Jin-Liang Du1,2,Li-Ping Cao1,2,Rui Jia3, Guo-Jun Yin1,2,3*

THEPROTECTION OF GLYCYRRHIZA POLYSACCHARIDE ON 2,3,7,8–TETRACHLORODIBENZO-P-DIOXIN INDUCED JIAN CARP（CYPRINUS CARPIO VAR.JIAN）LIVER INJURYUSINGPRECISION-CUT LIVER SLICES

Jin-Liang Du1,2,Li-Ping Cao1,2,Rui Jia3, Guo-Jun Yin1,2,3*

1.Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences，Wuxi 214081, China;

2.International Joint Research Laboratory for Fish Immunopharmacology, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China;

3.Wuxi FisheriesCollege, Nanjing Agricultural University, Wuxi 214081, China

ABSTRACTExposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) represents a potential health risk and hepatotoxicity. The aim of this study wastoevaluate the protective effects of Glycyrrhiza polysaccharide (GPS) against2,3,7,8–tetrachlorodibenzo-p-dioxin (TCDD)-induced hepatotoxicity in Jian carp using Precision-Cut Liver Slices (PCLS).In this study, PCLS were divided into six groups, five treatment groups (pre-treatment group, post-treatment group, pre and post-treatment group, TCDD group, GPS control group), and one blank control group, GPS (0.2, 0.4 and 0.8 mgmL-1) was added to the PCLS before (pretreatment), after (post-treatment) and both before and after (pre- and post-treatment) the incubation of the PCLS with TCDD at 0.3μgL-1 in the culture medium. The results showed, after PCLS treated with GPS, it was indicated that pre- and post-treatment group had the best effect on relieving TCDD damage, it could significantly inhibited the elevation of glutamate pyruvatetransaminase (GPT), glutamate oxalate transaminase (GOT), lactate dehydrogenase (LDH), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and immunoglobulin M(IgM), and significantly increased the level of superoxide dismutase (SOD) and glutathione S-transferase(GST). GPS can also reduce the expression of AhR2, ARNT2, CYP1A mRNA in different degrees. The present findings might provide new insight into the development of therapeutic and preventive approaches of TCDD toxicity.

KEYWORDS:2,3,7,8-tetrachlorodibenzo-p-dioxin,Precision-Cut Liver Slices,Glycyrrhiza polysaccharide,Aryl hydrocarbon (AhR2), Aryl hydrocarbon receptor nuclear translocator(ARNT2),Cytochrome P4501A (CYP1A)

1.INTRODUCTION

In recent years, the toxicity mechanism of dioxins hasbecomeahotresearch topic.Dioxins areorganic pollutants whichcan exist insoil,airand water, and originatemainly from industrialgarbageandwaste incineration[1].It usually enriches in adipose tissue, and can be gradually amplified by the food chains.It seriously interferes with the normal function of the endocrine system of the organism and displays a wide spectrum of toxiceffects, including dermal toxicity, reproductive toxicity, immunotoxicity, and hepatotoxicity[2, 3].Previousexperiments have shown that many chemicals, such asmicrocystin, dioxin, and lipopolysaccharide (LPS) can causeliver damage[4].The current research on TCDD mainly relates to the environment and mammals.In humans, short-term exposure to highlevels of TCDD often presents as liver damage and chloracne,while low-dose long-term exposure has been linked to immune deficiency, diabetes, and various cancer type[5-8]. Only less reports about the fish, such asthe report about the effects of 2,3,7,8–tetrachlorodi benzo-p-dioxin on the growth of fish[9].

Typically, TCDD is biotransformation in the liver, so liver is the main storage site and target organ.The liver has long been thought to play the major role indrug metabolism, first pass metabolism was almost exclusively attributed to the liver,the metabolic capacity of the intestine has beenincreasingly recognized. Nowadays, PCLS is widely used in pharmaceuticaldrug discoveryand development as an approach for toxicological screening of new drugs[10-12],although the procedure of PCLS was easy adapted to many species, this technology has rarely been used in fish, especially the Jian Carp.

At present, for TCDD therapy, some researchers found that astaxanthin prevented the suppression of antioxidant enzymes in the livers of animals exposed to TCDD[13].Some researchers also studied L-glutamine (Gln) in alleviating the toxicity of TCDD in liver of rats[14]. There are no reports about used Chinese herbal medicine to treat and prevent TCDD toxicity.Chinese medicinal herbs have been widely used in animal disease,Glycyrrhiza was one of the commontraditional Chinese herbal medicine, Glycyrrhiza polysaccharide(GPS)was the active polysaccharides extracted from Glycyrrhiza, GPS mainly consists of rhamnose, glucose, arabinose, galactose, which could adjust the immunity, anti-tumor, anti -virus, antioxidant and so on.In the latest research,GPS has good scavenging effect on DPPH, OH-, O2-.[15]. GPS, one of the major active components, can bolster the body’s ability to fight disease by activating proteins in the immune system[16]. Although numerous studies have reported itsbeneficial effects in mammals, there is a lack of reports on its function in aquaticanimals.

In this study, a liver damage model of Cyprinuscarpio var. Jian (Jian carp) was establishedby TCDDinduction, and the effects of GPS on changes in the liver indices, liver-associated enzymes, and the expression of AhR2, ARNT2, CYP1A mRNA in the liverwereinvestigated.

2. MATERIALS AND METHODS

2.1.Chemicals

L-15 medium, dimethylsulfoxide (DMSO),2,2-diphenyl-1-picrylhydrazyl (DPPH),EDTA,Dulbecco’s phosphate-buffered saline (D-PBS), gentamicin sulfate, insulin, streptomycin/penicillin and heparin were purchased from SigmaCompany (Sigma-Aldrich Co. LLC., santaclara,CA, USA). Fetal bovine serum(FBS) and cell culture plates were ordered from GibcoCompany (GibcoCompany,Carlsbad, CA,USA). TNF-α, IL-1β, IgMwerepurchased from Shanghai Zhaorui bio-tech Co., Ltd (Shanghai Zhaorui bio-tech Co., Ltd, Shanghai, CN). TCDDwas the product of Toronto Research Chemicals (Toronto Research Chemicals,Toronto, Ontario，CAN) (CAS1746-01-6,purity 98%).Glycyrrhiza polysaccharide(GPS)was purchased from Nantong universal plantextract co., Ltd (Nantong universal plantextract co., Ltd.,Nantong, CN).All other reagentsused in the experiment were of analytical grade.

2.2.Fish

Jian carp were obtained from the Freshwater Fish Research Center of Chinese Academy of Fishery Sciences, Wuxi, China. Fish were reared at 26℃in a recirculation system and fed adlibitum twice a day with diets containing 40% crude protein, 10%crude lipid, 10% ash and an energy content of 21 kJ g-1DM.

2.3.Preparation and culture of slices

In sterile conditions, the livers were immediately excised and placed in cold L-15 medium containing with 25 mM D-glucose, 50μgml-1gentamicin, and 2.5 mgmL-1fungizone previously gassed with 95% O2 , 5% CO2 by basic method, the liver was cut into pieces of 5 mm × 5 mm × 5 mm and embedded in a low melting point agarose. The slices were prepared using a ZQP-86 tissue slicer (Zhejiang JinhuaKedi Instrumental Equipment Co.,Ltd., Jinhua, CN) and cultured in 24-well plate in oxygenated L- 15 at 27℃.

(1)Thickness

The slices are fixed to 200, 300, 400and 500 μm, respectively. Different thickness of slices were cultured in 24-well plate for 0, 8, 16, 24 and 48 hours at 27℃. Then the biochemical indices were measured.

⑵PH

The slices of 300μm thickness were incubated in L-15 medium with different pH (6.8, 7.0 and 7.2) for 0, 8, 16, 24 and 48 hours at 27 ℃. Then the biochemical indices were measured.

⑶Viability assay

PCLS viability was estimated by adenosine triphosphate (ATP) content. The viability of the tissue slices were measured via ATP determination using the ATP Determination Kit(Nanjing Jiancheng Bioengineering Institute,Nanjing, CN). The results are expressed as µmol ATPg protein-1.

2.4.DPPH assay

To determine the radical scavenging activity of GPS, DPPH assay was conducted according to the methods described previously. Different methanolic dilutions of extract were prepared (0.05- 0.35 mgmL-1). Briefly, 2.0 mL GPSextract was added to 2 mL DPPH solution (90 μM in methanol) as the free radicalsource. The mixtures were shaken vigorously and allowed to stand at roomtemperature for 30 min. The decrease of solution absorbance due to proton donatingactivity of components of each extract was determined at 519 nm. Lower absorbanceof the reaction mixture indicates higher free radical scavenging activity. The DPPHwith corresponding solvents (without plant material) serves as control. The DPPHradical scavenging activity was calculated using the following formula: DPPH radicalscavenging activity (%) = [(A0 – A1 /A0) ×100], where A0 is the absorbance of the control, and A1 is the absorbance of extract or standard sample.

2.5.Treatments ofPrecision-Cut Liver Slices with GPS

The hepatoprotective effect of GPS was investigatedusing an in vitro model of TCDD-induced PCLS injury.Six groups were set up asfollows:

⑴Control: slices were cultured in L-15 for 3 h.

⑵GPS control: slices were cultured withGPS at 0.8 mgmL-1 in L-15 for 3 h.

⑶TCDDcontrol: slices were cultured with0.3μgL-1 in L-15 for 3 h.

⑷Pre-treatment group:The PCLS were firstly incubatedwith 0.2, 0.4, 0.8 mgmL-1 of GPSfor 3 h, then washed and incubatedwith TCDDat the concentration of 0.3μg/L for another 3 h.

⑸Post treatment group:The PCLS wereincubatedwith TCDDat the concentration of 0.3μgL-1 for3 h,then washed and incubated with GPSat the concentrations of 0.2, 0.4, 0.8 mgmL-1 foranother 3h.

⑹Pre and posttreatment group:The PCLS were first incubated with 0.2, 0.4, 0.8 mgmL-1 of GPSfor 3 h, and then, TCDD was added at a finalconcentration of 0.3μgL-1; with 3 h incubation of TCDD, the PCLS were further treated with GPS at the concentrations of 0.2, 0.4, 0.8 mgmL-1 foranother 3h.

For each set of conditions, six experiments wereperformed. At the end of experiment, the supernatantsand slices from each individual well were collected and stored at - 20 ℃.

2.6.Biochemical indexassays

GPT, GOT,TNF-α, IL-1β, IgM in the culture supernatants and LDH, ATP, MDA, SOD,GST, in the liver slice homogenates were measured usingkits from Nanjing Jiancheng Bioengineering Institute(Nanjing Jiancheng Bioengineering Institute, Nanjing, CN). Protein content in the liver slice homogenate was determined using BCA test kit. GPT, GOT were expressed as U L-1, SOD, GSH-ST were expressed as Umg-1 protein,TNF-α, IL-1βwere expressed as ngL-1,IgMwas expressed asngmL-1, LDH was expressed as Ug-1 protein, ATP were expressed as μmolg-1 protein, and MDA was expressed as nmolmg-1protein.

2.7.Western blotting assay

The levels of CYP1A protein were determined byimmunoblotting. Protein samples were boiled for 5 min in loading buffer (BeyotimeInstitute of Biotechnology, Haimen, CN), loaded onto a 12% SDS polyacrylamide gel, separated electrophoretically, and transferred to a cellulose acetate membrane. The cellulose acetate membrane was blocked with sealing fluid and the proteins were probed with a mouse anti-fish CYP1A monoclonal antibody (Biosense, Thane West, India; 1:3000dilution). The β-actin protein was used as the internal control. Alkaline phosphatase-labeled anti-mouse IgG antibody (Beyotime, Haimen, China; 1:3000 dilution) was used as the secondary antibody in the staining reaction. The amount of immunoreactive product in each lane was determined using Quantity One software (BioRad Laboratories, Hercules, CA, USA).

2.8.Gene expression analysis of CYP1A, AhR2,ARNT2

Total RNA was extracted from fish liver using a fast pure RNA kit (TaKaRa BiotechnologyCo., Ltd., Dalian, CN), according to the manufacturer’s instructions. The RNA quality was detected by gel electrophoresis, the RNA concentration was determined by GeneQuant 1300 (GE Healthcare Biosciences, Piscataway, NJ, USA), and normalized to a common concentration with DEPC treated water (Invitrogen,Carlsbad, CA, USA) before proceeding to cDNA synthesis. The procedure for reverse transcription was carried out according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA), and the products (cDNA) were then stored at -20℃for qRT-PCR.

Real-time quantitative PCR (qRT-PCR) was performed to detect the gene expression of AhR, ARNT, and CYP1A in fish liver using SYBR Premix Ex Taq (TaKaRa BiotechnologyCo., Ltd., Dalian, CN), and the reaction was performed on an ABI PRISM 7500 Detection System (Applied Biosystems,Foster city, CA, USA). The program was set to run for one cycle at 95℃for 30 s, 33 cycles at 95℃for 5 s and at 60℃for 34 s. The primers used in this study are listed in Table l. The specificity of PCR amplification was confirmed by agarose gel electrophoresis and melting curve analysis. The gene expression results were analyzed using the 2-∆∆Ct method.

2.9.Statistical analysis

The data were analyzed with one-way ANOVA using theSPSS 17.0 software and expressed as means ± standard deviation of the means.*P <0.05 and **P <0.01 were taken as statistically significance.

3. RESULTS

3.1.Effects of different cultured conditions on the biochemical indices

The level of ATP in liver slices of different thickness at various time points are presented in Figure 1a. The results showed that there was no detectable depletion of ATP in slices at 300μm and 500μmfor 16 hoursculture, indicating that the slices at 300μm and 500μm can keep normal energy metabolism with no significant change for up to 16 hours.

As shown in Figure 1(b, c, d), there were no significant increase in both GOT and GPT leakage from the liver slices at 300μm and 400μm after cultured 16 hours, and LDH leakage from slices at 300μm showed significant increase after cultured for 24 hours, indicating that the slices at 300μm can remain stable for more than 16 hours.

The level of ATP in liver slices at 300μm for different time is presented in Figure 2a. There was a remarked depletion of ATP in slices at pH7.2 after cultured for 16 hours, however, no detectable depletion of ATP in slices at pH6.8, 7.0 after cultured for 16 hours was observed,demonstrating that the slices can keep stableenergy metabolism for up to 16 hours.

The leakages of GOT, GPT and LDH in liver slices at each cultured time are presented in Figure2 (b, c, d).There were no significant increase in GOT leakage from the liver slices at pH6.8 and 7.0 after cultured 16 hours, but LDH and GPT leakage from slices at pH6.8 showed significant increase after cultured for 16 hours, indicating that the slices at pH7 can remain stable for more than 16 hours.

3.2.Radical scavenging activity of GPS

In order to verify the antioxidant power of GPS and its free radical scavenging potency, the DPPH assay was performed. The results showed that GPS even at lower concentration exerted a potent radical scavenging activity(Figure 3).

3.3.Effects of GPS on GOT, GPT andLDHactivities

As shown inFigure 4, cultured PCLS treated with TCDD,the activities of GOT, GPT, LDHin PCLSculture mediumincreasedsignificantly(P<0.01).When PCLS were treated with GPS at 3 different concentrations, pre-treatment, post-treatment and pre- andpost-treatment of the PCLS significantly reduced theincreasing activities of GPT, GOT and LDH induced by TCDD.

3.4.Effects of GPS on hepatic antioxidant enzyme activities

As shown inFigure 5,compared with the control group, the SODactivityandamount ofGSTreducedsignificantly when PCLS were treated with TCDD alone(P<0.01). Pre-treatmentwith GPS at 0.2, 0.8mgmL-1significantly increasedSODactivity,post-treatmentwith GPS at 0.4, 0.8mgmL-1significantly increasedSODactivityandamount ofGST,pre- andpost-treatment with GPS at 0.4mgmL-1significantly increasedSODactivityandamount ofGST.

3.5.Effects of GPS on lipid peroxidation

As shown in Figure 6, cultured PCLS treated with TCDD aloneshowed a threefold increase in the amount of MDAreleased into the medium(P<0.01).The inhibited MDA formation was significantly inhibited inpre-treatment,post-treatment and pre- and post-treatment.

3.6.Effects of GPS on TNF-α, IL-1β, IgM amounts

As shown in Figure 7, cultured PCLS treated with TCDD, the amounts of TNF-α,IL-1β,IgMinPCLShad an increased trend(P<0.01or P<0.05).In 3 treatment groups, GPS at the concentration of 0.2, 0.4 and 0.8mgmL-1 could obviously decrease the amounts of TNF-α,IL-1β,IgM todifferent degrees(P<0.01or P<0.05).

3.7.Effects of GPS on CYP1A protein expression

As shown inFigure 8,the expression of CYP1A was low in control group and GPS control group, aftertreated with TCDD, the expression of CYP1Ain PCLS increased; when precision cut liver slices were treated with 3 different concentrations of GPS, in pre-treatment group,with the concentration increased, the protein expression of CYP1A decreased，these results showed that GPS had some effect on anti liver damage which induced by TCDD.In post-treatment, GPS at the concentration of 0.2, 0.8 mgmL-1could decrease the protein expression of CYP1A; and in pre- and post-treatment,GPS at the concentrationof 0.2, 0.4 and 0.8mgmL-1 could decrease theprotein expression of CYP1A.

3.8.Effects of GPS on CYP1A , AhR2 and ARNT2 gene expression

As shown inFigure 9,AhR2,ARNT2,CYP1AmRNA expression increasedsignificantly in TCDD treatment group compared to the normal control.When PCLS were treated with 3 different concentrations of GPS,in pre-treatment group, GPS at the concentration of 0.8mgmL-1 had the best effect on decreasing the expression of CYP1A mRNA;inpost-treatment group,GPS at the concentration of 0.4, 0.8mgmL-1 had the best effect on decreasing the expressions of AhR2,ARNT2,CYP1A mRNA;inpre- andpost-treatment group,GPS at allthe three concentrationssignificantly decreased the expression of AhR2,ARNT2,CYP1A mRNA to different degrees.

4.DISCUSSION

In the past decades, the development of in vitro models to study the toxicity of manmade chemicals in fish species often used hepatocytes and hepatoma cell lines [17]. But these methods have limited ability to research the in vivo toxicity,they couldn’taccurately reflect theeffects of drugsorpoison on fish liver.In this study,PCLS were used to screendrugsfor relieving TCDD induced liver injury in Jian carp for the first time.PCLS have been confirmed by microarrays that PCLS more closely predicted in vivo toxicity than isolated hepatocytes or established cell lines[18].The best preparation and cultivation condition of liver slices is the key to the success of this experiment, so we set different thickness and medium pH to evaluate the slices viability and measured different biochemical indices to analyze the optimal slice thickness and medium pH.The results showed that the slices at 300μm in the pH 7 mediumkept good bioactivity for more than 16 hours.

About the toxicity mechanism of TCDD, the study mainly focus on oxidative stress.Oxidative stress is an important mechanism of chemical toxicity including TCDD, CCl4, ethanol. Normally, the antioxidant system made production and elimination of activated oxygen in a balanceable and dynamic state, it could prevent excessive ROS caused oxidative stress,kept the body balance, and maintained the normal structure and function of cells.when the bodymetabolismimbalance,it will causeoxidative damage tothe body.

GOT, GPT, LDH are the most sensitive indicator of liver diseases, theirvalues also reflect the degree of injury[19]. In this experiment, GOT, GPT, LDH activitiesin model group were significantly higher than those in control group, after treated with 3 different concentrations of GPS, GPS at all the three concentrations significantly reduced the activities of GOT, GPT, LDH (P<0.01). Our results were consistent with the report fromCao[20]thatGPS could markedly inhibit the levels of GOT, GPT and LDH induced by t-BHP.

Lipid peroxidationwas an important parameter ofoxidative stress, and it usually was reflected by levelsof MDA [21]. MDA usually combined with SOD or GST to reflect the degree of injury of oxygen free radical on the cells. The antioxidant enzymes (SOD, GST) play an important rolein defending the cells against free radical-mediated oxidative damage[22],SOD is one of the enzymes on free radical scavenging, inhibition of free radical reaction in vivo, it can prevent xanthinedehydrogenase into xanthineoxidase, reduce the production of free radicals, protect liver cells. When oxidative damage or free radicals occurred, the activity of SOD decreased. Therefore, the activities of SOD, GST reflect the degree of oxidative damage. In this study,after treated with different concentrations ofGPS,the increasing of MDAcaused by TCDDeffectively reduced(P<0.01) and the activity of SOD also increasedsignificantly (P<0.01),3 different concentrations of GPS can reduce the content of MDA caused by TCDD(P<0.01). The activity of SOD increased significantly(P<0.01), which showed thatGPS can anti theliver injury caused by TCDD.The similar results have been shown inSD rats[23].