| ObjectiveEthanol is one of the well-known hepatotoxins, and alcoholic liver disease (ALD) caused by excessive ethanol consumption is a worldwide health problem. ALD and its complications are the leading causes for death in western countries. In China, the incidence of ALD has been increasing in the recent years, and ethanol ranks as the 2nd cause for the liver damage. Although new insights have been proposed for the molecular mechanisms of ALD, no breakthrough is made in the prevention and treatment of the disease. More or less problems exist for the current drugs used for ALD therapy. Thus, it is important to explore more effective and safety medicines to cope with ALD.Garlic oil (GO), derived from garlic, is composed of more than 30 volatile organosulfur compounds. Pharmacological studies illuminate its dramatic antioxidant ability and modulation on the metabolic enzymes, such as cytochrome P4502E1 (CYP2E1). Since oxidative stress and the CYP2E1 activation play an important role in the initiation and progression of ALD, GO is theoretically a potential medication for ALD treatment.In the current study, the Male KM mice were treated with different doses of GO prior to exposure to a single dose of ethanol. The serum and hepatic biochemical parameters as well as the histopathological changes were determined to evaluate the protection of GO. The hepatic antioxidant system, mitochondrial function, hepatic ethanol metabolizing enzymes, and the fat metabolism-associated factors were examined for the mechanisms exploration. In addition, human normal cell line, LO2, was used to assess the protection of GO against ethanol-induced injury in LO2 cells.Methods1. Male healthy KM mice were divided into 3 groups treated with different doses of GO (50,100,200 mg/kg bw) for 30 d,7 d, and 1 time, respectively. Then the mice were exposed to 4.8 g/kg bw ethanol (50%, v/v,12 ml/kg bw) to induce acute alcoholic liver injury. The serum alanine aminotransferase (ALT) and aspartate aminotransferases (AST) activities, the ratio of liver weight to body weight, hepatic triglyceride (TG) levels, and the pathological changes, were examined to assess the protection of GO.2. Preparing 10% liver homogenate for the examination of the malondialdehyde (MDA) and reduced glutathione (GSH) contents, and the activities of the superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), and glutathione S-transferase (GST).3. Isolation and purification of mitochondria by differential centrifugation for the determination of the mitochondrial membrane potential (△Ψ) and Ca2+-induced membrane permeability transition (MPT). The mitochondrial MDA and GSH contents as well as Mn-SOD and GPx activities were detected for the antioxidant system assay.4. Preparing 10% liver homogenate for the measurement of the ADH and ALDH activities.5. Isolation and purification of microsome by ultracentrifugation for the determination of the activities and protein levels of CYP2E1,1A2, and 3A; CYP2E1 mRNA levels were determined by RT-PCR.6. The ratio of NAD+/NADH was reflected by cytosolic lactate/pyruvate ratio; serum very low density lipoprotein (VLDL) levels were determined using ELISA kits; adipose mobilization was reflected by measuring the serum free fatty acid (FFA) levels; the protein levels of AMP dependent kinase (AMPK), acetyl CoA carboxylase (ACC), peroxisome proliferator activated receptor alpha (PPAR-a), sterol regulating element binding protein 1 (SREBP-1), and fatty acid synthase (FAS), as well as the serum adiponectin and tumor necrosis alpha (TNF-a) levels were determined for investigation of the molecular mechanism of ethanol-induced fatty liver.7. Measuring the ALT and AST activities in the medium and the MDA, GSH, and TG contents in the LO2 cells, as well as the protein levels of CYP2E1 and caspase-3 to evaluate the protection of GO against ethanol-induced LO2 damage.Results1. The protective effects of GO against acute alcoholic liver injury1.1 GO pretreatment for 30 d effectively attenuated acute alcoholic liver injuryCompared with the normal group, the serum ALT and AST activities in ethanol group were increased by 14.47%(P<0.05) and 38.37%(P<0.01), respectively. Compared with ethanol group, the ALT activities in three GO groups were decreased by 3.57%,8.93%, and 17.86% (P<0.05), respectively, while the AST activities were decreased by 12.39%,18.21%, and 24.36%(P<0.05*或P<0.01), respectively.Compared with the normal group, the TG level in ethanol group was increased by 177.99% (P<0.01). Compared with ethanol group, the TG levels in three GO groups were decreased by 39.39%,53.66%, and 54.33% (P<0.01), respectively. Histopathological examination showed that the liver sections of ethanol group were filled with fat droplets stained in yellow, which was obviously inhibited by 30 d of GO pretreatment.1.2 GO pretreatment for 7 d effectively attenuated acute alcoholic liver injuryCompared with the normal group, the ALT and AST activities in ethanol group were increased by 48.76% and 71.64% (P<0.01), respectively. Compared with ethanol group, the ALT activities in three GO groups were decreased by 27.50%, 38.21%, and 39.29% (P<0.01), respectively, while the AST activities were decreased by 29.96%,40.01%, and 43.62% (P<0.01), respectively.Compared with the normal group, the TG content in ethanol group was increased by 120.99% (P<0.01). Compared with ethanol group, the TG contents in three GO groups were decreased by 24.9%,33.7%, and 47.5% (P<0.01), respectively. Histopathological examination showed that the liver sections of ethanol group were filled with fat droplets stained in yellow, which was obviously inhibited by 7 d of GO pretreatment.1.3 Single dose of GO effectively attenuated acute alcoholic liver injuryCompared with the normal group, the ALT and AST activities in ethanol group were increased by 55.89% and 75.05% (P<0.01), respectively. Compared with ethanol group, the ALT activities in three GO groups were decreased by 26.8%, 36.5%, and 37.1% (P<0.05 or<0.01), respectively, while the AST activities were decreased by 31.8%,40.0%, and 40.3% (P<0.01), respectively.Compared with the normal group, the TG cotent in ethanol group was increased by 130.01%(P<0.01). Compared with ethanol group, the TG contents in three GO groups were decreased by 18.50%,28.21%, and 47.47% (P<0.01), respectively. Histopathological examination showed that the liver sections of ethanol group were filled with fat droplets stained in yellow, which was obviously inhibited by single dose of GO pretreatment. 1.4 Effects of GO administered at different time on acute alcoholic liver injuryCompared with the normal group, the ALT and AST activities in ethanol group were increased by 52.35% and 53.45% (P<0.01), respectively. Compared with ethanol group, the ALT activities in GO pretreatment, simultaneous treatment and post-treatment groups were decreased by 33.33%,26.67%, and 26.15% (P<0.01), respectively, while the AST activities were decreased by 30.66%,23.68%, and 18.20%(P<0.01), respectively.Compared with the normal group, the TG content in ethanol group were increased by 142.51% (P<0.01). Compared with ethanol group, the TG levels in GO pretreatment, simultaneous treatment and post-treatment groups were decreased by 38.17%,30.56%, and 21.03% (P<0.01), respectively. Histopathological examination showed that the liver sections of ethanol group were filled with fat droplets stained in yellow, which was obviously inhibited by GO pretreatment and simultaneous treatment.2. Effects of GO on the hepatic antioxidant system2.1 GO pretreatment for 30 d enhanced the hepatic antioxidant systemCompared with the control group, the hepatic MDA content in ethanol group was increased by 94.83%(P<0.01), while the GSH content was decreased by 18.41% (P<0.05). GO pretreatment for 30 d effectively suppressed the MDA increase and GSH depletion, and the MDA contents were restored to the normal value in GO (100, 200 mg/kg bw) groups.Compared with the control group, the SOD and GR activities in ethanol group were decreased by 13.94% and 22.36% (P<0.05), respectively. GO pretreatment for 30 d enhanced the activities of SOD, GR, and CAT. Compared with the ethanol group, the activities of SOD, GR, and CAT were decreased by 27.51%,33.57%, and 18.58% (P<0.05 or<0.01), respectively.2.2 GO pretreatment for 7 d enhanced the hepatic antioxidant systemCompared with the control group, the hepatic MDA content in ethanol group was increased by 66.36%(P<0.01), while the GSH content was increased by 10.66% (P<0.05). Compared with the ethanol group, the MDA contents in three GO groups were decreased by 13.82%,20.32%,28.35%(P<0.01or<0.05), respectively.Compared with the control group, the GR and CAT activities in ethanol group were decreased by 24.09%(P<0.01) and 23.90%(P<0.05), respectively. Compared with the ethanol group, the GR activities in three GO groups were increased by 42.23%,41.24%, and 59.58%(P<0.01), respectively; the SOD activities were increased by 8.58%,13.10%(P<0.05 or<0.01), respectively, while CAT activities was increased by 14.72%and 45.09%(P<0.01), respectively.2.3 Single dose of GO enhanced the hepatic antioxidant systemCompared with the control group, the hepatic MDA content in ethanol group was increased by 82.58%(P<0.01), while the GSH content was increased by 10.40% (P<0.05). Compared with the ethanol group, the MDA contents in three GO groups were decreased by 14.18%,29.80%,32.75%(P<0.01or<0.05), respectively, while GSH contents were increased by 6.63%and 9.01%(P<0.05), respectively.Compared with the control group, the GR activity in ethanol group was decreased by 28.32%(P<0.01). Compared with the ethanol group, the GR activities in three GO groups were increased by 28.56%,37.93%, and 50.45%(P<0.05 or <0.01), respectively; the SOD activities in GO 100,200 mg/kg bw groups were increased by 14.48%,17.00%(P<0.05 or<0.01), respectively, while GST was increased by 19.92% and 42.13% (P<0.01), respectively.2.4 Effects of GO on hepatic mitochondria function and the antioxidant systemCompared with the control group, the decrease of A540 nm of ethanol group were increased by 45.70% and 42.47% (P<0.01), respectively, at 8 and 16 h time points, while those were significantly increased in GO pretreated groups (P<0.01).The decline of fluorescence in each group did not differ significantly at 4 h time point, indicating that mitochondrial membrane potential was not impaired. Compared with the control group, the decline of fluorescence was decreased by 8.76%(P<0.05) and 8.96%(P<0.01), respectively, at 8 and 16 h time points. Compared with ethanol group, the decline of fluorescence was increased by 5.05% and 5.89% (P<0.01), respectively, at 8 and 16 h time points.Compared with the control group, the hepatic MDA contents in ethanol group were increased by 73.94%,311.02%,279.86% (P<0.01), respectively, at 4,8,16 h time points. Compared with the ethanol group, the MDA contents in three GO groups were decreased by 47.06%,49.80%,55.70%(P<0.01) at 8 h time point, respectively, while those were decreased by 42.60%,61.10%,60.59%(P<0.01) at 16 time point, respectively.Compared with the control group, the hepatic GSH contents did not alter significantly at 4 and 8 h time point, but was decreased by 24.20%(P<0.01). Compared with the ethanol group, the GSH contents in three GO groups were increased by 32.92%,41.45%, and 41.17%(P<0.01) at 8 h time point, respectively, while that in 200 mg/kg bw GO group was increased by 37.6%(P<0.01) at 16 h time point.Compared with the control group, the SOD activity in ethanol group was increased by 46.39%(P<0.01) at 4 h time point, but decreased by 27.09%at 8 h time point. Compared with the ethanol group, the SOD activities were dose-dependently enhanced by GO pretreatment at 8 h time point.No significant alteration of the GPx activity exhibited in each groups (P>0.05).3. Effects of GO on the hepatic ethanol metabolizing enzymes3.1 Effects of GO on the hepatic ADH and ALDH activitiesThe activities of ADH and ALDH were not significantly altered by GO pretreatment (P>0.05).3.2 Effects of GO on the activity, protein and mRNA levels of CYP2E1Compared with control group, the CYP2E1 activities in ethanol group were increased by 47.38%,61.96%, and 17.87%(P<0.01), respectively, at three time points. Compared with ethanol group, the CYP2E1 activities in three GO groups were decreased by 33.64%,38.57%, and 39.53%(P<0.01), respectively, at 4 h time point, and decreased by 52.85%,58.95%, and 62.26%(P<0.01), respectively, at 8 h time point, while those were decreased by 25.14%,30.48%, and 41.75%(P<0.01), respectively, at 16 h time point.Compared with control group, the CYP2E1 protein levels in ethanol group were increased by 44.44%and 141.61%(P<0.01), respectively, at 4 and 8 h time points. Compared with ethanol group, the CYP2E1 protein levels in three GO groups were decreased by 44.32%,70.96%, and 80.26%(P<0.01), respectively, at 4 h time point, and decreased by 27.86%,40.88%, and 59.98%(P<0.01), respectively, at 8 h time point, while those were decreased by 44.38%,72.67%, and 88.42%(P<0.01), respectively, at 16 time point.Compared with control group, the CYP2E1 mRNA levels in ethanol group were decreased by 67.88%and 26.83%(P<0.01), respectively, at 4 and 16 h time points. Compared with ethanol group, the CYP2E1 mRNA levels in three GO groups were increased by 311.24%,191.11%, and 54.75%(P<0.01), respectively, at 4 h time point, and increased by 36.25%,65.71%, and 153.15%(P<0.01), respectively, at 8 h time point, while those were increased by 64.04%,54.78%, and 26.44%(P<0.05), respectively, at 16 time point. 3.3 Effects of GO on the activity and protein levels of CYP1A2Compared with control group, the CYP1A2 activities in ethanol group were not significantly altered (P>0.05), at three time points, while those of GO (50,200 mg/kg bw) groups were increased by 47.51%(P<0.05) and 56.76%(P<0.01), respectively, at 4 h time point. CYP1A2 activities in each group did not differ significantly at 8 and 16 h time points.Compared with control group, the CYP1A2 protein levels were not significantly altered at three time points. Compared with ethanol group, the CYP1A2 protein levels in GO (200 mg/kg bw) group was increased by 41.74%(P<0.01) at 4 h time point, while that in three dose of GO groups were decreased by 48.17%,45.04%, and 23.98%(P<0.01), respectively, at 16 h time point.3.4 Effects of GO on the activity and protein levels of CYP3AThe activities of CYP3A did not significantly differ in each group (P>0.05).Compared with control group, the CYP3A protein levels in ethanol group were not significantly altered at three time points; CYP3A protein level in GO (200 mg/kg bw) group was increased by 13.57%(P<0.05) at 4 h time point, while that in three dose of GO groups were increased by 17.25%,16.54%, and 49.39%(P<0.05 or <0.01), respectively, at 8 h time point.3.5 The response of CYP2E1,1A2 and 3A to single dose and 60d of GO treatmentCompared with the respective control group, the CYP2E1 protein levels were decreased by 88.41%(P<0.01) and 60.60%(P<0.01) in single dose and 60 d GO groups, respectively, while the activities were decreased by 33.22%(P<0.01) and 22.25%(P<0.05), respectively.Compared with the respective control group, the CYP1A2 protein levels were decreased by 70.76%and 41.49%(P<0.01) in single dose and 60 d GO groups, respectively, while the activity in single dose GO group was decreased by 23.71% (P<0.01).The activities and protein levels of CYP3A in single dose and 60 d GO groups did not significantly differ compared to the respective control group (P>0.05).4. Effects of GO on the factors involved in alcoholic fatty liver4.1 Effects of GO on the serum VLDL contentsThe serum VLDL contents were not significantly altered by ethanol or GO treatment (P>0.05).4.2 Effects of GO on the ratio of NAD+/NADH Compared with the control group, the ratio of NAD+/NADH in ethanol group was decreased by 23.20%(P<0.01). Compared with the ethanol group, the ratio of NAD+/NADH in three doses GO groups were increased by 11.42%(P>0.05), 24.67%(P<0.01), and 26.19%(P<0.01), respectively.4.3 Effects of GO on the serum and hepatic FFA levelsCompared with the control group, the serum FFA level in ethanol group was increased by 17.91%(P<0.01). Compared with the ethanol group, serum FFA levels in three doses of GO groups were decreased by 19.69%,31.76%, and 29.08% (P<0.01), respectively.Compared with the control group, the hepatic FFA level in ethanol group was increased by 43.29%(P<0.01), at 8 h time point. Compared with the ethanol group, hepatic FFA levels in three doses of GO groups were decreased by 9.87%,13.09%, and 13.48%(P>0.05), respectively, at 4 h time point, and by 14.56%(P>0.05), 24.85%(P<0.01), and 21.13%(P<0.05), respectively, at 8 h time point, while those were decreased by 16.54%(P>0.05),20.63%(P<0.05), and 25.25%(P<0.05) at 16 h time point, respectively.4.4 Effects of GO on the serum adiponectin and TNF-a levelsCompared with the control group, serum adiponectin levels in ethanol group were decreased by 75.62%,17.07%, and 30.69%(P<0.05 or<0.01). Compared with the ethanol group, serum adiponectin levels in three doses of GO groups were increased by 178.42%,248.07%, and 248.34%(P<0.01), respectively, at 4 h time point, while those were increased by 22.87%,22.22%, and 31.69%(P<0.05), respectively, at 16 h time point.Compared with the control group, serum TNF-a levels in ethanol group were increased by 105.45% and 45.54%(P<0.01), respectively, at 4 and 8 h time points. Compared with the ethanol group, serum TNF-a levels in three GO groups were decreased by 32.38%,46.63%, and 41.53%(P<0.01), respectively, at 4 h time point, and by 7.50%(P>0.05),22.56%(P<0.05), and 16.72%(P<0.01), respectively, at 8 h time point, while those were not significantly altered at 16 h time point (P>0.05).4.5 Effects of GO on the protein levels of mature SREBP-1, PPAR-a, p-AMPK, p-ACC, and FAS.Compared with the control group, hepatic SREBP-1 levels were increased by 24.39% and 10.91% (P<0.05 or<0.01). Compared with the ethanol group, hepatic SREBP-1 levels in three doses of GO groups were decreased 34.28%,45.41%, and 53.53%(P<0.01), respectively, at 8 h time point, while those were decreased by 16.99%,29.52%, and 47.76%(P<0.01), respectively, at 16 h time point.Compared with the control group, hepatic PPAR-a levels in ethanol group were decreased by 27.13%and 51.14%(P<0.01), at 4 and 8 h time points. Compared with the ethanol group, hepatic PPAR-a levels in three doses of GO groups were increased by 78.83%,105.45%, and 120.04%(P<0.01), respectively, at 4 h time point, while those were increased by 69.52%,62.66%, and 70.22%(P<0.01), respectively, at 16 h time point.Compared with the control group, hepatic p-AMPK protein level in ethanol group was increased by 46.70% (P<0.01), at 8 h time point. Compared with the ethanol group, hepatic p-AMPK protein levels in three doses of GO groups were increased by 26.97%,56.31%, and 86.89% (P<0.05或<0.01), respectively, at 4 h time point. Hepatic p-AMPK protein levels in three doses of GO groups were significantly higher than that of control group but lower than that of ethanol group at 8 h time point, while those were not significantly altered at 16 h time point.Compared with the control group, hepatic p-ACC protein levels in ethanol group were decreased by 17.65% (P<0.01), but increased by 80.91%(P<0.01), at 8 h time point. Compared with the ethanol group, hepatic p-ACC protein levels in three doses of GO groups were increased by 32.48%,19.64%, and 47.89%(P<0.05 or<0.01), respectively, at 4 h time point. Hepatic p-ACC protein levels in three doses of GO groups were significantly higher than that of control group but lower than that of ethanol group at 8 h, while those were not significantly altered at 16 h time point.Compared with the control group, hepatic FAS protein level in ethanol group was increased by 12.93%(P<0.05), at 8 h time point; FAS protein levels in three doses of GO groups were decreased by 40.00%,53.45%,70.06%(P<0.01), respectively, at 4 h time point, decreased by 36.87%,47.55%,66.83%(P<0.01), respectively, at 8 h time point, and decreased by 10.62%,35.59%,52.12%(P<0.01), respectively, at 16 h time point.5. Effects of GO against ethanol-induced LO2 cell damage5.1 Effects of GO on the ALT and AST activities and TG content in the mediumGO significantly suppressed the increase of the aminotransferase activities and TG content in the medium. Compared with the control group, the ALT, AST activities and TG content in ethanol group were increased by 34.10%(P<0.05),33.99%(P<0.01) and 45.74%(P<0.01), respectively. Compared with ethanol group, the ALT activities were decreased by 21.23%(P<0.05),28.71%(P<0.05), and 31.09% (P<0.01), respectively; the AST activities were decreased by 15.05%(P<0.01),22.37%(P<0.01) and 23.01%(P<0.01), respectively, while TG contents were decreased by 15.12%, 37.35%(P<0.01), and 35.80%(P<0.01), respectively.5.2 Effects of GO on the contents of MDA and GSH in LO2 cellsCompared with the control group, the MDA level in ethanol group was increased by 95.23%(P<0.01), while GSH level was decreased by 20.80%(P<0.01); GSH level in GO group was increased by 68.47% (P<0.01). Compared with ethanol group, the MDA level in GO and ethanol co-treatment group was decreased by 28.86%, 31.30%, and 46.26%(P<0.01); GSH level was increased by 36.67%(P<0.01),55.39%(P<0.01)和115.34%(P<0.01).5.3 Effects of GO on the fat accumulation induced by ethanol in LO2 cellsCompared with the control group, the TG level in ethanol group was increased by 37.71%(P<0.01). Compared with ethanol group, the TG levels in GO (5,10 mg/L) and ethanol co-treatment group were decreased by 12.94%(P<0.05) and 19.51% (P<0.01), respectively. Much fat droplets stained in red by Oil-red O in ethanol group were suppressed obviously by GO treatment.5.4 Effects of GO on the protein levels of CYP2E1 and caspase-3 in LO2 cellsCompared with the control group, the protein level of CYP2E1 in GO group was decreased by 13.40% (P<0.01), while the protein levels of CYP2E1 and caspase-3 in ethanol group were increased by 36.27%(P<0.01) and 27.47%(P<0.01), respectively. Compared with ethanol group, the protein levels of CYP2E1 in GO and ethanol co-treatment group were decreased by 10.43%(P<0.05),17.71%(P<0.01), and 18.70%(P<0.01), respectively, while caspase-3 protein levels in GO and ethanol co-treatment group were decreased by 8.30%,13.81%(P<0.01), and 15.28% (P<0.01), respectively.Conclusion1. GO could effectively attenuate acute ethanol (4.8 g/kg bw)-induced acute liver injury in mice and ameliorate 100 mM ethanol-induced injury in LO2 cells.2. GO could enhance the antioxidant capacity to cope with the oxidative stress caused by ethanol and sustain the mitochondrial function, which contributes to its protection against acute alcoholic liver injury. 3. The protection of GO against acute alcoholic liver injury might be associated with the inhibition of CYP2E1.4. The enhancement of the fatty acid oxidation and the reduction of fatty acid synthesis were involved in the anti-fatty liver effects of GO.5. The suppression of GO on CYP2E1 and 1A2 might be gradually weakened along with the dosing time.6. Go has no influence on CYP3A.
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