Hydrogen-Rich Saline Protects Against Hepatocellular Apoptosis And Mitochondrial Injury In Rats With Obstructive Jaundice

Background and Aims:Obstructive jaundice(OJ)is a frequently observed condition caused by occlusion ofthe common bile duct or its tributaries. Surgical, endoscopic, and interventionalradiographic decompression are the principal treatments of biliary obstruction, butdecompression alone may not be sufficient to prevent the development of life threateningcomplications such as liver injury, endotoxemia, systemic inflammatory response, andmultiple organ dysfunction that carry a high risk for mortality[1,2]. The mechanismsresponsible for the pathogenesis of cholestatic liver injury from acute biliary obstructionremain largely unknown, although intrahepatic accumulation of reactive oxygen species(ROS) is thought to be an important cause. Experimental and clinical studies havedemonstrated the pivotal role of oxidative stress associated with overproduction of ROS,which may cause lipid peroxidation and disturb the integrity of cellular membranes in thepromotion of hepatic injury in obstructive jaundice[3-5].Molecular hydrogen (H2) possesses antioxidative effects through selective reductionof the levels of hydroxyl radical. Previous studies have demonstrated that H2in the form ofgas or dissolved in water can suppress tissue injuries in brain, liver, heart and intestinecaused by oxidative stress following ischemia-reperfusion. Hydrogen also suppresses theinflammation induced in tissue-destructive diseases such as colitis, hepatitis, andtransplantation-induced intestinal graft injury.Hydrogen-rich saline provides a high concentration of hydrogen that can be easily andsafely applied. We here aim to investigate the effects of hydrogen-rich saline on theprevention of liver and mitochondria injury induced by obstructive jaundice in rats.Methods:1. H2concentration in organs in vivo was detected by hydrogen microelectrode for thefirst time.2. We observed the change of survival rate and weight, and collected blood and liversamples10days after BDL. 3. Liver function was measured using an autoanalyzer4. HE staining, naphthol AS-D chloroacetate esterase technique, immunohistologicalstaining were used to observe the change of liver morphology and neutrophilsaccumulation.5. We observed hepatocyte apoptosis and mitochondrial injury by transmission electronmicroscope.6. Hepatic MDA, SOD, and CAT levels were assessed spectrophotometrically toinvestigate the degree of oxidizing reaction in liver.7. Levels of TNF-α, IL-1β, IL-6, and HMGB1were measured with a commercial ELISAkit, the MPO activity was determined spectrophotometrically, endotoxin concentrationswere measured by the chromogenic LAL assay.8. The JNK and ERK1/2phosphorylation were detected by immunohistochemistry andwestern blot9. We separated mitochondria and cytosol by differential centrifugation10. Mitochondrial MDA, GSH, and GSSH levels were assessed by spectrophotometry.11. The electrical transmembrane potential of mitochondria was monitoredspectrophotometrically with the cationic dye, rhodamine123at the concentration of0.5μM. Mitochondrial swelling was estimated by changes in light scattering as monitoredspectrophotometrically at540nm. ATP content was measured with Sigma ATPbioluminescent assay kit based on the luciferin-luciferase method by achemiluminescence analyzer. Oxygen consumption of isolated mitochondria wasdetermined polarographically with a Clark oxygen electrode.12. The Bax and Bcl-2expression were detected by Western blot, Caspase-3and caspase-9activities were measured in the cytosolic fraction of liver tissues, using a colorimetricassay.Results:1. Concentrations of molecular H2peaked approximately5min following HS injection inliver and kidney, and returned to normal levels40min later2. HS treatment significantly reduced BDL-induced liver damage. 1) Administration of5ml/kg of HS considerably suppressed the release of ALT and ASTfrom the liver by60.03%and57.81%, respectively, compared with NS-treated rats. Thetreatment with10ml/kg HS decreased cholestasis-induced ALT and AST levels by66.89%and64.88%, respectively.2) Moderate hepatic cell edema, necrosis, and neutrophil cell infiltration were seen in HStreated groups and the increased ductular proliferation observed after bile duct ligation wassignificantly diminished after hydrogen treatment.3. HS ameliorated BDL-induced liver damage by reducing oxidative stress andinflammatory reaction in liver tissue.1) HS treatment dramatically suppressed the production of MDA in rats with OJ.Treatment with HS markedly increased the hepatic SOD and CAT activities in BDL rats2) When ligation of common bile duct was followed by HS treatment, plasma endotoxinconcentrations were significantly reduced compared with NS group. Administration of5ml/kg or10ml/kg HS reduced the hepatic levels of MPO in BDL rats by65.63%and75.56%, respectively. HS administration markedly lowered OJ-induced elevation ofhepatic TNF-α, IL-1β and IL-6concentrations. Treatment with HS significantly attenuatedHMGB1levels in serum and liver of BDL rats. Immunohistochemical and western blotanalyses demonstrated that JNK and ERK1/2were less activated in the liver tissuesobtained from HS-treated rats than in those from NS-treated rats4. HS reduced BDL-induced mitochondrial damage and oxidizing reaction1) HS dramatically decreased the level of mitochondrial MDA and GSSG in rats with OJand markedly increased the mitochondrial GSH content in BDL rats2) Administration of HS reduced mitochondrial swelling. Depletion of mitochondrialmembrane potential and ATP content were partially prevented in the mice treated with HS.HS promoted a significant increase of RCR and P/O compared to rats treated by NS.5. HS suppressed hepatocyte apoptosis by blocking the mitochondrial pathway ofapoptosis signaling.1) We observed that apoptosis of HS treated hepatocyte was obviously less than those ofcontrol group by TEM and TUNEL staining. 2) Western blot analyses demonstrated that Bax was less activated, whereas Bcl-2expression was increased in the liver from HS-treated rats.3) The activities of caspase-3and caspase-9were declined in the liver of HS treated rats.Conclusions:Hydrogen-rich saline attenuates BDL-induced liver damage, possibly by reduction ofinflammation and oxidative stress and inhibition of the JNK and ERK1/2pathway.Hydrogen-rich saline can also reduce BDL-induced mitochondria injury and hepatocyteapoptosis.