began to increase significantly after hepatic 1/R, peaked at 6h after 1/R. In the HTS group, the TNF-a expression was inhibited. In addition, we found marked evaluated IL-10 and STAT3 phosphorylation expressions in the HTS pretreatment group compared with the IR group, but no significant difference could be found in the expression of STAT3 between the two groups. Histological assessmentSwelling of hepatocytes, structural derangement, necrosis and inflammatory cell infiltration could be found in the hepatic lobes after I/R. Compared with IR group, HTS pretreatment showed moderate hepatocytes swelling and few neutrophils infiltration.ConclusionsPretreatment of HTS before 1/R can induce a marked release of the endogenous IL-10, which can inhibit TNF-a release and the infiltration of neutrophils into the liver to protect the liver from I/R injury. The activation of STAT3 resulted in the induction of endogenous IL-10 release.Part IIIEffects of hypertonic saline on expression of heme oxygenase enzyme 1 in hepatic ischemia reperfusion injuryIntroductionThe heme oxygenase (HO) system is the rate-limiting step in the conversion of heme into biliverdin, carbon monoxide (CO) and free iron (Fe2"). Three HO isoforms have been identified: HO-1, an inducible heat shock protein 32;HO-2, which is found in brain and testis;and the less well-characterized HO-3. HO-1 is highly induced and confers protection effects in the oxidative stress response both in vivo and in vitro.Up-regulatiort of HO-1 activity with a local Ad-based HO-1 gene therapy or treatment with cobalt protoporphyrin (CoPP) as HO-1 inducer was effective in the prevention of I/R injury in heart, liver, and kidney. Tian et al. reported that hypertonic stress could induce the expression of HO-1. Therefore we hypothesized that HO-1 might also play an important role in the protective effect of HTS pretreatment during hepatic I/R injur. This study was designed to analyze the effect of HTS pretreatment and the role of HO-1 in the hepatic I/R injury.Materials and MethodsIn vivo model of partial warm hepatic I/R injuryRat partial hepatic ischemia reperfusion injury model. Experimental groupsThe rats were divided into five groups. In Sham group, the rats only underwent laparotomy and isolation of the vessels, without the occlusion of the portal venous and hepatic arterial branches to the left and median hepatic lobes. In ZnPP group, the rats were pretreated with the HO-1 inhibitor zinc protoporphyrin at a dose of 5mg/kg administrated 24 hr before the same operation as the Sham group. In IR group, the rats received 4ml/kg volume of saline intravenous1 hour before the clamp of the vessels. In HTS pretreatment group, the rat received 4ml/kg volume of HTS (7.5%) intravenous 1 hour before the clamp of the vessels. In ZnPP intervention group, the rats were pretreated with ZnPP before the clamp of the vessels and then subjected to the same process as the HTS pretreatment group. The rats were sacrificed at the time of 6 hr after reperfusion in each group. Each group had 5 rats. Assessment of serum aspartate aminotransferase (ALT). Chemokine determinationsRat TNF-a and IL-10 chemokines were determined by enzyme linked immiinosorbent assay (ELISA) using commercially available reagents. Chemokine levels were reported in pg/ml. IMyeloperoxidase (MPO) assayThe MPO activity of the liver was analyzed by chromatometry using the kit according to the instructions. Endothelin 1 (ET-1) radio-immunoassayET-1 was analyzed using ET-1 radi-oimmunoassay kit. Reverse transcription polymerase chain reaction (RT-PCR) analysisRT-PCR was used to assess HO-1 mRNA expression in the liver. Western blot analysisTo study the protein expression of TNF-a, ICAM-1, HO-1 and IL-10, HistologyThe morphology of hepatocytes, infiltration of neutrophils, and the structure of hepatic cords were examined in the sections stained with hematoxylin and eosin. The structures of sinusoid were examined by TEM. Statistical analysisAll values were expressed as mean±standard deviation of the mean (SD). Analysis of variance (ANOVA) and / test of independent means were used for statistical analysis. Data were considered significant at a P level of P<0.05.Results HTS pretreatment induces HO-1 expressionWe used competitive-template RT-PCR to analyze the expression of mRNA for HO-1.The expression of HO-1 only increased slightly in livers after I/R. After HTS pretreatment. the expression of HO-1 increased significantly, as compared with sham controls. To confirm the translation into the protein, the expression of HO-1 in livers was also evaluated by Western blot. Consistent with mRNA levels, we found weak expression of HO-1 in the livers of sham controls, a slightly increase expression in the hepatic I/R group and a marked elevated expression in the HTS pretreatment group. HO-1 inhibitor (ZnPP) abolishes the beneficial effects of HTS pretreatmentAdministration of ZnPP alone without 1/R had no effect on ALT levels. After the usage of ZnPP, HTS pretreatment could not increase the expression of HO-1 mRNA and HO-1 protein. And we found augmented ALT levels, MPO activity and ET-1 levels in this group. Histological damage was comparable with the hepatic 1/R group, as evidenced by hepatocellular swelling, necrosis and sinusoidal congestion. All these data showed that HTS pretreatment lost its beneficial effects in the hepatic I/R. The effect of HO-1 inhibitor on cytokines expression after HTS pretreatmentAdministration of ZnPP alone without I/R had no effect on IL-IO and TNF-a expression. HTS pretreatment could also augment the serum IL-10 levels and 1L-10 expression in livers after I/R though these animals was pretreated with ZnPP. But we found no inhibitory effect of TNF-a expression in these animals. IL-10 showed no anti-inflammatory effect after the blockade of HO-1.ConclusionsHTS pretreatment could induce a marked expression of HO-1 and IL-10, which showed potent protective effects in the hepatic I/R injury. But the anti-inflammatory of IL-10 maybe depend on HO-1 expression.