Quantitative proteomics analysis of Fructus Psoraleae-induced hepatotoxicity in rats

LI Zhi-Jian1,2Δ, Abudumijiti Abulizi2,3Δ, XU Deng-Qiu1, Youlidouzi Maimaiti2, Silafu Aibai2,JIANG Zhen-Zhou1,5, ZHAO Guo-Lin1, WANG Tao1,6, Aiximujiang Refukati2, Zulikaer Maimaiti2,

Yiliyaer Simayi2, CAO Chun-Yu2,8, *, ZHANG Lu-Yong1,4,6,7, *

1 Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China;2 Department of Toxicology Laboratory, Xinjiang Institute of Traditional Uyghur Medicine, Urumqi 830049, China;3 State Key Laboratory of Natural and Biomimetic Drugs, Department of Pharmacology, School of Basic Medical Sciences, Pek-ing University, Beijing 100191, China;4 Center  for  Drug Screening and Pharmacodynamics  Evaluation,  School  of  Pharmacy,  Guangdong Pharmaceutical  University,Guangzhou 510006, China;5 Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Nanjing 210009, China;6 Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, China;7 State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;8 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

Available online 20 Feb., 2020

[ABSTRACT] Fructus Psoraleae, which is commonly consumed for the treatment of osteoporosis, bone fracture, and leucoderma, in-duces liver injury. This study investigated the pathogenesis of the ethanol extract of Fructus Psoraleae (EEFP)-induced liver injury inrats. EEFP (1.35, 1.80, and 2.25 g·kg–1) was administrated to Sprague Dawley (SD) rats for 30 d. We measured liver chemistries, histo-pathology, and quantitative isobaric tags for relative and absolute quantitation (iTRAQ)-based protein profiling. EEFP demonstratedparameters suggestive of liver injury with changes in bile secretion, bile flow rate, and liver histopathology. iTRAQ analysis showedthat a total of 4042 proteins were expressed in liver tissues of EEFP-treated and untreated rats. Among these proteins, 81 were upregu-lated and 32 were downregulated in the treatment group. KEGG pathway analysis showed that the drug metabolic pathways of cyto-chrome P450, glutathione metabolism, glycerolipid metabolism, and bile secretion were enriched with differentially expressed pro-teins. The expression of key proteins related to the farnesoid X receptor (FXR), i.e., the peroxisome proliferators-activated receptor al-pha (PPAR-α), were downregulated, and multidrug resistance-associated protein 3 (MRP3) was upregulated in the EEFP-treated rats.Our results provide evidence that EEFP may induce hepatotoxicity through various pathways. Furthermore, our study demonstrateschanges in protein regulation using iTRAQ quantitative proteomics analysis.

[KEY WORDS] Hepatotoxicity; iTRAQ Analysis; Fructus Psoraleae; FXR; MRP3[CLC Number] R965 [Document code] A [Article ID] 2095-6975(2020)02-0123-15

Introduction

Fructus Psoraleae (FP), a herbaceous plant of theleguminosae family and commonly referred to as "Boh-Gol-Zhee" or Psoralea corylifolia L. [1], is commonly used in Asia

as a traditional medicine, specifically in China, Korea, andJapan. It is commonly prescribed in traditional Chinese medi-cine as Bu-Shen-Yi-Nao-Pian, Qing-Er-Wan, Si-Shen-Wan,and Zhuang-Gu-Guan-Jie-Wan, for its active biological con-stituents such as coumarins, flavonoids, and monoterpenes [2-4],which impart positive therapeutic effects on osteoporosis,bone fracture, osteomalacia, and vitiligo [5]. A total of 35chemicals have been isolated from FP, including resins, fattyoils, essential oils, bakuchiol, corylifolinins, psoralidins,psoralens, isopsoralens, and bavachins [6].

FP has potent effects on various diseases; however, the

[Received on] 21-Aug.-2019[*Corresponding author] E-mail: caocy@foxmail.com (CAO Chun-Yu); Tel/Fax: 86-25-83271142, E-mail: lyzhang@cpu.edu.cn(ZHANG Lu-Yong)∆These authors contributed equally to this work.These authors have no conflict of interest to declare.

Available online at www.sciencedirect.com

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potential hepatotoxicity of herbal remedies is often ignored.Numerous clinical and experimental studies have revealedthat FP is associated with the occurrence of hepatotoxicitywhen ingested, thereby restricting its application. Thereare also reports of liver dysfunction that were related to theuse of proprietary FP medicines such as Bu-gu-zhi in-tramuscular injection, Xin-Cu-Hei-Su-Sheng-Zhang-Ji, andQu-Bai-Ba-Bu-Qi-Pian [5]. Some researchers have revealedthe hepatotoxicity effects on C57 mice induced by pso-ralens whose synthetic forms are 5-methoxypsoralen (ber-gapten) or 8-methoxypsoralen (xanthotoxin) [1]. A previousstudy showed that mice that orally received the psoralenisopsoralen exhibited impaired liver function, which wasmediated by a disruption of hepatic cytochrome P450(CYP450) [7]. High-dose Bakuchiol is toxic to mouse kid-neys [8] and causes a significant decrease in transaminaseactivity in HepG2 cells in  vivo [9]. The State Food and DrugAdministration of China has reported adverse reactions toZhuang-Gu-Guan-Jie-Wan and Bai-Shi pills, which are tradi-tional Chinese compounds whose main ingredients includeFP, have caused cholestatic liver damage [10-11](National Cen-ter for ADR Monitoring, China. 2008. ADR Notification Nos.1 and 9).

The active or toxic compounds of FP that contribute toacute cholestatic hepatic injury mainly include the low-polar-ity compounds of Psoralea  corylifolia L. such as psoralens,bakuchiol, and isopsoralens [5]. Our research team has repor-ted the expression of mRNAs and proteins related to the bio-synthesis and transport of bile acids in EEFP-treated rats.However, due to methodological limitations, past researchershave only focused on single or several different expressedproteins in the liver, and systemic liver changes at the mo-lecular level under the process of injury remain largely unex-plored.

Proteomic techniques based on isobaric tags for relativeand absolute quantification (iTRAQ) labeling offer a high-throughput approach to analyze differentially expressed pro-teins in various physiological and pathological states. In thisstudy, we analyzed alterations in the rat liver proteome usingiTRAQ, which provided additional details that can improveour understanding of the mechanism of action of FP.

Materials and Methods

Herbal plant collection and storageSeeds of FP were purchased from Xinqikang Pharma-

ceutical Co., Ltd. (Batch No. 20151027; Region: Shanxi). Avoucher specimen was reserved at the Laboratory of Toxico-logy, Xinjiang Institute of Traditional Uygur Medicine (BatchNo. 20151027), Xinjiang, China.Plant extraction and main constituent analysis

Seeds of FP (l kg) were extracted with 95% ethanol(3 × 10 L, each 1 h). The combined ethanol extracts wereconcentrated to produce crude extracts (yield of the extract:27.03%) under reduced pressure and stored at 4 °C. For

the determination of chemical constituents, EEFP (BatchNo. 20160310) was separated and analyzed by HPLC.We detected three main peaks, and the UV spectra andretention time that matched reference compounds wereidentified as psoralen (8.00 mg·g–1 of the total extract), isop-soralen (6.20 mg·g–1 of the total extract), and bakuchiol(179.18 mg·g–1 of the total extract), which were measured bythe external standard method.Experimental animals

Male and female specific pathogen-free (SPF) SpragueDawley (SD) rats (n = 90; weight: 120 –150 g; age: 6 –7weeks old) were obtained from the Xinjiang Laboratory An-imal Centre. The animals were housed in an SPF animalroom at the Center for Evaluation of Drug Safety, XinjiangInstitute of Traditional Uygur Medicine under standard con-ditions, with a 12 h/12 h dark and light cycle. Standard waterand food were available ad libitum. The protocols for the ex-periments were approved by the Xinjiang Institute of Tradi-tional Uygur Medicine Ethics Committee on Animal Experi-mentation and strictly conducted in accordance with interna-tional laboratory animal use and care guidelines (ApprovalNumber WYS-2017-09-01). Observations were conducteddaily for signs of toxicity and mortality, and weekly for foodintake and body weight.Experimental design

After stabilization, the SD rats were randomly assignedto four groups, with equal numbers of males and femaleseach group, and were fed and treated for 30 consecutive days.Group 1 was the control (untreated) group (n = 20) that re-ceived the vehicle solution only (0.5% CMC-Na and 2%Tween 80 solution). Treatment groups 2 (n = 20), 3 (n = 20),and 4 (n = 30) respectively received low (1.35 g·g–1), middle(1.80 g·g–1), and high doses (2.25 g·g–1) of EEFP each dayand were treated as 1 mL per 100 g according to body weight.After this period, the rats’ general symptoms were observedsuch as behavior, appearance, breathing, and glandular secre-tion. The body weight of each animal was weighed weekly,and differences in body weight and food intake among groupswere compared. Immediately after 30 d of gavage, the ratswere fasted for 12 h and anesthetized with pentobarbital.Blood samples were collected from the abdominal aorta foranalysis of biochemical indicators. No anticoagulants wereused, and the serum was analyzed after centrifugation at 3500r·min–1 at 4 °C for 10 min.Collection  of  bile  and  determination  of  bile  total  bile  acid(TBA) and total bilirubin (TBIL)

The rats were anaesthetized with urethane. Cannulationsof the common bile duct and femoral vein were perfor-med [12]. After a 3-min equilibration, bile was collected for2 h to determine to total bile acid output and flow rate. Thecontents of total bile acid and bilirubin were determined us-ing an automated biochemical analyzer (7100, Hitachi Ltd.,Tokyo, Japan), and all kits were purchased from ShenzhenMindray Biomedical Electronics Co., Ltd., Shenzhen, China.

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Serum assays for liver toxicitySerum biochemical and electrolyte index analyses were

performed with an automated biochemical analyzer (7100,Hitachi Ltd., Tokyo, Japan) according to the manufacturer’sprotocol. Recorded measurements included the following: as-partate aminotransferase (AST), alanine aminotransferase(ALT), albumin (ALB), total cholesterol (TC), triglycerides(TG), alkaline phosphatase (ALP), TBIL, TBA, and gamma-glutamyl transferase (GGT). All kits were purchased from Shen-zhen Mindray Biomedical Electronics Co., Ltd., Shenzhen,China.Histopathology

Observations of all organs included their color, size,shape, and position, as well as any gross lesions. Livers wereweighed and collected. Buffered formaldehyde solutions(10%) were used to fix the fresh tissue slices from each or-gan, which were then treated using ethanol-benzene and em-bedded in paraffin. A microtome was used to prepare sec-tions of 0.2 to 0.5 μm in thickness, which were then stainedwith haematoxylin and encoded and examined under a lightmicroscope by a pathologist.Protein extraction, digestion, and iTRAQ labeling

According to the liver physiological performance andanalysis, we selected the control and high-dose treatmentgroups for further iTRAQ studies. Approximately 50 mg ofprotein from each sample were dissolved in 1 mL of SDTbuffer (100 mmol·L–1 Tris-HCl, 4% SDS, pH 7.6, 1 mmol·L–1

DTT) and homogenized (60 s, twice). The lysates were sonic-ated at 80 W for 10 times on ice, and then boiled for 15 min.The lysates were cleared by centrifugation at 14 000 g for 15min at 4 °C. The supernatant was filtered by a 0.22-m filtermembrane, and the concentration was determined using aBCA kit (Beyotime Biotech, Nanjing, China). The quality ofliver lysate was examined by SDS-PAGE. Briefly, 30 μg ofeach sample were boiled in 100 mmol·L–1 DTT for 5 min.Protein samples were loaded into 200-μL Merck Millipore ul-trafiltration tubes with 30-kDa cut off and washed twice withUA buffer. Protein samples were then alkylated with 100mmol·L–1 iodoacetamide in UA buffer for 30 min at roomtemperature (RT) in the dark and centrifuged at 14 000 g for15 min at 4 °C. Protein samples were washed twice with100 μL of UA buffer and a dissolution buffer containing0.5 mol·L–1 triethylammonium bicarbonate (pH 8.5). Thesamples were digested with 40 μL trypsin (4 μg sequencing-grade modified trypsin dissolved in 40 μL dissolution buffer)at 37 °C for 18 h. The tryptic peptides were collected by cent-rifugation at 14 000 g for 15 min and then washed once witha dissolution buffer. Subsequently, protein samples werelabeled with iTRAQ Regent 8-plex Multiplex Kit (AB SCI-EX, Framingham, MA, USA), following the manufacturer ’srecommendations. Isobaric tags were solubilized in 70 mLisopropanol. Peptides from the control group were labeledwith iTRAQ tags and to profile changes of protein expres-sion levels, iTRAQ labeling was performed in three inde-

pendent experiments to ensure reproducibility.Strong  cation-exchange  fractionation  and  LC-MS/MS  ana-lysis

The labeled peptides were mixed and graded using theAgilent 1260 infinity II HPLC system. The solvent systemwas composed of phase A (10 mmol·L–1 HCOONH4, 5%ACN, pH 10.0) and phase B (10 mmol·L–1 HCOONH4, 85%ACN, pH 10.0). Peptides were eluted by a multistep gradient:0 –25 min, 0% B; 25 –30 min, 7% B; 30 –65 min, 40% B;65 –70 min, 60% B; 70 –85 min, 100% B; and 60 –75 min,100% A at a flow rate of 1 mL·min–1. A total of 36 fractionswere collected and pooled into 16 fractions, which served asthe base for the chromatographic profile of peptides. Thefractions were lyophilized by a rotary vacuum concentratorand desalted by a C18 cartridge (66872-U, Sigma, St. Louis,MO, USA).

Separation of SCX fractions was performed using anEASYnLC1000 column (Thermo Scientific). Peptide mix-tures were first loaded onto pre-column (100 Å, 100 μm i.d. ×20 mm, 5 μm-C18, Thermo Scientific) and then separated onanalytical EASY columns (100 Å, 75 μm i.d. × 15 cm, 3 μm-C18, Thermo Scientific). Solvent system was 0.1% formicacid in water for mobile phase A, and 80% acetonitrile plus0.1% formic acid for mobile phase B. Tryptic peptides wereeluted through multistep gradient from 0% to 38% buffer Bwith a linear gradient at 0 –50 min; 38% to 100% buffer Bwith a linear gradient at 50–55 min; 100% buffer B at a flowrate of 0.3 mL·min–1 at 55–60 min.

The eluted tryptic peptides were directly interfaced witha Q-Exactive Mass Spectrometer (Thermo Fisher Scientific,Waltham, MA, USA). Data were acquired in positive ionmode with a selected mass range of 350 –1800 mass/charge(m/z). Q-Exactive survey scans were obtained at 70 000 (m/z200) with 50 ms maximum ion injection time and 40 s dy-namic exclusion for higher-energy collisional dissociationspectra, followed by 10 data-dependent MS/MS scans at17 500 (m/z 200). The maximum ion injection time was fixedat 50 ms, the normalized collision energy was 30 eV, and theunder-fill ratio was defined as 0.1%. Raw mass spectrometrydata was identified and quantified using the software Mascot2.5 and Proteome Discoverer 2.1. Functional annotations ofdifferentially expressed proteins were predicted using GeneOntology. The Kyoto Encyclopedia of Genes and Genomes(KEGG) was performed for major pathways. This projectused the database (http://www.uniprot.org) for analysis. A P-value ≤ 0.05 was considered significant enrichment in the GOand KEGG pathways.Protein isolation and western blotting assay

Total proteins were extracted from the livers of differentgroups by homogenization in tissue protein lysis buffer sup-plemented with a protease inhibitor cocktail (Key gen Bi-otech, Nanjing, China). The concentrations of the protein ex-tracts were determined using Bicinchoninic Acid (BCA) pro-tein assay reagent kits (Beyotime Biotech, Nanjing, China).

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Before the same concentration of protein were denatured, anequal volume of the sample with the 2 × loading buffer wasmixed. Equal amounts of proteins were loaded and separatedby SDS-PAGE, then transferred into polyvinylidene difluor-ide membranes (PVDF) (Millipore Corp., Bedford, MA,USA). After blocking nonspecific binding sites for 2 h with5% dried skim milk (Becton, Dickinson and Co., MD, USA),the membranes were incubated overnight at 4 °C with homo-logous primary antibodies: goat polyclonal anti-FXR anti-body (1 : 200, Santa Cruz Biotechnology, USA), rabbit poly-clonal anti-PPARα antibody (1 : 500, Santa Cruz Biotechno-logy), and goat polyclonalanti-MRP3 antibody (1 : 500, SantaCruz Biotechnology) were implemented overnight at 4 °C.Samples were washed with 0.1% Tween-20 Tris-buffered sa-line on the second day, the membranes were incubated atroom temperature for 2 h with the homologous secondary an-tibodies, and the ECL detection reagents (Perkin Elmer,USA) were used to detect signals. Densitometric scanning ofband intensities were quantified by Quantity One (Bio-Rad,USA). β-Actin (1 : 500, Santa Cruz Biotechnology) was usedfor data normalization.Statistical analyses

Statistical analysis was performed with one-way analys-is of variance (ANOVA), and quantitative variables wereanalyzed using Tukey’s test based on at least three replicatesof each experiment. A P-value < 0.05 was considered statist-ically significant.

Results

The effect of EEFP on body weight and food intakeMales that were treated by EEFP showed a significant re-

duction in body weight. Food intake in the female EEFPhigh-dose group significantly increased compared to the con-trol group after 30 days of feeding (Figs. 1 and 2).Liver to body weight

Liver to body weight significantly increased in all EEFPgroups of males and females compared to the control group(P < 0.001) (Fig. 3).Collection of bile and determination of bile TBA and TBIL

Fig. 4 shows that the volume of bile and flow rate signi-ficantly increased in the EEFP-treated groups of bothgenders. There were no obvious changes in the levels of TBAand TBIL, but these decreased in the female and male EEFP-middle dose groups (P < 0.05).Serum biochemical indices of hepatic function

Compared to the control group, ALT increased in thefemale EEFP low- and middle-dose groups and in themale EEFP middle-dose group (P < 0.05). GGT, TBIL, andTBA increased in the female and male EEFP high-dosegroup (P < 0.05 or 0.01). TC and TG decreased markedly inboth gender groups (P < 0.05, 0.01, or 0.001) (Fig. 5).Histopathological changes

Compared to the control group, H&E staining resultsdemonstrated that the administration of EEFP caused grosshistopathological changes, including hepatic steatosis, bile

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t/d Fig. 1 Effects of 30-day administration of EEFP on rat body weight. The data are expressed as the mean ± SD ( each sex: Con-trol, EEFP-L, EEFP-M, n = 10; EEFP-H, n = 15). *P < 0.05, **P < 0.01 vs vehicle control. One-way ANOVA followed by Dunnett’s t-test. EEFP-L: ethanol extract of Fructus Psoraleae, low dose; EEFP-M: ethanol extract of Fructus Psoraleae, middle dose;EEFP-H: ethanol extract of Fructus Psoraleae, high dose

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duct dilatation, inflammatory cell infiltration, and liver cho-lestasis in a dose-dependent manner (Fig. 6).iTRAQ-based quantitative proteomic analysis  and functionalclassification

We used the iTRAQ technology coupled with LC-MS/MS to detect differentially expressed proteins in liversamples from EEFP-treated groups to determine the molecu-lar mechanisms of EEFP. Mascot identified a total of 43 237peptides and 4042 proteins. Among these, 113 differential ex-pressed proteins (P < 0.05) were identified in the liver by

Mascot 2.2 (Matrix Science, London, UK) against the Swiss-Prot database (last accessed in May 2016) from UniProt web-site (http://www.uniprot.org). Of the 113 selected differen-tially expressed proteins, 81 were upregulated (Table 1) and32 were downregulated (Table 2).

The results of cluster analysis of the 113 significantlydifferentially expressed proteins are shown in Fig. 7A. A heatmap was used to group the proteins into four main clusters tobetter explain the altered biological processes in their distri-bution across the proteomics database. Our results suggestthat considerable variability existed in proteome-wide expres-sion between the control and EEFP groups (Fig. 7A). Thesedifferential expression data are presented graphically inFig. 7B, which shows the fold difference for each individualprotein identified in at least four mice plotted against the Pvalue. To further elaborate the biological significance of reg-ulated proteins, enrichment analysis was performed to func-tionally annotate these into related biological processes andpathways. GO enrichment analysis using biological pro-cesses indicated that the differentially expressed proteinswere significantly related to monocarboxylic acid, lipid,carboxylic acid, oxoacid, and organic acid metabolic pro-cesses (Fig. 7C). KEGG pathway analysis indicated that thecategories of metabolism of xenobiotics by cytochrome P450,metabolic pathways, drug metabolism-cytochrome P450, ret-inol metabolism, steroid hormone biosynthesis, glutathionemetabolism, glycerolipid metabolism, and bile secretion weremainly involved in the response to EEFP-induced liver in-

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t/d Fig. 2 Effects of 30-day administration of EEFP on food intake in rats. The data are expressed as the mean ± SD (each sex: Con-trol, EEFP-L, EEFP-M, n = 10; EEFP-H, n = 15). *P < 0.05, **P < 0.01 vs vehicle control. EEFP-L: ethanol extract of FructusPsoraleae, low dose; EEFP-M: ethanol extract of Fructus Psoraleae, middle dose; EEFP-H: ethanol extract of Fructus Psoraleae,high dose

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Fig. 3 Effects of 30-day administration of EEFP on liver tobody weight in rats. Symbols and bars represent the mean ±SEM (each sex: Control, EEFP-L, EEFP-M, n = 10; EEFP-H,n = 15), respectively. ***P < 0.001 vs vehicle control, one-wayANOVA followed by Dunnett ’s t-test. Con: vehicle control;LD: ethanol extract of Fructus Psoraleae low dose; MD: eth-anol extract of Fructus Psoraleae middle dose; HD: ethanolextract of Fructus Psoraleae high dose

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jury (Fig. 7D).Hepatic gene and protein expressions in EEFP-induced liverinjury

Western blotting was performed (Fig. 8) to evaluate theprotein expression levels of FXR, MRP3, and PPAR-α in ratsthat received EEFP for 30 d. The levels of protein expressionof FXR and PPARα decreased, whereas that of MRP3 signi-ficantly increased.

Discussion

FP, a widely used Traditional Chinese Medicine, con-tains various bioactive compounds such as coumarin, benzo-furan, flavonoids, and monoterpenes. In traditional usage, FPshould be processed to reduce toxicity. There are 40 kinds ofChinese patent medicines containing FP in the Chinese Phar-macopoeia; these medicines are more widely used in clinicalprescriptions. Although clinical application found that FP hasa certain toxic effect on the liver [13], no systematic studieshave not been conducted to date.

Traditional medicines have been historically used in thetreatment of various diseases in Asia, and research studies onthe mechanism of hepatotoxicity have increased in the pastdecade [14-16]. The liver is the principal site for detoxificationand drug metabolism. More than 80% of liver blood flowstems from the gastrointestinal tract [17], and for this reason,the liver is highly susceptible to toxicity effects. With usageincreases, liver injury reports due to herbal medicines, includ-ing Folium sennae, Polygonum multiflorum, and Rheum pal-

matum L., have been reported [18-21].Adverse drug reactions (ADRs) are one of the five main

causes of death worldwide, and hepatotoxicity is regardedas the most common type of ADRs because the liver is theleading organ associated with the clearance of toxic sub-stances [22]. Drug-induced liver damage can be classified asacute hepatitis, hepatic cells apoptosis, cholestatic, oxidativestress injury, autoimmune, steatohepatitis, cirrhosis, or hepat-ic adenoma [23].

Results of the present study, we show that the adminis-tration of EEFP causes a significant elevation in the markersof cholestatic liver injury, particularly cholestasis and serumlevels of TBIL, TBA, and GGT (Fig. 5). In addition, the ad-ministration of EEFP results in increases in bile flow and bili-ary bile acid output as well as decreases in TC and TG in ratscompared to the control group (Fig. 4). UDP-glucuronyltrans-ferase 1A1 (UGT1A1) is a key enzyme responsible for themetabolic elimination of bilirubin. FP extract and its maincomponents have an inhibitory effect on UGT1A1. Both theextract of FP and five major components of FP displayedevident inhibitory effects on human UGT1A1 in human livermicrosomes (HLM). Among these five identified naturallyoccurring inhibitors, bavachin and corylifol A were found tobe strong inhibitors of UGT1A1 with inhibition kinetic para-meters (Ki) values lower than 1 μmol·L– 1, whereas neo-bavaisoflavone, isobavachalcone, and bavachinin displayedmoderate inhibitory effects against UGT1A1 in HLM. Thesefindings suggested that FP contains natural compounds with

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Fig. 4 Effects of repeated administration of EEFP on serum bile volume and flow rate in rats for 30 days. Symbols and barsrepresent the mean ± SEM (each sex: Control, EEFP-L, EEFP-M, n = 10; EEFP-H, n = 15), respectively. *P < 0.05, **P < 0.01,***P < 0.001 vs vehicle control, one-way ANOVA followed by Dunnett’s t-test. Con: vehicle control; LD: ethanol extract of FructusPsoraleae, low dose; MD: ethanol extract of Fructus Psoraleae, middle dose; HD: ethanol extract of Fructus Psoraleae, high dose;TBA: Total bile acid; TBIL: Total bilirubin

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potent inhibitory effects against human UGT1A1 [24].The results of GO (biological function) and pathway

(KEGG) analysis showed that the differentially expressedproteins in the EEFP group were mainly manifested in PPARsignaling pathway, bile secretion, lipid metabolism, drugmetabolism, and cytochrome P450 pathway. The formation,secretion, and excretion mechanisms of bile are highly com-

plex, and cholestasis can be induced by various causes suchas the formation, secretion, and excretion dysfunction ofbile [25]. Bile secretion to form bile flow can be divided intotwo parts: the level of liver cells and the level of bile duct.Each part completes the secretion of bile through the corres-ponding transporter to form bile flow [26]. FXR is a bile sensorand the nuclear receptor that regulates gene transcription in

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Con LD MD HD

******

******

*****

FemaleMale

Fig. 5 Effects of 30-day administration of EEFP on serum biochemical in rats. Symbols and bars represent the mean ± SEM (each sex: Control, EEFP-L, EEFP-M, n = 10; EEFP-H, n = 15), respectively. *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle control,one-way ANOVA followed by Dunnett’s t-test. CON: vehicle control; LD: ethanol extract of Fructus Psoraleae, low dose; MD:ethanol extract of Fructus Psoraleae, middle dose; HD: ethanol extract of Fructus Psoraleae, high dose; ALT: alanine amino-transferase; AST: aspartate aminotransferase; ALB: albumin; TBIL: total bilirubin; TBA: total bile acid; GGT: gamma-glutamyl transferase; TC: total cholesterol; TG: triglycerides

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the liver. Bile acid is the endogenous ligand of FXR and canactivate FXR to prevent the genetic transcription of CYP7A1and activate the secretion and transport of bile acid to protectthe liver and bile duct from accumulating toxic bile acid [27].

The results of this study showed that EEFP reduced theprotein expression of FXR, which may be explained by twoplausible pathways. One is that EEFP has a direct downregu-latory effect on FXR, which disrupts the negative feedbackpathway of bile acid and thereby induces cholestasis. The

other possibility is that under the condition of cholestasis invivo, the continuous stimulation of a high bile acid level in-duces the expression of FXR in the decompensated periodand inhibits its expression, suggesting that EEFP can inhibitthe expression of FXR and disrupt the regulation of bile acidand cholesterol homeostasis. In addition, EEFP upregulatedthe protein expression of MRP3, which is compensatory andhelps hepatocytes to excrete accumulated bile acid, and re-duces its toxic damage, which may thus be the self-protect-

50 μm

50 μm

50 μm

50 μm 50 μm

50 μm

50 μm

50 μm

a b

c d

e f

g h

Fig. 6 Optical micrographs of liver pathology after 30-day of EEFP administration to rats (200 ×, H&E staining). (a) male con-trol; (b) male LD; (c) male MD; (d) male HD; (e) female control; (f) female LD; (g) female MD; (h) female HD. Hepatic steatosis(1); liver cholestasis (2); bile duct dilatation (3); inflammatory cell infiltration (4). LD: ethanol extract of Fructus Psoraleae, lowdose; MD: ethanol extract of Fructus Psoraleae, middle dose; HD: ethanol extract of Fructus Psoraleae, high dose

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Table 1 Differentially expressed proteins in the liver of EEFP-treated rats upregulated proteins

Accession Gene name Description Fold-change in expression level P value

Q6T5E9 Ugt1a6 UDP-glucuronosyltransferase 4.812 555 0.008 06

Q68G32 Ugt1a7c Ugt1a7c protein (Fragment) 2.990 373 0.000 706

G3V786 Akr1b8 Aldo-keto reductase family 1 member B10 2.246 113 0.005 038

Q80UE2 Cyp1a1 Cytochrome P450 2.234 421 0.014 059

P04903 Gsta2 Glutathione S-transferase alpha-2 1.843 301 0.000 45

A1XF83 Ugt2b UDP-glucuronosyltransferase 1.740 882 0.029 983

P04799 Cyp1a2 Cytochrome P450 1A2 1.631 754 0.004 636

P04905 Gstm1 Glutathione S-transferase Mu 1 1.621 869 0.002 423

P09875 Ugt2b1 UDP-glucuronosyltransferase 2B1 1.596 655 0.000 269

A0A0H2UHF8 Orm1 Alpha-1-acid glycoprotein 1.587 354 0.020 147

P46418 Gsta5 Glutathione S-transferase alpha-5 1.575 758 0.000 137

P51556 Dgka Diacylglycerol kinase alpha 1.556 892 0.018 852

F1LMN1 Cyp2b12 Cytochrome P450 2B12 1.549 987 0.005 736

P38918 Akr7a3 Aflatoxin B1 aldehyde reductase member 3 1.544 077 0.000 504

Q5M827 Pir Pirin 1.511 111 0.001 365

A0A0G2K038 Rexo2 Oligoribonuclease, mitochondrial 1.497 775 0.044 62

F7FBM8 Cyp2b2 Cytochrome P450 2B2 1.490 805 0.012 28

A0A0G2K1S6 Me1 Malic enzyme 1.475 177 0.000 351

A0A0G2JTB1 Gsta3 Glutathione S-transferase 1.471 575 0.025 015

F7EQ04 RGD1312005 RCG46944, isoform CRA_c 1.449 047 0.047 452

D4A7F6 Colec10Collectin sub-family member 10 (C-type lectin)(Predicted)

1.436 527 0.029 599

P07687 Ephx1 Epoxide hydrolase 1 1.432 701 0.017 912

Q68FT6 Rhbg Ammonium transporter Rh type B 1.421 997 0.00009

Q9QYK2 Ppargc1aPeroxisome proliferator-activated receptor gammacoactivator 1-alpha

1.421 121 0.001 636

D4A1W7 Nphp3 Nephrocystin 3 1.420 455 0.003 016

P02634 S100g Protein S100-G 1.410 502 0.014 719

A0A0G2K1W9 Ldhd Lactate dehydrogenase D 1.401 123 0.011 625

Q4KLZ0 Vnn1 Vanin 1 1.397 59 0.017 971

P23606 Tgm1 Protein-glutamine gamma -glutamyltransferase K 1.390 967 0.011 612

F1LZB7 Frmd4b FERM domain-containing 4B 1.387 497 0.014 024

F1LM03 LOC100361492Cytochrome P450, family 2, subfamily c, polypeptide55-like

1.365 584 0.005 598

B6DYQ2 Gstm2 Glutathione S-transferase 1.359 114 0.001 413

Q32KK2 Arsa Arylsulfatase A 1.352 369 0.000 228

A9UMW1 Gsta4 Glutathione S-transferase alpha 4 1.348 123 0.044 072

Q7TP14 Chid1 Cc1-9 1.340 409 0.002 317

Q6IRJ7 Anxa7 Annexin 1.334 956 0.000 3

D3ZMK3 Uncharacterized protein 1.333 777 0.037 39

Q6TA48 Mptx1 Mucosal pentraxin 1.329 087 0.036 601

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Continued Accession Gene name Description Fold-change in expression level P value

Q66H59 Npl N-acetylneuraminate lyase 1.316 677 0.021 603

P16573 Ceacam1Carcinoembryonic antigen-related cell adhesionmolecule 1

1.308 98 0.008 965

Q5XIE6 Hibch 3-hydroxyisobutyryl-CoA hydrolase, mitochondrial 1.308 133 0.000 804

Q02759 Alox15 Arachidonate 15-lipoxygenase 1.303 048 0.043 524

Q9QYW3 Mob4 MOB-like protein phocein 1.300 021 0.010 161

D3ZM21 Comtd1Catechol-O-methyltransferase domain containing 1(Predicted), isoform CRA_a

1.291 103 0.009 749

B5DEM7 Supt4h1 Transcription elongation factor SPT4 1.282 338 0.040 189

G3V7A7 RGD1306941 Similar to CG31122-PA 1.275 528 0.042 835

P51647 Aldh1a1 Retinal dehydrogenase 1 1.271 456 0.022 019

P14668 Anxa5 Annexin A5 1.268 728 0.001 056

P05178 Cyp2c6 Cytochrome P450 2C6 1.267 228 0.002 719

Q05175 Basp1 Brain acid soluble protein 1 1.265 322 0.024 86

G3V7Q6 Psmb5 Proteasome subunit beta type 1.264 775 0.000 92

D3ZF64 Abcc3 Canalicular multispecific organic anion transporter 2 1.257 547 0.001 198

P05545 Serpina3k Serine protease inhibitor A3K 1.256 338 0.001 201

P02767 Ttr Transthyretin 1.254 056 0.007 246

B5DF65 Blvrb Biliverdin reductase B 1.248 34 0.001 122

F1LRJ9 Selenbp1 Selenium-binding protein 1 1.247 634 0.006 474

P32822 Trypsin V-B 1.247 173 0.041 616

Q4QQV0 Tubb6 Tubulin beta chain 1.246 689 0.009 16

P12346 Tf Serotransferrin 1.242 389 0.016 01

F1LX91 Rgs22 Regulator of G-protein-signaling 22 1.242 114 0.005 042

A0A0G2JSV2 Cbr1 Carbonyl reductase [NADPH] 1 1.237 327 0.001 541

Q9Z2Y0 Keg1 Glycine N-acyltransferase-like protein Keg1 1.237 068 0.009 498

G3V6H9 Nap1l1 Nucleosome assembly protein 1-like 1 1.235 821 0.003 149

P05370 G6pdx Glucose-6-phosphate 1-dehydrogenase 1.235 409 0.012 828

P16296 F9 Coagulation factor IX 1.229 005 0.011 551

Q64721 apo Apolipoprotein BL (Fragment) 1.227 507 0.044 588

P33274 Cyp4f1 Cytochrome P450 4F1 1.226 798 0.022 356

P05544 Serpina3l Serine protease inhibitor A3L 1.223 661 0.000 565

D3ZNK1 Mtx3 Metaxin 3 1.223 592 0.002 705

P70580 Pgrmc1Membrane-associated progesteronereceptor component 1

1.223 561 0.021 214

Q920A6 Scpep1 Retinoid-inducible serine carboxypeptidase 1.221 45 0.020 951

F1LPB8 Entpd5 Ectonucleoside triphosphate diphosphohydrolase 5 1.220 917 0.022 562

D3ZXL1 Arih1 Ariadne RBR E3 ubiquitin protein ligase 1 1.220 784 0.002 611

B2RZD7 Lyrm9 LYR motif-containing protein 9 1.216 786 0.003 167

A0A0G2JX01 Ttc7a Tetratricopeptide repeat domain 7A 1.214 126 0.020 62

A0A0G2JT90 Ash2lASH2-like histone lysine methyltransferasecomplex subunit

1.212 698 0.019 341

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Continued Accession Gene name Description Fold-change in expression level P value

Q62862 Map2k5 Dual specificity mitogen-activated protein kinase kinase 5 1.209 74 0.036 569

Q811X6 Cryl1 Lambda-crystallin homolog 1.208 286 0.039 668

Q4V882 Epn3 Epsin-3 1.206 813 0.047 94

D3ZLR6 Ugt2b15 UDP-glucuronosyltransferase 1.204 465 0.002 089

D3ZA85 Nfu1Histone cell cycle regulation defective interactingprotein 5 (Predicted), isoform CRA_a

1.200 55 0.020 028

Table 2 Differentially expressed proteins in the liver of EEFP-treated rats downregulated proteins

Accession Gene name Description Fold-change in expression level P value

M3ZCQ0 Sult2a1 Sulfotransferase 0.830 221 0.014 809

P20761 Igh-1a Ig gamma-2B chain C region 0.829 261 0.002 254

Q6AYT9 Acsm5 Acyl-coenzyme A synthetase ACSM5, mitochondrial 0.827 363 0.047 665

Q01129 Dcn Decorin 0.825 623 0.027 595

F1LZW6 Slc25a13 Solute carrier family 25 member 13 0.823 126 0.002 765

P70552 Gchfr GTP cyclohydrolase 1 feedback regulatory protein 0.822 558 0.012 763

P21213 Hal Histidine ammonia-lyase 0.820 343 0.001 976

O35913 Slco1a4 Solute carrier organic anion transporter family member 1A4 0.810 338 0.002 554

Q4G068 Tmem176a Transmembrane protein 176A 0.808 419 0.004 619

P02692 Fabp1 Fatty acid-binding protein, liver 0.806 635 0.006 235

P08009 Gstm3 Glutathione S-transferase Yb-3 0.806 098 0.009 796

Q6AYI2 Klhdc3 Kelch domain-containing protein 3 0.804 882 0.010 698

D3ZCF8 Abca8a ATP-binding cassette, subfamily A (ABC1), member 8a 0.799 598 0.009 523

O88867 Kmo Kynurenine 3-monooxygenase 0.797 567 0.001 145

P43278 H1f0 Histone H1.0 0.796 362 0.013 217

G3V709 Naprt Nicotinate phosphoribosyltransferase 0.792 915 0.006 538

A0A0H2UHH2 Apcs Serum amyloid P-component 0.792 533 0.002 968

F1M7E5 Cdh5 Cadherin 5 0.792 524 0.010 454

P28492 Gls2 Glutaminase liver isoform, mitochondrial 0.787 029 0.042 124

A0A0G2JTN8 Atp5g2 ATP synthase F (0) complex subunit C2, mitochondrial 0.775 768 0.017 58

P04041 Gpx1 Glutathione peroxidase 1 0.77 0.000 889

P24008 Srd5a1 3-oxo-5-alpha-steroid 4-dehydrogenase 1 0.751 403 0.011 479

Q4PLF2 CAT2 Cationic amino acid transporter-2 0.737 594 0.024 572

M0RDI0 Cyp3a9 Cytochrome P450 3A9 0.736 78 0.013 772

D4A5Q9 Gldc Glycine decarboxylase 0.718 417 0.003 484

A0A0G2KAH4 Dock6 Dedicator of cytokinesis 6 0.710 405 0.045 226

Q5FVS1 Fth1 Ferritin (Fragment) 0.704 778 0.035 724

Q4QR86 Depdc7 DEP domain-containing protein 7 0.674 242 0.011

A0A0G2K5J0 Sult1c3 Sulfotransferase 0.657 967 0.020 476

G3V7I5 Aldh1b1 Aldehyde dehydrogenase X, mitochondrial 0.599 38 0.000 594

B4F7E5 Atp2a1 Calcium-transporting ATPase 0.462 07 0.046 948

G3V7J5 Ces2e Carboxylic ester hydrolase 0.431 894 0.000 24

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ive response of hepatocytes [28-29].Many studies have reported that some drug-induced liv-

er injury can also lead to the occurrence and development ofdiseases by affecting the function of MDR3, such as ticlopid-ine, itraconazole, ketoconazole, verapamil, ritonavir, andchlorpromazine [30-32]. These reports suggest that drug-in-duced impairment of MDR3 function is an important target ofdrug-induced cholestasis. This type of drug-induced cho-lestasis has the following clinical features: serum levels ofALP, GGT, and TBIL are significantly increased, and thesecharacteristics are almost consistent with the results of EEFP-induced hepatotoxicity.

Our research results showed that protein expression ofPPARα in hepatocytes was inhibited by the administration ofEEFP. PPARα is a ligand-activated transcription factor that ishighly expressed in the liver and can be activated by fattyacids and various lipids as well as peroxidase proliferators [33].PPARα is a major regulator of lipid metabolism in the liverand suppresses inflammation and acute phase reactions. ThemRNA expression of PPAR in human and mouse liver is sim-ilar, and the expression of PPAR in liver is lower in patientswith various liver injuries. PPARα can effectively inducemany lipid metabolic pathways involved in the expression ofseveral genes, including microsomes, peroxidase and mito-

B2 B1 C1 C2B3

1.5

1.0

0.5

0

−0.5−1.0−1.5

4

3

2

1

0

0 2 4−2−4

−log

10 P

val

ue

log2 fold change

Volcano plot

0 10 20 30 40

Percentage of sequences (%)

Monocarboxylic acid metabolic processXenobiotic metabolic process

Cellular response to xenobiotic stimulusResponse to xenobiotic stimulus

Iron ion bindingIipid metabolic process

Cellular hormone metabolic processGlutathione metabolic process

Hormone metabolic processObsolete glutathione conjugation teaction

Glutathione transferase activityAndrogen metabolic processUronic acid ,etabolic process

reduced flavin or flavoprotein as one donor,and incorporation of one atom of oxygen

Glucuronate metabolic processSteroid metabolic process

Carboxylic acid metabolic processMonooxygenase activity

Arachidonic acid metabolic process

Glutathione derivative metabolic processGlutathione derivative biosynthetic process

Glucuronosy ltransferase activityCellular glucuronidationDrug metabolic process

Retinoic acid bindingOxoacid metabolic process

Organic acid metabolic processIcosanoid metabolic process

Fatty acid derivative metabolic processTransferase acticity, transferring

alkylor aryl(other than methyl) groups

Enric

hed

GO

term

s (To

p 30

)

Difference setReference set

A

C

B

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chondrial fatty acid oxidation, fatty acid activation, fattyacids and desaturation, triglyceride synthesis and decomposi-tion, Lipoprotein metabolism, gluconeogenesis, and bile acidmetabolism, among others. PPARα not only regulates dyslip-idemia, but its agonists have also been used in the treatmentof cholestasis [34], nonalcoholic fatty liver disease and/or type2 diabetes. PPARα binds with peroxisome proliferator re-sponse elements (PPREs) in gene promoters of 4-hydroxynonene aldehyde (4-HNE) metabolism of fatty aldehyde de-hydrogenase key enzymes (FALDH) [35]. The expression ofFALDH was upregulated to accelerate the scavenging of 4-HNE [36]. PPAR inhibition can accelerate lipid peroxidationand further aggravate liver injury.

The adipocytokine signaling pathway is one of the differ-entially expressed proteins that is related to the PPAR signal-ing pathway. PPAR is primarily expressed in tissues withhigh levels of fatty acid catabolism such as the liver, heart,and muscles. PPAR-α regulates the expression of many ma-jor genes for lipid and lipoprotein metabolism. In the liver,PPAR-α regulates fatty acid oxidation by directly promotingthe expression of multiple genes that control lipid metabol-

ism [37]. PPAR-α regulates the activity of all three fatty acidoxidation systems, i.e., the mitochondria, peroxisomal oxida-tion and microsomal oxidation [38]. FXR is the primary sensorof intracellular bile acids and controls bile acid synthesis andtransport. With the regulation of bile acid metabolism, FXRis also directly and indirectly involved in several importantmetabolic pathways in the body such as the regulation ofglucose and lipid metabolism. Therefore, the activation or in-hibition of FXR can have a significant impact on metabolichomeostasis [39].

In addition, genetic variants of FXR are associated withmetabolic diseases, including intrahepatic cholestasis duringpregnancy [40] and cholesterol cholelithiasis [41]. FXR ligandshave been proposed for the treatment of metabolic diseasessuch as cholestasis [42], liver fibrosis [43], type 2 diabetes [44],atherosclerosis [45] and erectile dysfunction [46]. Thus, EEFPcan induce the disruption of bile acid and lipid metabolism,resulting in cholestatic liver injury. FXR, PPAR, andCYP7A1 are closely related to the occurrence and develop-ment of hepatic metabolic dysfunction and cholestasis causedby EEFP.

Hepatocellular carccinoma

Metabolism of xenobiotics by cytochrome P450

Glycerolipid metabolism

Linoleic acid metabolism

Retinol metabolism

Fluid shear stress and atherosclerosis

Metabolic pathways

Ascorbate and aldarate metabolism

Bile secretion

Tryptophan metabolism

Steroid hormone biosynthesis

Pentose and glucuronate interconversions

Arachisonic acid metabolism

Platinum drug resistance

Ferroptosis

Drug metabolism-cytochrome P450

Drug metabolism-other enzymes

Glutathione metabolism

Porphyrin and chlorophyll metabolism

Chemical carcinogenesis

Mineral absorption

Enri

ched

KE

GG

pat

hw

ays

0 10 20 30 40

Percentage of sequences (%)

Difference setReference set

D

Fig. 7 Proteomics analysis of EEFP-treated rats. (A) Heat map of the significantly altered proteins in the control and EEFPgroups. Red indicates upregulation and green represents downregulation in protein expression. The proteins were clustered intofour broad clusters. (B) Volcano plots of the proteins quantified during iTRAQ analysis comparisons. Each point represents thedifference in expression (fold-change) between the two groups of mice plotted against the level of statistical significance. Dottedhorizontal lines represent differential expression differences, and the dotted vertical line represents a significance level of P <0.05. (C) Enriched Gene Ontology term statistics of differentially expressed proteins between the EEFP and control groups. GOenrichment analysis using biological processes indicated that proteins were significantly related to the monocarboxylic acid, lipid,carboxylic acid, oxoacid, and organic acid metabolic processes. (D): Enriched KEGG pathway (C: control group; B: EEFPgroup). KEGG pathway analysis indicated that the categories of metabolism of xenobiotics by cytochrome P450, metabolic path-ways, drug metabolism-cytochrome P450, retinol metabolism, steroid hormone biosynthesis, glutathione metabolism, glycerol-ipid metabolism, and bile secretion were mainly involved in the response to EEFP-induced liver injury.

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Acknowledgements

This work was supported by the the National Key Re-search and Development Program, Specialized Research onModernization of TCM (Nos. 2018YFC1708006 and2018YFC1708003), National Major New Drug Creation Spe-cial Project “National Drug New Varieties Research and De-velopment and Its Key Innovative Technology Topics” (No.2017ZX09301060-005), the National Natural Science Found-ation of China (No. 81320108029); Tianshan Cedar Plan (No.2018XS21), National “Major Scientific and TechnologicalSpecial Project for Significant New Drugs Creation” Project(No. 2015ZX09501004-002-004), and Specific Fund for Pub-

lic Interest Research of Traditional Chinese Medicine, Min-istry of Finance of China (No. 201507004-002).

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1.5

1.0

0.5

0

Rel

ativ

e pro

tein

lev

el

Con EEFP-L EEFP-H

*

FXR

2.5

2.0

1.5

1.0

0.5

0

Rel

ativ

e pro

tein

lev

el

Con EEFP-L EEFP-H

***

*

MRP3

1.5

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*

PPAR-α

Fig. 8 Western blotting analysis of liver samples from dif-ferent rat groups. Symbols and bars represent the mean ±SEM (n = 3), respectively. *P < 0.05, ***P < 0.001 vs vehiclecontrol. EEFP-L: ethanol extract of Fructus Psoraleae, lowdose; EEFP-M: ethanol extract of Fructus Psoraleae, middledose; EEFP-H: ethanol extract of Fructus Psoraleae, highdose; PPAR-α: peroxisome proliferators-activated receptoralpha; FXR: farnesoid X receptor; MRP3: multidrug resist-ance-associated protein 3

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Cite this article as: LI Zhi-Jian, Abudumijiti Abulizi, XU Deng-Qiu, Youlidouzi Maimaiti, Silafu Aibai, JIANG Zhen-Zhou,ZHAO Guo-Lin, WANG Tao, Aiximujiang Refukati, Zulikaer Maimaiti, Yiliyaer Simayi, CAO Chun-Yu, ZHANG Lu-Yong.Quantitative proteomics analysis of Fructus Psoraleae-induced hepatotoxicity in rats [J]. Chin J Nat Med, 2020, 18(2): 123-137.

LI Zhi-Jian, et al. / Chin J Nat Med, 2020, 18(2): 123–137

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