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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2017 Mar 16;174(8):672–688. doi: 10.1111/bph.13729

Schisandrol B protects against cholestatic liver injury through pregnane X receptors

Hang Zeng 1, Yiming Jiang 1, Pan Chen 1, Xiaomei Fan 1, Dongshun Li 1, Aiming Liu 2, Xiaochao Ma 3, Wen Xie 3, Peiqing Liu 1, Frank J Gonzalez 4, Min Huang 1, Huichang Bi 1,✉
PMCID: PMC5368048  PMID: 28128437

Abstract

Background and Purpose

Currently, ursodeoxycholic acid and obeticholic acid are the only two FDA‐approved drugs for cholestatic liver diseases. Thus, new therapeutic approaches need to be developed. Here we have evaluated the anti‐cholestasis effects of Schisandrol B (SolB), a bioactive compound isolated from Schisandra sphenanthera.

Experimental Approach

Hepatoprotective effect of SolB against intrahepatic cholestasis, induced by lithocholic acid (LCA), was evaluated in mice. Metabolomic analysis and gene analysis were used to assess involvement of pregnane X receptor (PXR). Molecular docking, cell‐based reporter gene analysis and knockout mice were used to demonstrate the critical role of the PXR pathway in the anti‐cholestasis effects of SolB.

Key Results

SolB protected against LCA‐induced intrahepatic cholestasis. Furthermore, therapeutic treatment with SolB decreased mortality in cholestatic mice. Metabolomics and gene analysis showed that SolB accelerated metabolism of bile acids, promoted bile acid efflux into the intestine, and induced hepatic expression of the PXR‐target genes Cyp3a11, Ugt1a1, and Oatp2, which are involved in bile acid homeostasis. Mechanistic studies showed that SolB activated human PXR and up‐regulated PXR target genes in human cell lines. Additionally, SolB did not protect Pxr‐null mice from liver injury induced by intrahepatic cholestasis, thus providing genetic evidence that the effect of SolB was PXR‐dependent.

Conclusion and Implications

These findings provide direct evidence for the hepatoprotective effects of SolB against cholestasis by activating PXR. Therefore, SolB may provide a new and effective approach to the prevention and treatment of cholestatic liver diseases.

Abbreviations

ALP

alkaline phosphatase

ALT

alanine aminotransferase

AST

aspartate transaminase

CA

cholic acid

CAR

constitutive androstane receptor

CCND1

cyclin D1

CDCA

chenodeoxycholic acid

CMC

carboxymethylcellulose sodium

CYP

cytochrome P450

DCA

deoxycholic acid

FXR

farnesoid X receptor

HDCA

hyodeoxycholic acid

LCA

lithocholic acid

LXR

liver X receptor

MDR

multidrug resistance

MRP

multidrug resistance‐associated protein

NR

nuclear receptor

OATP

organic anion transporter polypedtide

OPLS‐DA

orthogonal partial least squares discriminant analysis

PBC

primary biliary cirrhosis

PCA

principal components analysis

PCN

pregnenolone 16α‐carbonitrile

PCNA

proliferating cell nuclear antigen

PXR

pregnane X receptor

SolB

Schisandrol B

TBA

total bile acid

Tbili

total bilirubin

TCA

taurocholic acid

TCDCA

taurochenodeoxycholic acid

TDCA

taurodeoxycholic acid

THDCA

taurohyodeoxycholic acid

T‐β‐MCA

tauro‐β‐muricholic acid

UDCA

ursodeoxycholic acid

UGT

UDP‐glucuronosyltransferase

Tables of Links

TARGETS
Enzymes a
CYP3A4
Nuclear hormone receptors b
FXR, farnesoid X receptor
Liver X receptor
PXR, NR1I2
Transporters c
MRP3, ABCC3
OATP1A2, SLCO1A2
LIGANDS
CA, cholic acid
DCA, deoxycholic acid
LCA, lithocholic acid
OCA, obeticholic acid
PCN, pregnenolone 16α‐carbonitrile
Rifampicin
T0901317
TCA, taurocholic acid
UDCA, ursodeoxycholic acid
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These Tables list key protein targets and ligands in this article that are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (a,b,cAlexander et al., 2015a,b,c).

Introduction

Cholestasis is defined as a disturbance of bile formation or secretion leading to intrahepatic accumulation of toxic bile acids (Trauner et al., 1998). Retained toxins will induce liver damage followed by chronic liver disease with development of biliary fibrosis, cirrhosis and, finally, end‐stage liver disease requiring liver transplantation (Ghonem et al., 2015). For a long period of time, ursodeoxycholic acid (UDCA) was the only FDA‐approved drug to treat a limited group of cholestatic liver diseases such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (Lazaridis et al., 2001). However, the effectiveness of UDCA in PBC has been questioned, and its efficacy is limited to early stages of PBC (Wunsch et al., 2014). Until recently the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) was approved by FDA to treat biliary liver injury diseases (http://www.accessdata.fda.gov/drugsatfda_docs/nda/2016/207999Orig1s000TOC.cfm). Overall, there is a clear need to develop more effective drugs for the treatment of cholestatic liver diseases.

The underlying molecular mechanisms of cholestasis are mediated mainly at a transcriptional level via a network involving nuclear receptors (NRs) such as the FXR, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which target overlapping sets of enzymes and transporters participating in bile acid metabolism and transportation(Goodwin et al., 2000; Stedman et al., 2005; Wagner et al., 2005; Wagner et al., 2010). The therapeutic strategy against cholestasis is mainly focused on the elimination of excess toxic bile acids. Because NRs are the central regulators of bile acid synthesis, transport and detoxification, specific targeting of NRs represents an innovative approach for the treatment of cholestasis. Among these NRs, PXR and its human homologue, steroid and xenobiotic receptor, have emerged as promising therapeutic targets in cholestatic disorders, as alterations in PXR signalling may contribute to the pathogenesis and progression of cholestasis as well as bile acid homeostasis (Kliewer et al., 1998). The protective effect of PXR in cholestasis is due to its ability to inhibit bile acid synthesis and to activate detoxification pathways (Xie et al., 2000; Staudinger et al., 2001). Previous reports have shown that pregnenolone 16α‐carbonitrile (PCN), a rodent PXR agonist, can prevent lithocholic acid (LCA)‐induced hepatotoxicity in wild‐type rodents, but fails in Pxr‐/‐ mice (Staudinger et al., 2001; Xie et al., 2001). Consistent with this finding, hepatic damage is increased in Pxr −/− mice after bile duct ligation (Stedman et al., 2005). Furthermore, some natural products, including glycyrrhizin and tanshinone IIA that activate PXR, have been reported to protect against LCA‐induced cholestasis in mice (Wang et al., 2012; Zhang et al., 2015). These findings suggest that specific targeting of PXR could represent a new therapeutic approach to treat cholestatic liver disease and that PXR agonists may exert protection against cholestasis.

Pharmacological studies have significantly expanded to include massive screening of herbal products in the search for novel drug candidates. Schisandrol B (SolB) is one of the bioactive lignans isolated from Schisandra sphenanthera, a well‐known herbal medicine widely used in China, Japan and Korea for its protective effect on liver, kidney and heart (Panossian and Wikman, 2008). We recently reported that the ethanolic extracts of Schisandra sphenanthera protected against acetaminophen‐induced acute liver damage, via several mechanisms including the activation of detoxification systems and the promotion of compensatory liver regeneration (Bi et al., 2013; Fan et al., 2015; Fan et al., 2014a,b). Most recently, we found that SolB exerted the greatest, among the six lignans of Schisandra sphenanthera, and dose‐dependent (6.25, 25 and 100 mg·kg−1), hepatoprotection against acetaminophen‐induced acute liver damage (Jiang et al., 2015a,b). However, whether SolB could exert hepatoprotection against cholestasis‐induced liver damage remained unknown. Therefore, this study investigated if SolB could protect against LCA‐induced intrahepatic cholestasis in mice, and assessed the role played by PXR signalling in the hepatoprotective effects of SolB, against cholestatic disorders.

Methods

Wild‐type mice and treatment

All animal care and experimental studies were approved by the Institutional Animal Care and Use Committee at Sun Yat‐sen University, Guangzhou, China. All animal studies are reported in accordance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). The entire study adhered to the principles of replacement, refinement or reduction (the 3Rs). Adult male C57BL/6J mice (8 weeks old, 20–25 g) were purchased from Guangdong Medical Laboratory Animal Center, which were introduced from Jackson Laboratories (Bar Harbor, ME, USA). Animals were group‐housed (max. 6 per cage) in M2 cages, bedded with wood pieces at the animal facility of Sun Yat‐sen University in a specific pathogen free room at 22–24°C with a light/dark cycle of 12/12 h and 55–60% relative humidity with free access to standard rodent food and water.

Mice were randomly divided into different groups for all experiments. A single‐blind study design was applied during the experiment and data analysis. SolB (6.25, 25 and 100 mg·kg−1, p.o.) was dispersed in 0.5% CMC, while LCA (125 mg·kg−1, i.p.) and PCN (50 mg·kg−1, i.p.) were dissolved in corn oil as described previously (Staudinger et al., 2001). Mice were treated with SolB (bid) or PCN (qd) for 7 days, and treatment with LCA (bid) was initiated from the fourth day. Mice in the group treated with SolB alone were given the highest dose of SolB, once daily for 7 days. Animals were monitored twice a day throughout the whole experiment. No treated mice lost more than 20% of their initial weight or became moribund, during the study. Twelve hours after the last LCA injection, mice were killed by CO2 asphyxia. Serum and liver tissue samples were collected and snap‐frozen in liquid nitrogen, then stored at −80°C until use. Some samples were lost due to LCA‐induced deaths and, therefore, the sizes of LCA–treated groups were almost twice the size of others in order to maintain sufficient power.

Serum biochemical and histology evaluation

Serum alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bile acid (TBA) and total bilirubin (Tbili) levels were analysed by using commercially available kits (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer's instructions. For histological studies, tissues were immediately fixed in formaldehyde, embedded in paraffin wax, sectioned and stained for haematoxylin and eosin (H&E). H&E‐stained liver sections were examined using an Olympus BX41 microscope.

Therapeutic effect of SolB against LCA‐induced cholestasis

Adult male C57BL/6J mice were kept in a room at 22–24°C with a light/dark cycle of 12/12 h and 55–60% relative humidity with free access to standard rodent food and water. LCA in corn oil (125 mg·kg−1, bid, i.p.) was administered for 3 days to develop cholestasis in mice. From day 4, the mice were also treated with SolB (100 mg·kg−1, bid) or PCN (50 mg·kg−1, qd). The animals were humanely killed once they had lost 15–20% of their weight during the whole experiment. The survival of all groups was recorded from the beginning of SolB/PCN treatment until the end.

Liquid chromatography/mass spectrometry (LC/MS) and metabolomic analysis

Serum samples were thawed and 20 μL added to a tube containing 180 μL 67% aqueous acetonitrile. The samples were vortexed for 30 s each and centrifuged at 18000 × g for 20 min at 4°C to remove proteins. Approximately 20 mg liver or intestine were added to 50% aqueous acetonitrile (400 μL). The samples were homogenized at 8000 rpm for 8 s, then vortexed for 30 s each and centrifuged at 18000 × g for 20 min at 4°C to remove proteins. Samples of faeces (20 mg) were added to PBS (400 μL). These samples were vortexed and centrifuged at 18 000 × g for 20 min at 4°C. The supernatant (20 μL) was added to a tube containing 67% aqueous acetonitrile (180 μL) and the samples were vortexed for 30 s each and centrifuged at 18000 × g for 20 min at 4°C to remove proteins and particulates.

The supernatant was transferred to an UPLC vial and a 5 μL aliquot of deproteinized serum sample was injected into Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, CA) interfaced with Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Chromatographic separations were performed on Waters XTerra®MS C18 5 μm column (100 × 2.1 mm, Thermo Fisher Scientific, Waltham, MA). The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (acetonitrile). The gradient programme was as follows: 0 min 0% B, 1 min 0% B, 9 min 70% B, 16 min 83% B, 17 min 90% B, 19 min 100% B, 20 min 100% B, 25 min 0% B and 25 min stop. The flow rate was 0.3 mL·min−1. Electrospray negative ionization mode was used for analysis. The Spray voltage was set to 2.8 kV. Capillary and aux gas heater temperatures were set at 325 and 350°C respectively. Nitrogen was used as both sheath gas flow rate 40 arb and aux gas flow rate 10 arb.

The mass spectral data were aligned by using SIEVE 2.2 (Thermo Fisher Scientific, Waltham, MA). The multivariate data matrix was further exported into SIMCA‐P+13 software (Umetrics, Kinnelon, NJ). Either unsupervised principal components analysis (PCA) or supervised orthogonal partial least squares discriminant analysis (OPLS‐DA) models were constructed to analyse the data from serum, liver, intestine and faeces samples. Ions that were significantly altered were further confirmed by comparing the retention times and fragmentation patterns with authentic standards of bile acids.

RT‐qPCR analysis

Total RNA from mice liver tissues or HepG2 cells was isolated using Trizol reagent according to the manufacturer's instruction (Invitrogen, Grand Island, NY). Approximately 1 μg RNA was purified and randomly reverse‐transcribed to cDNA by using PrimeScript RT reagent kit with gDNA eraser (TaKaRa Biotech, Kyoto, Japan). RT‐qPCR analysis for specific genes was performed using SYBR Premix Ex Taq II (Tli RnaseH Plus) kit (TaKaRa Biotech, Kyoto, Japan) in Applied Biosystems 7500 Real‐Time PCR System. Gapdh for mice or GAPDH for human HepG2 cells was used as loading control. All the target genes are normalized to GAPDH. Values shown represent normalized relative fold changes of mRNA levels. The gene‐specific primer sequences are listed in Supporting Information Table S1.

Western blot analysis

Western blot analysis was performed as described in our previous report (Chen et al., 2014). Proteins extracted from mouse liver were prepared using RIPA lysis buffer (Biocolor BioScience and Technology, China) according to the manufacturer's directions. Protein concentration was determined by BCA assay (Thermo Scientific, Rockford, IL). Approximately 40 μg of protein extracts was separated by 8–12% SDS‐PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, USA). After blocking with 5% non‐fat dry milk in Tris‐buffered saline, membranes were incubated overnight with primary antibodies, including CYP3A (L‐14), p21 (F‐8) (Santa Cruz Biotechnology, Santa Cruz, CA); UGT1A1 (ab62600), p53 (pab 240, ab26) (Abcam, Cambridge, MA); GAPDH(14c10) (Cell Signaling Technologies, Danvers, MA, USA); CCDN1 (AB20509b), proliferating cell nuclear antigen (PCNA) (AB20014) (Sangon, China). Subsequently, a secondary horseradish peroxidase‐conjugated anti‐rabbit, anti‐mouse or anti‐goat IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was applied, and then specific bands were visualized using ECL detection kit (Engreen Biosystem, China). GAPDH was used as loading control and the protein bands intensities were analysed by Quantity One software (Bio‐Rad Laboratories, Hercules, USA).

Molecular docking

To rationalize the binding mode of SolB on hPXR, docking calculations were carried out with the CDOCKER protocol of Accelrys Discovery Studio 2.5 (Accelrys Software Inc., San Diego, CA). The pdb file about the crystal structure of hPXR complexing with its classic agonist Rif (PDB : 1SKX) was obtained from the RCSB protein data bank (http://www.pdb.org). The hydrogen positions were optimized with CHARMm force field. The molecules of SolB was generated with ChemBio3D Ultra 12.0 and optimized with MMFF94 method. After the end of molecular docking, selecting the lowest ranks from 10 molecular docking poses in the CDOCKER Interaction Energy tactic and visually examining the binding conformation of the docking poses by Discovery Studio Visualizer 3.5.

Dual‐luciferase reporter gene assays

HEK293T cells (from ATCC) were maintained in DMEM containing 10% FBS and 100 U penicillin/streptomycin. Cells were seeded in 96‐well plates at a density of 1.5 × 104 cells per well without antibiotics. For PXR‐reporter gene transactivation assays, each well contained 100 ng pGL3‐CYP3A4‐XREM‐Luc, 50 ng pSG5‐hPXR and 5 ng pRL‐TK. The transfection procedure was followed by Lipofectamine 2000 instruction (Invitrogen, Grand Island, NY). Six hours later, the transfection mixtures were removed and replaced with phenol red free DMEM containing 10% charcoal‐stripped delipidated FBS. Transfected cells were then treated with Sol B (2.5, 10 and 40 μM) and hPXR positive agonist Rif (10 μM) for 24 h. Luciferase activity was assayed in an Amersham Pharmacia Biotech luminometer using the Dual Reporter Assay System (Promega, Madison, WI) according to the manufacturer's instructions. Renilla activity was employed as control and firefly luciferase activity was normalized to Renilla activity for each well.

Drug treatment on hepatocarcinoma cell lines

HepG2 and Huh7 cells (from ATCC) were maintained in DMEM containing 10% FBS and 100 U penicillin/streptomycin. Cells were seeded in 12‐well plates at a density of 5 × 105 cells per well. Subsequently, adherent cells were exposed to SolB (2.5, 10 and 40 μM) or the hPXR positive agonist rifampicin (10 μM) for 24 h. The total RNA was isolated, purified, and transcribed to cDNA, and then RT‐qPCR analysis for specific genes was performed as described above.

Data and statistical analysis

The data and statistical analysis in this study comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). All data were expressed as mean ± SEM. Data were analysed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA). Unpaired Student's t‐test or nonparametric Mann–Whitney U‐test was used for statistical analysis between two groups. For comparison of more than two groups, one‐way ANOVA as well as nonparametric Kruskal–Wallis Test was performed and followed by Dunnett's multiple comparison post hoc test, only when F achieved P < 0.05 and there was no significant variance inhomogeneity. Differences in ALT, AST, ALP, TBA and Tbili levels among the different groups were analysed by one‐way ANOVA, followed by Duncan's multiple range post hoc test. Differences between group means were considered significant if P < 0.05.

Materials

SolB was supplied by Shanghai Winherb Medical Science and Technology Development Co., Ltd. (Shanghai, China,http://www.winherb.cn/). LCA (purity >98%) and corn oil were purchased from Aladdin Company (Shanghai, China). PCN was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Carboxymethylcellulose sodium (CMC), cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), UDCA, hyodeoxycholic acid (HDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), tauro‐β‐muricholic acid (T‐β‐MCA), taurochenodeoxycholic acid (TCDCA), taurohyodeoxycholic acid (THDCA), rifampicin and T0901317 (T0) were purchased from Sigma‐Aldrich (St.Louis, MO). The pSG5‐hPXR expression vector was generously provided by Dr. Steven Kliewer (University of Texas Southwestern Medical Center, Dallas, TX, USA). The pGL3‐CYP3A4‐XREM luciferase reporter construct containing the basal promoter (−362/+53) with the proximal PXR response element (ER6) and the distal xenobiotic responsive enhancer module (XREM, −7836/−7208) of the CYP3A4 gene 5′‐flanking region inserted to pGL3‐Basic reporter vector was generously provided by Dr. Jeff Staudinger (University of Kansas, Lawrence, KS, USA). The FXR‐responsive reporter tk‐EcRE‐Luc and expression vector for FXR, as well as the liver X receptor (LXR)α‐responsive reporter pGL3‐LXRE‐Luc and expression vector for LXRα, were kindly provided by Dr. Wen Xie (University of Pittsburgh, PA, USA). pRL‐TK Rotylenchulus reniformis control vector was obtained from Promega (Madison, WI, USA). All other chemicals and solvents were commercially available and of analytical grade.

Results

SolB prevents LCA‐induced cholestasis and liver injury

Morphological and histopathological examinations revealed that treatment with LCA for 4 days resulted in severe hepatic necrosis, diffuse vacuolization, infiltrating neutrophils and gall bladder enlargement (Figure 1A,B). This pattern was significantly attenuated by pre‐treatment with SolB (100 mg·kg−1) or PCN (50 mg·kg−1), as also confirmed by histological assessment of the extent of liver necrosis (Figure 1A,B). Consistent with the histopathology analysis, LCA treatment caused a marked elevation in serum ALT, AST and ALP levels (Figure 1C). SolB treatment dose‐dependently reversed LCA‐induced increase of ALT; AST and ALP (Figure 1C). Similarly, serum TBA and Tbili levels were strikingly increased by LCA treatment, which was 18.3‐ and 15.4‐fold higher than that of the vehicle group respectively (Figure 1D). SolB treatment dose‐dependently reversed the serum TBA and Tbili levels raised by LCA (Figure 1D). More importantly, treatment with PCN or the highest dose of SolB returned all these serum parameters to normal levels, i.e., those in the vehicle group. Taken together, these data clearly demonstrated that SolB protected against LCA‐induced intrahepatic cholestasis and hepatoxicity, in a dose dependent manner.

Figure 1.

Figure 1

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Hepatoprotective effect of SolB against LCA‐induced cholestatic liver damage in wild‐type mice. (A) Morphology of representative livers. Gall bladders were marked by arrows. (B) Representative H&E stained liver sections (10 × 10). Areas of severe liver necrosis are indicated with arrows. (C) Serum ALT, AST and ALP activities as well as (D) serum TBA and Tbili levels elevated by LCA, were significantly reduced by pretreatment with SolB or PCN. (E) LCA‐induced mortality was decreased by therapeutic treatment with SolB. Mice were treated with LCA for 3.5 days, followed by co‐treatment with 100 mg·kg−1 SolB or 50 mg·kg−1 PCN (n = 10). Survival was recorded every 12 h after the first treatment with SolB/PCN. Data are the mean ± SEM (n = 6 for LCA alone group, n = 8 for other groups). One‐way ANOVA for C and Kruskal–Wallis Test for D were performed. *P < 0.05, significantly different from vehicle; # P < 0.05, significantly different from LCA alone.

SolB exerts therapeutic effects against LCA‐induced cholestasis

In an earlier study, a significant elevation of serum TBA was observed after 3 days of treatment with LCA, although severe toxicity was observed only after 4 days of LCA (data not shown). We, therefore, started therapeutic treatment with SolB (100 mg·kg−1) or PCN (50 mg·kg−1), on day 4 and survival over the next 3 days was determined. Treatment with this dose of LCA alone increased mortality (including those mice humanely killed when they had lost >15–20% of their weight) to 90% at 5 days, while adding SolB or PCN treatment from day 4 onwards markedly decreased mortality (Figure 1E). This result suggested SolB could still protect the animals even when given after LCA had induced cholestasis, i.e. a potentially therapeutic effect.

SolB alters the metabolomic spectrum of bile acids in samples of serum, liver, intestine and faeces of cholestatic mice

In order to investigate the effect of SolB on the dynamic changes of endogenous bile acids, targeted metabolomic analysis of bile acids was performed in samples of serum, liver, intestine and faeces. Total ion chromatography profiles suggested that the abundance and quantity of endogenous molecules in serum and liver samples of the LCA alone group were higher than those from animals of the SolB‐treated group (Figure 2A). The opposite was observed in intestine and faeces (Figure 2A), indicating that some endogenous molecules were transferred from serum and liver to intestine and faeces after SolB treatment. Furthermore, PCA analysis revealed a distribution pattern of the LCA‐treated group in the scores scatter plot that was clearly separated from the SolB (100 mg·kg−1) treatment group, indicating significant difference of endogenous metabolome between these two groups (Figure 2B). The above observations were consistent with the hepatotoxic status of these groups.

Figure 2.

Figure 2

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The effect of pretreatment with SolB (100 mg·kg−1) on serum metabolome after LCA. (A) Total ion chromatography profiles analysed by UHPLC‐Q‐Exactive orbitrap mass spectrometer in bio‐samples from LCA‐treated mice with or without SolB (100 mg·kg−1). (B) PCA analysis in bio‐samples between LCA group and LCA+SolB group. (C) S‐plot of OPLS‐DA recognized metabolome in LCA treated mice with or without SolB (100 mg·kg−1). (D) Heat maps of bile acid profiles of serum, liver, intestine and faeces samples.

Further analysis of the loadings scatter plot of OPLS‐DA models showed many ions contributed to the separation of the metabolomes of the LCA and SolB groups. The identities of these ions were confirmed after carrying out comparison of retention time and tandem mass spectra to authentic standards of bile acids (Figure 2C). A significant difference in the amounts of bile acids was noted in all samples. In serum, TCA, TDCA, T‐β‐MCA, TCDCA and THDCA in SolB treatment group were significantly lower than that in LCA alone group, while CA, DCA and HDCA showed no difference. In livers, the TCA and T‐β‐MCA in the SolB treatment group were lower than in the LCA group, while THDCA was higher, and no difference was found for TDCA or TCDCA. In intestines, TCA in the SolB treatment group was lower than than in the LCA group, while CA, CDCA and T‐β‐MCA were higher, and no difference noted for DCA, TCDCA and THDCA. In faeces, CA, DCA, UDCA, HDCA and T‐β‐MCA in SolB group were all significantly higher than than in the LCA group. The heat‐map clearly revealed the abundance of these bile acids transferred from serum or liver to the intestine or faeces (Figure 2D).

Taken together, these results revealed that SolB treatment could significantly affect the dynamic changes of endogenous bile acids in LCA‐induced cholestasis. Specifically, SolB increased oxidation of LCA in liver, and increased the transfer of bile acids from serum and liver, to intestine and faeces.

SolB up‐regulates PXR genes involved in bile acid homeostasis

A range of enzymes and transporters are involved in the regulation of bile acid homeostasis. SolB alone up‐regulated hepatic levels of mRNAs for Cyp3a11, Ugt1a1, Oatp2 and Mrp3 (Figure 3A). LCA also increased Cyp3a11, Oatp2 and Mrp3 mRNAs, whereas no induction was found of Ugt1a1 mRNA (Figure 3B). However, SolB co‐treatment only significantly up‐regulated Ugt1a1 expression, while no further induction effect was found for Cyp3a11, Oatp2 or Mrp3 mRNAs compared with the LCA group. In addition, the known mouse PXR agonist, PCN, induced a similar induction pattern in these genes. To confirm these mRNA results, the respective protein levels were determined using Western blot analysis. Hepatic CYP3A11 and UGT1A1 proteins were remarkably consistent with the mRNA levels (Figure 3C). Taken together, these data indicate that the protective effect of SolB on cholestasis may be due to its induction of hepatic PXR‐regulated genes such as Cyp3a11, Ugt1a1, Oatp2 and Mrp3, which contribute to increased transport and metabolism of toxic bile acids.

Figure 3.

Figure 3

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Effect of SolB (100 mg·kg−1) on expression of hepatic genes (mRNAs) and proteins involved in bile acid homeostasis. RT‐qPCR analysis was used to detect hepatic gene expressions in mice relative to house‐keeping gene GAPDH. (A) A wide range of gene expressions was compared between vehicle and SolB treatment groups (n = 6). (B) Cyp3a11, Ugt1a1, Oatp2 and Mrp3 gene expressions were compared among all five groups (n = 6). (C) Levels of CYP3A11 and UGT1A1 protein in mouse livers were determined by Western blot analysis (n = 6). Data are the mean ± SEM. Unpaired Student's t‐test for A, and one‐way ANOVA for B and C. *P < 0.05, significantly different from vehicle; # P < 0.05, significantly different from LCA alone.

Molecular docking predicts that SolB can bind to the LBD of human PXR

In vivo studies showed that SolB induces mPXR‐regulated genes such as Cyp3a11, Ugt1a1, Oatp2 and Mrp3. To further demonstrate that SolB also has potential in binding to the ligand binding domain (LBD) of hPXR and then activating hPXR, we performed docking calculations in silico. The binding modes of SolB and rifampicin with the hPXR complex are illustrated in Figure 4A, and the CDOCKER Interaction Energy values were −72.8 and −36.8 kcal·mol−1 respectively. The hydroxyl of SolB could form a hydrogen bond with His407 with distances of 5.0 Å and the methyl could form two hydrogen bonds with Gln105, with distances of 5.0 and 5.1 Å. Moreover, the benzene rings of SolB formed one potential π interaction with Met243 (Figure 4B). Therefore, docking results show that SolB can bind to the LBD of human PXR and suggest SolB could, potentially, transactivate hPXR and induce hPXR‐regulated genes.

Figure 4.

Figure 4

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Identification of binding and transactivation effect of SolB on hPXR. (A) Docking of SolB into the human PXR‐LBD in the agonist conformation (Molsoft ICM). The three‐dimensional diagram shows the binding conformation of rifampicin (green) and SolB (red) with hPXR. The haem of hPXR is shown in grey. (B) The two‐dimensional diagrams displayed the docking model of SolB in active site of hPXR. The protein–ligand hydrogen bond interaction is displayed as dashed arrows. (C) Effect of rifampicin (RIF) or SolB on PXR NR agonism by dual‐luciferase reporter gene assay conducted in HEK293T cells (n = 5). (D) Regulation by SolB of CYP3A4, UGT1A1, Oatp2 and Mrp3 mRNA in HepG2 cells (n = 5). Data are the mean ± SEM. *P < 0.05, significantly different from vehicle control; one‐way ANOVA.

SolB transactivates hPXR and modulates hPXR‐regulated genes in human cell lines

To further examine whether SolB has the ability to transactivate human PXR, a dual‐luciferase reporter gene assay was performed in HEK293T cells via transient transfection with reporter plasmids. Rifampicin, a classical human PXR agonist, significantly enhanced the luciferase activity of the hPXR reporter gene by 3.8‐fold higher than that of the control group, whereas SolB concentration‐dependently enhanced the hPXR reporter gene activity by 3.6‐fold at 40 μM (Figure 4C). To examine whether SolB has the ability to modulate PXR‐regulated genes involved in bile acid metabolism and transport, HepG2 cells were incubated with SolB and mRNA expression of CYP3A4, UGT1A1 and OATP2 was measured by qRT‐PCR. Exposure to SolB significantly and dose‐dependently increased CYP3A4, UGT1A1 and OATP2 mRNA levels (Figure 4D). SolB (10 μM) significantly induced CYP3A4 mRNA by 2.9‐fold, UGT1A1 mRNA by 2.6‐fold, OATP2 mRNA by 12‐fold, effects similar to those of rifampicin. Similar inducible effects of SolB on the target genes were observed in Huh7 cells (Supporting Information Figure S3). These results indicate that SolB can transactivate human PXR and modulate PXR‐regulated genes.

SolB could not activate FXR or LXRα

A dual‐luciferase reporter gene assay was used to examine whether SolB could activate other NRs such as FXR and LXRα that are involved in regulating bile acid homeostasis. SolB could neither activate hFXR‐EcRE nor hLXRα‐LXRE reporter genes at 2.5, 10 and 40 μM concentrations, while the corresponding bonafide agonist used as positive controls were active (Supporting Information Figure S2A, B). These results indicated that SolB does not activate FXR or LXRα.

SolB protection against LCA‐induced cholestasis is PXR dependent

To determine the role of PXR signalling in the anti‐cholestasis effect of SolB, we tested SolB against LCA‐induced cholestasis using Pxr‐/‐ mice (Supporting Information Figure S4). The drug treatment and modelling were both the same as the previous study in wild‐type mice. SolB treatment did not prevent enlargement of the gall bladder or the liver damage and necrosis induced by LCA in Pxr‐/‐ mice (Figure 5A,B). Interestingly, SolB co‐treatment still significantly decreased ALT and AST, suggesting that it might exhibit some hepatoprotection against LCA‐induced liver injury, independent of PXR (Figure 5C). However, ALP, TBA and Tbili, which are more related to cholestasis status, were not significantly reversed by LCA in Pxr‐/‐ mice (Figure 5D).

Figure 5.

Figure 5

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The effect of SolB treatment (100 mg·kg−1) on LCA‐induced cholestatic liver damage and serum parameters in Pxr −/− mice. (A) Morphology of representative livers. Gall bladders were marked by arrows and the apparent subcapsular necrotic foci in the liver could be seen in both LCA and SolB treatment group. (B) Representative H&E stained liver sections (10 × 10). Areas of severe liver necrosis were indicated with arrows. (C) Serum ALT and AST activities elevated by LCA were significantly reduced by pretreatment with 100 mg·kg−1 SolB. (D) The elevation of ALP, TBA and Tbili levels by LCA was not reversed by SolB. Data are the mean ± SEM (n = 6 for LCA group, n = 7 for LCA+SolB group). Mann–Whitney U‐test was performed. *P < 0.05, significantly different from LCA alone.

PCA analysis showed no significant differences in serum, liver, intestine or faeces samples of Pxr‐/‐ mice between the SolB‐treated group and LCA‐treated group (Figure 6A). Likewise, targeted metabolomic analysis of bile acids demonstrated SolB did not affect the bile acid metabolites in Pxr‐/‐ mice (Figure 6B), while these bile acids were markedly altered by SolB in wild‐type mice (as shown in Figure 2D). These observations were consistent with the corresponding histological and biochemical analysis and indicated that SolB did not significantly protect against LCA‐induced cholestasis in Pxr‐/‐ mice, suggesting A critical role of PXR signalling in the hepatoprotection of SolB. Therefore, these findings provide genetic evidence that the effect of SolB is PXR‐dependent.

Figure 6.

Figure 6

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The effect of SolB treatment (100 mg·kg−1) on serum, liver, intestine and faeces metabolome in LCA‐treated Pxr −/− mice. (A) PCA analysis of samples from the LCA group and LCA+SolB group. (B) The comparison for the effect of SolB treatment (100 mg·kg−1) on bile acids in bio‐samples between LCA‐treated/untreated Pxr −/− mice. The relative amount of bile acid was displayed as log intensity of peak area. Data are expressed as means ± SEM (n = 6 for LCA group, n = 7 for LCA+SolB group). Student's t‐test was performed. *P < 0.05, significantly different as indicated.

Figure 7.

Figure 7

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Proposed mechanism of hepatoprotective effect of SolB against cholestatic liver injury through PXR.

SolB exhibits effects similar to those of PCN on liver regeneration‐related genes.

The liver regeneration process was delayed after partial hepatectomy in Pxr‐/‐ mice, indicating that PXR also participates in liver regeneration (Dai et al., 2008). To further confirm the role of PXR in the hepatoprotection of SolB against cholestatic liver injury, the expression of liver regeneration‐related proteins such as CCND1, PCNA, and their up‐stream regulators p53 and p21 were measured in the SolB‐treated LCA model. Expression of p53 and p21 was dramatically elevated in the group treated with LCA alone (Supporting Information Figure S5), which confirmed the morphological and histological liver damage induced by LCA treatment. SolB co‐treatment restored the raised expression of p53 and p21 to normal levels. CCND1 and PCNA expression were also induced by LCA treatment to a slight extent (Supporting Information Figure S5), suggesting that the liver regeneration process was activated after liver damage. However, SolB co‐treatment showed a much more significant induction of CCND1 and PCNA. In addition, SolB alone in vehicle group also significantly induced CCND1 and PCNA. Interestingly, the mouse PXR agonist PCN showed similar effects on expression of these proteins. Taken together, these findings suggest that SolB and PCN exhibited similar effect on the liver regeneration‐related pathways and genes during cholestatic liver injury.

Discussion

Currently, therapy for cholestatic liver diseases is limited to UDCA and OCA (Lazaridis et al., 2001; Samur et al., 2016). Thus, the development of new therapeutic approaches and novel drug candidates is urgently needed. Bile acid homeostasis is regulated to a large extent at the transcriptional level via NRs that play an important role in the regulation of transport and synthesis (Wagner et al., 2010). NRs are promising therapeutic targets for cholestatic liver injury. Notably, PXR represents an attractive target for therapy of cholestasis because of its central role in bile acid transport and detoxification. PXR behaved as a bile acid sensor, due to its induction of the metabolizing enzymes CYP3A, UGT1A1, the uptake transporter OATP2 and the output transporter MRP3 leading to clearance of bile acids from the liver (Lehmann et al., 1998; Staudinger et al., 2001; Xie et al., 2001; Guo et al., 2002; Chen et al., 2003; Wagner et al., 2005; Zhou et al., 2005). In the present study, SolB dose‐dependently exerted significant hepatoprotection effect against LCA‐induced intrahepatic cholestasis. Specifically, a 100 mg·kg−1 dose prevented liver damage and maintained all the cholestatic parameters at normal levels. Therapeutic treatment of SolB can significantly reverse LCA‐induced hepatic toxicity. Therapeutic treatment with PCN also displayed improvement in liver damage, indicating the therapeutic effectiveness of PXR agonists in reducing LCA‐induced mortality.

Moreover, the mechanism of SolB hepatoprotection against LCA‐induced cholestasis was further investigated by targeted metabolomic analysis of bile acids. Metabolomic analysis revealed dynamic changes of bile acids in serum, liver, intestine and faeces. Notably, SolB co‐treatment promoted the transport of most of the detected bile acids from serum/liver to intestine/faeces. Interestingly, the levels of HDCA and THDCA, which are hydroxylated metabolites of LCA via CYP3A oxidation, was higher in liver after SolB co‐treatment(Xie et al., 2001; Zhang and Klaassen, 2010), providing direct evidence that SolB activated hepatic CYP3A, which is a main target gene of PXR. The induction of PXR‐target genes including Cyp3a11, Ugt1a1, Oatp2 and Mrp3 (Lehmann et al., 1998; Guo et al., 2002; Xie et al., 2003; Teng and Piquette‐Miller, 2005; Wagner et al., 2005) by SolB revealed that the drug accelerated detoxification and transport across membranes in vivo. The induction of Cyp3a11 was consistent with the observation that SolB increased LCA metabolism via CYP3A as revealed by metabolomics analysis. Interestingly, SolB as well as ethanol extracts of Schisandra sphenanthera was reported to exert a dual effect on CYP3A; long‐term induction of expression and short‐term transient inhibition of enzyme activity (Mu et al., 2006; Jin et al., 2010; Su et al., 2013; Qin et al., 2014). For long‐term administration, the net effect depends on the administration interval (Lai et al., 2009). At early stages after administration, the inhibition effect is dominant, while the induction effect gradually has a major role after drug elimination in vivo. As SolB has a fast elimination in vivo with a half‐life less than 1 h (Wei et al., 2010), the inductive effect of SolB was active during most of the administration interval of 12 h, as revealed in the present study.

As PXR agonists exhibit species differences between rodents and human, docking and cell experiments were conducted to confirm the similar activation effect of SolB on human PXR. The docking results revealed potential binding sites and interaction bonds of SolB with LBD of human PXR. Dual reporter gene assays indicated the transactivation effect of SolB on human PXR, which was consistent with an earlier study on compounds isolated from Schisandra chinensis (Mu et al., 2006). What is more, the present studies further observed an induction effect of SolB on expression of human PXR‐targeted genes including CYP3A4, UGT1A1, and OATP2, which are the human homologues for mouse Cyp3a11, Ugt1a1 and Oatp2. The results suggest that SolB could also function as a potential human PXR agonist to regulate bile acid‐metabolizing and transporter genes. Unexpectedly, in HepG2 cells, SolB did not induce MRP3, a PXR‐regulated gene. But a weak induction of SolB on MRP3 was observed in Huh7 cells. As there are other transcription factors that can affect MRP3 apart from PXR, as well as possible differences in signalling pathways between liver and HepG2 cells, SolB may also affect some other negative regulators of MRP3 in HepG2 cells, and exhibit no regulation of MRP3 overall.

To determine the role of PXR on the anti‐cholestasis effect of SolB, the protective effects of SolB against LCA‐induced cholestasis were examined in Pxr‐/‐ mice. Although there was no hepatoprotection by SolB against LCA‐induced cholestasis in Pxr‐/‐ mice, paradoxically, SolB still decreased serum ALT and AST levels. Considering the fact that SolB as well as extracts of Schisandra sphenanthera display hepatoprotective effects against various types of liver injury other than cholestasis (Maeda et al., 1985; Yamada et al., 1993; Ko et al., 1995), the decrease in ALT and AST by SolB may be independent of the PXR pathway. Overall, the data for gall bladder enlargement, as well as ALP, TBA, Tbili and bile acids metabolomic analysis, which are more direct cholestatic indexes, clearly demonstrated that the central role of the PXR pathway in the anti‐cholestasis effect of SolB.

Additionally, the levels of ALP and Tbili in LCA‐treated Pxr‐/‐ mice were both significantly lower than those in wild‐type mice, while ALT, AST and TBA displayed little difference. Previous studies observed similar findings with potential mechanistic explanations. Basal expression of CAR could be up‐regulated in Pxr‐/‐ mice as a compensatory effect, resulting from the inhibitory effects of PXRs on CAR (Stedman et al., 2005; Teng and Piquette‐Miller, 2007). As the key target gene of CAR (Sugatani et al., 2001), the basal expression of UGT1A1, a classic bilirubin metabolizing enzyme (Huang et al., 2003), is also up‐regulated in Pxr‐/‐ mice and these mice possess exhibit higher rates of bilirubin elimination, compared to wild‐type mice. Another major toxic compound elevated during cholestasis is bilirubin which also contributes to bile duct injury, as reflected by ALP. Therefore, less elevation of ALP and Tbili was observed in LCA‐treated Pxr‐/‐ mice.

Apart from regulation of bile acid homeostasis, increased liver regeneration after liver damage could be another therapeutic strategy against cholestatic liver injury. Liver regeneration is activated after partial hepatectomy, as well as after acute liver damage (Jeong et al., 2001; Fan et al., 2014a,b). The mechanism of liver regeneration involves activation of cell cycle related pathways, with CCND1 and PCNA as indicator proteins, which are negatively regulated by the p53/p21 pathway (Wu et al., 1996). The present study revealed that SolB up‐regulated CCND1 and PCNA, and reduced p53 and p21, compared with the LCA‐treated group. More importantly, SolB treatment could also induce CCND1 expression. These results suggested that SolB may promote liver regeneration after cholestatic liver injury. Interestingly, it was reported that PXR may also participate in liver regeneration. In a previous study, the liver regeneration process was delayed after partial hepatectomy surgery in Pxr‐/‐ mice, when compared with wild‐type mice (Dai et al., 2008). Furthermore, in the present study, PCN co‐treatment displayed a regulation pattern of these proteins, very similar to that of SolB, indicating a potential promotion by PXR agonists of liver regeneration. These findings indicate that the promotion of liver regeneration by SolB may also be mediated via PXR.

To further investigate the specificity of the transactivation of PXR by SolB, other NRs were examined including FXR and LXRα. SolB did not activate FXR or LXRα, consistent with the observation that SolB treatment could not induce the hepatic expression of the bile salt export pump, a specific FXR target gene (Xu et al., 2002). In a previous study, SolB was found to activate the Nrf‐2‐ARE pathway (Fan et al., 2014a,b), which is also a promising therapeutic target of cholestasis. Mrps are NRF‐2 targeted genes involved in bile acid homeostasis (Maher et al., 2007). In the present study, SolB did induce hepatic expression of Mrp3 mRNA in mice. However, Mrp3 is also a PXR targeted gene. Thus, the current data still cannot determine a role for Nrf‐2 in the anti‐cholestasis effect of SolB. Therefore, the possible effect of SolB on other anti‐cholestasis molecular targets including the Nrf‐2 pathway remain to be investigated.

In summary, we have provided evidence that SolB dose‐dependently protected against LCA‐induced intrahepatic cholestasis, and decreased LCA‐induced mortality, when given as a therapeutic treatment. The hepatoprotective effects of SolB was due to its activation of PXR signalling which, in turn, accelerated the detoxification and efflux of toxic bile acids from the liver, and possibly due to the increase of liver regeneration, which is also related to the PXR pathway. This study reveals SolB as a natural product which could possibly be developed into a promising anti‐cholestasis drug. Apart from this, our data support the notion that development of potent PXR agonists might be beneficial in the treatment of cholestatic liver diseases in clinical practice.

Author contributions

H.Z. and Y.J. contributed equally to this work.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Table S1 Primer sequences for RT‐qPCR.

Figure S1 Chemical structure of Schisandrol B (SolB).

Figure S2 Effects of SolB on hFXR, hLXRα nuclear receptor agonism. Relative luciferase activity was detected to reflect activation by SolB on (A) FXR‐EcRE or (B) LXRα‐LXRE reporter genes in HEK293T cells. HEK293T cells were transiently transfected with respective plasmids as described in the Methods, then the cells were treated with different concentrations of SolB (2.5, 10, 40 μM) or the positive agonist CDCA (100 μM) for hFXR or T0 (10 μM) for LXRα, 24 h before harvesting. Data are the mean ± S.E.M. (n = 5). ***P < 0.001, versus vehicle.

Figure S3 Effects of SolB on CYP3A4, UGT1A1, OATP2 and MRP3 mRNA in Huh7 cells (n = 5). Data are the mean ± SEM. *P < 0.05, versus vehicle control.

Figure S4 Phenotype identification of WT and Pxr−/− mice. RT‐qPCR analysis was conducted to determine Pxr gene expression in mouse livers. (WT: n = 6, Pxr−/−: n = 13) N.D.: Undetected (Ct value bigger than 35).

Figure S5 SolB (100 mg·kg−1) induced hepatic CCND1 and PCNA protein expression, as well as inhibiting the activation of p53/p21 pathway by LCA. Protein levels of CCND1, PCNA, p53 and p21 in mouse livers for LCA model were detected by Western blot analysis. Data are the mean ± SEM; n = 5. *P < 0.05, versus vehicle; #P < 0.05, versus LCA alone.

Click here for additional data file. (825.5KB, pdf)

Acknowledgements

We would like to thank Steven A. Kliewer, Department of Pharmacology, UT Southwestern Medical Center who provided the Pxr‐null mice. This work was supported by the Natural Science Foundation of China (grants: 81573489, 81522047, 81373470 and 81320108027), the 111 project (grant no.: B16047), the Key Laboratory Foundation of Guangdong Province (grant: 2011A060901014) and the Guangzhou Health Care Collaborative Innovation Program (grant: 201508020250).

Zeng, H. , Jiang, Y. , Chen, P. , Fan, X. , Li, D. , Liu, A. , Ma, X. , Xie, W. , Liu, P. , Gonzalez, F. J. , Huang, M. , and Bi, H. (2017) Schisandrol B protects against cholestatic liver injury through pregnane X receptors. British Journal of Pharmacology, 174: 672–688. doi: 10.1111/bph.13729.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1 Primer sequences for RT‐qPCR.

Figure S1 Chemical structure of Schisandrol B (SolB).

Figure S2 Effects of SolB on hFXR, hLXRα nuclear receptor agonism. Relative luciferase activity was detected to reflect activation by SolB on (A) FXR‐EcRE or (B) LXRα‐LXRE reporter genes in HEK293T cells. HEK293T cells were transiently transfected with respective plasmids as described in the Methods, then the cells were treated with different concentrations of SolB (2.5, 10, 40 μM) or the positive agonist CDCA (100 μM) for hFXR or T0 (10 μM) for LXRα, 24 h before harvesting. Data are the mean ± S.E.M. (n = 5). ***P < 0.001, versus vehicle.

Figure S3 Effects of SolB on CYP3A4, UGT1A1, OATP2 and MRP3 mRNA in Huh7 cells (n = 5). Data are the mean ± SEM. *P < 0.05, versus vehicle control.

Figure S4 Phenotype identification of WT and Pxr−/− mice. RT‐qPCR analysis was conducted to determine Pxr gene expression in mouse livers. (WT: n = 6, Pxr−/−: n = 13) N.D.: Undetected (Ct value bigger than 35).

Figure S5 SolB (100 mg·kg−1) induced hepatic CCND1 and PCNA protein expression, as well as inhibiting the activation of p53/p21 pathway by LCA. Protein levels of CCND1, PCNA, p53 and p21 in mouse livers for LCA model were detected by Western blot analysis. Data are the mean ± SEM; n = 5. *P < 0.05, versus vehicle; #P < 0.05, versus LCA alone.

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