Evaluation of the effect of pyrrolidine dithiocarbamate in

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1 PO Box 2345, Beijing 123, Chin World J Gstroenterol 27 Mrch 21; 13(11): World Journl of Gstroenterology ISSN wjg@wjgnet.com 27 The WJG Press. All rights reserved. BASIC RESEARCH Evlution of the effect of pyrrolidine dithiocrbmte in suppressing inflmmtion in mice with dextrn sodium sulfte-induced colitis Ichiro Hirt, Shingo Ysumoto, Ken Toshin, Tkuy Inoue, Tkshi Nishikw, Noko Murno, Mitsuyuki Murno, Fng-Yu Wng, Ken-ichi Ktsu Ichiro Hirt, Deprtment of Gstroenterology, Fujit Helth University, Jpn Shingo Ysumoto, Ken Toshin, Tkuy Inoue, Tkshi Nishikw, Noko Murno, Mitsuyuki Murno, Ken-ichi Ktsu, Second Deprtment of Internl Medicine, Osk Medicl College, Jpn Fng-Yu Wng, Deprtment of Gstroenterology, Jinling Hospitl, Nnjing University School of Medicine, Nnjing 212, Jingsu Province, Chin Correspondence to: Dr. Ichiro Hirt, Deprtment of Gstroenterology, Fujit Helth University, 1-98 Dengkugkubo Ktsukke-Cho, Toyoke, Aichi , Jpn. ihirt@fujit-hu.c.jp Telephone: Fx: Received: Accepted: Abstrct AIM: To evlute the effect of pyrrolidine dithiocrbmte (PDTC; n NF-κB inhibitor) dministered t low (5 mg/kg) nd high (1 mg/kg) doses in suppressing colitis in mice with dextrn sodium sulfte (DSS)-induced colitis. METHODS: Mice were divided into DSS-untreted group (norml group), DSS-treted control group, DSS+ (low-dose group), nd DSS+ (high-dose group). In ech group, the disese ctivity index score (DAI score), intestinl length, histologicl score, nd the levels of ctivted NF-κB nd inflmmtory cytokines (IL-1β nd TNF-α) in tissue were mesured. RESULTS: The DSS+ exhibited suppression of shortening of intestinl length nd reduction of DAI score. Activted NF-κB level nd IL-1β nd TNF-α levels were significntly lower in DSS+PDTCtreted. CONCLUSION: These findings suggest tht PDTC is useful for the tretment of ulcertive colitis. 27 The WJG Press. All rights reserved. Key words: Ulcertive colitis; DSS-induced colitis; Pyrrolidine dithiocrbmte; NF-κB; Mice Hirt I, Ysumoto S, Toshin K, Inoue T, Nishikw T, Murno N, Murno M, Wng FY, Ktsu K. Evlution of the effect of pyrrolidine dithiocrbmte in suppressing inflmmtion in mice with dextrn sodium sulfte-induced colitis. World J Gstroenterol 27; 13(11): INTRODUCTION Nucler fctor-κb (NF-κB) is trnscription ctivtion fctor tht moves from the cytoplsm into the nucleus following pproprite extrcellulr signling. It is thought to ply roles in inflmmtion, immune rections, crcinogenesis nd poptosis. NF-κB is composed of fmily of genes with high degree of homology. Five subtypes of NFκB hve been identified, including NF-κB 1 (p5), NFκB 2 (p52), Rel A (p65), Rel B, nd c-rel, which form hetero- or homo-dimers. Hetero-dimers of p65 nd p5 re most bundnt in cells. NF-κB is usully found in the cytoplsm conjugted to n inhibitory protein termed IκB. Phosphoryltion of IκB by IκB kinse (IKK) following inflmmtory signl trnsduction leds to degrdtion of IκB vi proteosome, resulting in the trnsfer of NK-κB into the nucleus nd its ctivtion there [1]. Pyrrolidine dithiocrbmte (PDTC) is NF-κB inhibitor. Vrious studies hve been performed in ttempts to suppress inflmmtion medited by the NF-κB pthwy [2,3]. Németh et l reported tht tretment of intestinl epithelil cells with PDTC in vitro suppressed the ctivity of NF-κB [4]. All studies thus fr reported, involving evlution of the efficcy of PDTC in suppressing intestinl inflmmtion, were performed in vitro, nd no such in vivo study hs been reported. The present study ws undertken to evlute the efficcy of intrperitonelly dministered PDTC in suppressing inflmmtion in mice with dextrn sodium sulfte (DSS)-induced colitis in vivo. MATERIALS AND METHODS Preprtion of mouse model of DSS-induced enteritis Six-week-old femle BALB/c mice (SLC, Shizuok) were used for this study. DSS with moleculr weight of 5 (Ncli Tesque, Kyoto) ws dissolved in tp wter to obtin 5% DSS solution. Mice were llowed free ccess to

2 Hirt I et l. Effect of pyrrolidine dithiocrbmte in DSS colitis 1667 Tble 1 Disese ctivity index score Score Weight loss (%) Stool consistency Occult/grossbleeding (-) Loose Guic (+) > 15 Dirrhe Gross bleeding The disese ctivity index = (combined score of weight loss, stool consistency nd bleeding)/3. stools = well formed pellets; Loose = psty stool which do not stick to the nus; Dirrhe = liquid stools tht stick to the nus. Tble 2 Histologicl disese score group group PDTC (5 mg/kg) PDTC (1 mg/kg) Sline Sline Sline Wter Sline 5% DSS PDTC (5 mg/kg) 5% DSS PDTC (1 mg/kg) 5% DSS Scrifice Grde Grde 1 Grde 2 Grde 3 Grde 4 colonic mucos Loss of one-third of the crypts Loss of two-third of the crypts The lmin propri is covered with single lyer of epithelium nd mild inflmmtory cell infiltrtion is present Erosions nd mrked inflmmtory cell infiltrtion re present d d 3 d 7 Figure 1 Method of DSS colitis model nd dministrtion of PDTC. scoring reported by Cooper et l [6] (Tble 2). Rndomly selected 8 fields (mgnified 1 times) in ech section were inspected nd grded s bove by pthologist who ws blinded to the tretment protocol. The men in ech section ws clculted by scoring the grdes in 8 fields. this solution s drinking wter for 7 d to prepre mouse model of DSS-induced colitis. PDTC (Sigm), dissolved in distilled wter, ws dministered intrperitonelly to mice t dose levels of 1 nd 5 mg/kg. Mice were divided into four groups. In the DSS-untreted group (norml group, n = 5), ech mouse ws llowed free ccess to tp wter for 7 d. In the DSS-treted control group (control group, n = 5), ech niml ws llowed free ccess to 5% DSS solution, supplied s drinking wter, for 7 d, nd underwent intrperitonel dministrtion of physiologicl sline immeditely before nd 3 d fter the strt of DSS tretment. In the DSS+ (lowdose group, n = 5), ech niml ws llowed free ccess to 5% DSS solution for 7 d nd underwent intrperitonel dministrtion of PDTC (5 mg/kg) immeditely before nd 3 d fter the strt of DSS tretment. In the DSS+ (high-dose group, n = 5), ech niml ws llowed free ccess to 5% DSS solution for 7 d nd underwent intrperitonel dministrtion of PDTC (1 mg/kg) immeditely before nd 3 d fter the strt of DSS tretment (Figure 1). Anlysis of DAI score of colitis, evlution of intestinl shortening, nd histologicl evlution On the d 7 of the experiment, ech niml ws weighed to check for weight loss, nd the ppernce of feces nd severity of bloody stool were lso checked, followed by clcultion of the DAI score ccording to the method reported by Murthy et l [5] (Tble 1). Ech mouse ws then scrificed nd the lrge intestine ws immeditely removed for mesurement of intestinl length nd evlution of intestinl shortening. Furthermore, the histologicl score of HE-stined specimens of the distl segment of the colon ws determined in ccordnce with the method for Mesurement of cytokine nd NF-κ B ctivity in colorectl tissue On the d 7 of the experiment, protein extrct from the distl segment of the intestine of ech scrificed mouse ws obtined, using PARIS Kit (Ambion). The levels of the inflmmtory cytokines interleukin-1β (IL-1β) nd tumor necrosis fctor-α (TNF-α) in the extrct were mesured by ELISA, using the Quntkine Mouse IL-1β/ IL-1F Immunossy Kit nd Mouse TNF-α/TNFSF1A Immunossy Kit (R&D Systems, Minnepolis, MN, USA). The experiment ws repeted on other mice in the sme fshion s described bove, except for the timing of scrifice (the d 6 insted of the d 7 of the experiment). A nucler extrct from the distl segment of the intestine of ech scrificed mouse ws obtined using Nucler/ Cytosol Frctiontion Kit (Biovision). The level of NF-κB in the extrct ws mesured by ELISA, using Mercury TM TrnsFctor Kit (NF-κB p65, BD Biosciences), to evlute ctivtion of NF-κB. Sttisticl nlysis The dt ws nlyzedsis using the t-test. men ± SE vlues re presented for ech prmeter. RESULTS Evlution of enteritis (DAI score nd intestinl shortening) Compred to the control group, both DSS+ nd DSS+ exhibited mrked reduction of weight loss, improvement in ppernce of feces, nd llevition of bloody stool. DAI score, n indictor of the severity of intestinl inflmmtion, ws in the norml group, 2.73 ±.16 in the control group, 1.73 ±.27 in DSS+, nd.67 ±.3 in DSS+. Thus, significnt suppression of inflmmtion ws noted in DSS+ group nd DSS+ compred to the control group (P <.5). Suppression ws strongest in DSS+ (P <.1) (Figure 2).

3 1668 ISSN CN /R World J Gstroenterol Mrch 21, 27 Volume 13 Number men ± SE DAI score ± ±.27 b.67 ±.3 group group Figure 2 The effect of PDTC on clinicl indices (DAI). P <.5, b P <.1, vs control group. Figure 3 Comprison of length of colon in different groups. (cm) 12 men ± SE A B 1 8 NS ± ± ± ±.3 2 C D Figure 4 The effect of PDTC on length of colon. P <.5, P = NS, vs control group. NS: not significnt. DAI score ± ±.21 men ± SE.5 ±.9 Figure 6 The effect of PDTC on histologicl disese score. P = NS vs control group. NS: not significnt. The effect of DSS on the severity of intestinl shortening cused by intestinl inflmmtion ws evluted. Pictures of intestine from the norml group, the control group, nd DSS+ groups nd re shown in Figure 3. Intestinl length ws 1.5 ±.17 cm in the norml group, 7.3 ±.5 cm in the control group, 8.2 ±.3 cm in DSS+, nd 8.6 ±.3 cm in DSS+. Thus, shortening of the intestine ws not significntly suppressed in the NS Figure 5 Histologicl findings of distl colon in mouse. A: norml; B: control; C: ; D:. DSS+, but ws in the DSS+PDTCtreted (P <.5) (Figure 4). Histologicl evlution The histologicl score for the distl segment of the colon ws in the norml group, 1.2 ±.48 in the control group,.75 ±.21 in the DSS+, nd.5 ±.9 in the DSS+. Compred to the norml group (Figure 5A), the control group exhibited mrked erosion of the lmin propri mucose, disppernce of glndulr epithelium, inflmmtory cell infiltrtion, nd other relted findings (Figure 5B). In the DSS+ (Figure 5C), the findings of evlution of inflmmtion did not differ mrkedly from those in the control group. In the DSS+PDTCtreted (Figure 5D), erosion, disppernce of glndulr epithelium, inflmmtory cell infiltrtion, nd other bnormlities tended to be less severe thn those in the control group, lthough none of these differences ws sttisticlly significnt (Figure 6). Activted NK-κ B level in colonic tissue NF-κB levels in nucler extrct from distl colonic tissue were compred mong groups. The level ws.79 ±.191 ng/μg in the norml group,.938 ±.229

4 Hirt I et l. Effect of pyrrolidine dithiocrbmte in DSS colitis (ng/μg).79 ±.191 ng/μg in the control group,.175 ±.27 ng/μg in the DSS+, nd.127 ±.24 ng/μg in the DSS+. This prmeter ws thus slightly higher in the control group thn in the norml group, nd significntly lower in the DSS+ (P <.5) nd the DSS+ (P <.1). These findings suggest tht tretment with PDTC suppressed ctivtion of NF-κB (Figure 7). IL-1β nd TNF-α levels in colonic tissue Levels of inflmmtory cytokines IL-1β nd TNF-α in distl intestinl tissue were compred mong groups. IL-1β level ws.8 ±.3 pg/μg in the norml group,.143 ±.52 pg/μg in the control group,.14 ±.32 pg/μg in the DSS+, nd.77 ±.23 pg/μg in the DSS+. Thus, this prmeter tended to be lower in the DSS+ thn in the control group, lthough this difference ws not sttisticlly significnt. However, this prmeter ws significntly lower in the DSS + PDTC treted (P <.5) thn in the control group (Figure 8A). TNF-α level ws.4 ±.6 pg/μg in the norml group, 4.55 ± 2.41 pg/μg in the control group, 1.63 ± 1.12 pg/μg in the DSS + PDTC treted, nd 1.79 ±.94 pg/μg in the DSS+. Thus, this prmeter ws significntly lower in the DSS + PDTC treted group nd the DSS+ thn in the control group (P <.5) (Figure 8B). DISCUSSION.983 ± ± ±.24 men ± SE Figure 7 The effect of PDTC on NF-κ concentrtion. P <.5, b P <.1, vs control group. Ulcertive colitis is n inflmmtory bowel disese whose for which the etiology hs not yet been fully clrified. Mny studies of it hve been crried out using niml models of enterocolitis. Numerous reports hve been published concerning experiments using niml models of ulcertive colitis, i.e. rodent with DSS-induced colitis [7-1]. It is thought tht NF-κB is ssocited with the expression of vrious cytokines, chemokines, nd dhesion fctors in inflmmtion. NF-κB p65 protein hs been reported to exhibit incresed expression in nucler extrcts from locl mucosl tissue specimens of ptients with inflmmtory bowel disese [11,12]. Recently reports hve been b (pg/μg) ± A B (pg/μg).4 ± ± ± 1.8 published to evlute the inflmmtion suppressing effect of ntisense oligonucleotide for NF-κB (p65) dministered to mice with TNBS-induced enteritis ( model of Crohn s disese) [13] nd mice with DSS-induced colitis ( model of ulcertive colitis) [14] or ischemi-reperfusion injury of the smll intestine [15]. It hs been reported tht NF-κB binds to the DNA sequence termed κb sequence nd induces expression of the genes regulted by this sequence. NF-κB is usully present in n inctive form, bound to IκB, within the cytoplsm. If IκB is detched, NF-κB cn move into the nucleus to become ctivted. Of the IκB fmily members, IκB-α hs recently been shown to ply roles in the signling triggering the entry of κb into the nucleus [16]. PDTC is kind of ntioxidnts known to inhibit NF-κB. PDTC hs been reported to suppress NF-κB ctivity more powerfully thn ny other drug of the dithiocrbmte fmily. Cuzzocre et l reported tht PDTC suppresses the trnsfer of NF-κB from the cytoplsm into the nucleus by inhibition of the IκB-α degrdtion. [17,18]. In the present study, BALB/c mice were treted with 5% DSS solution to induce cute colitis. DAI score ws mrkedly higher in the control group thn in the norml group. Histologiclly, the rectum of the control group exhibited mrked inflmmtory cell infiltrtion nd erosion. As in previous study using rts with DSSinduced colitis [19], the present study reveled mrked increses in IL-1β nd TNF-α, known to produce primrily by ctivted mcrophges in the control group. In the DSS+ (mice intrperitonelly NS.14 ±.1 men ± SE.77 ±.14 men ± SE 1.63 ± ±.42 Figure 8 A: The effect of PDTC on IL-1β cytokine concentrtion. P <.5, P = NS, vs control group. NS: not significnt; B: The effect of PDTC on TNF-α cytokine concentrtion. P <.5 vs control group.

5 167 ISSN CN /R World J Gstroenterol Mrch 21, 27 Volume 13 Number 11 dministered PDTC t high dose, 1 mg/kg), suppression of intestinl shortening nd improvement of DAI score were noted, ccompnied by reduction of inflmmtory cytokines IL-1β nd TNF-α. Suppression of inflmmtion ws most mrked in DSS+, in which NF-κB level in rectl nucleus extrct ws lower thn in ny other group. These findings demonstrte tht tretment with PDTC suppresses the ctivity of NF-κB in the intestine, nd suggest tht tretment with PDTC reduces intestinl NF-κB ctivity nd suppresses the production of IL-1β nd TNF-α, resulting in llevition of DSS-induced colitis. However, on histopthologicl exmintion, no significnt suppression of inflmmtion ws noted in the DSS+ or the DSS+PDTCtreted. It hs been reported tht NFκB is ssocited with cyclin D during the cell cycle nd tht p65/p5 hetero-dimer ( NF-κB molecule) binds to the NFκB binding site of the cyclin D promoter, possibly resulting in pproprite regultion of cyclin D nd triggering of the strt of the cell cycle [2,21]. This indictes tht suppression of NF-κB with PDTC will suppress progression of the cell cycle, resulting in dely of cell prolifertion nd tissue repir. Thus, in the present study, in which inflmmtion ws not stisfctorily controlled, the mucosl dmge rising from inflmmtion ws found not to hve been llevited when exmined histologiclly, becuse suppression of NF-κB hd suppressed regene-rtion of the dmged mucos. These findings suggest tht suppression of NF-κB ctivity by PDTC cn dely the heling of mucosl tissue defects (erosions or ulcers) rising from inflmmtion, but tht it cn strongly suppress the expression of inflmmtory cytokines (IL-1β nd TNF-α), resulting in significnt llevition of colitis. We conclude tht PDTC is promising s drug cliniclly useful for the tretment of ulcertive colitis. COMMENTS Bckground In inflmmtory bowel disese, the inflmmtory cytokines IL-1β nd TNF-α ply importnt roles in inflmmtory rections in the intestinl mucos. NF-κB is one of the fctors tht induces expression of these inflmmtory cytokines. The present study ws imed to evlute the effect of NF-κB inhibitor PDTC dministered t low (5 mg/kg) nd high (1 mg/kg) doses in suppressing DSS-induced colitis in mice. Reserch frontiers Vrious studies hve been performed in ttempts to sup-press inflmmtion medited by the NF-κB pthwy. Németh et l reported tht tretment of intestinl epithelil cells with PDTC in vitro suppressed the ctivity of NF-κB. All studies thus fr reported, involving evlution of the efficcy of PDTC in suppressing intestinl inflmmtion, were performed in vitro, nd no such in vivo study hs been reported. The present study ws undertken to evlute the efficcy of intrperitonelly dministered PDTC in suppressing inflmmtion in mice with DSS-induced colitis in vivo. Innovtions nd brekthroughs These findings suggest tht suppression of NF-κB ctivity by PDTC cn dely the heling of mucosl tissue defects (erosions or ulcers) rising from inflmmtion, but tht it cn strongly suppress the expression of inflmmtory cytokines (IL-1β nd TNF-α), resulting in significnt llevition of colitis. Applictions Therefore, PDTC my be promising s drug cliniclly useful for the tretment of ulcertive colitis. Terminology Pyrrolidine dithiocrbmte (PDTC) is NF-κB inhibitor. Vrious studies hve been performed in ttempts to suppress inflmmtion medited by the NF-κB pthwy. Nucler fctor-κb (NF-κB) is trnscription ctivtion fctor which moves from the cytoplsm into the nucleus following pproprite extrcellulr signling. It is thou-ght to ply roles in inflmmtion, immune rections, crcinogenesis nd poptosis. Peer review This study is well constructed nd well documented. They clerly presented the effects of PDTC on DSS-induced colitis in mice. REFERENCES 1 Isrel A. The IKK complex: n integrtor of ll signls tht ctivte NF-kppB? Trends Cell Biol 2; 1: Long SM, Lubch VE, Tribble CG, Kz AK, Fiser SM, Cssd DC, Kern JA, Kron IL. Pyrrolidine dithiocrbmte reduces lung reperfusion injury. J Surg Res 23; 112: Bruck R, Aeed H, Schey R, Mts Z, Reifen R, Ziger G, Hochmn A, Avni Y. Pyrrolidine dithiocrbmte protects ginst thiocetmide-induced fulminnt heptic filure in rts. J Heptol 22; 36: Nemeth ZH, Deitch EA, Szbo C, Hsko G. Pyrrolidinedithio crbmte inhibits NF-kppB ctivtion nd IL-8 production in intestinl epithelil cells. Immunol Lett 23; 85: Murthy SN, Cooper HS, Shim H, Shh RS, Ibrhim SA, Sedergrn DJ. Tretment of dextrn sulfte sodium-induced murine colitis by intrcolonic cyclosporin. Dig Dis Sci 1993; 38: Cooper HS, Murthy SN, Shh RS, Sedergrn DJ. 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