| Background:Radiation-induced Liver Damage（RILD) is a kind of frequently observeddisease.With the various forms of radiotherapy to the application of new technology in thetreatment of hepatic malignancies and other abdominal tumor,its incidence increased。Deteriorating liver function often leads to liver acute systemic inflammatory response, aseries of complications and multiple organ dysfunction syndrome[1,2]. But,the exactmolecular mechanisms responsible for the pathogenesis of RILD remain unknown。Free radicals are unpaired electrons of atoms,radicals or molecules.In the metabolicprocesses,they continue to be generated and cleared,and are necessary to sustain life. Theyare maintained in a state of dynamic equilibrium.A variety of factors to break this balancewill lead to oxidative stress, resulting in releasing many inflammatory cytokines such asTGF-β, IL-6and causing apoptosis or necrosis. Hydroxyl radical and nitrite anion is themost important medium in the process of oxidative stress. ROS is considered to play acrucial role in the progress. Lots of researches have proved that ROS can lead to kinds ofdisease[3-5].But,molecules or substances that can effectively remove them have not beenfound.Molecular hydrogen (H2),the simplest elment in the nature, and possess mildantioxidative effects.Numerous studies show that hydrogen can be selectively scavengehydroxyl radicals and nitrite anions.H2has signaling effects in hydrogen peroxide,buthas no toxic side effects,and it is invalid to nitric oxide. A variety of animal model studieshave confirmed that hydrogen obstructive jaundice,cerebral ischemic injury,heart andischemia reperfusion in intestine transplantation.Previous researches have proved that H2in the form of gas or liquid can restrain injury in obstructive jaundicebrain, cerebralischemic,heart and ischemia-reperfusion in intestine caused by oxidative stress. Hydrogenalso can mitigate the inflammation in many tissues,such as kidney and intestines.Based on the unique selectivly antioxidant activity for hydrogen saline andmechanism of radiation induced liver damage.Radioactive liver injury rats model will beestablished to validate the protective effect of hydrogen saline against radioactive liverinjury.It will explore possible molecular mechanisms of H2, and provide theoreticalbasis for prevention and clinical treatment of radiation induced liver damage.Experimental content and methods:1.The protective effect of Hydrogen saline on radiation induced liver damage rats (1) Hydrogen concentration was detected by hydrogen microelectrode in liver tissues ofSD rats through intraperitoneal injection of hydrogen saline.(2) The change of survival rate and weight was observed, and blood and liver tissues of SDrats was collected and detected10days after radiotherapy.(3)We measure the liver function of rats in three groups by using an autoanalyzer2. Research possible mechanisms of hydrogen saline alleviating radiation inducedliver damage.(1)The alteration of hepatic morphology or neutrophils accumulation was detected by HEstaining.(2)We detected the level of Hepatic MDA, SOD, and CATs by spectrophotometrically towatch the level of oxidizing reaction in rat livers(3)We measured the levels of TNF-α, IL-1β, IL-6, and HMGB1using the ELISAkit.Then,the MPO expression was detected spectrophotometrically, and the chromogenicLAL assay was used to measure concentrations of endotoxin.(4)We detected the level of JNK and ERK1/2phosphorylation by the method of IHC andWB(5) Using differential centrifugation,we separated Mitochondria and cytosol(6)Hepatocyte apoptosis was detected by transmission electron microscope,andmitochondrial injury was observed in the same way.(7)We assessed the levels of Mitochondrial MDA, GSH, and GSSH spectrophotometrily.(8)We monitored the electrical transmembrane potential of mitochondria,and estimated thelevel of Mitochondrial swelling with the spectrophotometer.(9) We also detected ATP content in the mitochondria of RILD hepatic cells by thechemiluminescence analyzer..(10)Using a colorimetric assay, we measured activities of Caspase-3and caspase-9in thecytosolic fraction of liver tissues,then we detected the level of Bax and Bcl-2by WB.Results:1. HS treatment significantly protected liver function against RILD.(1)After intraperitoneal injection of Hydrogen saline, hydrogen concentration in the ratlivers reached the peak levels about5minutes later, and then decreased gradually returnedto normal levels after about40minutes.(2)After intraperitoneal injection of Hydrogen saline,we detected reduction of theexpression of ALT and AST in serum. 2.HS treatment significantly reduced the levels of Oxidative stress and inflammation(1)After radiotherapy was significantly diminished in hydrogen treatment,we detectedincreased level of the cell swelling, necrosis and osmotic pressure.(2) HS alleviated radioation-induced liver damage by lessening the level of oxidativestress and inflammatory reaction.1) HS treatment significantly restrained the MDA expression in RILD groups. HS sharplyimproved the level of SOD and CAT in liver tissues.2) HS treatment dramatically reduced plasma endotoxin levels compared to that in NStreatment rats. HS decreased the hepatic expression of MPO in RILD rats. HSadministration markedly reduced the concentrations of TNF-α, IL-1β and IL-6in liver. HStreatment dramaticlly reduced HMGB1concentrations in serum or liver of RILD rats. IHCand WB analyses showed that the levels of JNK and ERK1/2were more phosphorylated inthe hepar in NS-treated groups than that in HS-treated groups.(3)HS reduced radiological mitochondrial injury and oxidizing reaction1) HS significantly lowed the concentrations of mitochondrial MDA and GSSG in RILDrats and significantly promoted the mitochondrial GSH levels.2)HS ameliorated mitochondrial swelling.,and partially inhibited Depletion ofmitochondrial membrane potential and ATP level in the rats. HS sharply increased thelevel of RCR and P/O in rats NS treatment groups.3. Hydrogen-rich saline protects against hepatocellular apoptosis and Mitochondrialdamage in RILD rats(1)HS protected mitochondrial morphological Structure in the hepatic cells1)The decreased electron-dense granules in the intramitochondrial matrix were detectedin rats with HS treatment.2)A little-amplitude swelling was observed with mitochondria in groups with HStreatment.(2)HS reduced hepatocyte apoptosis by suppressing the expressionof apoptosis signaling in mitochondrial pathway.1)Hepatocyte apoptosis were less in HS-treated groups compared with control groups.2) WB analyses showed that the level of Bax was less activated, while Bcl-2expressionwas promoted in the liver of groups with HS treatment.3) The expression of caspase-3and caspase-9were decreased in HS treatment comparedwith NS treatment groups. Conclusions:Hydrogen-rich saline can protect liver function against radiation-induced liverdamage.In this process,HS possibly suppress the level of inflammation and oxidativestress.One the other way,HS maybe block the JNK and ERK1/2pathway. HS may lessenradiological mitochondria injury and hepatocyte apoptosis.This provided theoretical basisfor prevention and clinical treatment of radiation induced liver damage. |
