Lipidomics investigation of reversal effect of glycyrrhizin (GL) towards lithocholic acid (LCA)-induced alteration of phospholipid profiles

Abstract

Context: Glycyrrhizin (GL), the major ingredient isolated from licorice, exerts multiple pharmacological activities.

Objective: To elucidate the protective mechanism of GL towards lithocholic acid (LCA)-induced liver toxicity using lipidomics.

Materials and methods: GL (200 mg/kg) dissolved in corn oil was treated intraperitoneally for 7 d. On the 4th day, 200 mg/kg LCA was used to treat mice (i.p., twice daily) for another 4 d. The protective role of GL towards LCA-induced liver toxicity was investigated through evaluating the liver histology and the activity of alanine transaminase (ALT). The complete lipid profile was employed using UFLC-Triple TOF MS-based lipidomics.

Results: Intraperitoneal (i.p.) administration of 200 mg/kg GL can significantly protect LCA-induced liver damage, indicated by alleviated histology alteration and prevention of the ALT elevation. Lipidomics analysis can well separate the control group from LCA-treated group, and three lipid components were major contributors, including LPC 16:0, LPC 18:0, and LPC 18:2. GL treatment can significantly prevent LCA-induced reduction of these three lipid compounds, providing a new explanation for GL's protection mechanism towards LCA-induced liver toxicity.

Discussion and conclusion: The recent study highlights the importance of lipidomics in elucidating the therapeutic mechanism of herbs.

• Alanine transaminase
• bile acids
• herbs
• licorice
• metabolomics database
• principal component analysis

Introduction

Bile acids, synthesized from cholesterol, have been considered to be the important components required for the absorption and excretion of lipophilic metabolites such as cholesterol (Vlahcevic et al., 1991). The accumulation of bile acids in liver will disrupts liver function, including disruption of homeostasis of bile acids and induction of liver inflammation (Matsubara et al., 2012). Lithocholic acid (LCA) is the most potent endogenous chemical inducing liver toxicity and has been frequently utilized to make animal model of intrahepatic cholestasis (Hofmann et al., 2004). After treatment with LCA, many metabolic pathways can be affected, and lipid metabolic pathway can be significantly disturbed (Matsubara et al., 2011).

Licorice is one of the most commonly prescribed herbs in Eastern traditional medicines (Messier et al., 2012). Glycyrrhizin (GL, Figure 1), the major ingredient isolated from licorice, has been demonstrated to exert multiple pharmacological activities. For example, GL has emerged as a promising drug candidate for cancer therapy due to its significant pro-apoptotic effect on tumor cells, and this effect might be related with its anti-angiogenic activities (Kim et al., 2013). Additionally, GL have anti-inflammatory, antiviral, and neuroprotective role (Ming & Yin, 2013). The liver protective role has been frequently reported in the recent literatures. For example, carbon tetrachloride (CCl4)-induced liver injury can be well protected by the treatment of GL through induced expression of heme oxygenase-1 and down-regulation of pro-inflammatory mediators (Lee et al., 2007).

Figure 1. The molecular structure of glycyrrhizin (GL).

Lipidomics is a technology used to describe the complete lipid profile within a cell, organ, tissue, or biofluid (Lam & Shui, 2013). The present study aims to investigate the protective mechanism of GL towards LCA-induced liver toxicity through analysis of lipid profile using lipidomics, trying to indicate the contribution of lipid profile alteration towards protective role of GL towards LCA-induced liver toxicity.

Materials and methods

Chemicals and reagents

GL (purity>95%) and lithocholic acid (LCA, purity>97%) were purchased from Sigma Chemical Co. (St. Louis, MO). All the reagents with highest grade commercially available were obtained from Sigma-Aldrich (St. Louis, MO). Alanine transaminase (ALT) activity assay kits were purchased from Cayman Chemical company (Ann Arbor, MI).

Animals and treatment

About 6–8 weeks old male C57BL/6N mice (weight from 20 to 25 g) were purchased from the animal center of Academy of Military Medical Sciences (Beijing, China). Mice were maintained under a standard 12 h light/12 h dark cycle with food and water ad libitum. Mice were divided into three groups, including the control group, the LCA-treated group, and the LCA + GL-treated group. GL (200 mg/kg) dissolved in corn oil was treated intraperitoneally for 7 d. On the 4th day, 200 mg/kg LCA was used to treat mice (i.p., twice daily) for another 4 d. Blood samples were collected in BD microtainer serum separator tubes (Franklin Lakes, NJ) by retro-orbital bleeding. Serum was obtained through 15 min centrifugation at 8000 × g. The liver was taken after sacrifice and fixed in a 4% formaldehyde-phosphate-buffered saline solution. And then, the samples were embedded in paraffin, sectioned into 4 μm slices, and stained with hematoxylin and eosin as previously described (Fang et al., 2013). The activity of liver-specific enzyme ALT was determined using commercial kits according to the manufacturer's instructions.

UFLC-Triple TOF MS-based lipidomics study

Ice-cold chloroform/methanol (v/v = 2:1) (200 μL) with 10 μM LPC 17:0 as an internal standard was used to extract the lipid components in 50 μL serum samples. After vortexing for 5 min, centrifugation was carried out at 13 000 × g for 5 min to separate the water phase from organic phase. After removing the upper phase, the careful collection of lower organic phase was performed through piercing protein carefully. The organic phase was evaporated using nitrogen, and then dissolved with chloroform/methanol (v/v = 1:1). Before analysis, the samples were diluted with 10 × the original volume of plasma with acetonitrile/water (v/v = 50:50). The aliquots of samples were analyzed using an ultra-fast liquid chromatography (Shimadzu Corporation UFLC XR; Kyoto, Japan) system connected to a triple time-of-flight mass spectrometer (AB SCIEX Triple TOF 5600; Foster City, CA) with a duo spray ionization source. Waters Xbridge C18 column (2.1 × 150 mm, 3.5 μm) was used to separate all the samples. A binary solvent gradient consists of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) at a flow rate of 0.5 ml/min. The following gradient conditions were used: 99% A for 0.5 min, and then increased to 99% B over the next 7.5 min, and returned to 99% A in last 3 min. The settings of nitrogen gas for nebulizer, 50 psi; curtain gas, 25 psi; drying solvent, 50 psi, temperature, 600 °C, and ion spray voltage at 5500 V. An IDA method was also created to acquire both MS and MS/MS data. The IDA criteria were as follows: peak intensity, more than 600 cps; excluding time for former target ion, 8 s; excluding isotope, 4.0 Da. The accuracy and precision for each concentration were more than 95%. The ions were output, and the data were preprocessed using the Microsoft Excel software (Microsoft, Redmond, WA). Principal component analysis (PCA) was performed using MetATT (http://metatt.metabolomics.ca/MetATT).

Results

The protection role of GL towards LCA-induced liver toxicity

Compared with the control group, the liver from LCA-treated mice showed large areas of focal necrosis, steatosis of liver, and the infiltration of inflammation cells, and the treatment of GL significantly alleviated the histology alteration induced by LCA-induced liver damage (Figure 2A). Additionally, the activity of ALT (the representative marker of liver damage) significantly increased (p < 0.001), and while co-treatment with GL totally inhibited the elevation of ALT activity caused by LCA toxicity (p < 0.001) (Figure 2B).

Figure 2. Protection of glycyrrhizin (GL) towards lithocholic acid (LCA)-induced liver toxicity. (A) Histology of liver section from control, LCA-treated, and LCA + GL-treated mice. (B) The activity of ALT in different groups, including control (CTRL), LCA-treated (LCA), and LCA + glycyrrhizin-treated (LCA + GL) groups. Data are given as mean ± S.E.M. (n = 4–5 in each group). ***p < 0.001.

The reversal influence of GL towards lipidomics profile alteration induced by LCA treatment

A total of 1064 ions can be extracted from mass spectrum for the lipidomics analysis. After entering these ions into MetATT website for principal component analysis (PCA), the good separation was observed between the control group and the LCA-treated group (Figure 3A), and the lipid components are the major contributors for this separation. Heat map indicated the alteration trend of two representative lipid ions for separation, including LPC 16:0 and LPC 18:0 (Figure 3B). In detail, LCA treatment significantly decreased the serum level of LPC 16:0, LPC 18:0, and LPC 18:2 (Figure 4). The treatment of GL can markedly reverse this change to make all these components nearly reach the normal level. All these ions were identified through comparison of the retention time and mass spectrum with the human metabolomics database (http://www.hmdb.ca/) and the previous literature (Fang et al., 2013; Matsubara et al., 2012; Ollero et al., 2011).

Figure 3. Lipidomics analysis results. (A) Principal component analysis (PCA) of serum lipidomics of the control and the LCA-treated group. (B) Heat map of representative lipid components. LPC16:0 and LPC 18:0 were given as representative lipid components.

Figure 4. Serum exposure levels of LPC 16:0, LPC 18:0, and LPC 18:2 in different groups, including control (CTRL), LCA-treated (LCA), and LCA + glycyrrhizin-treated (LCA + GL) groups. The peak area is used, and data are given as mean ± S.E.M. (n = 4–5 in each group). *p < 0.05; **p < 0.01, ***p < 0.001.

Discussion

Phospholipids play key role in constructing cell membrane through forming lipid bilayers. Besides the structural function, phospholipids also exhibit other important functions. For example, lysophosphatidylcholine (LPC) exhibited pro-inflammation function through activating various immune cells, including monocytes and neutrophils (Huang et al., 1999; Kume et al., 1992; Liu-Wu et al., 1998). Therefore, the alteration of phospholipids has been investigated as a major reason for LCA-induced liver toxicity in which inflammation factors mainly contributed to the pathogenesis mechanism (Matsubara et al., 2011).

The therapeutic role of GL towards many liver diseases is very obvious; however, the mechanism remains unclear. Multiple possible mechanisms were speculated. The experiment performed by Tsuruoka et al. (2009) demonstrated the contribution of inhibition of inducible nitric oxide synthase (iNOS) towards the liver-protection role. Down-regulation of matrix metalloproteinase-9 (MMP-9) has also been considered a major reason for the protection of GL towards lipopolysaccharide/D-galactosamine-induced liver injury (Abe et al., 2008). The previous literature has indicated the possible reason of GL's protection towards LCA toxicity to be the induction of CYP3A11 and inhibition of CYP7A through PXR activation (Wang et al., 2012). However, only one mechanism pathway cannot explain the strong protection role of GL towards LCA-induced liver damage. The reversal effect of GL towards LCA-induced phospholipid profile alteration was observed in the present study, which provides another possible mechanism which can be used as a new explanation for GL's protection towards LCA toxicity. The previous literatures have reported that LCA disrupted the homeostasis of phospholipids through regulation of the activities of some enzymes, such as lysophosphatidylcholine acyltransferases, phospholipase D₁, and phospholipase D₂, and choline kinase (Matsubara et al., 2011). GL might affect the phospholipids profile through preventing the alteration of these enzymes' activity.

In conclusion, the reversal effect of GL towards LCA-induced lipidomics alteration was firstly reported in the recent study, which can be regarded as a new explanation for the protective mechanism of GL towards LCA-induced liver toxicity. It should be noted that the molecular mechanism of GL's protection towards LCA-induced liver toxicity might also be applied for elucidation of the protection role of GL towards other liver toxicity, such as lipopolysaccharide/d-galactosamine, concanavalin A, and acetaminophen-induced liver injury.

Declaration of interest

There is no conflict of interest.