Chronic excessive alcohol consumption (more than 40–80 g/day for males and more than 20–40 g/day for females) could induce serious liver injury. In this study, effects of lemon juice on chronic alcohol-induced liver injury in mice were evaluated. The serum biochemical profiles and hepatic lipid peroxidation levels, triacylglycerol (TG) contents, antioxidant enzyme activities, and histopathological changes were examined for evaluating the hepatoprotective effects of lemon juice in mice. In addition, the in vitro antioxidant capacities of lemon juice were determined. The results showed that lemon juice significantly inhibited alcohol-induced increase of alanine transaminase (ALT), aspartate transaminase (AST), hepatic TG, and lipid peroxidation levels in a dose-dependent manner. Histopathological changes induced by alcohol were also remarkably improved by lemon juice treatment. These findings suggest that lemon juice has protective effects on alcohol-induced liver injury in mice. The protective effects might be related to the antioxidant capacity of lemon juice because lemon juice showed in vitro antioxidant capacity.
Alcohol abuse and alcoholism could lead to serious health and socioeconomic problems worldwide. Chronic excessive alcohol consumption (more than 40–80 g/day for males and more than 20–40 g/day for females) could lead to several illnesses, such as gastrointestinal damage, pancreatitis, alcoholic liver disease, neurologic disorders, diabetes mellitus, and cancer [
Chronic alcohol consumption can generate abundant reactive oxygen species (ROS), including superoxide anion radical (
Lemon is a popular fruit consumed as juice and contains high contents of vitamins and polyphenols (mainly flavonoids), such as hesperidin, eriocitrin, naringin, neohesperidin, rutin quercetin, chlorogenic acid, luteolin, and kaempferol [
The compounds 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tri(2-pyridyl)- S-triazine (TPTZ), quercetin, gallic acid, and Folin–Ciocalteu’s phenol reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Assay kits for the determination of SOD, lipid peroxidation, CAT, and TG contents were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other chemicals were of analytical grade.
Lemon was obtained from markets in Guangzhou, China. The fruit was cleaned with deionized water. The edible portion was weighed precisely and mixed with deionized water (1 : 1, m/v), and the mixture was ground into a homogenate with a homogenizer. Then, the homogenate was centrifuged at 5,000
Male C57BL/6 mice (20–25 g) were employed in this study. Thirty mice were randomly divided into 5 groups, each group containing 6 mice. They were maintained in a SPF laboratory animal room, which kept a 12 h light/dark cycle at
The levels of ALT, AST, and TG in serum were determined by a Hitachi-7180 automated biochemistry analyzer (Hitachi, Japan) with the corresponding reagent kit.
The levels of TG, SOD, and CAT in liver tissue were measured using the commercial detection kits according to the manufacturer’s instructions.
The levels of lipid peroxidation in liver tissue were measured by thiobarbituric acid (TBA) method using the commercial detection kits according to the manufacturer’s instructions. The reference standard was malondialdehyde (MDA), and the results were expressed as nmol MDA equivalent/mg prot.
The liver tissue fixed in 4% paraformaldehyde was embedded in paraffin, sectioned into 5
The FRAP assay was performed based on the method described in the literature [
The TEAC assay was carried out according to the procedure in the literature [
TPC were measured according to the literature [
TFC were measured according to the literature [
Statistical analysis was carried out by one-way analysis of variance (ANOVA) with post hoc LSD test using SPSS 13.0 software.
As shown in Figure
Effects of lemon juice on the levels of AST (a) and ALT (b) in serum of mice. Control: normal group; Model: alcohol group; LL: alcohol and low dose of lemon juice group; LM: alcohol and medium dose of lemon juice group; LH: alcohol and high dose of lemon juice group.
Triacylglycerol (TG) content in serum was significantly (
Effects of lemon juice on TG contents in serum (a) and liver (b). Control: normal group; Model: alcohol group; LL: alcohol and low dose of lemon juice group; LM: alcohol and medium dose of lemon juice group; LH: alcohol and high dose of lemon juice group.
The lipid peroxidation levels in liver tissue are shown in Figure
Effects of lemon juice on hepatic lipid peroxidation level in mice. Control: normal group; Model: alcohol group; LL: alcohol and low dose of lemon juice group; LM: alcohol and medium dose of lemon juice group; LH: alcohol and high dose of lemon juice group.
Figure
Effects of lemon juice on the levels of several biochemical parameters.
Parameters | Control | Model | LL | LM | LH |
---|---|---|---|---|---|
AST (U/L) | 103 ± 10.45 | 136.53 ± 19.94 |
117.88 ± 15.37 | 113.5 ± 7.7 | 98.85 ± 10.94# |
ALT (U/L) | 40.5 ± 3.89 | 54.32 ± 4.76 |
54.05 ± 7.18 | 41.32 ± 6.25# | 34.68 ± 2.71# |
Serum TG (nmol/L) | 0.4 ± 0.06 | 1.01 ± 0.12 |
1.09 ± 0.04 | 1.03 ± 0.05 | 0.82 ± 0.08# |
Liver TG (mmol/g prot) | 0.07 ± 0.01 | 0.1 ± 0.02 |
0.09 ± 0.01 | 0.07 ± 0.01# | 0.06 ± 0.01# |
Lipid peroxidation (nmol MDA equivalent/mg prot) | 0.64 ± 0.14 | 1.26 ± 0.22 |
0.88 ± 0.12# | 0.84 ± 0.15# | 0.72 ± 0.13# |
SOD (U/mg prot) | 89.6 ± 3.42 | 97.51 ± 3.96 |
85.27 ± 5.57# | 83 ± 9.28# | 81.03 ± 6.65# |
CAT (U/mg prot) | 6.55 ± 0.41 | 6.29 ± 0.39 | 5.55 ± 0.64# | 5.47 ± 0.28# | 5.17 ± 0.51# |
Effects of lemon juice on the activities of SOD (a) and CAT (b) in liver. Control: normal group; Model: alcohol group; LL: alcohol and low dose of lemon juice group; LM: alcohol and medium dose of lemon juice group; LH: alcohol and high dose of lemon juice group.
Histopathology assessment of the liver was carried out for all groups (Figure
The photomicrographs of liver sections taken from mice. (a) Normal group; (b) alcohol group; (c) alcohol and low dose of lemon juice group; (d) alcohol and medium dose of lemon juice group; (e) alcohol and high dose of lemon juice group. Arrow indicates a condition of small fat droplets changes, and the circle indicates hepatocytes necrosis, which mainly occurs in alcohol model group.
The in vitro antioxidant activities of lemon were evaluated using ferric-reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays. The FRAP and TEAC values were
Alcohol use disorder causes substantial diseases, and the liver is the most adversely affected organ. In the present study, the effects of lemon juice on chronic alcohol-induced liver injury in mice were investigated. Ethanol induced impairment of liver in mice was evidenced by increased AST and ALT levels. Treatment with lemon juice lowered the increased levels of AST and ALT in serum. The return of the activities of aminotransferases (AST or ALT) in serum to normal indicates the regeneration of hepatocytes and the healing of hepatic parenchyma; therefore, lemon juice had a protective effect on alcohol-induced liver injury. The results were in agreement with previous reports that showed lemon possessing a hepatoprotective effect on liver injury induced by carbon tetrachloride and acute exercise [
Various factors and mechanisms are associated with the pathological progress of alcohol-induced liver injury, and oxidative stress was one of them [
Liver steatosis is the earliest disease of the liver on account of chronic ethanol consumption, with the characteristic of fat accumulation. It is generally accepted that, in the development of hepatic steatosis, ethanol exposure increases the ratio of reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide in hepatocytes, which disturb mitochondrial fatty acid
The antioxidant enzymes, such as SOD and CAT, represent the defense response system to excessive ROS. SOD catalyzes the dismutation of two superoxide anions to hydrogen peroxide and oxygen, and then CAT degrades two hydrogen peroxide molecules to water and oxygen [
Lemon contains numerous beneficial bioactive compositions, including phenolic compounds (mainly flavonoids), vitamins, carotenoids, essential oils, minerals, and dietary fiber [
In this study, lemon juice revealed a protective effect on chronic alcohol-induced liver injury. Due to the fact that lemon contains a variety of bioactive ingredients, the hepatoprotective effect might be the result of joint action of multiple mechanisms, and it is difficult to clarify the specific mechanism of effect. The medium in vitro antioxidant capacities of lemon and reduced in vivo MDA levels indicated that lemon might reduce the oxidative stress induced by ethanol, thus exerting hepatoprotective effects. This study has found that lemon juice has a strong hepatoprotective effect, which provides valuable information for the general public to reduce harm of alcohol consumption. In the future, active components in lemon juice should be separated and identified, and the mechanism of action of the purified compound should be explored, including the action on the small intestine.
Chronic alcohol consumption could induce liver injury. Lemon juice is readily available as a widely consumed beverage. In this study, we found that treatment with lemon juice exerted hepatoprotective effects on alcohol-induced liver injury in mice through decreasing the levels of serum ALT and AST as well as hepatic TG and lipid peroxidation. In addition, the in vitro antioxidant experiment of lemon showed that lemon had medium in vitro antioxidant capacities. Therefore, we speculate that the hepatoprotective effects might be related to the antioxidant capacities of lemon juice. The results showed that lemon juice might be a potential dietary supplement for the prevention and treatment of liver injury related to chronic alcohol consumption.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Tong Zhou and Yu-Jie Zhang contributed equally to this work.
This work was supported by the National Natural Science Foundation of China (no. 81372976), Key Project of Guangdong Provincial Science and Technology Program (no. 2014B020205002), and the Hundred-Talents Scheme of Sun Yat-sen University.