Research Article Investigation of Sesamol on Myeloperoxidase and Colon Morphology in Acetic Acid-Induced Inflammatory Bowel Disorder in Albino Rats
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1 e Scientific World Journl Volume 2014, Article ID , 7 pges Reserch Article Investigtion of Sesmol on Myeloperoxidse nd Colon Morphology in Acetic Acid-Induced Inflmmtory Bowel Disorder in Albino Rts Phni Krishn Kondmudi, Hemlth Kovelmudi, Geeth Mthew, Pwn G. Nyk, Mllikrjun C. Ro, nd Rekh R. Shenoy Deprtment of Phrmcology, Mnipl College of Phrmceuticl Sciences, Mnipl University, Mnipl, Krntk , Indi Correspondence should be ddressed to Rekh R. Shenoy; rekh.shenoy@mnipl.edu Received 26 August 2013; Accepted 20 November 2013; Published 30 Jnury 2014 Acdemic Editors: M. Gluppo nd J. M. Reid Copyright 2014 Phni Krishn Kondmudi et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Bckground. Inflmmtory bowel disese (IBD) is chronic inflmmtory disorder of gstrointestinl trct of immune, genetic, nd environmentl origin. In the present study, we exmined the effects of sesmol (), which is the ctive constituent of sesme oil in the cetic cid () induced model for IBD in rts. Methods. The groups were divided into norml control, control,, nd sulfslzine (). On dy 7, the rts were killed, colon ws removed, nd the mcroscopic, biochemicl, nd histopthologicl evlutions were performed. Results. The levels of MPO, TBARS, nd tissue nitrite incresed significntly (P < 0.05) in the group wheres they reduced significntly in the nd treted groups. Serum nitrite levels were found to be insignificnt between the different groups. Conclusions. The mucosl protective effects of sesmol in IBD re due to its potentil to reduce the myeloperoxidse nd nitrite content. 1. Introduction Inflmmtory bowel disese (IBD) with its two min forms: Crohn s disese (CD) nd ulcertive colitis (UC), usully ffects the qulity of life of the ptients. The pthologicl findings include loss of mucosl integrity nd inflmmtory cell infiltrtion [1]. The pthophysiology remins elusive [2 11]. Aprt from these, rective oxygen species (ROS) lso ply n importnt role in the mucosl dmge nd proved to ply n importnt role in the progression of the disese [12, 13]. In cute colitis ptients, long with epithelil cell dmge, there is mrked infiltrtion of inflmmtory cells. The dmged epithelil cells re rpidly repired by restitution, prolifertion, nd differentition. The most criticl process for mucosl heling is restitution [14, 15]. The drug therpy is limited to immunity nd microbes instigted IBD nd hs lot of limittions due to their toxicities [16, 17]. Moreover, some drugs like NSAIDs, isotretinoin, ntibiotics, orl contrceptives, mycophenolte mofetil, ipilimumb, rituximb, nd sodium phosphte ct s custive gents for inflmmtory bowel like diseses [18]. Nowdys, the use of medicinl plnts or their ctive ingredients hve been incresed in the tretment of IBD to those which re unresponsive to the stndrd therpy. In prticulr, the focus hsbeenshiftedtothereltivelynontoxicfooddditives but lck of sufficient scientific understnding relted to their mechnism of ction limits their use into the min strem of medicl cre. The present drug, sesmol (), is componentoftrditionlhelthfoodinvriousasincountries[19]. It protects ginst therosclerosis, hypertension, nd ging [20, 21]. It hs lso been explored for wound heling [22], ntioxidnt [23], nti-inflmmtory [24], nd free rdicl scvenging ctivity [25]. The nti-inflmmtory effect of on dinitrochlorobenzene (DNCB) model of colitis hs been studied [26].Incutemodelsliketrinitrobenzenesulphonic cid (TNBS) induced colitis lso, the ctivity of sesme oil hs been explored [27] but not the ctive constituent sesmol. So, the present study investigtes the nti-inflmmtory effect of sesmol in n cute model of colitis. Of severl models for the induction of IBD, we hve used cetic cid becuse
2 2 The Scientific World Journl intrrectl dministrtion of dilute cetic cid into rodents or rbbits leds to epithelil injury nd incresed permebility followed by n cute mucosl/trnsmurl inflmmtion in dose-dependent mnner [28]. The mimicking fetures which this model shows while correlting with humn IBD re vsopermebility, prolonged neutrophils infiltrtion, nd incresed production of inflmmtory meditors [29]. So, the present study involves the nti-inflmmtory ctivity long with mucosl heling of in cetic cid () induced IBD. 2. Methods 2.1. Chemicls. Acetic cid, 2-thiobrbituric cid, nd trichloro-cetic cid wrere obtined from (HiMedi Lbortories, Mumbi, Indi). Sesmol, O-dinisidine dihydrochloride, nd Griess regent were obtined from Sigm-Aldrich, St. Louis, MO, USA. Totl protein kit ws obtined from Thermo Fisher Scientific Inc., Rockford, IL USA. Anlyticl grde chemicls were used Animls. Helthy inbred femle lbino rts of Wistr strin ( g) were used. The rts were kept in irconditioned room mintined t temperture of 23 ± 2 C with 24 hr light-drk cycle. The nimls hd free ccess to stndrd pellet diet nd wter d libitum. The experiments were pproved by Institutionl Animl Ethicl Committee (IAEC) (vide no. IAEC/KMC/88/ ) nd were crried ccording to the guidelines of the Committee for the Purpose of nd Supervision of Experiments on Animls (CPCSEA), Government of Indi. Intrrectl dministrtion of ws crried out under ketmine nesthesi Induction of Colitis nd Tretments. The protocol nd dosing strtegies were tken from the previous study with slight modifiction [30]. 24 rts were divided into 4 groups of 6 nimls ech s follows: norml control, control, Sesmol treted (), nd sulfslzine treted (). From dy 1 to dy 3, nd treted groups were given 100 mg/kg p.o. wheres treted group ws given 0.3% CMC. On dy 4, ll the nimls except control group were given cetic cid (3% v/v) by intrcolonicl dministrtion, 2 h fter dministrtion of the drugs to the nimls in their respective groups. Volume of cetic cid dministered ws 2 ml of 3% (v/v) by intrcolonicl route. After nesthetizing the rts with ketmine, n intrvenous cnnul (21G) ws inserted into the rectum [6-7 cm from the nus]. Animls were kept in tht position for few minutes nd lter wshed with sline to remove the remining cetic cid solution. (Mkesurethttheinstilledceticcidshouldnotcome outsitcuseswrithingctivityintherectum.)thecontrol nimls were instilled with distilled wter. On dy 6, drug dministrtion ws continued to the respective groups nd finlly on the 7th dy ll the nimls were scrificed. The entire colon strting from cecum ws tken nd plced on slb for mesuring the length nd weight. Around 6-7 cm of proximl prt of colon ws tken for biochemicl estimtion which includes nitrite, TBARS, nd MPO by plcing them in physiologicl buffer ph 7.4 until the homogeniztion of the smples ws crried out. A smll prt of proximl colon ws tken for histopthologicl study nd stored in 10% formlin until the histologicl studies were crried out. Before killing the nimls, blood ws collected, serum nd plsm were seprted individully from ech rt, nd the smples were estimted for nitrite levels Homogeniztion of Smples. The smples were homogenized in n ice continer t concentrtion of 10% (w/v) in 11.5 g/l solution of potssium chloride by using glss homogenizer. After this, the homogenized smples were centrifuged t 10,000 rpm for 15 min t 4 C. The superntnt ws pipetted out with microtiter pipette nd seprted into liquots for individul biochemicl estimtions Assy of Colonic MPO. Myeloperoxidse (MPO) is n enzyme found in the intrcellulr grnules of neutrophils which cn be utilized s n indirect mesure of the neutrophil content of the tissue smple [31]. The entire estimtion ws crried out in 96-well plte nd the redings were tken on microplte reder (ELx800, BioTek Instruments, Inc., Winooski, VT, USA) t 490 nm. 50 μl ofsmplews tkeninduplicte.tothis,250μl ofoda-h 2 O 2 ws dded which comprises of mg of potssium dihydrogen orthophosphte in 100 ml of distilled wter nd the ph ws djusted to 6.0. ODA solution includes mg of ODA in 1mLofphosphtebufferofpH6.0.Finlly,ODA-H 2 O 2 ws prepred by dding 1 ml of 30% of H 2 O 2 in 1 ml of ODA solution. After ddition, the reding ws noted t 5 min nd 15 min. After this, 4 M H 2 SO 4 ws dded to stop the rection ndoncegintheredingwsnoted.theconcentrtions of MPO t subsequent time intervls were determined from stndrd plot which uses horse rdish peroxidse s stndrd. Note tht H 2 O 2 nd ODA solutions re light sensitive, so they were wrpped in luminum foil. The entire experiment ws done under drk conditions especilly ddition of ODA- H 2 O 2 solution Assy of Lipid Peroxides in Colonic Homogentes. Mlondildehyde (MDA) which ws formed by the brekdown of polyunsturted ftty cids (PUFA) serves s n index for determining the extent of peroxidtion rection [32]. 250 μl of TBA-TCA regent ws dded to 250 μl of colonic homogente. The regent comprises of 15% (w/v) of trichlorocetic cid (TCA); 0.375% (w/v) of 2-thiobrbituric cid (TBA); 15 mg of butylted hydroxytoluene (BHT); nd 200 μl of 0.25 N hydrochloric cid. The solution ws kept in sonictorforhlfnhourndgentlyhetedonmgnetic stirrer for bout 1 hr to ssist the dissolution of TBA. After ddition, these smples were heted on wter bth for bout 40 min t 80 C. After heting, the smples were centrifuged t 10,000 rpm for 10 min t 4 C. The superntnt ws trnsferred to 96-well plte for mesuring the bsorbnce t 432 nm. The concentrtions of MDA were determined by constructing stndrd plot by 1,1,3,3-tetrmethoxypropne Nitrite Assy. During inflmmtion, mcrophges nd neutrophilic grnulocytes of intestinl mucous membrne re
3 The Scientific World Journl 3 ctivted nd relese lrge mounts of toxic NO [33], which would dmge the intestinl mucous membrne or even rect with superoxide nion (O 2 )ndproducemorective oxidizing substnce clled oxidized nitrous cid (OONO ). The cell membrnes nd orgnelles contin proteins nd lipids which re oxidized by these oxidizing species nd destruct the tissue in terms of free rdicl chin rection so tht the integrity of mucus membrnes s brrier is destroyed. In this ssy, 100 μl of smple(serum or colonic tissue homogente) ws tken nd to this 100 μl ofgriess regent ws dded in 96-well plte. The bsorbnce ws mesured t 540 nm by plcing the plte undisturbed in drk for10min.theconcentrtionswereclcultedwithstndrd plot by using sulfnilmide s stndrd Sttistics. The results were expressed s men ± SEM of six redings. Sttisticl significnce ws clculted by nlysis of vrince (ANOVA) followed by posthoc Tukey s multiple comprison test by using Prism 5.03 Demo Version (GrphPd Softwre, Inc., L Joll, CA, USA). P < 0.05 ws considered to be significnt. 3. Results 3.1. Body Weight. Figure 1 shows tht the intrcolonicl dministrtion of cetic cid cused the body weight to decrese from the 4th dy onwrds nd continued until the 7th dy, tht is, the dy of scrifice. Compred to control there ws significnt weight loss in nd groups which were found to be ± 5.0, ± 7.95,nd184.4 ± 2.4 g, respectively, t P < 0.05, wheres there ws no significnt weight loss in group which ws found to be ± 2.32.It ws lso found tht there ws significnt difference between thetestdrug()ndstndrddrug()tp < Colon Weight. From Figure 2, itcnbeseenthtweight of the colon incresed significntly t P < 0.05 which ws found to be ± 0.029, ± 0.091,nd1.655 ± g in,, nd tretment groups, but when compred to group, none of the tretments meliorte this effect MPO Estimtion. Figure 3 depicts significnt rise in the levels of MPO t P < 0.05 in the group which ws found to be ± μg/mg of tissue. There ws significnt decrese in the levels of MPO in drug treted groups ( nd ) when compred to group t P < 0.05,whichws found to be ± nd ± 3.33 μg/mg of tissue, respectively. In between the stndrd nd test drug tretment groups, no significnt difference ws observed Tissue Nitrite Estimtion. It is evident from Figure 4 tht when compred to the control group there ws significnt rise in the levels of tissue nitrite t P < 0.05 in the group which ws found to be 0.97 ± ng/μg ofprotein. Compred to group, both nd treted groups showed significnt decrese in the levels of tissue nitrite which were found to be 0.58 ± 0.042, nd0.69 ± 0.064, respectively. Body weight (g) Body weight Dy Figure 1: Effects of sesmol nd sulfslzine on the body weight of lbino rts. P < 0.05 s compred to positive control. (g) Colon weight Figure 2: Effects of sesmol nd sulfslzine on colon weight of rts. P < 0.05 s compred to positive control TBARS Estimtion. Figure5 exhibits significnt rise in the levels of MDA in the group t P < 0.05 which is found to be ± 3.89 nm/mg of protein. When compred to group, the levels of MDA were not significntly reduced t P < 0.05 in nd tretment groups nd were found to be ± 2.468, nd14.25 ± nm/mg of protein, respectively Estimtion of Serum Nitrite. Results were expressed s concentrtion of nitrite in μg nd lso percentge decrese with respect to control in the serum. As shown in Figure 6, when compred to control only, induced group only showed decrese in the levels of nitrite in the serum, wheres in the remining groups the decrese ws not found to be significnt.
4 4 The Scientific World Journl 250 MPO Lipid peroxidtion 30 MPO conc. (μg/mg of tissue) , b, b MDA (nm/mg of protein) Figure 3: Effect of sesmol nd sulfslzine on the levels of MPO in tissue homogentes. P < 0.05 s compred to positive control; b P < 0.05 s compred to group only. 0 Figure 5: Effect of nd on the concentrtion of MDA in the colonic tissue homogentes; P < 0.05 s compred to positive control. 1.5 Tissue nitrite 2.5 Serum nitrite Conc. (ng/μg of protein) b b Cocn. (μg) Figure 4: Effects of sesmol nd sulfslzine on the levels of tissue nitrite. P < 0.05 s compred to positive control; b P < 0.05 s compred to group only. 0.0 Figure 6: Effect of nd on serum nitrite concentrtion; P < 0.05 s compred to positive control Histopthologicl Studies. The norml histology of the colonwsnotedinthecontrol(figure 7()). In the treted group, colonic shrinkge of villi nd mucosl lyer ws clerly seen (Figure 7(b)). Sesmol nd sulfslzine treted groups were similr to control (Figures 7(c) nd 7(d), resp.) depicting norml crypts nd few inflmmtory cell infiltrtion. 4. Discussion Inflmmtory bowel disese is disorder in which both utoimmune nd immune medited disorders re involved [34]. Of the two forms, especilly in UC, n utontigen nmed humn tropomyosin isolted form 5 (htm5) plys n importnt role in the ctivtion of humorl nd cellulr medited responses [35]. Modifiction of fctors ssocited with IBD results in provision of relief to the ptients. Aprt from these fctors, rective oxygen species (ROS) lso ply n importnt role in the progression of the disese [36, 37]. Hence, we hve selected sesmol () which hs proved nti-inflmmtory [24] nd ntioxidnt ctivity [23] but its role in cetic cidinduced model of IBD hs not been found out. The mechnism by which induces colitis involves the entry of protonted form of cid into the epithelium where it dissocites to liberte protons cusing intrcellulr cidifiction tht might ccount for the epithelil injury [38].
5 The Scientific World Journl 5 () (b) (c) (d) Figure 7: Histopthologicl studies; () norml control; (b) group; (c) treted; nd (d) treted. In the present study, the ctions of ws ssessed depending upon the gross (body weight nd colon weight) nd biochemicl prmeters (MPO, lipid per-oxidtion nd nitrite). Weight loss is minly due to bdominl pin nd norexi [39]. There ws significnt reduction in the body weight in the nd tretment groups when compred to control which ws meliorted in the tretment group. Weight of colon is rised due to the inflmmtion nd lso becuse of the incresed ctivity of the fibroblsts leding to the overgrowth of musculris mucos [40]. All the groups showed significnt rise in the weight of colon when compred with control. Myeloperoxidse ctivity gives quntittive mesure of disese severity nd method of evluting drug ction in niml models of intestinl inflmmtion [31]. In our experiment, myeloperoxidse ctivity in the inflmed colon ws determined. The drug ws ble to produce reduction in the MPO ctivity, which cn be considered s mnifesttion of the nti-inflmmtory ctivities of the test compound in the model. Inthepresentstudy,inducedgroupshowedsignificnt rise in the lipid peroxides which is indictive of oxidtive stress [41]. The test drug ws ble to combt oxidtive stress by reducing the colonic tissue contents of lipid peroxides but not in significnt mnner. Nitric oxide plys n importnt role in the pthogenesis of IBD [42, 43] ndthelevelsofnohvebeenfoundtobe rised in UC, CD, nd toxic megcolon [44, 45]. NO levels in the colon of groups were found to be significntly rised when compred with norml control group which is in ccordnce with the erlier reports [46]. The tretment groups ( nd ) showed significnt decrese in the levels of NO which indicted the mucosl protective ctivity of these compounds. This ws further confirmed with histopthology findings. In contrst the levels of serum NO levels were found to be decresed in the group which is not correlting with the previously mentioned report due to unknownreson[46]. Sesme oil is shown to ccelerte the heling ctivity in TNBS induced colitis model [27]. One of the constituents of rosted sesme oil, sesmol, proved tht this is the min ctive constituent, becuse of its effective ntioxidnt [23], free rdicl scvenging properties [25] ble to suppress MPO, TBARS, nd NO. So, we might conclude tht the drug, nmely, showed comprble ctivity with nd lso protected the mucos from the hrmful effects of cetic cid. Abbrevitions CD: Crohn s disese : Sesmol : Acetic cid IBD: Inflmmtory bowel disese MPO: Myeloperoxidse ROS: Rective oxygen species : Sulfslzine TBARS: Thiobrbituric cid rective substrte UC: Ulcertive colitis.
6 6 The Scientific World Journl Conflict of Interests The uthors declre tht there is no conflict of interests regrding the publiction of this pper. Acknowledgments The uthors re thnkful to Deprtment of Phrmcology, Mnipl College of Phrmceuticl Sciences, Mnipl University, for providing the fcilities to crry out the project. They would lso like to cknowledge All Indi Council of Technicl Eduction (AICTE), New Delhi, through RPS (Reserch Promotion Scheme) for microplte reder used in the present study. References [1] R. J. Xvier nd D. K. Podolsky, Unrvelling the pthogenesis of inflmmtory bowel disese, Nture, vol. 448, no. 7152, pp , [2] P.Asquith,P.Mckintosh,ndP.L.Stokes, Histocomptibility ntigens in ptients with inflmmtory bowel disese, The Lncet,vol.303,no.7848,pp ,1974. [3] K. W. Somerville, R. F. A. Logn, M. Edmond, nd M. J. S. Lngmn, Smoking nd Crohn s disese, British Medicl Journl,vol.289,no.6450,pp ,1984. 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8 Tropicl Medicine Scientific The Scientific World Journl Autoimmune Diseses Interntionl Antibiotics Toxins Volume 2014 Anesthesiology Reserch nd Prctice Submit your mnuscripts t Advnces in Phrmcologicl Sciences Toxicology MEDIATORS of INFLAMMATION Emergency Medicine Interntionl Pin Reserch nd Tretment Addiction Stroke Reserch nd Tretment Vccines BioMed Reserch Interntionl Phrmceutics Interntionl Medicinl Chemistry Drug Delivery
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