Protective Effect Of Hydrogen-rich Saline Against Liver Injury Caused By Acetaminophen In Mice

BackgroundDrug-induced liver injury (DILI) is defined as a liver injury caused by variousmedications and herbs or their metabolic product leading to liver dysfunction and evenhepatic failure, finally can cause patients died[1]. There is evidence thatamoxicillin/clavulanate isoniazid, and nonsteroidal anti-inflammatory drugs (NSAIDs) areamong the most common causes of DILI[2]. In western countries, NSAIDs are widelyconsumed and constitute the most major causes of DILI[3]. In fact, about10%of total DILIis NSAID related[4]. NSAIDs may be the most frequent source of DILI in United States,accounting for about41%of all cases[5].Acetaminophen (paracetamol, APAP) is a kind of important NSAIDs is the mostcommon cause of DILI and acute liver failure, especially in the USA[6]which accounts formore than50%of cases of acute liver failure in adults[7]. Acetaminophen is a prototypicalexample of dose-dependent hepatotoxic drugs with involving the formation of a highlyreactive, intermediate metabolite, N-acetyl-pbenzoquinone imine (NAPQI)[8]. However,there is emerging evidence that the host immune response might also be involved in theetiopathogenesis of acetaminophen hepatotoxicity[8].At therapeutic doses, acetaminophenis mainly metabolized in the liver by the second phase reaction conjugation reactionsnamely sulfation and glucuronidation[8]. Only a small proportion of the drug administeredis metabolized initially by the first phase reaction reactions of cytochrome P450(CYP)-mediated oxidation reaction and subsequently by second phase reaction conjugationreactions[8]. Under physiologic circumstances oxidation of acetaminophen by means ofCytochrome P450enzyme system namely CYP2E1and CYP3A4isoenzymes can lead toamounts of NAPQI being formed, which is then conjugated to glutathione (GSH) anddetoxified to mercapturic acid and eliminated[8]. When excessive doses of acetaminophenare ingested, the sulfation and glucuronidation pathways become saturated to cause moreNAPQI to be formed rapidly and can lead to the depletion of intrahepatic GSH stores andreduced detoxification of the drug[9]. Prolonged fasting and chronic alcohol use canincrease the sensitivity of acetaminophen hepatotoxicity by the reduction of intrahepaticglutathione stores and possibly through increased expression of CYP2E1, an enzymewhose activity is induced by ethanol[10]. Accumulated NAPQI covalently binds to cellularmacromolecules such as nucleinic acid, protein and interfere with mitochondrial andnuclear function, also causes production of reactive oxygen species, leads to oxidative stress and ultimately lead to apoptosis and centrilobular necrosis in the liver[11].Many antioxidants have been proved that play a protective part in APAP-induced liverinjury, such as glutathioned[12], Schisandrin B[13], Vitamin E[14], beta-carotene[15],melatonin[16].Hydrogen(H2), a novel antioxidant, Ohsawa I and other people have demonstratedthat the inhalation of H2gas markedly suppressed brain injury induced byischemia-reperfusion or inflammation via selectively reduceing the hydroxyl radical andthe most cytotoxic of reactive oxygen species (ROS) to effectively protecte cells, whichshow here that H2gas has potential as an antioxidant in preventive and therapeuticapplications[17]. There are many reports proved that H2gas not only has a protective rolefor brain injury induced by ischemia-reperfusion or inflammation or inflammation[17], butalso has a treatment for ischemia-reperfusion such as heart[18], liver[19], kidney[20].However, Hydrogen-rich water (H2gas dissolved in normal saline at0.4MPa, HS) ismore safe and convenient and has the same functions of like H2gas which plays aprotective role for injury caused by ischemia-reperfusion injury or inflammation[21-25].Therefore, the aim of the present study was to evaluate the protective effects of HS onAPAP-induced liver injury in mice via liver function markers in the serum as well as onantioxidant enzyme levels and hepatic histopathology.Experimental content and methods:1. Model of acetaminophen-induced liver injury and the experimental group.(1) The experimental modelExperimental animals were purchased from the Second Military Medical Universitylaboratory animal center. Before the experiment, animals were raised a week and werefasted12hours. Liver injury was induced by an intraperitoneal injection of500mg/kgacetaminophen.(2) The experimental modelForty BALB/C male mice were divided randomly into four groups: normal saline(NS), APAP, APAP+NS and APAP+HS groups. The NS group received a single dose NS(500mg/kg) intraperitoneal injection. In the APAP group, liver injury was induced by anintraperitoneal injection of500mg/kg APAP. Otherwise, in the APAP+NS and APAP+HSgroups, with exception of APAP(i.p.500mg/kg), HS (6ml/kg) or an equivalent volume ofNS as control was given intraperitoneally1h after the administration of acetaminophen and either HS (6ml/kg) or an equivalent volume of NS was given intraperitoneally every3h after that.2. Examination index of acetaminophen-induced liver injury.(1) Examination of liver function, such as ALT, AST, ALP.(2) Examination of oxidant stress, such as MPO, MDA, GSH.(3) Examination of inflammatory cytokine, such as IL-6、TNF-.(4) Observation of histopathology by H&E.3. The possible mechanisms of acetaminophen-induced liver injury.(1) HS reduces plasma ALT, AST and ALP levels may be because that HS can alliviateliver cell necrosis degree. Thus, HS reduces the leakage of ALT, AST and ALP from livercells to the blood.(2) HS decreases MPO and MDA and increases GSH levels may be because HS has notonly remove ROS and RNS to reduce damage of mitochondrial ROS和RNS and maintainnormal structure and function of mitochondria, but also can reduce lipid peroxidation andinhibit oxidative stress to protect liver cells from oxidative stress and the damage of freeradicals ultimately.(3) The study has shown that cytokines TNF alpha and IL-6take participate inAPAP-induced liver injury. HS significantly reduced the plasma TNF alpha and IL-6level and liver tissue TNF alpha and IL-6gene expression. The study shows that thehydrogen-rich saline can protect the liver against injury be suppressing inflammatory cellsto secrete inflammatory cytokine.(4) The experiment demonstrates that APAP gives rise to the centrilobular hepatocytenecrosis with the increase of dose may develop to liver failure and even death in mice. HSnot only significantly keeps a good liver shape, but also reduces hepatocyte area.Result1. HS can reduce plasma ALT, AST and ALP levels in acetaminophen-induced liver injuryin mice.2. HS can maintain the structure and function of mitochondria and inhibit oxidative stressin acetaminophen-induced liver injury in mice.3. HS can significantly reduce inflammatory reaction induced in acetaminophen-inducedliver injury in mice.4. HS not only significantly keeps a good liver shape, but also reduces hepatocyte area inacetaminophen-induced liver injury in mice. ConclusionThe experiment takes BALB/C male mice as the research object to research theprotective role in acetaminophen-induced liver injury. The study proves that hydrogensaline can reduce plasma ALT, AST and ALP levels, inhibit oxidative stress, reduceinflammation, and effectively alleviate the severity of liver cell necrosis.