Protective Role And Potential Mechanism Of Carbon Monoxide Against Alcoholic Liver Damage

Part1Protective effect and potential mechanism of carbon monoxide againstethanol-induced damage on rat primary hepatocytesObjective: To investigate the potential protective effect and the key signalingpathways of carbon monoxide against ethanol-induced damage on rat primaryhepatocytes.Methods:1. Primary hepatocytes were isolated from Sprague-Dawley rat using a two-stepcollagenase perfusion procedure and incubated with200mmol/L ethanol for24h toinduce oxidative damage. Meanwhile, quercetin (100μmol/L), as well as Hb(hemoglobin10μmol/L), etc was introduced into the culture medium. The redoxparameters were detected by spectrophotometry, which included the leakage of ASTand LDH, the activity of SOD, as well as the levels of MDA, GSH, and ROS.2. The ethanol-intoxicated hepatocytes were treated with different doses ofCORM-2(5-100μmol/L) or inactivated CORM-2(iCORM-2) for24h. Theparameters mentioned above were detected to observe the dose-response ofexogenous CO against ethanol-derived oxidative stresss on rat heaptocytes.3. Ethanol-incubated rat hepatocytes were co-treated by exogenous inflammatoryfactors (tumor necrosis factor a/TNF-a,<5ng/ml> plus interleukin-6/IL-6<2ng/ml>)and/or CORM-2(20μmol/L), and the redox indices were detected.4. Specific signaling inhibitors, including ODQ (sGC inhibitor,40μmol/L),SB203580(p38inhibitor,15μmol/L), SP600125(JNK inhibitor,30μmol/L), and PD98059(ERK inhibitor,15μmol/L), were introduced into medium whenheaptcocytes were treated by ethanol and/or CORM-2. The change of redox statuswas assayed following24h treatment.5. Main MAPK proteins, including p38, JNK, ERK and their phosphoratedisoforms were detected by Western blot when hepatocytes were treated by ethnoland/or CORM-2for24h.Results:1. Quercetin inhibited the over-production of MDA and ROS, decreasedenzymatic leakage of AST and LDH, and suppressed GSH depletion and SODinactivation in heaptocytes induced by ethanol. Hemoglobin, an inhibitor of carbonmonoxide, abolished the protective effect of quercetin on ethanol-treated hepatocytes.2. Exogenous carbon monoxide, at the moderate or high concentration (20μmol/L-100μmol/L), manifested an evident protection by inhibiting GSH depletionand SOD inactivity, decreasing in the formation of MDA and ROS, and lowering theenzymes leakage from hepatocytes induced by ethanol. Twenty μmol/L of CORM-2,thus, was choosed in the subsequent studies.3. Compared with the alcohol exposure alone, the co-treatment of ethanol andinflammatory factors aggravated the enzymes leakage of AST and LDH, SODinactivity, GSH depletion, as well as the generation of MDA and ROS. Suchdetrimental effect was also significantly inhibited by CORM-2.4. CORM-2inhibited oxidative damage substantially and increased theantioxidant capacity in heaptocytes damaged by ethanol, but ODQ and SB203580abolished the protective effect of CORM-2.5. Ethanol upregulated the phosphorylated levels of p38, JNK, ERK. CORM-2further stimulated p38phosphorylation, whereas JNK or ERK was not influenced.Conclusions:Carbon monoxide prevented rat primary hepatocytes from ethanol-elicitedoxidative damage and inflammatory stress through activating the activity of sGC andp38MAPK pathway. Part2Protective effect and potential mechanism of carbonmonoxide against alcoholic liver injury on miceObjective: To further explore the potential protective effect and the potentialsignaling pathways of carbon monoxide against alcohol-induced liver damage onmice.Methods:Male Balb/c mice (18~20g) were randomized into the following groups:normal control, alcohol, alcohol+CORM-2, and CORM-2control. Liver damage wasinduced by continous three doses (5.0g/kg.bw.) of ethanol (50%ethanol v/v，0.125ml/10g) with12h interval. CORM-2were administered systemically (8.0mg/kg; i.v.,tail vein) immediately after ethanol intake. Four hours later, all animals weresacrificed and the degree of liver oxidative injury were monitored byspectrophotoemtry, which included the level of hepatic MDA, GSH, SOD and ROS,as well as enzymatic leakage of ALT, AST and the level of proinflammatorycytokines (TNF-a, IL-6) in the serum. The phosphorated levels of p38, JNK, ERK andtheir phosphorated isoforms were detected by Western blot.Results:1. Compared to normal control, oral administration of ethanol caused significantincrease in AST, ALT in serum. Depletion of GSH, SOD and enhancement of MDA,ROS were observed in liver tissues. However, CORM-2exhibited potenthepatoprotective activity by decreasing oxidative stress.2. CORM-2significantly supressed the upregulation of suerum TNF-a and IL-6induced by ethanol. Moreover, CORM-2exerted substantial hepatoprotective role bydownregulation of TNF-a, IL-6and upregulation of IL-10mRNA expression in livertissue compared with ethanol-dosed mice alone.3. CORM-2selectively affected the alcohol-induced activation of p38MAPKwhile having no effect on either the ERK or JNK MAPK as comparison with ethanol-treated mice. Conclusions:Carbon monoxide protected mouse liver from ethanol-elicited oxidative damageand inflammatory stress through activating p38MAPK pathway.