Prevention Of Alcoholic Fatty Liver In Rats By Ethanol Extract Of Portulaca Oleracea And Its Mechanism

Steatosis, the earliest and most response of the liver to alcohol comsumption. Fatty liverdevelops in more than90%heavy drinkers. Some researchers indicate that alcoholic fatty lievr(AFL) is not what has been considered a benigh disease condition for a long time. If alcohol consumption iscontinued, steatosis can progress to irreversible hepatic injury. Recently increasing evidence suggests thatthe high energy consumption by hepatocytes accentuates hepatocellular mitochondrial respiratory chain sothat hepatic oxygen consumption is increased, with a smaller increase in hepatic oxygen delivery, resultingin central venous hypoxia. This sensitizes the liver to other insults. Moreover, hepatomegaly can oppresshepatic sinusoid when AFL happens so that hepatocytes are forced in an ischemic and hypoxia condition.Surveys show that20%-40%of AFL patients develop more severe fibrosis and8%-10%develop advanced cirrhosis. These advanced alcoholic liver diseases are so severe that it’sdifficult to cure. Therefore, the prevention of AFL disease is widely thought highly of.The pathogenesis of AFL is not very clear now, however, available targeted therapies arestill lack. Silymarin well known as a hepatoprotector has been used in clinic. It was foundeffective clinically to treat a variety of liver disorders, including alcoholic liver disease. Butthe effectiveness of Silymarin as liver disease remedy was discounted by its poor watersolubility and low bioavailability after oral administration. The American Association ofHepatology and Gastroenterology announced that silymarin has no persuasive effect onpatients with acute or chronic alcoholic liver diseases and only used in clinical trials, whichwas published in Hepatology in2010. Other drugs for AFL are still in the experience.Therefore, it is very important to study and exploit the anti-AFL drugs.The Portulaca oleracea L.(PO) belonging to Portulaceae and Portuaca is one of the wildplants not only as a kind of food but also a drug approved by Ministry of Public health. In ourprevious research, we found that the ethanol extract of Portulaca oleracea L.(EEPO)significantly protected against hypoxia-induced neuro damage and oxidant stress. Moreover,we also noticed that EEPO exhibited potent protective property in hepaticischemia-reperfusion injury in mice. Recent pharmacological studies have reported that a widerange of pharmacological effects of PO, such as anti-inflammatory, anti-oxidant, anti-aging,regulating serum lipids and so on. Other researches also show that it can protect chemical-,drug-and ethanol-induced hepatic injury. Therefore, the aim of the present study is toinvestigate prevention of alcoholic fatty liver in rats by EEPO. Furthermore, we studied thepossible mechanism by which EEPO ameliorates lipid metabolism. ObjectiveTo study the prevention of ethanol induced fatty liver in rats by EEPO and the possibleunderlying mechanism by which EEPO ameliorates lipid metabolism.Method1. The replication of AFL model in rat1.1Experimental animals and treatmentThe20male Wistar rats weighting160to180g were randomly assigned to control groupand model group(n=10) according to body weight after the first5days’ acclimation. All rats ofthe model group were given52°wine0.5g/100g(body weight) and high-fat diet in fastingstate by gavage in the morning while the rats of control group were given physiological salineand standard diet. All rats were on empty stomach after eight every evening. Rats wereweighted every three days and sacrificed in fast state after75days. The whole liver wereexcised and weighted, the same portion preserved in10%neutral-buffered formalin and forhistopathological analysis and other portions snap-frozen in liquid nitrogen for biochemicalassays. Blood was collected and serum was separated for biochemical analysis.1.2Calculation of the ratios of liver weight/body weightLiver weight(g)/body weight(100g) ratios mean the ratios of liver weight(g) to the lastbody weight(100g) of rats.1.3Determination of Hepatic TriglyceridesHepatic triglycerides(TG) were determined enzymatically using a commercially availableenzymatic kit according to the manufacturer’s protocol.1.4Histopathological Analysis.Livers were fixed with10%neutral formalin, embedded in paraffin, and stained withhematoxylin/eosin and analyzed via microscopy.1.5Measurement of serum TG, TC, ALT and ASTSerum levels of TG, total cholesterol(TC), alanine aminotransferase (ALT) and aspartateaminotransferase (AST) were measured by standard techniques in the Clinical of ChanghaiHospital, Shanghai, China. 2. Protective effect of EEPO on ethanol induced fatty liver in rats2.1Preparation of EEPO and SilymarinEEPO was prepared and provided by Department of Traditional Chinese Medicine,Changhai Hospitial, Shanghai. EEPO was dissolved in0.5%sodium carboxymethyl cellulosesolution and prepared three different doses (1.8g/ml,3.5g/ml,7.0g/ml). Silymarin was alsodissolved in0.5%sodium carboxymethyl cellulose solution(20mg/ml).2.2Experimental animals and treatmentAfter the first5days’ acclimation, the70Male Wistar rats weighting160to180g wererandomly assigned to seven groups(n=8): normal control group(standard pellet diet plusphysiological saline), AFL model group(high-fat diet plus ethanol), solvent group(high-fatdiet plus ethanol and0.5%sodium carboxymethyl cellulose solution), Silymaringroup(high-fat diet plus ethanol and200mg/kg Silymarin), low-dose EEPO group(high-fatdiet plus ethanol and1.8g/100g EEPO), medium-dose EEPO group(high-fat diet plus ethanoland3.5g/100g EEPO) and high-dose EEPO group(high-fat diet plus ethanol and7.0g/100gEEPO). All rats except the normal control group were given52°wine0.5g/100g(body weight)and high-fat diet in fasting state by gavage in the morning while the rats of control group weregiven physiological saline and standard diet for75days. The rats of EEPO, Silymarin andsolvent groups were orally administrated with EEPO, Silymarin or0.5%sodiumcarboxymethyl cellulose solution1.0ml/100g body weight per day by gavage in the evening.Rats were sacrificed after the last drug administration. At the conclusion of the feeding, ratswere weighted and euthanized. The whole liver were excised and weighted, the same portionpreserved in10%neutral-buffered formalin and other portions snap-frozen in liquid nitrogenfor biochemical assays. Blood was collected and serum was separated for biochemicalanalysis.2.3Calculation of liver indexSame as in part1.2.4Determination of Hepatic TriglyceridesSame as in part1.2.5Histopathological AnalysisLivers were fixed with10%neutral formalin, embedded in paraffin, and stained with hematoxylin/eosin and analyzed via microscopy. For Oil Red O staining, frozen liver tissueswere embedded in OCT Compound. The8μm cryosections were cut and fixed to microscopeslides, allowed to air dry overnight at room temperature and stained with fresh Oil Red O for15minutes, rinsed in water, and then counterstained with hematoxylin.2.6Measurement of serum TG, TC, ALT, AST and GGTSame as in part1. The measurement of GGT is same to ALT and AST.3. Study on mechanism of the protective of EEPO on alcoholic fatty liver in rats3.1Animal Experiment3.11Experimental animals and treatmentSame as in part2.3.12Determination of hepatic ACC, FAS,SREBP-1c, CPT-1, PPARα and HIF-1αmRNA levels in ratsReal time PCR was performed using IQ5Real-Time PCR Detection System. Two stepRT-PCR method was performed using Real Time PCR Master Mix. Primers used to analyzeall the transcripts have been reported else where.3.13Determination of hepatic ACC and phosphoryl-ACC protein levels in ratsHepatic ACC and phosphoryl-ACC protein levels in rats were determined by Westernblot.3.2Cell study3.21Preparation of herb-containing serumThe male Wistar rats weighting120g were randomly assigned to control group, silymaringroup and EEPO group for the preparation of herb-containing serum being orallyadministrated accordingly with0.5%sodium carboxymethyl cellulose solution, silymarin andEEPO, twice a day for five days. The rats were sacrificed one hour after the last drugadministration. The blood was collected and centrifuged for15min at3000rpm. The serumwas collected and inactivated at56℃for half an hour and then stored at-20℃for use.3.22Arnt1siRNA transfection and EEPO interventionHuman L02hepatic cells were transfected with Arnt1siRNA. Cells were divided into control group, silymarin group and EEPO group administrated respectively0.5%sodiumcarboxymethyl cellulose solution, silymarin and EEPO. The final serum concentration were1%,2%,5%and10%separately.3.23Determination of ACC, FAS, SREBP-1c mRNA levels in L02hepatic cellsSame as in part2.4. Statistical analysesThe results are expressed as means±SD. Statistical analysis was carried out by usingSPSS16.0statistical software. T-test, One-way analysis of variance (ANOVA) and nonparametric testwere performed for the analysis of the biochemical indices. Differences were considered significantlyat P<0.05level.Results1. The replication of AFL model in rat1.1Comparation of the ratios of liver weight/body weightThe liver weight/body weight ratios of rats were significantly higher in AFL model groupcompared with control group(P<0.05).1.2Comparation of hepatic triglyceridesHepatic triglycerides were significantly higher in AFL model group than control group(P<0.001)1.3Histopathological AnalysisHE staining revealed steatosis with varied aizes of lipid deposition in diffuse distribution.No lipid drops were seen in control group.1.4Comparation of serum TG, TC, ALT, AST levelsSerum TG and TC levels were significantly higher in AFL model group compared withcontrol group (P<0.05). Serum ALT was also higher than control group, but no significantdifference was shown between two groups.2. Protective effect of EEPO on ethanol induced fatty liver in rats2.1Effects of EEPO on body weight and the ratios of liver weight/body weight in AFL ratsAt the end of experiment, The body weight of rats showed no significant differences in allgroups(P>0.05). The liver weight/body weight ratios of rats were significantly higher in AFLmodel group and solvent control group compared with normal control group(P<0.001). Afteroral administration of EEPO for75days, The liver weight/body weight ratios decreased inlow-dose EEPO, middle-dose-EEPO and high-dose EEPO compared with AFL model groupand solvent control group(P<0.001). Data showed that the EEPO had a dose-dependent effecton decreasing liver index. It was also decreased in silymarin group compared with AFL modelgroup and solvent control group. There is no significant difference within three doses EEPOgroup and silymarin group.2.2Effects of EEPO on hepatic triglycerides in AFL ratsHepatic triglycerides were significantly higher in AFL model group and solvent controlgroup compared with normal control group (P<0.001), but no significant difference of hepatictriglycerides was observed within the two groups(P>0.05). Hepatic TG levels decreased inlow-dose EEPO, middle-dose-EEPO and high-dose EEPO compared with AFL model groupand solvent control group (P<0.05). Data showed that the EEPO had a dose-dependent effecton decreasing hepatic TG. Hepatic TG was also decreased in silymarin compared with AFLmodel group and solvent control group (P<0.05).2.3Histopathological AnalysisHE and Oil Red staining revealed alleviative steatosis with varied aizes of lipid depositionin alcoholic-induced fatty liver of rats adiministrated orally EEPO for75days compared tocorresponding control group, respectively.2.4Effects of EEPO on serum TG, TC, ALT, AST and GGT in AFL ratsSerum TG and TC levels were significantly higher in AFL model group and solventcontrol group compared with normal control group (P<0.001), but no significant differencewas observed within the two groups (P>0.05). Serum TG and TC levels decreased inlow-dose EEPO, middle-dose-EEPO and high-dose EEPO group compared with AFL modelgroup and solvent control group (P<0.001). Data showed that the EEPO had a dose-dependenteffect on decreasing serum lipids. Serum lipids were also decreased in silymarin groupcompared with AFL model group and solvent control group(P<0.001). Serum ALT, AST andGGT showed no significant differences in all groups. 3. Study on mechanism of the protective of EEPO on alcoholic fatty liver in rats3.1Animal Experiment3.11Effects of EEPO on hepatic ACC, FAS, SREBP-1c, CPT-1, PPARα and HIE-1αmRNA levels in rats liverThe real-time PCR results showed that EEPO had no effect on PPARα and CPT-1mRNAlevels but decreased significantly SREBP-1c, ACC, FAS and HIF-1α mRNA levels comparedwith AFL model group and solvent control group (P<0.05). This was also observed inSilymarin group.3.12Effects of EEPO on hepatic ACC and phosphoryl-ACC protein levels in ratsBoth of hepatic ACC and phosphoryl-ACC protein levels were significantly higher in AFLmodel group and solvent control group compared with normal control group (P≤0.001), butno significant differences of body weight were observed within the two groups (P>0.05).After oral administration of EEPO for75days, hepatic ACC and phosphoryl-ACC proteinlevels decreased in EEPO groups compared with AFL model group and solvent control group(P<0.05). EEPO showed a dose-dependent effect on hepatic ACC and phosphoryl-ACCprotein levels.3.2Cell study3.21Effects of ARNT1siRNA on ARNT1mRNA levels in L02hepatic cellsThe ARNT1mRNA levels were decreased in L02cell with siRNA transfection thancontrol group(P<0.05).3.22Effects of EEPO-contained serum on ACC, FAS, SREBP-1c mRNA levels in L02hepatic cells with siRNA interferenceThe ACC, FAS and SREBP-1c mRNA levels in siRNA interference group showed nodifference compared with control group without siRNA interference(P>0.05) but weredecreased in EEPO groups compared to control group with siRNA interference(P<0.05).Conclusions1. lipid accumulation in liver, increased hepatic TG and serum TG and TC levels were found in rats intragastric wine and given the high-fat diet in a fixed time every day for75days,which indicated the AFL model of rat were replicated successfully.2. EEPO decreased hepatic TG and serum TG and TC levels and ameliorated lipidaccumulation in ethanol-induced fatty liver, which indicated that EEPO had preventiveeffect on AFL.3. EEPO may affect fat metabolism by decreasing fatty acid synthesis in hepatocytes viasuppressing transcription of SREBP-1c gene and subsequently down-regulate expression ofACC activity and FAS mRNA and thus ameliorates lipid accumulation in ethanol-inducedfatty liver.4. HIF-1α mRNA expression increased in AFL model group compared with normal controlgroup. However the effect of EEPO on of HIF-1α in AFL is still be studied. For thecomplexity of the pathogenesis of AFL, the preventive effect of EEPO on AFL mightthrough not one target. Further study will be necessary.