Anti-proliferative and pro-apoptotic effects of tectorigenin on hepatic stellate cells

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1 Online Sumissions: doi:1.3748/wjg.v16.i World J Gstroenterol 21 August 21; 16(31): ISSN (print) 21 Bishideng. All rights reserved. ORIGINAL ARTICLE Anti-prolifertive nd pro-poptotic effects of tectorigenin on heptic stellte cells Jun-Hu Wu, Yu-Rong Wng, Wu-Yng Hung, Ren-Xing Tn Jun-Hu Wu, Yu-Rong Wng, Wu-Yng Hung, Ren-Xing Tn, Institute of Functionl Biomolecules, Stte Key Lortory of Phrmceuticl Biotechnology, Nnjing University, Nnjing 2193, Jingsu Province, Chin Author contriutions: Wu JH nd Wng YR performed the mjority of experiments; Hung WY did dt nlysis nd mnuscript modifiction; Tn RX nd Wu JH designed nd finnced the study. Supported y The Ntionl Nturl Science Foundtion of Chin, No. NSFC381417; Nturl Science Foundtion of Jingsu Province, No. BK28267; nd Doctorl Fund of Ministry of Eduction of Chin, No. RFDP Correspondence to: Ren-Xing Tn, Professor, Institute of Functionl Biomolecules, Stte Key Lortory of Phrmceuticl Biotechnology, Nnjing University, Nnjing 2193, Jingsu Province, Chin. rxtn@nju.edu.cn Telephone: Fx: Received: Ferury 27, 21 Revised: April 2, 21 Accepted: April 27, 21 Pulished online: August 21, 21 Astrct AIM: To investigte the effect of tectorigenin on prolifertion nd poptosis of heptic stellte cells (HSC)-T6 cells. METHODS: HSC-T6 cells were incuted with tectorigenin t different concentrtions, nd their prolifertion ws ssessed y romodeoxyuridine incorportion ssy. Apoptosis ws detected y flow cytometry ssy with Hoechst stining. Also, genertion of rective oxygen species (ROS), intrcellulr [C 2+ ]i, potentil of mitochondril memrne, ctivities of cytochrome c nd cspse-9 nd -3 were investigted to explore conceivle poptotic pthwy. RESULTS: Tectorigenin suppressed the prolifertion of HSC-T6 cells nd induced poptosis of HSC-T6 cells in time- nd dose-dependent mnner. Tectorigenin t the concentrtion of 1 μg/ml gretly inhiited the viility of HSC-T6 cells nd induced the condenstion of chromtin nd frgmenttion of nuclei. When treted for 48 h, the percentge of cell growth nd poptosis reched 46.3% ± 2.37% (P =.4) nd 5.67% ± 3.24% (P =.3), respectively. Furthermore, tectorigenin-induced poptosis of HSC-T6 cells ws ssocited with the genertion of ROS, incresed intrcellulr [C 2+ ]i, loss of mitochondril memrne potentil, trnsloction of cytochrome c, nd ctivtion of cspse-9 nd -3. CONCLUSION: Tectorigenin inhiits prolifertion of HSC-T6 cells nd induces poptosis of HSC-T6 cells. 21 Bishideng. All rights reserved. Key words: Tectorigenin; Apoptosis; Heptic stellte cells; Heptic firosis; Mitochondri; Prolifertion Peer reviewers: Richrd Hu, MD, MSc, Division of Gstroenterology, Deprtment of Medicine, Olive view-ucla Medicl Center, Olive View Drive, Los Angeles, CA 91342, United Sttes; Dr. Ktj Breitkopf, Deprtment of Medicine II, University Hospitl Mnnheim, University of Heidelerg, Theodor-Kutzer- Ufer 1-3, Mnnheim, Germny; Murizio Prol, Professor, Deprtment Medicin e Oncologi Sperimentle, University of Torino Corso Rffello 3, 1125 Torino, Itly Wu JH, Wng YR, Hung WY, Tn RX. Anti-prolifertive nd pro-poptotic effects of tectorigenin on heptic stellte cells. World J Gstroenterol 21; 16(31): Aville from: URL: htm DOI: INTRODUCTION Heptic firosis, common wound-heling response to diseses such s chronic heptitis nd lcoholic liver dmge, is result of destruction in rchitecture of the liver prenchym nd n imlnce etween firogenesis 3911 August 21, 21 Volume 16 Issue 31

2 Wu JH et l. Effects of tectorigenin on heptic stellte cells nd firolysis forming scrs or firous tissues. Heptic stellte cells (HSC), the mjor cells in heptic firosis, re responsile for the development of firosis [1,2]. Activtion nd prolifertion of HSC re the key to firogenesis while poptosis of HSC is ssocited with the resolution of firosis. So, HSC hve ttrcted incresing ttention due to their essentil role in liver firosis. With etter understnding of their iologicl properties, inhiiting the ctivtion nd prolifertion of HSC nd inducing poptosis of ctivted HSC hve een proposed s potentil nti-firosis strtegies. In trditionl Chinese medicine, Iris tectorum (I. tectorum) hs een used in tretment of liver injury for long time. It hs een shown tht tectorigenin, n importnt ioctive compound isolted from I. tectorum, hs ntioxidnt, nti-inflmmtory, nd nticncer ctivities [3-5]. Experiments on niml models hve lso demonstrted tht tectorigenin exhiits heptoprotective effect on CCl4- induced or t-bhp-induced heptic injury in rts [6,7]. Since heptic firosis is common wound-heling response to liver injury, we studied whether tectorigenin hs ntifirosis potentils nd exhiits its heptoprotective effect y plying role in liver firosis. The present study ws therefore performed to investigte the effects of tectorigenin on prolifertion nd poptosis-relted events of ctivted HSC-T6 cells nd disclose its possile mechnism underlying poptosis of HSC-T6 cells. MATERIALS AND METHODS Mterils Tectorigenin ws isolted from I. tectorum with purity of over 98% s confirmed y high-performnce liquid chromtogrphy (HPLC) nlysis. Cell culture HSC-T6 cells, n immortlized rt heptic stellte cell line, exhiit n ctivted HSC phenotype [8]. The cells, purchsed from Cncer Institute nd Hospitl, Chinese Acdemy of Medicl Sciences (Beijing, Chin), were cultured in Dulecco s-modified Egle s medium (DMEM; Gico, NY, USA) supplemented with 1 U/mL penicillin, 1 μg/ml streptomycin, nd 12% new ovine serum (Hngzhou Sijiqing Co., Ltd., Hngzhou, Chin), in humidified tmosphere contining 5% (v/v) CO2 t 37. L2 cells ( humn heptocyte cell line), purchsed from Xingy Centrl Experiment Lortory, Centrl South University, Chin, were cultured in DMEM medium (Gico, NY, USA) supplemented with 1 U/mL penicillin, 1 μg/ml streptomycin, nd 1% fetl ovine serum (Hngzhou Sijiqing Co., Ltd., Hngzhou, Chin), in humidified tmosphere contining 5% (v/v) CO2 t 37. Cell viility ssy Cells were plted in 96-well pltes t density of cells/well nd grown for 24 h. Tectorigenin t different concentrtions ws dded to the cells while only DMSO (solvent) ws dded s negtive control. After growing for 12, 24, nd 48 h, cell viility ws evluted y the reduction of 3-(4, 5-dimethylthizol-2-yl)-2, 5-diphenyl tetrzolium romide (MTT; Amresco, OH, USA) [9]. Bromodeoxyuridine uptke HSC-T6 cells were incuted for 48 h with 2, 4, nd 1 μg/ml tectorigenin, respectively. Two hours efore the cells were hrvested, romodeoxyuridine (BrdU; GenMed Scienfics Inc., USA) ws dded. The cells were fixed in 4% prformldehyde nd stined with Hoechst following the mnufcturer s protocol [1]. Morphologicl oservtion of nucler chnge HSC-T6 cells were incuted for 48 h with tectorigenin t 2, 4, nd 1 μg/ml, respectively. Nucler morphologicl chnge ws ssessed using Hoechst stining [11]. In rief, cells were fixed in 4% prformldehyde for 1 min, wshed three times with pre-chilled PBS nd exposed to 5 μg/ml of Hoechst t 37 in drk for 15 min. Smples were oserved under fluorescent microscope (Nikon UFX-Ⅱ, Jpn). Cells showing cytoplsmic nd nucler shrinkge, chromtin condenstion or frgmenttion, were defined s poptotic cells. Flow cytometry nlysis To quntify poptotic cells, HSC-T6 cells were hrvested fter exposed to tectorigenin for 24 nd 48 h, respectively, wshed twice with cold PBS, resuspended in PBS contining fluorescein isothiocynte (FITC)-conjugted nnexin V nd propidium iodide (PI) for 1 min, nd mesured using FACScn flow cytometer [12,13] (Becton Dickinson, Frnklin Lkes, NJ, USA). Mesurement of ROS genertion Intrcellulr ROS ws quntified with fluorescence plte reder using 2, 7-dichlorodihydrofluorescein dicette (DCFH-DA, Sigm) [14]. The cells on lck 96-well pltes were treted with tectorigenin t with tectorigenin t 2, 4, nd 1 μg/ml for 1, 3, 6 nd 24 h, respectively, nd incuted with DCFH-DA t 37 for 3 min. After DCFH-DA ws removed, the cells were wshed with phosphte uffered sline (PBS). DCFH-DA-loded cells were red on Sfire fluorescence plte reder (Tecn, Crilsheim, Germny). Mesurement of intrcellulr [C 2+ ]i [C 2+ ]i ws monitored using fluorescent C 2+ -sensitive dye, Fur 2-cetoxymethyl ester (Fur 2-AM) [15]. Cells were cultured nd treted with tectorigenin for 1, 3, 6 nd 24 h, respectively, nd preloded with 1 μmol/l Fur2-AM for 3 min in drk t 37 in humidified incutor. After loding with Fur2-AM, cells were collected, gently rinsed three times with D-Hnks solution, nd resuspended in D-Hnks solution contining.2% BSA t 1 6 cells/ml. Intrcellulr [C 2+ ]i ws mesured t n emission wvelength of 51 nm nd n excittion wvelength of 34 nd 38 nm on Sfire fluorescence 3912 August 21, 21 Volume 16 Issue 31

3 Wu JH et l. Effects of tectorigenin on heptic stellte cells A T6 cell viility h 24 h 1 48 h Inhiition rtes (fold of control) Concentrtions (mg/ml) Concentrtions (mg/ml) B L2 cell viility h 24 h 48 h Concentrtions (mg/ml) Figure 1 Effects of tectorigenin on the viility of heptic stellte cell -T6 (A) nd -L2 cells (B). Cells were treted for 12, 24 nd 48 h with tectorigenin t 1, 2, 4,, 8 nd 1 μg/ml, respectively, followed y ssessing the cell viility reltive to tht of untreted cells (= control). Brs represent men ± SD. plte reder (Tecn, Crilsheim, Germny). The rtio of fluorescence intensity t 34 to 38 nm (F34/F38) ws used to estimte intrcellulr free clcium. Mesurement of mitochondril memrne potentil Chnge in mitochondril memrne potentil (MMP) ws monitored using Rhodmine 123 (Rh-123) [16]. In rief, Rh-123 ws dded to cells to ttin finl concentrtion of 3 μg/ml. After incuted t 37 for 3 min, cells were collected, wshed twice with PBS, nd nlyzed with FACScn flow cytometer (Becton Dickinson, Frnklin Lkes, NJ, USA). Western lotting nlysis HSC-T6 cells were seeded into -mm dishes (1 1 6 cells/dish). After treted on the next dy for 48 h with tectorigenin t, 2, 4, nd 1 μg/ml, respectively, HSC-T6 cells were hrvested, resuspended in n ice-cold lysis uffer consisting of 5 mmol/l Tris-HCl, ph 8., 5 mmol/l KCl, 5 mmol/l DTT, 1 mmol/l EDTA,.1% SDS,.5% Triton X-1, nd protese inhiitor cocktil tlets (Roche, IN), incuted for 1 min on ice, disrupted in micro ultrsonic cell disrupter for 1 s nd centrifuged t 75 g for 15 min t 4. The superntnt (cytosolic frction) ws removed nd mintined t Figure 2 Prolifertive inhiition of tectorigenin on heptic stellte cells-t6 cells. Cells were treted for 48 h with tectorigenin t, 2, 4, nd 1 μg/ml, respectively. Dt re expressed s fold increse over tht of untreted cells. Brs represent men ± SD ( P <.5, P <.1). -8. The pellet contining mitochondri ws resolved in lysis uffer. Protein level ws mesured using stndrd colorimetric ssy kit (BCA kit). Proteins were seprted y polycrylmide/sds gel electrophoresis nd trnsferred onto polyvinylidene fluoride (PVDF) memrnes (Roche, IN). The memrnes were proed with ntiodies (cytochrome c nd cspse-9 diluted t 1:1, Cell Signling Technology, MA, USA) overnight t 4, nd incuted with HRP coupled secondry ntiody (HRP; 1:5, Cell Signling Technology, MA, USA). Detection ws performed using LumiGLO chemiluminescent sutrct system (KPL, Guildford, UK). β-ctin (1:2, Boster, Wuhn, Chin) s loding control. Results were quntified with scnning densitometer (Bio- Rd, USA). Mesurement of cspse-3 ctivity Activity of cspse-3, the min execution cspse, ws detected with cspse-3 colorimetric ssy kit (KenGen Biotech, Nnjing, Chin) ccording to its mnufcturer s instructions. Cultured HSC-T6 cells were wshed twice with cold PBS, resuspended in lysis uffer nd left on ice for 2 min. Lyste ws centrifuged t 1 r/min for 3 min t 4. Superntnts were collected nd protein concentrtions were mesured with BCA kit. Proteins (1 μg) were incuted for 4 h t 37 with rection uffer in totl volume of 15 μl contining 5 μl cspse-3 sustrte, nd detected with fluorescence microplte reder (Tecn, Crilsheim, Germny) t λ 45 nm. Sttisticl nlysis All dt were expressed s men ± SD. Origin Pro 7. sttisticl pckge ws used to determine sttisticl significnce. Difference etween two groups ws nlyzed y two-tiled Student s t-test, nd difference mong three or more groups ws nlyzed y one-wy ANOVA multiple comprisons. P <.5 or P <.1 ws considered sttisticlly significnt August 21, 21 Volume 16 Issue 31

4 Wu JH et l. Effects of tectorigenin on heptic stellte cells A B C D E Figure 3 Fluorescent stining of nuclei in heptic stellte cells-t6 cells (2 ). Cells were treted with tectorigenin for 48 h t μg/ml (A), 2 μg/ml (B), 4 μg/ml (C), μg/ml (D) nd 1 μg/ml (E), respectively. The rrows in B-E indicte the poptotic cells. Apoptotic cells (%) RESULTS 2 mg/ml 4 mg/ml mg/ml 1 mg/ml t /h Figure 4 Flow cytometry-evidenced poptosis of heptic stellte cells-t6 cells upon exposure to tectorigenin. Cells were incuted for 24 nd 48 h with tectorigenin t 2, 4, nd 1 μg/ml, respectively, followed y eing stined with fluorescein isothiocynte-conjugted nnexin Ⅴ nd propidium iodide. Brs represent men ± SD ( P <.5, P <.1). Effect of tectorigenin on viility of HSC-T6 nd L2 cells The percentge of cell growth ws significntly different etween tectorigenin-treted nd untreted groups. Tectorigenin inhiited the growth of HSC-T6 cells in dose-dependent mnner (Figure 1A). Incresed incution time of HSC-T6 cells decresed the percentge of cell growth, indicting tht tectorigenin lso inhiits the growth of HSC-T6 cells in time-dependent mnner. Tretment with tectorigenin t 1 μg/ml resulted in moderte cytotoxicity to L2 cells fter incuted for 48 h (Figure 1B). To further investigte the inhiitory effect of tectorigenin on prolifertion of HSC-T6 cells, BrdU incorportion, nother indictor of cell prolifertion, ws detected. Tectorigenin t 2-1 μg/ml could significntly inhiit the prolifertion of HSC-T6 cells (Figure 2). Tectorigenin induced poptosis of HSC-T6 cells We studied if tectorigenin cn induce poptosis of HSC-T6 cells. In rief, poptotic cells were visulized using DNA-inding Hoechst Regulr nd roundshped nuclei were oserved in control HSC-T6 cells (Figure 3). After treted for 48 h with tectorigenin t 4 nd μg/ml, condensed nuclei were found in HSC-T6 cells, which ecme smller in size, nd eventully frgmented into poptotic odies. Moreover, fter treted with 1 μg/ml tectorigenin, the nuclei of HSC-T6 cells were further condensed with the numer of poptotic odies shrply incresed. These dt suggest tht tectorigenin gretly induces the condenstion of chromtin nd frgmenttion of nuclei. The poptosis rte of HSC-T6 cells ws determined y flow cytometry nlysis with nnexin V-FITC nd PI stining. In the control group, most cells were vile. When HSC-T6 cells were exposed for 48 h to tectorigenin t nd 1 μg/ml, the percentge of poptotic cells incresed to 2.69% ± 2.57% nd 5.67% ± 3.24% (P =.3), respectively (Figure 4), which ws significntly higher thn tht of those not exposed to tectorigenin. In ddition, tectorigenin induced poptosis of HSC-T6 cells in dose- nd time-dependent mnner. Tectorigenin induced ROS genertion in HSC-T6 cells To determine whether tectorigenin is le to induce ROS genertion in HSC-T6 cells, the level of ROS, mesured using the fluorescence proe DCFH-DA, ws significntly 3914 August 21, 21 Volume 16 Issue 31

5 Wu JH et l. Effects of tectorigenin on heptic stellte cells DFC fluorescence intensity (fold of control) mg/ml 4 mg/ml mg/ml 1 mg/ml F34/F38 (fold of control) mg/ml 4 mg/ml mg/ml 1 mg/ml t /h t /h Figure 5 Tectorigenin produces rective oxygen species in heptic stellte cells-t6 cells. Cells were treted with tectorigenin for 1, 3, 6 nd 24 h, followed y 3-min incution t 37 with rective oxygen species detected y dichlorodihydrofluorescein dicette. Dt re expressed s fold increse over tht of untreted cells. Brs represent men ± SD ( P <.5, P <.1). Figure 6 Tectorigenin increses [C 2+ ]i in heptic stellte cells-t6 cells. Cells were treted with tectorigenin for 3, 6 nd 24 h. [C 2+ ]i ws detected y Fur-2/AM 38nm/34nm fluorescence rtio (F34/F38). Dt re expressed s fold increse over tht of untreted cells. Brs represent men ± SD ( P <.5, P <.1). 3 h 1 μg/ml 85.76% 79.26% Events M1 Events M Rh Rh123 6 h 1 μg/ml 24 h 1 μg/ml 71.7% 69.75% Events M1 Events M Rh Rh123 Figure 7 Tectorigenin reduces mitochondril memrne potentil in heptic stellte cells-t6 cells. After treted with tectorigenin t 1 μg/ml for 3, 6 nd 24 h, respectively, cells were hrvested nd stined with Rhodmine 123 to determine mitochondril memrne potentil y flow cytometry. higher in HSC-T6 cells thn in control cells fter treted with 1 μg/ml tectorigenin for 1, 3 (peked) nd 6 h (Figure 5). Tectorigenin incresed [C 2+ ]i in HSC-T6 cells To determine whether tectorigenin influences the level of intrcellulr C 2+, the level of intrcellulr C 2+ ws mesured with Fur 2-AM stining. After treted with 1 μg/ml tectorigenin for 3 h, the fluorescence rtio (F34/F38) incresed to 123.1% ± 7.18% compred to tht not treted with tectorigenin (Figure 6). In ddition, tectorigenin incresed cytoplsmic C 2+ in time-dependent mnner. When the incution time ws prolonged to 24 h, the F34/F38 vlue incresed from 123.1% ± 7.18% to 183.3% ± 8.64% (P =.2). Tectorigenin decresed mitochondril memrne potentil in HSC-T6 cells To determine whether tectorigenin decreses mitochondril memrne potentil in HSC-T6 cells, flow cytometry 3915 August 21, 21 Volume 16 Issue 31

6 Wu JH et l. Effects of tectorigenin on heptic stellte cells A TEC (mg/ml) Mitochondri Cytochrome c (15 kd) Cytosol Cytochrome c (15 kd) Pro-cspse-9 (47 kd) 37 kd 35 kd -ctin (46 kd) B 1. C.9 Cytochrome c/-ctin rtio Pro-cspse-9/-ctin rtio Concentrtions (mg/ml) Concentrtions (mg/ml) Figure 8 Western lotting nlysis. After 48-h exposure to tectorigenin t, 2, 4, nd 1 μg/ml, respectively, levels of cytochrome c nd cspse-9 in heptic stellte cells-t6 cells (A), long with those of cytochrome c (B) nd pro-cspse-9 (C) in cytosol, were evluted. Protein (3 μg) from ech smple ws resolved on 12% SDS-PAGE nd β-ctin ws used s loding control. Cspse-3 ctivity (fold of control) mg/ml 4 mg/ml mg/ml 1 mg/ml t /h Figure 9 Tectorigenin ctivtes pro-cspse-3 in heptic stellte cells-t6 cells. Cells were treted for 24 nd 48 h with tectorigenin t, 2, 4, nd 1 μg/ml, respectively. Dt re expressed s fold increse over tht of untreted cells. Brs represent men ± SD ( P <.5, P <.1). nlysis ws crried out using Rhodmine 123. Compred to control cells not treted with tectorigenin, HSC-T6 cells treted with 1 μg/ml tectorigenin for 24 h decresed the mitochondril memrne potentil (MMP) from 85.76% ± 6.39% to 69.75% ± 5.28% (P =.3) (Figure 7). Effect of tectorigenin on relese of cytochrome c nd ctivities of cspse-9 nd -3 To ssess whether tectorigenin-treted cells ccompny incresed cytosolic trnsloction of cytochrome c, the ctivted cspses-9 nd -3 were detected, nd the cytosolic nd mitochondril levels in cytochrome c nd intrcellulr cspses-9 nd -3 were mesured. The results revel tht tectorigenin releses mitochondril cytochrome c into cytosol in dose-dependent mnner (Figure 8B). Western lotting lso showed tht tectorigenin induced proteolytic clevge of pro-cspse-9 into the ctive form of HSC-T6 cells nd tectorigenin ctivted the cspse-9 in dosedependent mnner (Figure 8C). Tectorigenin (1 μg/ml) significntly incresed the ctivity of cspse-3 in HSC-T6 cells (Figure 9). DISCUSSION In the present study, tectorigenin, ioctive compound isolted from I. tectorum used trditionlly for severe liver disorder, suppressed the prolifertion of HSC nd induced poptosis of HSC in time- nd dose-dependent mnner. As n immortlized rt liver stellte cell line, HSC-T6 cell line exhiits n ctivted phenotype of HSC nd firolst-like morphology, which presents s useful tool in exploring heptic firosis [17]. MTT nd BrdU incorportion ssy demonstrted tht tectorigenin could inhiit the prolifertion of HSC-T6 cells, with lower cytotoxicity to humn heptocytes (L2). Furthermore, flow cytometry nlysis indicted tht tectorigenin could induce poptosis of HSC-T6 cells in dose- nd timedependent mnner. Clrifiction of the moleculr mechnism of tectorigenin underlying the discerned poptosis is of gret importnce. It hs een shown tht ROS genertion 3916 August 21, 21 Volume 16 Issue 31

7 Wu JH et l. Effects of tectorigenin on heptic stellte cells C 2+ ATpse C 2+ Figure 1 Tectorigenin produces rective oxygen species nd increses C 2+ concentrtion in cytoplsm of heptic stellte cells-t6 cells, leding to the depletion of mitochondril memrne potentil nd relese of cytochrome c from mitochondri. Cytochrome c fcilittes poptosis of heptic stellte cells-t6 cells y ctivting cspse cscde. ROS: Rective oxygen species. increses memrne permeility of mitochondri, triggers rupt mitochondril depolriztion nd relese of cytochrome c from inner mitochondril memrne [18]. Clcium homeostsis is n essentil mechnism underlying physiologicl process. Since incresed cytosolic clcium my induce mitochondri to tke up intrcellulr overloded C 2+, much lrger mount of C 2+ will ccumulte in mitochondri, which cn potentilly dmge the electron trnsfer chin, leding to filure in mintining the mitochondril memrne potentil [19-21]. Dmges to mitochondrion result in loss of its function. For exmple, relese of cytochrome c from mitochondri leds to cell poptosis [22]. In this study, tectorigenin incresed the ROS production nd C 2+ concentrtion in cytoplsm of HSC-T6 cells, the intrcellulr ccumultion of ROS nd C 2+ further induced the loss of MMP. The disruption of MMP cused relese of cytochrome c from mitochondri to cytosol. Cytosolic cytochrome c ctivted the pro-cspse-9 nd susequently, cspse-9 ctivted the downstrem effector cspses-3, eventully triggered poptosis of HSC-T6 cells (Figure 1). In conclusion, tectorigenin suppresses the prolifertion of HSC-T6 cells in dose- nd time-dependent mnner, nd produces slight cytotoxicity to L2 cells. More importntly, tectorigenin induces poptosis of HSC-T6 cells nd my hve nti-firosis potentils. COMMENTS Tec Tec Tec Tec Tec C 2+ ROS Bckground Liver firosis represents significnt helth prolem worldwide without ny currently ville nd effective therpeutic pproch. Advnced liver firosis results in cirrhosis, liver filure, nd portl hypertension, nd often requires liver trnsplnttion. The most chrcteristic feture of liver firosis is excess deposition of type I collgen. A gret numer of reserches hve een performed to understnd the moleculr mechnism underlying liver firosis. Activted heptic stellte cells (HSC) re the primry cell type responsile for the excess production of collgen. Reserch frontiers HSC re the mjor cells responsile for the development of liver firosis nd Tec Δy Cyt c Cyt c C 2+ C2+ C 2+ C 2+ C 2+ Cyt c Cyt c Mitochondri Cyt c Cspse-9 Cspse-3 Apoptosis C 2+ N + /C 2+ exchnger cirrhosis. Activted HSC re prolifertive nd firogenic, with ccumultion of extr cellulr mtrix, including α-smooth muscle ctin (α-sma) nd type I collgen. Suppression of ctivtion nd prolifertion, nd induction of poptosis in HSC hve een reported s the therpeutic strtegies ginst liver firosis. Tectorigenin, ioctive compound of Iris tectorum (I. tectorum), shows ntioxidnt, nti-inflmmtory, nticncer, nd heptoprotective ctivities, nd hs een used in therpy for liver injury. However, how tectorigenin works in live firosis hs not een unequivoclly ddressed. In this study, tectorigenin suppressed prolifertion of HSC nd induced poptotis of HSC. Innovtions nd rekthroughs Currently used drugs such s corticosteroids nd colchicine usully show vrious side effects such s immunosuppression or cytotoxicity. Tectorigenin, n ntioxidnt, nti-inflmmtory, nd heptoprotective compound isolted from I. tectorum, shows its inhiitory effect on prolifertion of HSC nd produces low toxicity. Furthermore, in vitro studies suggest tht tectorigenin produces its pro-poptotic effect on HSC is proly vi the mitochondril pthwy to some extent. Applictions By showing how tectorigenin works in HSC, this study my represent new nd future strtegy for mnging heptic firosis. Peer review This study showed tht tectorigenin inhiited the growth nd viility of rt heptic HSC-T6 cells in dose-dependent mnner. The uthors conclude tht the nti-prolifertive nd pro-poptotic effects of this component on HSC might explin its nti-firotic properties oserved in vivo. This is n interesting study. However, the mechnism of tectorigenin underlying the growth nd viility of HSC-T6 cells needs to e further studied. REFERENCES 1 de Villiers WJ, Song Z, Nsser MS, Deciuc IV, McClin CJ. 4-Hydroxynonenl-induced poptosis in rt heptic stellte cells: mechnistic pproch. J Gstroenterol Heptol 27; 22: Chong LW, Hsu YC, Chiu YT, Yng KC, Hung YT. Antifirotic effects of thlidomide on heptic stellte cells nd dimethylnitrosmine-intoxicted rts. J Biomed Sci 26; 13: Kng KA, Lee KH, Che S, Zhng R, Jung MS, Kim SY, Kim HS, Kim DH, Hyun JW. Cytoprotective effect of tectorigenin, metolite formed y trnsformtion of tectoridin y intestinl microflor, on oxidtive stress induced y hydrogen peroxide. 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Inhiitory effects of Ligusticum chunxiong on the prolifertion of rt heptic stellte cells. J Gstroenterol Heptol 26; 21: Ermk N, Lcour B, Drüeke TB, Vicc S. Role of rective oxygen species nd Bx in oxidized low density lipoproteininduced poptosis of humn monocytes. Atherosclerosis 28; 2: Shi YQ, Qu XJ, Lio YX, Xie CF, Cheng YN, Li S, Lou HX. Reversl effect of mcrocyclic isienzyl plgiochin E 3917 August 21, 21 Volume 16 Issue 31

8 Wu JH et l. Effects of tectorigenin on heptic stellte cells on multidrug resistnce in drimycin-resistnt K562/A2 cells. Eur J Phrmcol 28; 584: Yu N, Xu W, Jing Z, Co Q, Chu Y, Xiong S. Inhiition of tumor growth in vitro nd in vivo y monoclonl ntiody ginst humn chorionic gondotropin et. Immunol Lett 27; 114: Shi DH, Wu JH, Ge HM, Tn RX. Protective effect of hopehinol A, novel cetylcholinesterse inhiitor, on hydrogen peroxide-induced injury in PC12 cells. Environ Toxicol Phr 29: 28: Hong H, Liu GQ. Protection ginst hydrogen peroxideinduced cytotoxicity in PC12 cells y scutellrin. Life Sci 24; 74: Gong Y, Hn XD. Nonylphenol-induced oxidtive stress nd cytotoxicity in testiculr Sertoli cells. Reprod Toxicol 26; 22: Vogel S, Pintedosi R, Frnk J, Llzr A, Rockey DC, Friedmn SL, Blner WS. An immortlized rt liver stellte cell line (HSC-T6): new cell model for the study of retinoid metolism in vitro. J Lipid Res 2; 41: Kkkr P, Singh BK. Mitochondri: hu of redox ctivities nd cellulr distress control. Mol Cell Biochem 27; 35: Chkrorti T, Ds S, Mondl M, Roychoudhury S, Chkrorti S. Oxidnt, mitochondri nd clcium: n overview. Cell Signl 1999; 11: Orrenius S, Zhivotovsky B, Nicoter P. Regultion of cell deth: the clcium-poptosis link. Nt Rev Mol Cell Biol 23; 4: Wng X. The expnding role of mitochondri in poptosis. Genes Dev 21; 15: Xio Y, He J, Gilert RD, Zhng L. Cocine induces poptosis in fetl myocrdil cells through mitochondri-dependent pthwy. J Phrmcol Exp Ther 2; 292: 8-14 S- Editor Wng JL L- Editor Wng XL E- Editor M WH 3918 August 21, 21 Volume 16 Issue 31