14Deoxyandrographolide desensitizes hepatocytes to tumour necrosis factor-alpha-induced apoptosis...

RESEARCH PAPER

14-Deoxyandrographolidedesensitizes hepatocytes totumour necrosis factor-alpha-induced apoptosisthrough calcium-dependenttumour necrosis factorreceptor superfamilymember 1A release via theNO/cGMP pathwayDN Roy1, S Mandal1, G Sen1, S Mukhopadhyay2 and T Biswas1

1Cell Biology and Physiology Division, Indian Institute of Chemical Biology, A Unit of Council of

Scientific and Industrial Research, Kolkata, India, and 2Chemistry Division, Indian Institute of

Chemical Biology, A Unit of Council of Scientific and Industrial Research, Kolkata, India

CorrespondenceDr Tuli Biswas, Cell Biology andPhysiology Division, IndianInstitute of Chemical Biology,CSIR, 4, Raja S.C. Mullick Road,Pin - 700032, Jadavpur, Kolkata,India. E-mail:tulibiswas@iicb.res.in----------------------------------------------------------------

Keywordsandrographolide;14-deoxyandrographolide; TNF-a;TNFR1; TNFRSF1A; apoptosis;Ca-ATPase; calcium; nitric oxide;cGMP; hepatocytes----------------------------------------------------------------

Received29 January 2010Revised22 March 2010Accepted31 March 2010

BACKGROUND AND PURPOSEAndrographis paniculata (AP) has been found to display hepatoprotective effect, although the mechanism of action of theactive compounds of AP in this context still remains unclear. Here, we evaluated the hepatoprotective efficacy of14-deoxyandrographolide (14-DAG), a bioactive compound of AP, particularly its role in desensitization of hepatocytes totumour necrosis factor-alpha (TNF-a)-induced signalling of apoptosis.

EXPERIMENTAL APPROACHTNF-a-mediated ligand receptor interaction in hepatocytes in the presence of 14-DAG was studied in vitro in primaryhepatocyte cultures, with the help of co-immunoprecipitation, confocal microscopy and FACS analysis. Events associated with14-DAG-induced TNFRSF1A release from hepatocytes were determined using immunoblotting, biochemical assay andfluorimetric studies. Pulse-chase experiments with radiolabelled TNF-a and detection of apoptotic nuclei by terminaltransferase-mediated dUTP nick-end labelling were performed under in vivo conditions.

KEY RESULTS14-DAG down-regulated the formation of death-inducing signalling complex, resulting in desensitization of hepatocytes toTNF-a-induced apoptosis. Pretreatment of hepatocytes with 14-DAG accentuated microsomal Ca-ATPase activity throughinduction of NO/cGMP pathway. This resulted in enhanced calcium influx into microsomal lumen with the formation ofTNFRSF1A–ARTS-1–NUCB2 complex in cellular vesicles. It was followed by the release of full-length 55 kDa TNFRSF1A and areduction in the number of cell surface TNFRSF1A, which eventually caused diminution of TNF-a signal in hepatocytes.

CONCLUSION AND IMPLICATIONTaken together, the results demonstrate for the first time that 14-DAG desensitizes hepatocytes to TNF-a-mediated apoptosisthrough the release of TNFRSF1A. This can be used as a strategy against cytokine-mediated hepatocyte apoptosis in liverdysfunctions.

BJP British Journal ofPharmacology

DOI:10.1111/j.1476-5381.2010.00836.xwww.brjpharmacol.org

British Journal of Pharmacology (2010) 160 1823–1843 1823© 2010 The AuthorsJournal compilation © 2010 The British Pharmacological Society

Abbreviations14-DAG, 14-deoxyandrographolide; ActD, actinomycin D; AG, andrographolide; D-GalN, D-galactosamine; DISC,death-inducing signalling complex; DMSO, dimethyl sulphoxide; DTT, dithiothreitol; ER, endoplasmic reticulum;FADD, fas-associated death domain; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; L-NAME, L-nitro-argininemethyl ester; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-kb, nuclear factor kappa beta; NO,nitric oxide; OH, hydroxyl; PAF, platelet-activating factor; PMSF, phenylmethylsulphonyl fluoride; RPMI-1640, RoswellPark Memorial Institute-1640; SDS, sodium dodecyl sulphate; TNFRSF1A (TNFR1), tumour necrosis factor receptor 1;TNFRSF1B (TNFR2), tumour necrosis factor receptor 2; TNF-a, tumour necrosis factor-alpha; TRADD, tumour necrosisfactor receptor-associated death domain

Introduction

Andrographis paniculata (AP) (family: Acanthaceae) isextensively used as traditional herbal medicine inmany Asian countries (Tang and Eisenbrandt, 1992).Extracts of this plant are reported to exhibit a broadrange of therapeutic activities including anti-parasitic, anti-inflammatory, immunostimulant andhepatoprotective (Choudhury and Poddar, 1984;Kapil et al., 1993; Kumar et al., 2004; Sheeja et al.,2006; Jarukamjorn and Nemoto, 2008). Major bio-active diterpenoids of AP include andrographolide(AG) and 14-deoxyandrographolide (14-DAG) (Tanet al., 2005). These compounds vary widely in theirpharmacological activities, which might be relatedto the differences in their structures (Nanduri et al.,2004). The crude extract of AP was found to protectliver against apoptosis, although the individualeffects of active compounds of AP on hepatoprotec-tion are yet to be ascertained (Kapil et al., 1993;Visen et al., 1993). AG is a diterpenoid lactone con-taining an a-alkylidene g-butyrolactone moiety andthree hydroxyl groups at C-3, C-19 and C-14(Figure 1A). Computational chemistry studies onthe structural–activity relationship of AG showthat the 16-carbonyl, 12, 13-olefin bond and 14-hydroxyl on the alpha methylene lactone of AG are

the key structural moieties, which are responsiblefor its therapeutic activity (Nanduri et al., 2004).This molecule can act as a template, and any smallmodification is likely to have an impact on its bio-logical properties. Recently, the analogue of AG,14-DAG, with an endocyclic double bond D13(14) withno OH group at C-14 has been found to induce theactivation of the NO/cGMP pathway (Zhang andTan, 1999); this effect might contribute to theimmunomodulatory and anti-inflammatory proper-ties of AP extracts (Jarukamjorn and Nemoto, 2008).

Tumour necrosis factor-alpha (TNF-a), the pleio-tropic cytokine, has an important role in inflamma-tion and apoptosis in many cell types (Locksleyet al., 2001). The cellular signalling network used byTNF-a to cause apoptosis is complex and involves avariety of intermediates and protein–protein inter-actions (Locksley et al., 2001). The roles of TNF-asignalling molecules in hepatic disorders are wellunderstood (Schümann and Tiegs, 1999). AlthoughTNF-a can signal through two receptors, TNFRSF1A(TNFR1) and TNFRSF1B (TNFR2), the majority ofTNF-a-mediated biological events are mediatedthrough TNFRSF1A signalling (Hsu et al., 1996). Inall type II cells including hepatocytes, TNF-a-induced cell death is mediated by TNF-a–TNFRSF1Ainternalization, which triggers death-signallingpathways by initiating the engagement of TRADD,FADD and caspase-8, and forming the death-inducing signalling complex (DISC) (Locksley et al.,2001). When hepatocytes get aberrantly committedto the TNF-a-induced apoptotic pathway, it leads tomassive cell death resulting in hepatic fibrosis(Canbay et al., 2004). Here, we have evaluated theefficacy of both AG and 14-DAG as anti-apoptoticcompounds with a view to assessing their potencyagainst TNF-a-mediated hepatotoxicity. To ourknowledge, this is the first report demonstratingthat 14-DAG is better than AG at combating TNF-a-induced apoptosis of hepatocytes.

Thus, in the present study, we examined the roleof 14-DAG at inhibiting TNF-a-induced hepatocyteapoptosis. We found that 14-DAG suppressed DISCformation by reducing the number of cell surfacereceptors (TNFRSF1A) in the cell membrane. This

Figure 1Chemical structures of diterpenoids: (A) AG and (B) 14-DAG.

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1824 British Journal of Pharmacology (2010) 160 1823–1843

reduction in TNFRSF1A on the cell surface couldbe due to proteolytic cleavage of TNFRSF1A orrelease of exosome with full-length TNFRSF1A fromthe cells (Reddy et al., 2000; Hawari et al., 2004).TNFRSF1A released by the cells functions as TNF-a-binding proteins in plasma and competes with cellsurface TNFRSF1A, thereby decreasing the sensitiv-ity of cells to TNF-a (Nophar et al., 1990). TNFRSF1Areleased from cells also decreases the number ofavailable cell surface receptors for ligand binding(Cui et al., 2002). Intravesicular calcium homeosta-sis plays an important role in initiating release ofTNFRSF1A from cells. Nucleobindin-2 (NUCB2),a multifunctional protein that interacts withanother regulatory protein aminopeptidase regula-tor of TNFRSF1A shedding (ARTS-1) in signallingTNFRSF1A release from cells. The associationbetween TNFRSF1A and NUCB2–ARTS-1 complex isa calcium-dependent event occurring in the intra-cytoplasmic vesicles of the cell (Islam et al., 2006).We investigated the involvement of 14-DAG in theevents associated with an increase in vesicularcalcium that leads to the commitment of hepato-cytes to release TNFRSF1A.

Methods

ReagentsUnless otherwise noted, all chemicals were obtainedfrom Sigma (St Louis, MO, USA). Roswell ParkMemorial Institute (RPMI) Medium 1640 (Gibco)and fetal bovine serum (FBS) (Gibco) were obtainedfrom Invitrogen Corporation (Grand Island, NY,USA). Terminal transferase-mediated dUTP nick-endlabelling (TUNEL) assay kit (APODIRECT kit) waspurchased from Calbiochem (San Diego, CA, USA).Terminal deoxynucleotidyl transferase (TdT) in situapoptosis detection kit was obtained from R&DSystems (Minneapolis, MN, USA). All antibodieswere obtained from Abcam Inc (Cambridge, MA,USA). Rat TNF-a was from (recombinant, expressedin Escherichia coli, powder) Sigma. Fluorescentdyes were from Molecular Probes (InvitrogenCorporation); Ru360 was from Calbiochem (EMDBioscience, Gibbstown, NJ, USA); 1,3,4,6-tetrachloro-3a, 6a di phenyl glycoluril (chlorogly-coluril) (PIERCE, Rockford, IL, USA); antibodies ofTRADD, FADD were purchased from Santa Cruz Bio-technology Inc. (Santa Cruz, CA, USA); antibody ofTNFRSF1A was from Sigma. TNFRSF1A Quantikinesandwich ELISA kit was bought from R&D Systems.All drugs and molecular target nomenclatureconform with the BJP’s Guide to Receptors and Chan-nels (Alexander et al., 2009).

Isolation of 14-DAG14-DAG was isolated from the n-butanol extract ofAP by chromatography over silica gel using amixture of chloroform and methanol. The structureof 14-DAG was determined from the detailed spec-tral (IR, MS, 1H HMR, 13C NMR) analysis and firmlyestablished by X-ray crystallography (Fujita et al.,1984; Bhattacharyya et al., 2005).

Preparation of drugsAG (Aldrich) and 14-DAG were dissolved separatelyin a minimum quantity of dimethyl sulphoxide(DMSO) and further diluted with phosphate-buffered saline (PBS) (pH 7.4) to achieve therequired concentration for in vitro and in vivostudies. The final DMSO concentration in themedium of cell culture was less than 0.1%.

Primary hepatocyte cultureHepatocytes were isolated from male Sprague-Dawley rats (100 � 10 g) for hepatocyte culture(Seglen, 1976). Hepatocyte viability was determinedby trypan blue exclusion. Freshly isolated hepato-cytes (1 ¥ 107) were seeded into culture dishes inRPMI 1640 medium, supplemented with100 IU·mL-1 penicillin, 50 mg·mL-1 streptomycin,50 mg·mL-1 gentamicin and 10% FBS at 37°C in ahumidified 5% CO2–95% air atmosphere (Sasakiet al., 1997). After 24 h, the cells were further cul-tured with fresh medium containing 5% FBS andwere then used for the in vitro experiments.

Inhibition of TNF-a-induced cell deathThe inhibition of TNF-a-induced cell death wasassayed on the primary hepatocytes. Cells weretreated with different concentrations of either AG or14-DAG for 1 h at 37°C before TNF-a induction.Then, cells were further treated with TNF-a(10 ng·mL-1) along with actinomycin D (ActD)(200 ng·mL-1), and cultured for 12 h at 37°C in 5%CO2 (Leist et al., 1994). The number of live cells wasdetermined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay(Mosmann, 1983). Briefly, 50 mL of MTT solution(5 mg·mL-1 in PBS) was added to each well of ELISA

plate. After 2 h of incubation, the medium wasremoved and formazan crystal was solubilized with200 mL DMSO. The plate was then read on an ELISA

plate reader (Bio-Rad, Hercules, CA, USA) at 595 nm.Percentage of live cells was calculated with the fol-lowing formula (Qin et al., 2007):

OD ODOD OD

ActD TNF- drug ActD TNF-

ActD ActD TNF-

+ + +

+

−−

×α α

α100

Cells with AG or 14-DAG treatment were alsoincluded in this assay. Each experiment was

BJP14-DAG mediated TNFRSF1A release from hepatocytes

British Journal of Pharmacology (2010) 160 1823–1843 1825

repeated independently three times, and per experi-ment all treatments were done in triplicates.

ELISA assay for binding activity of TNF-ato TNFR1Briefly, 2 mg·mL-1 of rat TNFRSF1A in 0.1 M Na2CO3,pH 9.6, was incubated at 4°C overnight in a 96-wellELISA plate. The rat TNFRSF1A-coated plate wasblocked with PBS containing 1% BSA and 0.2%Tween 20 for 1 h at 37°C. Biotin-conjugated ratTNF-a (1 mg·mL-1) was added to these plates in thepresence of varying concentrations of 14-DAG, andincubated for 1 h at 37°C. The excess biotin-conjugated TNF-a was removed by washing theplates with PBS. Then, 0.1 mg·mL-1 streptavidin–horseradish peroxidase (HRP) was added to theplates and incubated for 15 min at room tempera-ture. Unbound streptavidin–HRP was removed bywashing with PBS. Absorbance of samples was mea-sured at 450 nm in an ELISA plate reader. The peptideWP9QY (100 mM) (Calbiochem) that binds withTNF-a and prevents interactions of TNF-a withTNFRSF1A was used as a positive control (Takasakiet al., 1997; Cao et al., 2009). Five replicates perexperiment were done from three independentexperiments. Percentage inhibition of TNF-abinding was calculated with the following formula(Qin et al., 2007):

OD ODOD

TNFRSF1A TNF- TNFRSF1A TNF- drug

TNFRSF1A TNF-

+ + +

+

−×α α

α100

Preparations of microsomeLiver was rinsed in 10 mM Tris–HCl, pH 7.2, 2 mMdithiothreitol (DTT) and 0.25 M sucrose at 4°C. Theliver was then homogenized in the same buffer witha Dounce homogenizer. The homogenate was cen-trifuged at 12 000¥ g for 20 min at 4°C. The super-natant obtained was then centrifuged at 105 000¥ gfor 60 min at 4°C. The pellet was collected asmicrosomes (Witter et al., 1981). No superoxide dis-mutase or catalase activities were found in themicrosomal suspension (data not shown).

Study of TNF-a receptor internalizationPretreated cells (1 ¥ 107 mL-1) with or without14-DAG were incubated with 10 ng·mL-1 biotiny-lated TNF-a (R&D Systems) in PBS (pH 7.4) for60 min at 4°C. Streptavidin–fluorescein isothiocyan-ate (FITC) reagent was added and cells were incu-bated for another 30 min at 4°C. Incubation wascontinued for the next 60 min at 37°C for studyingreceptor internalization (Schneider-Brachert et al.,2006). The cells were washed and fixed in paraform-aldehyde (4% in PBS, pH 7.4), and TNFRSF1A inter-nalization was viewed under confocal microscope

(Zeiss LSM 510 laser scanning microscope, StandortGöttingen, Germany). At least 10 fields per slide andthree independent sets were examined. Cellsshowing TNFRSF1A internalization were analysed bya flow cytometer (Becton Dickinson FACS Calibur,Becton Dickinson, San Jose, CA, USA). A minimum of10 000 cells were analysed for each sample. Sevenindependent sets of experiments were performed.

Measurement of endoplasmic reticulum(ER) calciumHepatocytes were washed with cold PBS (pH 7.4) andincubated with 10 mM Ru360 for 45 min to preventmitochondrial calcium uptake. Mag-fura 2-AM,10 mM, was then added and the cells were kept in thedark at room temperature for 75 min. The dye wasreleased from cytoplasm when the cells were trans-ferred into the plasma membrane permeabilizationbuffer (19 mM NaCl, 125 mM KCl, 10 mM HEPES,1 mM EGTA, 0.33 mM CaCl2, 8.14 mM digitonin, pH7.2 with KOH) so that the dye was restrained withinmembrane-bound organelles, especially in the ER.Refilling of calcium to ER was initiated by the addi-tion of the intracellular buffer (95 nM free calciumconcentration, 19 mM NaCl, 125 mM KCl, 10 mMHEPES, 1 mM EGTA, 0.33 mM CaCl2, 1.4 mM MgCl2,3 mM ATP, pH 7.2 with KOH). Different drugs wereadded after 30 s of observation, and the kinetics wasobserved up to 800 s with a spectrofluorimeter (Perki-nElmer, LS50, Llantrisant, UK). The cells were excitedat 340 and 380 nm, and emission was measured at510 nm (Kimberly et al., 2008). To validate thechanges in the ER calcium, the experiments wereperformed on a subset of four replicates over threeindependent experiments.

Measurement of Ca-ATPase activityHepatocytes were treated with different combina-tions of drugs for 10, 20 and 30 min separately, andmicrosomal fractions were prepared. Ca-ATPaseactivity of microsomal fractions was determined bythe hydrolysis of Pi from ATP in the presence andabsence of calcium (Diez-Femandez et al., 1996).The incubation medium contained, in a finalvolume of 1.0 mL, 100 mM KCl, 5 mM MgCl2,20 mM HEPES–KOH (pH 7.0), 1 mg oligomycin(mitochondrial Ca-ATPase inhibitor). For mediumrepresenting Mg-ATPase activity, 1 mM EGTA waspresent, and for the medium representing theCa–Mg-ATPase activity, 50 mM CaCl2 was present.The medium was allowed to incubate for 5 min at37°C prior to the addition of 1 mg of microsomalprotein, which was allowed to incubate at 37°C forapproximately 2 min. The reaction was initiated bythe addition of 1 mM ATP, and stopped after 10 minat 37°C by the addition of 0.5 mL of 5% TCA.

BJP DN Roy et al.


Following centrifugation, 1.0 mL of the supernatantwas removed and Pi was determined. The paireddifference between Pi hydrolysis with and with-out calcium represents Ca-ATPase activity innmol·min-1·mg-1 protein. Data represent theaverage of three parallel independent experimentsperformed in triplicate.

Real-time RT–PCR of TNFR1Total RNA was prepared from 14-DAG-treated and-untreated hepatocytes using the Trizol kit accord-ing to the manufacturer’s protocol (Invitrogen Cor-poration), and applied in real-time RT–PCR assay.The primers used for the real-time RT–PCR analyseshad the following sequences (Huang et al., 2006;Calamita et al., 2007):

TNFRSF1A (forward): 5′-GCCTCCCGCGATAAAGCCAACC-3′TNFRSF1A (reverse): 5′-GGACACCCACTTTCACCCGTTTC-3′b-Actin (forward): 5′-TTGTAACCAACTGGGACGAT-3′b-Actin (reverse): 5′-TAATGTCACGCACGATTTCC-3′

Real-time RT–PCR was performed on an iCycler(Bio-Rad Laboratories) with SYBR green reagent. ThePCR mixture (20 mL) contained 15 pmol of eachprimer, 8 mL of water and 12.5 mL of cDNA. Thesamples were placed in 96-well plates (Bio-Rad), andPCR amplification was performed by using iCycleriQ multicolor real-time PCR detection system(Bio-Rad). We used the comparative cycle thresholdmethod (DDCT method) for relative quantificationof gene expression (Leite et al., 2002). Finally, thearithmetic calibrator (2-DDCT) was used to calculatethe relative mRNA level expression of TNFRSF1A.Each experiment was repeated independently threetimes with two replicates.

Analysis of cell surface TNFRSF1A by flowcytometry and ELISA testHepatocytes (1 ¥ 107 cells·mL-1) were washed andincubated with blocking buffer (PBS containing 1%BSA) followed by incubation at 4°C with FITC-conjugated antibody at a saturating concentration.Cells were washed in PBS, and fluorescence wasanalysed by flow cytometry, collecting 10 000events per sample. The experiments were repeatedfour times independently.

For quantification of extracellular TNFRSF1Arelease by ELISA, the culture media were cleared ofcells and debris by sequential centrifugation at 200¥g for 10 min, 500¥ g for 10 min, 1200¥ g for 20 minand 10 000¥ g for 30 min at room temperature(Islam et al., 2006). The level of TNFRSF1A in culturemedia was determined using a sandwich ELISA kit.

Each assay was carried out with four replicates ofthree samples from independent experiments.

Measurement of NO and cGMPin hepatocytesHepatocyte generation of NO was determined byusing the specific fluorescent probe 4,5diaminofluo-rescein diacetate (DAF-2/DA) (Gumpricht et al.,2002). Cells were pre-loaded at 37°C for 30 min with10 mM DAF-2/DA for NO detection. Upon enteringthe hepatocyte, intracellular esterases hydrolyse thediacetate moiety, trapping free DAF-2 within the cellthat is covalently modified by NO. After the cells hadbeen loaded with dye, they were washed twice andresuspended in KRH/BSA, and incubated either withthe 14-DAG or 14-DAG along with NOS inhibitor(L-NAME). Measurements were not affected by sol-vents or added compounds. Cells were removed as analiquot at different times for fluorescence determina-tion at 495 nm excitation and at 515 nm emissionsfor DAF-2. Generation of NO from isolated rat hepa-tocytes was expressed as relative fluorescence unitsper 106 cells. The experiment was repeated four timeswith four wells per condition per replication.

For determination of cGMP, hepatocytes werewashed twice with ice-cold PBS and lysed with0.1 M HCl at 4°C. The lysates were centrifuged at100 000¥ g for 15 min at 4°C. The cGMP level of thelysate was determined with a commercially availablekit (cGMP Direct Immunoassay Kit, BioVision,Mountain View, CA, USA) following the manufac-turer’s instructions. All experiments were repeatedat least three times with four replicates.

DNA fragmentation assayThe DNA fragmentation pattern (DNA ladder) wasstudied by agarose gel electrophoresis. Cells (1 ¥106 mL-1) after different treatments were centrifugedat 1200¥ g for 10 min. The pellet was resuspended in1 mL of lysis buffer [50 mM Tris–HCl (pH 8.0),10 mM NaCl, 10 mM EDTA, 100 mg·mL-1 proteinaseK and 0.5% SDS] and incubated for 2 h at 50°C. DNAwas extracted with 1 mL of phenol, pH 8.0, followedby extraction with 1 mL of phenol/chloroform (1:1)and chloroform. The aqueous phase was precipitatedby overnight incubation with 2.5 volumes of ice-coldethanol and 1/10 volume of 3 M sodium acetate, pH5.2, at 20°C. The precipitates were collected by cen-trifugation at 13 000¥ g for 10 min. The pellets wereair-dried and resuspended in 50 mL of TE buffersupplemented with 100 mg·mL-1 RNaseA. DNA wasloaded onto a 2% agarose gel containing ethidiumbromide, electrophoresed in TAE buffer for 2 h at50 V and photographed under UV illumination. Theassay was done in duplicate and repeated five timeson separate days.



TUNEL assayIn the in vitro study, TUNEL assay was performedusing a commercially available kit, following themanufacturer’s instructions (Apo-Direct Kit, Calbio-chem). Briefly, after all treatments, hepatocytes werewashed twice with PBS and fixed with freshly pre-pared 4% paraformaldehyde in PBS (pH 7.4) for30 min at room temperature. The cells were perme-abilized with 0.1% Triton X-100 in 0.1% sodiumcitrate for 2 min on ice. To label DNA strand breaks,cells were incubated with 50 mL TUNEL reactionmixture containing TdT and fluorescein–dUTP inthe binding buffer, and incubated for 1 h at 37°Cin a humidified atmosphere. The cells were thenwashed twice with PBS and analysed by flow cytom-eteric analysis (Becton Dickinson FACS Calibur).

Determination of caspase activitiesCaspase-3 and caspase-8 activities were determinedusing the fluorometric substrates DEVD–AFC(caspase-3 substrate) and IETD–AFC (caspase-8 sub-strate), following the protocols of the Caspase Activ-ity Assay kit from BioVision, Inc. Hepatocytes wereisolated and suspended in 100 mL of lysis buffer (Bio-Vision Inc.) and incubated at 4°C for 10 min fol-lowed by centrifugation at 12 000¥ g for 10 min.Aliquots (50 mL) of the supernatant were removedand placed in a 96-well microplate containing reac-tion buffer (BioVision Inc.). Substrate was added,and the microplate was incubated at 37°C for30 min. The activity was monitored as the linearcleavage and release of the AFC side chain and com-pared with a linear standard curve generated on thesame microplate. The assay was done in duplicateand repeated three times on separate days.

Western blot analysisCells were washed once with PBS (pH 7.4). Nuclearand cytosolic fractions were collected from thetreated and untreated cell using the ProteoExtractSubcellular Proteome Extraction Kit (Calbiochem).Proteins from the cytosolic fraction were separatedby 10% SDS–PAGE and analysed by Westernblot using antibodies directed against caspase-3,TNFRSF1A (55 and 28 kDa) and glyceraldehyde-3-phosphate dehydrogenase (as loading control). Pro-teins from the nuclear fraction were also separatedby 10% SDS–PAGE, and polyclonal antibody againstnuclear factor kappa beta (NF-kb) (p65) was used forimmunoblot. Immunoreactive bands were incu-bated with a 1:5000 dilution of HRP conjugatedsecondary antibody. Binding signals were visualizedwith TMB substrate. Experiments were performed atthe times indicated in the figure legends.

Co-immunoprecipitationHepatocytes (5 ¥ 107 mL-1) were washed three timesin PBS (1000¥ g for 5 min) and lysed immediatelyin 500 mL of cold lysis buffer [10 mM Tris–HCl (pH7.5), 1% Triton X-100, 100 mM NaCl, 5 mM EDTA,1 mM DTT, 1 mM pepstatin A, 1 mM leupeptin,0.1 mM PMSF], and were kept on ice for 1 h.During this period, Protein G/Sepharose beads(40 mL) were incubated with 20 mL of anti-TNFRSF1A Ab in 500 mL of Tris buffer [10 mMTris–HCl (pH 7.5), 1% Triton X-100, 100 mM NaCl]and Complete protease inhibitor mixture (RocheApplied Science, Mannheim, Germany) for 1 h at4°C. The hepatocyte lysate was centrifuged(10 000¥ g for 15 min at 4°C) and the supernatantincubated with the Protein G/Ab complex for 3 h at4°C. The Sepharose beads were then washed threetimes with lysis buffer and twice with lysis bufferlacking Triton X-100. The precipitates were resus-pended in non-reducing Laemmli sample buffer,boiled, resolved by SDS–PAGE and transferred ontonitrocellulose membrane. After being blocked, themembrane was immunoblotted with the mono-clonal anti-TRADD Ab (1:500), anti-FADD Ab(1:500) and anti-caspase-8 (p43 fragment) Ab(1:400) overnight at 4°C, and visualized withAP-conjugated goat anti-mouse IgG using the NBT/BCIP as a substrate. Following the same procedure,co-immunoprecipitaion of NUCB2/CALNUC,ARTS-1 and TNFRSF1A was performed using anti-NUCB2 antibody, anti-ARTS-1 antibody and anti-TNFRSF1A antibody. All co-immunoprecipitationexperiments were repeated at least three timesindependently.

In vivo studiesAnimal treatment. Male Sprague-Dawley rats (100–120 g) were maintained under controlled conditions(22°C, 55% humidity, 12 h day/night rhythm) withfree access to solid pellet diet and water ad libitumthroughout the study. All animals were bred andhoused in the animal house of Indian Institute ofChemical Biology, CSIR and Kolkata. Animal experi-ments were started at 8:00 AM. The rats were treatedwith 14-DAG, 40 mg·kg-1 body weight, i.p., 2 hbefore TNF-a administration. ActD (800 mg·kg-1) orD-GalN (600 mg·kg-1) was injected i.p. with saline,and TNF-a (5 mg·kg-1 body weight) was injected in atotal volume of 300 mL mixed with saline into thetail vein 2 min after the administration of ActD orD-GalN (Hishinuma et al., 1990; Leist et al., 1994;Mignon et al., 1999). Survival of all animals afterTNF-a infusion was monitored until the end of a 5 hperiod, at which point in time no mortality wasrecorded. The animal experiments were performedthree times with four animals in each group.

BJP DN Roy et al.


Measurements of ALT and TNFRSF1A in plas-ma. Blood samples were collected from rat tailveins in heparin, and the plasma was obtained bycentrifugation at 1000¥ g for 10 min. Plasma ALTactivity was determined as a biochemical indicatorof hepatocellular damage. ALT activity was mea-sured colorimetrically at 340 nm according to stan-dard procedures, using a commercially availablediagnostic laboratory kit. Equal amounts of plasmaproteins (40 mg) were loaded into SDS/PAGE.Western blot analysis was performed to identify thepresence of TNFRSF1A in rat plasma after 14-DAGtreatment.

In situ apoptosis detection by TUNEL labelling. Apo-ptotic nuclei were detected in histological sectionsusing TdT in situ apoptosis detection kit (R&DSystems) according to the manufacturer’s instruc-tions. Briefly, cryostat sections of frozen liver tissues,5 mm in thickness, were fixed with 10% neutralbuffered formalin and dehydrated in PBS (pH 7.4)according to the standard protocol. TdT enzyme wasapplied to the sections and incubated for 1 h at37°C. Then, 50 mL of streptavidin–HRP solution wasadded, and the mixture was incubated for 10 min at37°C in a humidity chamber to avoid evaporation.DAB solution was added for 5 min, and the sectionwas counterstained with haematoxylin.

The TUNEL assay was performed in in vivo con-ditions after the hepatocytes had been isolatedusing a standard procedure. dUTP–FITC-labelledDNA fragmentation was assayed using an in situ celldeath detection kit (APO-DIRECT kit, Calbiochem).Hepatocytes were incubated at 37°C for 1 h with Tdtbuffer containing FITC-labelled dUTP, and counter-stained with PI. The cells were then analysed with aFACS scan (Becton Dickinson; FACS caliburequipped with 488 nm argon laser. Cells are dis-played as DNA area (linear red fluorescence) on theX-axis versus FITC–dUTP (log green fluorescence) onthe Y-axis. A horizontal gate was applied to thisdisplay to discriminate between apoptotic (FITCstaining) and non-apoptotic cells (Roy et al., 2009).The experiments were repeated at least three timesindependently.

Binding of 125I labelled TNF-a to hepatocytesIn vivo studies on binding of TNF-a to its receptorwere done by using 125iodine-labelled recombinantrat TNF-a ([125I]-TNF-a). TNF-a was iodinated usinga 2 mL tube coated with 25 mg of 1,3,4,6-tetrachloro-3a, 6a diphenylglycoluril (chlorogly-coluril) dissolved in 500 mL of chloroform. Afterevaporation of chloroform, 3 mg of TNF-a wasreacted with 18.5 MBq of Na125I in this tube at 0°C.[125I]-TNF-a was separated from free 125I by elution

with 0.1% BSA–PBS on a PD-10 column (Pharmacia,Uppsala, Sweden). The specific activity of the [125I]-TNF-a was 60–80 mCi·mg-1 (Scallon et al., 2002). Inaccordance with a protocol approved by the Institu-tional Animal Ethics Committee, rats were anaes-thetized by intramuscular injection of ketamine.[125I]-TNF-a (10 mCi) in 100 mL of lactated Ringer’ssolution was delivered in a bolus into the tail vein.At the end of the experiment, the rats were killedand liver was excised for hepatocyte isolation. Theisolated hepatocytes were dissolved in 500 mL of thelysis buffer (1% SDS, 0.2 N NaOH), and the boundlabel was quantified in a gamma counter (GammaRay Spectrometer, ECIL, Hyderabad, India). Theamount of bound [125I]-TNF-a in hepatocytes after90 min in the control group was considered as 100%binding.

Statistical analysisAll experimental values were first evaluated by theone sample Kolmogorov–Smirnov goodness of fittest to determine whether they followed a normaldistribution pattern. Depending on the results, mul-tiple associations with categorical data were exam-ined using one-way ANOVA or Student’s t-test, or itsnon-parametric equivalent Mann–Whitney U-tests.Significant differences were considered for prob-abilities <5% (P < 0.05). The statistical analysis wasdone by using GraphPad Instant Software (Graph-Pad, La Jolla, CA, USA).

Results

Anti-apoptotic effect of AG and 14-DAGA double bond at D12(13), C-14 hydroxyl group andlactone ring are the key moieties of AG (Figure 1A).The essential structural differences between the twolabdane diterpenoids AG and 14-DAG are theabsence of hydroxyl functionality in 14-DAG withthe migration of double bond at D12(13) in AG to D13(14)

in 14-DAG (Figure 1B). Despite their structuraldifferences, no cytotoxic effects of either AG or14-DAG could be observed at the concentrations2.5–15 nM when the hepatocytes were treated withthese compounds alone (Figure 2A). We determinedthe sensitivity of hepatocytes to TNF-a-inducedcytotoxicity in the presence of diterpenoid lactonesAG and 14-DAG (Figure 2A). Hepatocytes were pre-incubated with different concentrations of thesetwo drugs (2.5–15 nM) and exposed to TNF-a(10 ng·mL-1) along with ActD for 12 h. The doses ofthe diterpenoids were chosen for further study onthe basis of their efficacy in providing protectionagainst cytotoxicity. While both drugs offeredprotection against TNF-a-induced cell death,



14-DAG appeared to be more effective than AGgiving almost complete protection (7% cell death in14-DAG-treated group where as 35% cell death inAG-treated group) at a concentration of 10 nM. So,we chose to use 10 nM of these two compounds forour subsequent experiments. We examined the DNAfragmentation of cells by TUNEL using FACS analy-sis to optimize the time period of pretreatment withAG or 14-DAG. Cells were pretreated with AG or14-DAG at various time periods (15, 30, 45 and60 min) before 12 h of exposure to TNF-a (withActD) (Figure 2B). Flow cytometric analysis revealed

that 14-DAG was more effective than AG in produc-ing time-dependent inhibition of DNA strandbreaks in hepatocytes as compared to hepatocytestreated with TNF-a alone. Pretreatment with14-DAG for 60 min gave the most effective cytopro-tection (only 6% of the cells being TUNEL positive)to hepatocytes against TNF-a-induced apoptosis,and therefore, we used a 60 min (i.e. 1 h) incubationin all subsequent experiments. Our results suggestthat 14-DAG, with the exocyclic double bond D12 (13)

of AG isomerized to endocyclic double bond D13 (14)

along with simultaneous removal of OH at C-14, is

Figure 2Protective effect of AG and 14-DAG on TNF-a-induced cell death in hepatocytes. (A) Hepatocytes were pre-incubated in the presence of differentconcentrations of AG or 14-DAG in culture media for 1 h. Cells were washed in PBS, centrifuged and further incubated with or without TNF-a(10 ng·mL-1) along with ActD (200 ng·mL-1) for 12 h. Cell death was measured by the MTT assay. The data are shown as a percentage of cell death(mean � SD) of three independent experiments. *P < 0.05, **P < 0.02, ***P < 0.01 versus AG-treated cells. (B) Hepatocytes were pre-incubatedwith or without 10 nM of AG or 14-DAG at various time periods. Cells were harvested at indicated time (min) and further exposed to TNF-a(10 ng·mL-1) with ActD (200 ng·mL-1) for 12 h. The DNA fragmentation in hepatocytes was evaluated by performing a DNA nick-end labellingassay using FITC–dUTP incorporation, and these cells were enumerated on a flow cytometer. Results are representative of seven separateexperiments with similar results.

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more effective than AG at preventing hepatocytedeath, which made us select this compound for oursubsequent experiments.

14-DAG inhibits TNF-a-inducedTNFRSF1A-associated death domainWe next examined the influence of 14-DAG on theevents that lead to the apoptosis of TNF-a-sensitizedhepatocytes. Expression of active caspase-3 in thecytosolic fraction of cells was assessed by immuno-blotting (Figure 3A, left panel). 14-DAG inhibitedthe expression of active caspase-3. TNF-a-induced

caspase-3 activation in hepatocytes was inhibitedby pretreatment of hepatocytes with 14-DAG in aconcentration-dependent manner (Figure 3A, rightpanel). Caspase-3 activation in vitro is triggered byupstream events. Hence, we examined the effectof 14-DAG on the activation of caspase-8. 14-DAGalso suppressed the activity of caspase-8 in aconcentration-dependent manner (Figure 3B).

Next, we attempted to identify the upstreammolecular events that lead to 14-DAG inhibition ofapoptosis in TNF-a-sensitized hepatocyte. The use ofco-immunoprecipitation provided us with valuable

Figure 314-DAG inhibited TNF-a-mediated DISC formation. (A) Hepatocytes were pretreated with 14-DAG (5, 10 and 15 nM) for 1 h and then exposedto TNF-a/ActD for 12 h. Control cells were treated with the vehicle, DMSO. Immunoreactive bands of the active caspase-3 fragment were analysedby Western blot. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Caspase-3 activity was determined by using thefluorometric substrates DEVD–AFC. The data are shown as mean � SD of three independent experiments. *P < 0.02, **P < 0.01, versusTNF-a/ActD-treated cells. (B) Caspase-8 activity was also determined by using the fluorometric substrates IETD–AFC in different treatment groupssimilar to caspase-3. The data are shown as mean � SD of three independent experiments. *P < 0.02, **P < 0.01, versus TNF-a/ActD-treated cells.(C) Hepatocytes were pretreated with 14-DAG for 1 h at 37°C followed by treatment with TNF-a (10 ng·mL-1) and ActD (200 ng·mL-1) for 1 hat 37°C. TNFRSF1A was immunoprecipitated from the cell lysate, and the immunoprecipitates (IPs) were electrophoresed and immunoblottedwith anti-FADD, anti-TRADD and anti-caspase-8 (p43) antibodies. Densitometric analyses of IPs were performed. The data are shown as mean �

SD of three independent experiments. (D) Hepatocytes were pretreated with 14-DAG for 1 h followed by incubation in PBS (pH 7.4) containingbiotinylated TNF-a (10 ng·mL-1). TNFRSF1A internalization in hepatocytes was viewed under laser scanning confocal microscope usingstreptavidin–FITC. The magnification of the photomicrograph is 100¥. (E) Evaluation of TNFRSF1A internalization in the presence of 5, 10 and15 nM 14-DAG (experimental conditions were same as D) was quantified by FACS analysis using CELL Quest software. Results are representativeof seven independent experiments with similar results.



information regarding protein–protein interactionin DISC assembly (direct association of TRADD,FADD and caspase-8 with TNFR1). The presence ofTRADD and FADD could be detected in the anti-TNFRSF1A immune complex of TNF-a-sensitizedhepatocytes (Figure 3C, lane 2). 14-DAG inhibitedthe aggregation of TRADD, FADD and caspase-8,and restrained the formation of DISC in TNF-a-sensitized hepatocytes (Figure 3C, lane 3). Whenquantified, the decline in the relevant co-immunoprecipitated proteins in 14-DAG-treatedTNF-a-sensitized hepatocytes as compared to cellstreated with TNF-a alone was found to be statisti-cally significant. The lack of recruitment of DISCproteins in the presence of 14-DAG could be due tothe defective endocytosis of the receptor-boundligand. We used confocal microscopy to analysethe internalization of TNFRSF1A in hepatocytes,using biotin–TNF-a coupled with FITC–streptavidin(Figure 3D). After the hepatocytes had beenincubated with biotin–TNF-a coupled with FITC–streptavidin at 37°C for 60 min, endocytosis ofTNFRSF1A occurred, resulting in intracellular accu-mulation of labelled TNF-a (Figure 3D; row 1).In contrast, biotin–TNF-a did not enter into thehepatocytes, which had been pre-incubated with14-DAG (Figure 3D; row 2) for 1 h. These micro-graphs supported the concept that 14-DAG preventsthe endocytosis of TNF-a bound to TNFRSF1A.We further quantified these results by evaluatingthe endocytosis of TNFRSF1A by flow cytometry(Figure 3E). FACS analysis revealed a concentration-dependent effect of 14-DAG on TNFRSF1A endocy-tosis; 10 nM 14-DAG was chosen for furtherexperiments as it markedly inhibited the internal-ization process, with only 7.2% FITC-positive cellsas compared to 69.9% FITC-positive cells in hepato-cytes incubated with TNF-a alone.

14-DAG was administered before and after TNF-ato determine its mechanism of action. Post-treatment with 14-DAG of TNF-a-sensitized hepato-cytes failed to inhibit apoptosis of the cells asevident from DNA fragmentation into oligonucleo-somal ladders (Figure 4A, lane iii). However, thehepatocytes treated with 14-DAG for 1 h beforethe addition of the cytokine protected the cellsfrom TNF-a-mediated apoptosis (Figure 4A, lane iv).This finding concurred with the experiments thatshowed 14-DAG inhibited the endocytosis of theligand-bound receptor. Next, we determinedwhether 14-DAG inhibits the binding of TNF-a toTNFRSF1A (Figure 4B). WP9QY, which binds toTNF-a and prevents TNF-a from interacting withTNFRSF1A, was used as a positive control. The ELISA

assay, which measures the binding of TNF-a toTNFRSF1A, was carried out in the presence of

various concentrations of 14-DAG. Almost 100%binding was observed between TNF-a andTNFRSF1A in the presence of various concentrationsof 14-DAG. Hence, 14-DAG did not appear toinhibit the binding of TNF-a to TNFRSF1A in ourexperiments.

14-DAG induces the release of TNFRSF1Afrom hepatocytesIt was tempting to speculate that 14-DAG attenu-ated the movement of TNFRSF1A in the hepato-cytes, and that this might be responsible for thedecline in endocytosis of ligand-bound receptor inthe cells. So, experiments were performed to assesswhether 14-DAG modulates the release of solubleTNFRSF1A into the extracellular compartment.Figure 5A reveals the presence of 55 kDa TNFRSF1Ain the supernatants of hepatocyte culture mediatreated with 14-DAG. The densitometric scananalysis of the Western blot revealed that thesechanges were significant. Furthermore, the releaseof sTNFRSF1A (35%) from hepatocytes into theextracellular compartment after 14-DAG treatmentwas associated with a 39% decrease in total cellularTNFRSF1A (55 kDa) content. The interactionamong NUCB2, ARTS-1 and TNFRSF1A was assessed

Figure 4Studies on binding of 14-DAG to TNF-a. (A) The DNA ladder patternswere analysed in groups: (i) control hepatocytes were treated withvehicle (DMSO); (ii) hepatocytes were treated with TNF-a/ActD for12 h; (iii) hepatocytes were exposed to 10 nM 14-DAG 1 h aftertreatment with TNF-a/ActD for 12 h; and (iv) hepatocytes werepre-incubated with 10 nM 14-DAG for 1 h, then exposed to TNF-a/ActD for 12 h. Results are representative of five independent experi-ments. (B) The binding of TNF-a to TNFRSF1A (TNFR1) was studiedusing TNFRSF1A-coated ELISA plate. 14-DAG (at concentrations 5, 10,15, 20 nM) was added to TNFRSF1A-coated ELISA plates along withTNF-a (1 mg·mL-1). WP9QY is used as a positive control. W/O rep-resents nothing was there except TNF-a and TNFRSF1A in thewell. The data are shown as mean � SD of three independentexperiments.

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by the use of the co-immunoprecipitation assay.14-DAG induced the association of TNFRSF1A withARTS-1/NUCB2 in the hepatocytes, which favouredthe extracellular release of full-length TNFRSF1A(Figure 5B). The relative levels of precipitating

NUCB2 and ARTS-1 were quantified by densitomet-ric analysis of several independent experiments. Asignificant increase in the amount of these proteinswas observed after the hepatocytes had beentreated with 14-DAG. Extracellular release of

Figure 514-DAG induced release of cellular TNFRSF1A (TNFR1) from hepatocytes. (A) Cell lysates (40 mg) were collected from 14-DAG-treated (1 h at37°C) hepatocytes and untreated hepatocytes. Supernatants from the culture media were collected from same groups of treatment. Proteins wereseparated by SDS–PAGE, transferred into nitrocellulose membrane and reacted with antibodies against TNFRSF1A (55 kDa). Densitometricanalyses of immunoblots were done. The data are mean � SD of five independent experiments. (B) Hepatocytes were treated with 14-DAG for10 min at 37°C. The association among endogenous NUCB2, ARTS-1 and TNFRSF1A was observed in cell lysate by a co-immunoprecipitationexperiment. Anti-NUCB2, anti-ARTS-1 and anti-TNFRSF1A antibodies were used to detect the presence of NUCB2, ARTS-1 and TNFRSF1A proteinsin the complex. Densitometric analyses of immunoprecipitates were done. The data are shown as mean � SD of three independent experiments.(C) TNFRSF1A (55 kDa) released in the media after treatment with 14-DAG at different concentrations (5–20 nM) for 1 h was measured by ELISA.The data are shown as mean � SD of three independent experiments. **P < 0.02, ***P < 0.01 (D) FACS analysis of TNFRSF1A in membranes ofhepatocytes was done after different time periods (0, 15, 30 and 45 min, and 1, 12 and 24 h) of treatment with 14-DAG using anti-TNFRSF1Aantibody tagged with FITC. Results are representative of four independent experiments with similar results. (E) Levels of mRNA for TNFRSF1A (at0, 1, 10 and 24 h after 14-DAG treatment) in hepatocytes were quantified by RT–PCR assay using specific primers as described in Methods. Thedata are shown as mean � SD of three independent experiments. (F) One group of hepatocytes was incubated with PMA (10 ng·mL-1) for 1 h,and another group of hepatocytes was pre-incubated with TAPI-2 (10 mM) for 1 h before activation with PMA, the other group was treated with14-DAG (10 nM) for 1 h. Supernatants of culture media were collected from these three groups. Proteins were separated by SDS–PAGE, transferredinto nitrocellulose membrane and reacted with antibodies against TNFRSF1A (28 kDa). This experiment was performed three times independently.(G) TNFRSF1A (55 kDa) released from hepatocytes into the media after treatment with 14-DAG (10 nM) for 1 h, and in another group ofhepatocytes pre-incubated with TAPI-2 (10 mM) for 1 h before treatment with 14-DAG (10 nM) for 1 h, was measured by ELISA. The data areshown as mean � SD of three independent experiments. **P < 0.01 versus 14-DAG-treated group.



TNFRSF1A from the hepatocytes increased from 27to 50 pg·mL-1 as the concentration of 14-DAGincreased from 5 to 10 nM, and reached a plateauat 10 nM (Figure 5C), after which the rate ofrelease remained almost steady. The kinetics of thedecline in cellular TNFRSF1A due to its releasefrom the hepatocytes in the presence of 14-DAGwas analysed by flow cytometry analysis. Theresults showed that initially the cellular TNFRSF1Adecreases, but after a certain period of time(30 min) the cellular TNFRSF1A failed to declinefurther (Figure 5D). To verify these results, weanalysed the quantitative expression of TNFRSF1AmRNA in 14-DAG-treated hepatocytes by real-timeRT–PCR (Figure 5E). Real-time RT–PCR revealedthat the expression level of TNFRSF1A mRNA wasincreased significantly (1.4-fold) in 14-DAG-treatedhepatocytes after 10 h in comparison with 0 h, andafter 24 h the expression of TNFRSF1A hadregressed and reached normal levels. These resultsindicate that the release of TNFRSF1A from thehepatocyte in the presence of 14-DAG is a tran-sient event. With the loss of TNFRSF1A in the pres-ence of 14-DAG, the hepatocytes graduallyregained this receptor by increasing mRNA expres-sion. Next, we assessed the presence of theTNFRSF1A ectodomain cleaved fractions (28 kDa)in the media of 14-DAG-treated hepatocytes(Figure 5F). Phorbol myristate acetate (PMA) isknown to activate proteolytic cleavage ofTNFRSF1A in cells by activating the TACE (TNF-a-converting enzyme, a zinc metalloprotease) (Cooket al., 2008). Incubation of hepatocytes with PMAresulted in a significant release of cleavedTNFRSF1A ectodomain (28 kDa) from the cells intothe media (positive control, Figure 5F, lane 1). Thehepatocytes pretreated with the broad-spectrumzinc metalloprotease inhibitor, TAPI-2, did notrelease 28 kDa in the presence of PMA (negativecontrol, Figure 5F, lane 2). The 28 kDa cleavedTNFRSF1A ectodomain was also absent in themedia of 14-DAG-treated hepatocyte (Figure 5F,lane 3). These data demonstrate that TNFRSF1Areleased from hepatocytes in the presence of14-DAG was full-length TNFRSF1A (55 kDa). Wefurther investigated whether the release of55 kDa TNFRSF1A from hepatocytes in the pres-ence of 14-DAG required zinc metalloprotease(TACE) catalytic activity by treating hepatocyteswith the TAPI-2. TAPI-2 significantly attenuatedthe release of full-length TNFRSF1A (55 kDa) from14-DAG-treated hepatocytes (Figure 5G). Theseresults confirm the involvement of TACE in therelease of full-length of TNFRSF1A (55 kDa),although there was no ectodomain cleavage ofTNFRSF1A.

14-DAG increases intravesicular calciumin hepatocytesWe used mag-fura-2AM as free calcium indicator toelucidate the changes in calcium concentrationwithin the microsomal lumen (Figure 6A). In thepresence of an intracellular buffer containingcalcium, magnesium and ATP, there was a distinctincrease in the fluorescent ratio indicating anincrease in the calcium uptake in the lumen. Theresults of this study showed that the basal calciumlevel in ER lumen was the same in all groups. Underthe initial set of experimental conditions describedabove, the microsomal refill of calcium was greaterin the 14-DAG-treated group than the control hepa-tocytes. Hepatocytes pretreated with t-BuHQ, an

Figure 614-DAG treatment increased free calcium in microsomal lumen. (A)Microsomal lumen free calcium was measured using mag-fura-2 AMloaded cells as fluorescence ratio of 340/380 nm. Free calcium of ERwas traced in control hepatocytes, hepatocytes treated with 14-DAG(10 nM), hepatocytes treated with t-BuHQ (100 mM) and hepato-cytes simultaneously treated with 14-DAG (10 nM) and t-BuHQ(100 mM). This kinetic was followed for 800 s. Results are represen-tative of three independent experiments with similar results. (B)TNFRSF1A (TNFR1) in cellular lysate and that correspondinglyreleased in culture media were determined by Western blot. Thehepatocytes were treated with 14-DAG (10 nM) with or withoutt-BuHQ (100 mM) for 1 h at 37°C. Densitometric analyses of immu-noblots were done. The data are shown as mean � SD of threeindependent experiments.

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inhibitor of Ca-ATPase-driven calcium pump, failedto activate calcium uptake into the ER lumen. Whent-BuHQ was added along with 14-DAG, the calciumuptake by ER did not increase. Thus, the increasedcalcium uptake into the microsomal lumen inducedby the addition of 14-DAG could be attributed tothe activation of ER Ca-ATPase. We further immu-noblotted the cellular TNFRSF1A and extracellularTNFRSF1A (isolated from culture media) after thehepatocytes had been incubated in the presence of14-DAG and t-BuHQ (Figure 6B). Densitometricscanning of the immunoblots revealed no signifi-cant decrease in cellular TNFRSF1A after the hepa-

tocytes had been pretreated with 14-DAG alongwith t-BuHQ. This might be due to inactivation ofCa-ATPase pump; if the ER luminal calcium does notincrease, the TNFRSF1A cannot associate with theNUCB2 complex, which would result in their reten-tion in the cellular vesicles.

14-DAG increases Ca-ATPase activity inmicrosomes14-DAG-elicited increases in ER luminal freecalcium could be attributed to increased Ca-ATPaseactivity of ER (Figure 7A). Increased Ca-ATPaseactivity of the luminal surface of the microsome is

Figure 7NO/cGMP modulates ER Ca-ATPase activity in 14-DAG-treated cells. (A) Kinetics of Ca-ATPase activity was measured in microsome of hepatocytestreated with or without 14-DAG in the presence of KT5823 180 nM, L-NAME 100 mM, t-BuHQ 100 mM, 8-Br-cGMP 100 mM and Na-nitroprusside10 mM. All measurements were made at room temperature. The data represent mean � SD of three independent experiments. (B) Kinetics ofCa-ATPase activity were measured in microsome of hepatocytes treated with or without 14-DAG in the presence of L-NAME 100 mM and L-NIL100 mM for 30 min. All measurements were made at room temperature. The data represent mean � SD of three independent experiments. (C)NO was measured in 14-DAG-treated hepatocytes in the presence or absence of L-NAME (100 mM) using the membrane-permeable fluorescentindicator DAF-2/DA. Results are representative of four independent experiments. (D) cGMP was measured in hepatocytes in response to 14-DAGtreatment with or without KT5823 (180 nM). The data represent mean � SD of three independent experiments. (E) Cellular TNFRSF1A (TNFR1)and TNFRSF1A released in the media were determined where the hepatocytes were treated with 14-DAG, cGMP and 14-DAG in the presence ofKT5823. Densitometric analyses of immunoblots were performed. The data are shown as mean � SD of four independent experiments.



known to sequester calcium into the lumen fromcytosol (Meldolesi and Pozzan, 1998). IncreasedCa-ATPase activity was also observed in the presenceof nitroprusside (NO donor) and 8-Br-cGMP(membrane-permeable analogue of cGMP). 14-DAGfailed to accelerate the falling phase of theCa-ATPase activity in cells pre-incubated with theNO inhibitor (L-NAME) or the cGMP inhibitor(KT5823), suggesting that NO/cGMP have a role inthe 14-DAG-mediated stimulation of ER Ca-ATPase.L-NAME inhibits cNOS more effectively than iNOS(Misko et al., 1993). The effect of L-NAME onCa-ATPase is unlikely to be due to inhibition ofiNOS. So, we further incubated the hepatocytes inthe presence of L-NIL and 14-DAG which preferen-tially inhibits iNOS activity. Presence of L-NIL failedto attenuate Ca-ATPase activity, indicating thatiNOS is not involved in the increased activation ofCa-ATPase mediated by 14-DAG (Figure 7B).

We determined NO production using themembrane-permeable dye DAF-2/DA, which con-verts to fluorescent DAF-2T (triazolofluorescein) inthe presence of NO. A potential artefact attributableto photo-activation of DAF-2 was excluded, becausefluorescence was stable at baseline and did not pro-gressively increase (data not shown in the figure).NO production in hepatocytes increased on increas-ing the concentration of 14-DAG, and this declinedin the presence of L-NAME (Figure 7C). cGMP pro-duction also increased after 14-DAG treatment, andthis declined when the cells were simultaneouslyincubated in the presence of the cGMP inhibitor(Figure 7D). We also observed that a significantnumber of TNFRSF1A (55 kDa) were released fromcells into the media in the presence of 8-Br-cGMP(Figure 7E). Also, the release of TNFRSF1A fromhepatocytes during 14-DAG treatment significantly

declined in the presence of a cGMP inhibitor(KT5823), confirming that the NO/cGMP signallingpathway is involved in the cellular release ofTNFRSF1A (Figure 7E).

Desensitization of hepatocytes to theTNF-a signal by 14-DAG treatments:an in vivo studyIt is known that TNF-a induces apoptosis in hepa-tocytes as in other cell types, provided that thegene transcription is blocked with transcriptionalinhibitors such as ActD. This transcriptional in-hibition appears to specifically block numerousNF-kb-dependent genes induced by TNF-a. Liverdamage was induced in male rats by an intravenousinjection of TNF-a. Serum ALT, a marker of acutehepatic dysfunction, was increased significantly 4 hafter the TNF-a injection (Figure 8A). Pretreatmentwith 14-DAG, i.p., inhibited this TNF-a-mediatedhepatic dysfunction, as indicated by the serum ALT.Next, we used immunochemistry to evaluate celldeath in the liver (Figure 8B). A substantial increasein TUNEL-positive cells revealed increased DNAfragmentation in hepatocytes from TNF-a-treatedrats as compared to untreated groups. A consider-able reduction in the number of TUNEL-positivecells was observed in the 14-DAG-pretreated TNF-agroups, thereby showing that 14-DAG effectivelyameliorated TNF-a-mediated hepatocyte apoptosis.To determine the relative distribution of TNF-abinding in in vivo conditions, we injected a bolus of125I-labelled TNF-a into the rats. The TNF-a labelledwith 125I tracer significantly bound to liver cells90 min after its administration. Pretreatment with14-DAG reduced the retention of 125I-labelledTNF-a in the liver (12% binding at 90 min)(Figure 8C). Our in vitro studies had demonstrated

�Figure 814-DAG protects rats from TNF-a-mediated liver injury. Animals were administered 14-DAG (40 mg·kg-1 body weight, i.p.), followed 2 h later byActD (800 mg·kg-1, i.p.) together with TNF-a (5 mg·kg-1 body weight) infused in a bolus dose into the tail vein. (A) The ALT level in plasma wasmeasured after 4 h of TNF-a/ActD treatment. The data represent mean � SD of three independent experiments. (B) Photomicrographs of liversections from animals treated with TNF-a/ActD treatment were stained by TUNEL technique and counterstained with haematoxylin. Apoptoticnuclei are indicated by arrows. The magnification of the photomicrograph is 40¥. Results are representative of three independent experiments.(C) Animals were injected with 14-DAG (40 mg·kg-1 body weight, i.p.) and then 2 h later [125I]-TNF-a (10 mCi)/ActD was infused in a bolus doseinto the tail vein. Binding of 125I-labelled TNF-a in hepatocytes was evaluated by a gamma counter after 45 and 90 min. The data represent mean� SD of three independent experiments. **P < 0.02, ***P < 0.01 versus TNF-a-treated cells at corresponding time. (D) The percentage of apoptoticnuclei was quantified from the photomicrographs of liver sections, which were stained by TUNEL technique and counterstained with haema-toxylin, and was analysed in the following groups: (i) TNF-a/ActD induction; (ii) 14-DAG treatment followed by TNF-a/ActD induction; and (iii)t-BuHQ (a single i.p. dose of 1.0 mmoL·kg-1 body weight in 0.15 mL of ethanol) treatment for 1 h then 14-DAG treatment and followed byTNF-a/ActD induction. The data represent mean � SD of three independent experiments. The rats were treated with D-GalN (600 mg·kg-1, i.p.)and TNF-a (5 mg·kg-1 body weight, i.v.) after 2 h of 14-DAG treatment. (E) ALT levels in plasma were measured after 4 h of TNF-a/D-GalNtreatment. The data represent mean � SD of three independent experiments. (F) FACS analysis of DNA end labelling with FITC–dUTP inhepatocytes after 4 h of TNF-a/D-GalN treatment in rats. Cell aggregates were gated out with PI staining, and FITC fluorescence was measuredrelative to a horizontal gate set by analysis of apoptotic and non-apoptotic hepatocytes. The data are representative of three independentexperiments with similar result. (G) The release of TNFRSF1A (TNFR1) in plasma of animals treated with 14-DAG (40 mg·kg-1 body weight, i.p.).TNFRSF1A (55 kDa) present in the plasma at various time periods (30 min, and 1 and 2 h) after 14-DAG treatment was analysed by immunob-lotting. Densitometric analyses of immunoblots were done. The data are shown as mean � SD of three independent experiments.

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that 14-DAG induced the release of TNFRSF1Athrough events mediated by the activation of ERCa-ATPase. Pretreatment of rats with t-BuHQ sig-nificantly prevented 14-DAG-mediated desensitiza-tion of the hepatocytes to TNF-a-mediatedapoptosis. This confirms the essential role of ERCa-ATPase in the 14-DAG-mediated hepatoprotec-tion against this cytokine (Figure 8D). To furtherspecify the effect 14-DAG on TNF-a-mediated apo-ptosis in the liver, we induced transcriptional arrestby injecting the rats with D-GalN, which is knownto induce a selective transcriptional block in hepa-tocytes (Decker and Keppler, 1974). Hepatic ALT, a

known indicator of liver injury (Leist et al., 1994),increased markedly after 4 h in D-GalN/TNF-a-treated rats, whereas only a slight increase wasobserved in the 14-DAG-pretreated rats comparedto control levels (Figure 8E). Rats pretreated with14-DAG before the D-GalN/TNF-a injection wereresistant to the lethal effects of TNF-a, as revealedby FACS analysis of TUNEL-positive hepatocytes(Figure 8F).

To confirm our in vitro results that showed a 1 hpretreatment with 14-DAG induced the extracellularrelease of TNFRSF1A from hepatocytes, we studiedthis effect of 14-DAG in our in vivo model in rats



treated with 14-DAG for 2 h. An increased presenceof soluble 55 kDa TNFRSF1A appeared in the circu-lation of these rats with time (Figure 8G), therebyconfirming the role of 14-DAG in the release ofextracellular TNFRSF1A from liver cells.

To mimic the primary aspects of the responsegenerated by the release of pro-inflammatory cytok-ines during pathological conditions, we injectedrecombinant rat TNF-a into rats at a single dose of5 mg·kg-1 body weight. This was followed by an i.v.injection of 14-DAG at various time periods.Without transcriptional arrest, the IKKb-driven clas-sical pathway is important for cells, and TNFRSF1Agenerates anti-apoptotic signals upon ligation withTNF-a (Xu et al., 1998). Induction of NF-kb wasascertained by translocation of NF-kb heterodimerinto the nucleus of hepatocytes for initiation oftranscription in TNF-a-exposed rats (Figure 9). Ourresults revealed that post-treatment with 14-DAG

20 min after the TNF-a stimulus decreased NF-kb(p65) translocation into the nucleus, therebyshowing that 14-DAG can significantly thwart thecell signalling of TNF-a. However, 14-DAG failed toaffect NF-kb (p65) translocation into the nucleus40 min after the TNF-a injection; this can beexplained by the fact that once the TNF-a gets inter-nalized in the cells, 14-DAG fails to intervene in thesignalling process.

Discussion

The aerial part of the plant AP has caught the atten-tion of researchers because of its anti-apoptoticand hepatoprotective properties (Kapil et al., 1993;Jarukamjorn and Nemoto, 2008). AG is one of themain active compounds exerting anti-inflammatoryand immunostimulant effects (Kumar et al., 2004;Sheeja et al., 2006). Several studies have suggestedthat some of the new AG analogues exhibit betteractivity than the parent compound. When the twohydroxyl groups at C-3 and C-19 of AG were substi-tuted with aromatic aldehydes, the compound wasmore potent at inducing cell cycle arrest at the G1phase (Jada et al., 2008). Reports suggest that14-DAG is a PAF antagonist and NOS activator(Zhang and Tan, 1999; Burgos et al., 2005). However,the role of 14-DAG in the modulation of pro-inflammatory cytokines is yet to be established.Both of the active compounds (AG and 14-DAG)of AP are non-toxic at therapeutic dosage (Tanet al., 2005). Some studies on the structure–pharmacological activity relationship of AG and itsanalogues have indicated the importance of thelactone moiety and the conjugated double bondD12(13) for exerting anti-inflammatory properties(Hidalgo et al., 2005). Although our study did notinvestigate the structure–activity relationship, thedehydration of AG with concomitant isomerizationof the double bond from D12 (13) to D13 (14) resulted inthe formation of 14-DAG. The changes in structureof the AG molecule altered its ability to modulatevarious biological processes, with 14-DAG beingmore effective at attenuating the cell death signalgenerated by TNF-a than the parent compound.Hence, we used 14-DAG in this study to elucidatethe molecular events involved in the inhibition ofTNF-a-mediated apoptosis in hepatocytes, andfocused on the mechanism by which 14-DAG desen-sitizes the cells to cytokine challenge.

TNF-a is associated with apoptosis of liver cellsunder the metabolic conditions of arrested tran-scription and functional translation (Leist et al.,1994). Under these conditions TNF-a initiates DISCformation and sets the process of cell death in

Figure 9Effectiveness of 14-DAG post-treatment of rats is time dependent.Animals were administered TNF-a (5 mg·kg-1 body weight) infused ina bolus dose into the tail vein followed by an i.v. injection of 14-DAG(40 mg·kg-1 body weight), 20 and 40 min later. The liver was excised4 h after the initial stimulus (TNF-a). Nuclear fractions were collectedat 4°C. NF-kb (p65) was identified in the nuclear extract by Westernblot analysis. The bands corresponding to NF-kb (p65) were quan-tified by densitometry. The data are shown as mean � SD of threeindependent experiments.

BJP DN Roy et al.


motion (Locksley et al., 2001). Ligand-boundTNFRSF1A gets internalized within a short period,resulting in ligand-induced recruitment of TRADD,FADD and caspase-8 to the death domain ofTNFRSF1A (Schneider-Brachert et al., 2006). Weobserved that activation of the intracellular deathsignalling system of enzymes in hepatocytes sensi-tized to TNF-a was initiated by a selective interplaybetween the cytoplasmic domain of the TNF recep-tor and a number of TNF receptor-associated pro-teins, which were activated after endocytosis ofTNF-a. Pretreatment of hepatocytes with 14-DAGprevented the internalization of TNFRSF1A in hepa-tocytes, resulting in the failure of recruitment ofDISC components to the membrane. These resultsaccord with previous reports, indicating that thepharmacological inhibition of TNFRSF1A internal-ization prevents TNF-a-mediated apoptosis (Yu andMalek, 2001; Levine et al., 2005). If the cells weretreated with 14-DAG after the TNF-a had been inter-nalized, 14-DAG failed to intervene with the DISCformation and apoptosis of the cell. We also foundthat 14-DAG did not inhibit the binding of TNF-a toTNFRSF1A.

Under these conditions, it was tempting tospeculate that TNFRSF1A released from hepatocyteswas responsible for the decreased internalization ofthe receptor. However, quantification of the amountof cellular TNFRSF1A and that released to the mediaprovided insights into the 14-DAG-induced declinein the TNF-a response of hepatocytes. The reductionin TNFRSF1A in the hepatocytes in the presence of14-DAG was found to be a transient. Some endog-enous molecules are known to antagonize TNF-asignalling temporarily by depletion of TNFRSF1Apresent in cells, but the cells can revive, throughintracellular metabolic processes (Wang et al., 2003).The extracellular release of TNFRSF1A from hepato-cytes in the presence of 14-DAG resulted in adecrease in the number of receptors on the hepato-cyte surface, which made the hepatocyte insensitiveto receptor–ligand binding. This transient desen-sitization of hepatocytes induced by 14-DAGaccounted for the temporary loss of the lethal effectsof TNF-a.

TNFRSF1A is principally localized to the trans-Golgi network from which it gets recruited to theplasma membrane following TNF-a activation(Wang et al., 2003; Levine, 2008). TNFRSF1A isreleased into the extracellular space by one of twomechanisms, proteolytic cleavage leading to theshedding of soluble receptor ectodomains (28–34 kDa) or the release of full-length receptors withinexosome-like vesicles (55 kDa) (Brakebusch et al.,1994; Théry et al., 2002). Proteolytic cleavage of theTNFRSF1A is known to occur primarily in the spacer

region between Asn-172 and Val-173, with a minorsite between Lys-174 and Gly-175, which results inthe shedding of cleaved soluble receptors from thecell surface (Brakebusch et al., 1994). However,during the in vitro studies with 14-DAG treatment,we observed a reduction in intracellular TNFRSF1A(55 kDa) and the release of full-length constitutiveTNFRSF1A (55 kDa) into the media. This is an alter-native pathway by which soluble cytokine receptorsare generated in the cells, and is independent ofproteolytic cleavage of the receptor ectodomain(Hawari et al., 2004; Islam et al., 2006; Zhang et al.,2008). However, we found that TAPI-2 decreased therelease of full-length TNFRSF1A, and this confirmedthat catalytic activity of TACE is essential for therelease of full-length TNFRSF1A. This is in agree-ment with the hypothesis regarding involvementof TACE in the release of 55 kDa, even though itdoes not proteolytically cleave the ectodomain ofTNFRSF1A (Hawari et al., 2004).

The function of calcium in apoptosis is particu-larly fascinating, especially when we consider theimportance of calcium in regulating a multitude ofphysiological processes and the involvement of adisrupted cellular calcium homeostasis in the patho-genesis of hepatic disorders (Tsutsui et al., 2003;Jeschke et al., 2009). Nevertheless, until now thespecific mechanisms through which 14-DAG regu-lates calcium dynamics and exerts an anti-apoptoticsignal have been elusive. We found that 14-DAGtreatment specifically induced an increase in ERcalcium and the release of TNFRSF1A from hepato-cytes. We explored the initial events that incited thehepatocytes to release full-length TNFRSF1A fromcells in the presence of 14-DAG. Calcium is animportant regulator of extracellular TNFRSF1Arelease (Islam et al., 2006). Calcium-dependentendogenous protein NUCB2 is known to participatein this process (Lin et al., 1998; Cui et al., 2002).NUCB2 is a highly charged 420-amino acid proteinwith a leucine zipper motif, two helix-loop-helixmotif regions and EF-hand motifs, which binds withcalcium through its two EF-hand domains (Krollet al., 1999). With an increase in vesicular calcium,an interaction occurs between the NUCB2 calcium-binding EF-hand domain and extracellular domainof ARTS-1 (aminopeptidase regulator), which associ-ate with TNFRSF1A prior to their constitutive releasefrom cells (Wang et al., 2003; Islam et al., 2006). Theco-immunoprecipitation experiments revealed theco-localization of NUCB2–ARTS-1 with TNFRSF1Ain 14-DAG-treated hepatocytes, which committedthe TNFRSF1A to be released from the cell. Weneeded to elucidate the mechanism, which leads toan increase in the vesicular calcium that favours theformation of the NUCB2–ARTS1–TNFRSF1A com-



plexes in hepatocytes treated with 14-DAG. A rapidincrease in the calcium concentration of vesicles canbe brought about by an increase in the activity of ERCa-ATPase (Meldolesi and Pozzan, 1998). An extractof AP is known to increase Ca-ATPase activity (Wangand Zhao, 1994; Zhi-ling et al., 1995; Burgos et al.,2003). Our study revealed that in the presence of14-DAG, an increased influx of calcium from thecytosol into the lumen of the ER occurred alongwith the activation of ER Ca-ATPase. The Ca-ATPaseactivity of ER can be modulated by several intrinsicevents occurring in the cytosol, including anincrease in NO production (Lua et al., 2003; Qihanget al., 2005). Our findings concur with those fromprevious studies by other workers, who have indi-cated the role of 14-DAG in the activation of cNOS,which stimulates NO-sensitive guanylyl cyclase inthe cells (Burgos et al., 2003; Zhang and Tan, 2007).14-DAG enhanced cGMP production in hepato-cytes, which increased ER Ca-ATPase activity,leading to an increase in intravesicular calcium. Ourin vivo data lend credence to the in vitro findings,where it is noteworthy that 14-DAG could protectthe hepatocytes against TNF-a-induced toxicity. TheTNFRSF1A release (55 kDa) in plasma of 14-DAG-treated rats agrees with our in vitro findings.

In an attempt to further evaluate the beneficialeffect of 14-DAG under in vivo conditions, it wasadministered to rats after they had been treated withTNF-a. TNF-a-induced Ikb kinase (IKK), JNK andROS pathways are highly intertwined at severallevels, leading to cell death, inflammation and pro-liferation during various liver diseases (Schwabe andBrenner, 2006). Inhibition of ROS production andJNK activation has proven to be efficacious in liverinjury (Garcia-Ruiz and Fernández-Checa, 2007).However, the initial response to TNF-a-mediatedreceptor activation is often the activation of survivalstrategy of liver cells (Yuan, 1997). Ligation ofTNFRSF1A induces protein recruitment that doesnot result in cell death unless IKKb or NF-kb activi-ties have been compromised (Xu et al., 1998). Liga-tion of TNFRSF1A activates IKK complex thatphosphorylates two serine residues (Ser32 andSer36) in the N-terminal domain of Ikba, followedby its polyubiquitination and subsequent degrada-tion by the 26S proteasome in hepatocytes. Thisresults in the release of NF-kb heterodimer whichthen enters the nucleus and regulates gene expres-sion (Caamaño and Hunter, 2002). Post-treatmentof animals with 14-DAG within 20 min of the initialTNF-a stimulus could prevent activation of NF-kb tosome extent. However, delayed administration of14-DAG in rats (after 40 min of TNF-a stimulus)could not save the hepatocytes from activation ofNF-kb. This could be a reflection of the fact that

14-DAG failed to intervene in the signals of TNF-a,because the ligand–receptor interaction had alreadyoccurred.

Under in vivo conditions, the onset of hepatictissue injury is marked by the recruitment andmigration of mononuclear cells within the perisinu-soidal space of the liver, resulting in the increasedgeneration of pro-inflammatory cytokines (Knittelet al., 1999), which leads to the activation of NF-kb(Tsukamoto, 2002). This induces de novo expressionof adhesion molecules, which is a defence mecha-nism (Jobin et al., 1998). Reports suggest that de-pletion of immune cells from the liver duringchronic stress completely inhibits the inflammatoryresponse of hepatocytes, resulting in the regressionof fibrotic scars in the liver (Iredale, 2007). Modu-lating the cytokine response may be an attractiveapproach to experimental therapeutics in inflam-matory liver diseases; however, more studies arerequired to understand the effect of 14-DAG treat-ment that reduces the severity of cytokine stimuli.

The use of this active component of the medici-nal plant AP can be used as a strategy against TNF-a-induced inflammation in the liver, which is oftena major cause of morbidity in several pathophysi-ological conditions. This study represents a pre-viously unrecognized event in which an activecomponent of AP regulates the transient constitu-tive release of soluble cytokine receptors from hepa-tocytes, and so prevents the TNF-a signallingpathway.

Acknowledgements

This work was supported by the Council of Scientificand Industrial Research (CSIR), India, project NWP0009, and CSIR fellowships (to D.N.R. and S.M.). Wethank Mrs Banasri Das for confocal microscopicstudies, and Ms Tulika Mukherjee for extraction of14-DAG.

Conflict of interest

None.

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