Ameliorative Effects of Monoammonium- Glycyrrhizinate on Liver Lipid Metabolism and Antioxidant Capacity in Laying Hens

Authors’ Contribution MW and SH drafted the basic manuscript and analyzed the data. CZ, ZY and SH contributed to conception and design of the research and reviewed the manuscript. MW, HG, SC, SW, QS, WX, SM and PL participated in sample collection and laboratory testing. MW, SH, CZ and ZY participated in the data analysis and revising the manuscript. All authors read and approved the final manuscript.


INTRODUCTION
L icorice is widely used as a traditional Chinese herb. More than 100 kinds of flavonoids and 60 kinds of triterpenoid saponins, coumarin, alkaloids, and polysaccharide ingredients have been isolated from licorice (Tang et al., 2015). It is noteworthy that triterpene saponins is the main active components which possess hepatoprotective, anti-inflammatory, and even anti-cancer effects (Tang et al., 2015). Glycyrrhizic acid (GA), a triterpenoid saponin, consists of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid.

O n l i n e F i r s t A r t i c l e
has been reported that inhibition of Mrp2 by GA can achieve liver protection through increase in the content of glutathione (GSH) in liver cells (Xu et al., 2012). In Japan, GA has been used to treat chronic hepatitis for more than 25 years without inducing side effects (Nishimoto et al., 2010). Recent studies demonstrated that GA exhibit favourable hepatoprotective effects by enhancing the anti-inflammatory and antioxidant ability (Huo et al., 2011;Wang and Du, 2016). Liver damage is common in farm animals, especially in bovine and laying hens. Antioxidant could provide a way of protecting the liver from oxidative stress . The current study aims to find a specific antioxidant to enhance animal health and animal welfare, hence reducing the economic loss caused by oxidative damage in poultry production.
Although GA preparations are widely used in human clinical and small animal diseases, and have achieved positive results, but there are few studies related to the effects of MAG on the farm animals. The present study evaluates the effects of MAG on liver lipid metabolism and anti-oxidant capacity in laying hens.

Birds, experimental design and measurements
One hundred and eighty 320-day-old Hy-Line brown laying hens were obtained from local commercial hen farm. After 5 days of acclimatization, the hens were divided into five groups, 3 replicates per group and 12 laying hens per replicate. All hens were fed a basal corn-soybean meal diet (Table I), which were formulated mainly according to NRC (1994). On the sixth day, the treatments were named 0 mg/L group (control group), 25 mg/L group, 50 mg/L group, 100 mg/L group, 200 mg/L group respectively, based on the content of MAG in drinking water. Feed and water were supplied ad libitum, with 16 h light per day throughout the experimental period. Meanwhile, at the onset and end of the trial, the weight of each laying hen was recorded using an electronic scale. All experimental procedures were performed in strict accordance with animal ethics and approved by the Committee of Henan University of Science and Technology.

Sample collection
On the 21 st day, all birds were weighed following a 12-h fasting. The blood samples were collected into tubes without anticoagulant through brachial wing vein puncture and left to clot at room temperature. The collected blood was slightly tilted at 4 o C for 2 h before being centrifuged at 3,000 rpm for 10 min. Separated serum was stored into 1.5 mL Eppendorf tubes and frozen at -20 o C until further analysis. All the hens were slaughtered according to animal welfare regulations of China. The whole livers were collected, weighed and used for triglyceride estimation and liver histopathology analysis.

Liver histopathology
All liver tissues were placed in neutral formalin (10%, 4 o C, 24 ~ 48h), embedded in paraffin, and sliced into 2-3 sections (5 μm in thickness). Lipid accumulation in the liver was histologically analyzed by using the Oil Red O soluble dye, which dyed neutral lipid (mainly triglycerides) to orange-red tint. Hepatocyte apoptosis was (were) stained with TUNEL staining. After staining, histopathological changes in the liver tissues were observed under an optical microscope (Olympus, Tokyo, Japan) with the magnification of 200.

Hepatic triglyceride
The liver tissue (0.5 g) was homogenized in 4.5ml phosphate buffer (pH 7.4), and then was centrifuged at 3,000 rpm for 10 min. The supernatant was used for estimation of hepatic triglyceride with the help of kit according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Determination of antioxidant activity in serum
Lipid peroxidation degradation products were determined by the thiobarbituric acid method (Arukwe et al., 2016), which measures malondialdehyde, (MDA) and expressed as nmol/mL and lipid peroxidation product of lipid breakdown caused by oxidative stress using colorimetric diagnostic kit (A003-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Total antioxidant capacity (T-AOC), catalase (CAT), total superoxide dismutase (T-SOD) and TG in serum were measured by using some commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All these indices were determined with Microplate Reader (Thermo Fisher Scientific, Shanghai, China).

Statistical analysis
All detected data were analyzed by SPSS statistics software (Version 17.0, SPSS Inc, Chicago, IL, USA), and were presented as the means ± standard deviations (SD). Comparative analysis of the different groups was made using one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) Duncan test. P<0.05 was considered to be statistically significant. All results were repeated at least three times and representative histopathological results were shown.

MAG supplementation decreased liver weight and liver index
As shown in Table II, the liver wet weight and liver indices of all the experimental treatments were higher than the control group except for the 200 mg/L MAG group. Especially in the 25, 100 mg/L MAG groups liver weights and liver indices of laying hens were markedly higher than the control group (P<0.05).

MAG supplementation inhibited lipid deposition and apoptotic hepatocytes production
As shown in Figure 1, Oil Red O-stained lipid droplet was observed and the degree of steatosis in the MAG group showed a clear reduction compared with the control group.
Moreover, the liver lipid content of laying hens was also obviously reduced compared to the control group (P<0.05) (Fig. 1F). In comparison with the control, TUNEL-positive apoptotic hepatocytes in the MAG group were found to be decreased significantly ( Fig. 2A-2E).

MAG supplementation ameliorated serum biochemical parameters
In Table III, the levels of TP, ALB, and GLB in the MAG groups were decreased compared to the control group. Moreover, the ALB content in 200 mg/L group and the TP and GLB content in 25 mg/L group, 100 mg/L group and 200 mg/L group decreased significantly (P<0.05), but no significant changes were observed in serum ALB/GLB ratio, and GLU, HDLC, LDLC, TBIL, DBIL, IBIL levels during the experiment (Table III). In addition, the activity of ALT was significantly decreased in 25 and 200 mg/L groups (P<0.05) (Fig. 3A). In comparison with the control, the activities of AST and ALP were not effected in the MAG group (P>0.05).

MAG supplementation decreased malondialdehyde concentration and enhanced antioxidant enzymes activity in serum
The T-AOC and CAT concentrations were higher in the MAG groups than in the control group ( Fig. 3D and  3G). The MDA concentrations decreased slightly in Figure  3F (P>0.05), which indicated no obvious change in the concentrations of T-AOC, CAT, and MDA through MAG supplementation. However, the serum oxidative stress index T-SOD showed a significant increase in 25, 50 and 100 mg/L groups, (P<0.05) (Fig. 3E).

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Ameliorative Effects of Monoammonium-Glycyrrhizinate on Liver Fig. 3. Effect of different levels of MAG in serum parameters of laying hens. Data represent means±SD, one-way ANOVA, and different letters represent statistically significant differences (P < 0.05). ALT, alanine aminotransferase; AST, aspartate transaminase; ALP, alkaline phosphatase; T-AOC, total antioxidant capacity; T-SOD, total-superoxide dismutase; MDA, malondialdehyde; CAT, malondialdehyde; TG, serum triglycerides.

DISCUSSION
In this study, the results showed that liver weight, liver index, and liver lipid content significantly decreased compared with the control group. The levels of TP, ALB, and GLB decreased compared with the control group, but no significant difference was observed in A/G ratio. However, lipid parameters in MAG-treated groups were improved partly in serum samples. Compared with control group, TG and CHOL content in serum was decreased, but the difference was not significant. These findings were consistent with another study which found that obese rats with oral glycyrrhetinic acid 100 μg/g for 28 d, triglyceride, blood glucose, and total cholesterol in serum were decreased (Eu et al., 2010). It has also been reported by Chandramuli et al. (2011) that in high sucrose-fed rat after oral administration of glycyrrhetinic acid at 100 μg/g for 28 d, blood glucose was significantly reduced and blood lipids improved. In this study, by adding different levels of MAG in the drinking water of the laying hens, GLU was decreased and the blood lipid was improved, which was consistent with the previous research results. Moreover, the degree of liver steatosis in the MAG supplement group was significantly reduced, consistent with the results of liver triglyceride content. Lipid deposition decreases significantly in quadriceps femoris and abdominal muscle after oral 50 mg/kg of GA for one week in rats (Lim et al., 2009). The result indicated that MAG can reduce liver fat deposition by reducing liver TG levels, but blood lipids are not decreased significantly.
The number of TUNEL-stained apoptotic cells in liver was decreased significantly compared with the control group, which indicated MAG supplement with drinking water of laying hens can protect the liver by reducing oxidative stress and cell apoptosis effectively. Upon clinical examination and experimental studies, the increase of ALT and AST levels in serum is recognized as biochemical markers of hepatocellular injury (Scheig, 1996;ArAGon and Younossi, 2010). After hepatocellular damage, these enzymes are released into the bloodstream (Ramaiah, 2007). Previous studies reported that licorice can protect the rats effectively from CCl 4 -induced hepatic injury by decreased serum AST, ALT, and ALP activities (Nose et al., 1994;Gumpricht et al., 2005;Huo et al., 2011). Pre-treatment with 18 β-glycyrrhetinic acid can decrease the activity of ALT and AST in serum and suppress hepatic lipid peroxidation caused by CCl4 (Jeong et al., 2002). Kim et al. (2006) reported that O n l i n e

F i r s t A r t i c l e
M. Wang et al. liquiritigenin pretreatment with acetaminophen-induced rats can improve liver necrosis and reduce ALT activity. Moreover, 18-glycyrrhetinic acid (50 mg/kg) protects the liver tissue by reducing serum AST, ALT and ALP activity significantly to maintain the liver's structural integrity and less histopathological change. Liquiritigenin and 18-glycyrrhetinic acid belong to flavonoid and triterpene groups, respectively. In addition, low-dose glycyrrhetinic acid in triptolide rats significantly increased SOD/CAT activity but strengthened the liver defense system for antioxidant (Yang et al., 2017). Study also pointed out that the liver injury was positively correlated with ketoacidosis and the activity of AST and ALT. Ketosis cows usually show higher serum LDH content compared to normal cows (Du et al., 2017). Similarly, Gumpricht et al. (2005) indicated that glycyrrhetinic acid can inhibit hepatocyte apoptosis in rat by glycochenodeoxycholic acid-induced cytotoxicity. These results suggest that MAG supplement can suppress the activity of higher liver enzymes, thereby reducing liver damage. Under normal circumstances, there is a stable dynamic balance between the oxidation system and the antioxidant system of the animal's body. Once this balance is broken, it will lead to oxidative stress (Reuter et al., 2010). Many studies have found that oxidative stress plays a crucial function in liver damage such as fatty liver, liver fibrosis, viral hepatitis (Cederbaum et al., 2009;Tolooei and Mirzaei 2015). Although low concentrations of reactive oxygen species (ROS) are involved in cell proliferation as intracellular messengers, a large number of ROS has direct effects on different types of cell death, including apoptosis and necrosis Su et al., 2016). Furthermore, ROS is not cleared in time or is overproduced, which also causes oxidative stress to occur. (Yu et al., 2015). A study in rat heart mitochondria showed that glycyrrhetinic acid depending on its concentration, is able to prevent or to induce oxidative stress, thereby affecting ROS production (Battaglia et al., 2008). T-SOD is a vital antioxidant enzyme in organisms and is the primary material in vivo free radical scavenging. T-AOC can reflect the status of all antioxidants in body fluids and which is contributes to the balance of active oxygen (Ghiselli et al., 2000;Rajani et al., 2011). T-SOD and CAT play an important role in protecting cells from damage caused by ROS (Miao et al., 2017). Serum MDA is a soluble lipid degradation end-product (Lorente et al., 2015), and MDA contents can be used to monitor the extent of lipid peroxidation (Dragun et al., 2017). Licorice extract against carbon tetrachloride can markedly suppressed the elevated concentrations of MDA and increased levels of SOD in comon carp (Yin et al., 2011). Data of the present study suggest that MGA had a consistent and significant effect in decreasing serum MDA content versus control. The serum SOD content was increased significantly after using MAG supplementation, resulting in a reduced superoxide anion concentration in laying hens. We speculate that MAG strengthen the oxidative defenses through increased T-SOD contents and decreased MDA content in serum.

CONCLUSION
According to the results of this study, supplementation of MAG through drinking-water in chickens can enhance the antioxidant capacity, improve lipid metabolism and have a curative effect on liver injury.

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Ameliorative Effects of Monoammonium-Glycyrrhizinate on Liver O n l i n e