| ObjectiveAlcohol abuse is worldwide problem, and causes a large variety of diseases every year, as ethanol in alcohol beverage has been shown to be a typical hepatotoxin. Among these adverse effects of alcohol, alcoholic fatty liver disease (AFLD) can be listed at the top, which is able to gradually develop into many more severe healthy issues, such as alcoholic hepatitis, alcoholic hepatofibrosis and ultimate hepatosclerosis. In China, the consumption of alcohol is drastically increasing every year, along with which the number of patients suffering from AFLD is mounting greatly, arousing wide social concerns. However, the mechanisms underlying AFLD remains unknown. Thus, it is important to explore more effective and safety medicines to cope with AFLD.Garlic oil (GO), a product usually prepared by steam distillation, contains more than thirty types of organ sulfur compounds, including diallyl trisulfide (DATS), diallyl disulfide (DADS), diallyl sulfide (DAS) and so forth. These sulfur components provide protective effects against oxidative stress. Recent studies show that garlic oil is able to prevent acute experimental liver injury induced by alcohol, so GO is predicated as a potential medicne for AFLD.Therefore, the aim of the present study is to investigate the effects of GO on AFLD model mice induced by a high administration of ethanol. Here, serum, hepatic biochemical parameters as well as histopathological changes were determined to evaluate the protective effects of GO. Besides, hepatic antioxidant system, mitochondrial function, activities of hepatic ethanol metabolizing enzymes, and fat metabolism-associated factors were examined to exploit the underlying mechanisms.Methods1. Animal modelMale Kun-Ming mice, weighing 18-22g, were provided by the Laboratory Animal Center of Shandong University. AFLD models were established by feeding with high fat diet plus ethanol for 3 weeks. All the mice were maintained at approximately 22℃with a 12h light cycle, and had free access to commercial food and tap water.2. Animal treatmentThe protective effect of garlic oil on alcoholic fatty liverNinety mice were randomly divided into six groups with 15 in each:garlic oil groups (20,40,80 mg/kg), tiopronin (150 mg/kg) group, model group and control group. Apart from these in control group, mice were fed with high fat diet and ethanol (i.g.). Mice in garlic oil groups were administered orally with different doses of garlic oil once a day, while control and model group animals were treated with the same volume of corn oil. At the end of 3 weeks, blood and hepatic tissue were collected after the mice were euthanatized for biochemical analysis and histological tests.The curative effect of garlic oil on alcoholic fatty liverThe aim is to demonstrate whether GO can reverse AFLD by using 150 mice. Mice in model group were established by treatment with high fat diet plus ethanol for 3 weeks. After AFLD model was established, these mice were randomly divided into six group:GO groups (25,50,100 mg/kg), tiopronin (150 mg/kg) group, natural recovery group and control group. All animals were fed commercial food. GO1 groups were administered orally with different doses of GO once a day, while control and natural recovery group animals were treated with the same volume of corn oil. One week later, blood and hepatic tissue were collected after the mice were euthanatized for biochemical analysis and histological tests.3. Activity of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) in serum, liver index (the ratio of liver weight to body weight), hepatic triglyceride (TG) level, and hepatopathological changes were examined to assess the protective effect of GO.4.10% liver homogenate was prepared to examine the contents of malondialdehyde (MDA) and reduced glutathione (GSH), and the activity of the superoxide dismutase (SOD).5. Levels of CYP2W1 in 10% liver homogenate were determined using Western Blotting.Results:1. The protective effect of garlic oil on alcoholic fatty liver1.1 Effects of GO on serum ALT and ASTCompared with control group, the serum ALT and AST activities in model group were increased by 41.64%(P<0.01) and 51.80%(P<0.01), respectively. Compared with model group, the ALT activities in three GO groups (20,40,80 mg/kg) were decreased by 10.10%,10.84%, and 12.43%(P<0.05), respectively, while the AST activities were decreased by 22.34%,22.66%, and 25.80%(P<0.01), respectively.1.2 Effects of GO on serum TG and TC levelsCompared with the control group, the serum TC and TG level in model group were increased by 86.60%(P<0.01) and 41.64%(P<0.01), respectively. Compared with model group, the TG level in three GO groups(20,40,80mg/kg) and tiopronin group were decreased by 14.92%(P<0.01),21.55%(P<0.01),33.15%(P<0.01) and 11.05%(P<0.01), respectively; while the TC levels were decreased by 15.38%(P<0.01),18.90%(P<0.01),22.42%(P<0.01) and 23.30%(P<0.01), respectively.1.3 Effects of GO on hepatic TG and TC levelsCompared with the control group, the hepatic TC and TG level in ethanol group were increased by 301.31%(P<0.01) and 383.33%(P<0.01), respectively. Compared with model group, the TG level in three GO groups and tiopronin group were decreased by 23.12%(P<0.01),24.27%(P<0.01),27.85%(P<0.01) and 24.10%(P<0.01), respectively, while the TC levels were decreased by 12.93%, 22.70%(P<0.05),25.00%(P<0.01) and 21.84%(P<0.01), respectively.1.4 Effects of Go on hepatic FFA levelsCompared with the control group, the hepatic FFA levels in model group were increased 89.25%(P<0.01), respectively. Compared with model group, the FFA levels in three GO groups(20,40,80mg/kg) and tiopronin group were decreased by 25.13%(P<0.01),34.89%(P<0.01),41.13%(P<0.01) and 31.79%(P<0.01), respectively.1.5 Effects of GO on the serum and hepatic lipid peroxidationThe lipid peroxidation was determined by measuring the thiobarbituric acid-reactive substrates (TBARS) in homogenate and expressed as the MDA levels. The MDA level in serum and hepatic of the model group increased significant compared with control group (P<0.01), which was dramatically attenuated by GO pretreatment. Compared with the model group, the hepatic MDA levels decreased by 53.48%(P<0.01),57.75%(P<0.01) and 60.96%(P<0.01) in three GO groups(20,40,80mg/kg) and 43.85%(P<0.01) in tiopronin group, respectively. While the serum MDA levels were decreased by 15.52%(P<0.01),19.46%(P<0.01), (P<0.01) and 24.63%(P<0.01), respectively.1.6 Effects of GO on the hepatic GSH level and the activities of SOD.Compared with the control group, The level of hepatic GSH and activities of SOD was all prominently decreased by 50%(P<0.01) and 27.69%(P<0.01) in model group mice, However, administration of GO increased hepatic GSH and the activities of SOD in degree compared with the model group.1.7 Histological examinationFat droplets were found on the liver sections in ethanol model group through histopathological examination., which was obviously inhibited by GO pretreatment.1.8 Effects of GO on the protein levels of CYP2E1Compared with the control group, CYP2E1 protein levels in model group were increased by 72.00%(P<0.01). and CYP2E1 protein levels in three GO groups and tiopronin group were decreased by 43.60%(P<0.01),63.95%(P<0.01) and 76.16 %(P<0.01), respectively, while the tiopronin group were not significantly altered (P>0.05).2. The curative effect of garlic oil on alcoholic fatty liver2.1 The establishment of alcoholic fatty liver mice modelTo assess the degree of fatty liver in the 3 weeks model group, we detected hepatic TG content and performed Sudan III staining of mice liver. Compared with control group, hepatic TG levels and liver index in model group were increased by 259.31% and 58.87%(P<0.01). Histopathological examination showed that the liver sections of ethanol group were filled with fat droplets stained in yellow.2.2 Effects of GO on serum ALT and ASTCompared with the control group, serum ALT and AST activities in natural recovery group were increased by 30.29%(P<0.01) and 4.48%, respectively. Compared with natural recovery group, ALT activities in three GO groups(25,50,100) were decreased by 7.21%,16.22%(P<0.05) and 20.71%(P<0.01), respectively, while the AST activities were decreased by 2.82%,,5.57% and 8.72%(P<0.01), respectively. And the ALT,AST level of tiopronin group were decreased by 9.91%,1.64%.2.3 Effects of GO on serum TG and TC levelsCompared with the control group, serum TG and TC level in natural recovery group were increased by 52.75%(P<0.01) and 45.81%(P<0.01), respectively. Compared with natural recovery group, TG level in three GO groups(25,50,100) and tiopronin group were decreased by 20.14%(P<0.01),27.34%(P<0.01), 31.65%(P<0.01) and 20.86%(P<0.01), respectively, while the TC levels were decreased by 19.25%(P<0.01),20.61%(P<0.01),22.97%(P<0.01) and 25.34% (P<0.01), respectively.2.4 Effects of GO on hepatic TG and TC levelsCompared with the control group, the hepatic TG and TC level in natural recovery group were increased 241.06%(P<0.01) and 147.22%(P<0.05), respectively. Compared with natural recovery group, the TG level in three GO groups(25,50,100) were decreased by 22.91%(P<0.01),25.83%(P<0.01) and 32.43%(P<0.01), respectively; while the TC levels were decreased by 25.28%,35.39% and 47.75%.2.5 Effects of GO on hepatic FFA levelsCompared with the control group, the hepatic FFA levels in natural recovery group were increased by 115.55%(P<0.01), respectively. Compared natural recovery group, the FFA levels in three GO groups(25,50,100) and tiopronin group were decreased by 22.79%(P<0.01),21.63%(P<0.01),28.36%(P<0.01),25.28%(P<0.01), respectively.2.6 Effects of GO on the serum and hepatic MDA levelThe lipid peroxidation was determined by measuring thiobarbituric acid-reactive substrates (TBARS) in homogenate and expressed as the MDA levels. MDA levels in serum and hepatic of the natural recovery group increased significant, compared with control group (P<0.01), which was dramatically attenuated by GO pretreatment. Compared with natural recovery group, hepatic MDA levels decreased by 7.94%, 11.11% and 27.78% in three GO groups(25,50,100), respectively. While serum MDA levels were decreased by 4.93%,7.22% and 12.26%, respectively. And serum and hepatic MDA level in tiopronin group were decreased by 5.56% and 3.78%.2.7 Effects of GO on the hepatic GSH levelCompared with the control group, The level of hepatic GSH was all prominently decreased by 36.84% in natural recovery group; Compared natural recovery group, the SOD in three GO groups(25,50,100) and tiopronin group were increased by 91.67%(P<0.01),108.33%(P<0.01),141.67%(P<0.01) and 33.33%, respectively.2.8 Effects of GO on the activities of hepatic SODCompared with the control group, The activities of hepatic SOD was decreased by 8.75%(P<0.01) in natural recovery group; Compared natural recovery group, the SOD in three GO groups(25,50,100) and tiopronin group were increased by 12.58% (P<0.01),12.81%(P<0.01) 15.43%(P<0.01) and 7.73%, respectively.2.9 Histological examinationHistopathological examination showed that the liver sections of ethanol group were filled with fat droplets stained in yellow, which was obviously inhibited by GO pretreatment.2.10 Effects of GO on the protein levels of CYP2E1Compared with control group, CYP2E1 protein levels in natural recovery group were increased 58.00%(P<0.01). Compared with natural recovery group CYP2E1 protein levels in three GO groups were decreased by 23.42%(P<0.01), 44.94%(P<0.01) and 52.23%(P<0.01), respectively, while the tiopronin group were not significantly altered (P>0.05).Conclusion1. Garlic oil can prevent Alcoholic Fatty Liver Disease in mice.2. Garlic oil can cure Alcoholic Fatty Liver Disease in mice.3. The protective effect of GO against AFLD is associated with its antioxidant activity and the inhibition of CYP2E1 in liver. |
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