Investigation into the Anti-inflammatory and Antigranuloma Activity of Colchicum luteum Baker in...

Investigation into the Anti-inflammatory and AntigranulomaActivity of Colchicum luteum Baker in Experimental Models

Vinod Nair,1 Rohit Kumar,1 Surender Singh,1,2 and Y. K. Gupta1

Abstract—The present study evaluates the anti-inflammatory and antigranuloma activity of CLHE inexperimental models, viz. carrageenan-induced paw edema, subcutaneous cotton pellet implantation-induced granuloma formation, and complete Freund's adjuvant-induced stimulation of peritoneal mac-rophages in rats. Serum TNF-alpha, IL-6, and IL-1 beta levels were estimated as markers for globaleffects of inflammation. TNF-R1 protein expression was estimated in stimulated peritoneal macro-phages. There was a significant reduction (P<0.05) of paw edema in the CLHE-treated groups ascompared to control. In the cotton pellet-induced granuloma model, there was a significant (P<0.05)reduction in the dry granuloma weight and serum TNF-alpha, IL-6, and IL-1beta levels in the CLHE-treated group as compared to control. Immunoblot analysis for TNF-R1 also demonstrated a significantreduction in the receptor protein expression on stimulated macrophages. Result of the present study thusdemonstrates and validates the antigranuloma activity of CLHE.

KEY WORDS: carrageenan-induced paw edema; cotton pellet granuloma; inflammation; proinflammatorycytokines; macrophage activation; complete Freund's adjuvant.

INTRODUCTION

Macrophage activation and dysfunction are knownto be central players in the process of chronic inflam-matory disorders and granulomatous conditions [1].Rheumatoid arthritis (RA) is one such chronic inflam-matory disorder where persistent antigen stimulation andmacrophages activation/aggregation have been shown toplay an important role in both maintenance andprogression of the disease, as is evident by the elevatedlevels of predominantly macrophage-derived proinflam-matory cytokines viz. TNF-alpha, IL-6, and IL-1beta [2].Additionally, it has also been hypothesized that activationof these macrophages in RA and other granulomatousconditions is a global phenomenon involving both synovialas well as circulating monocytes [3].

In a recent study from our laboratory, we haddemonstrated the disease-modifying activity of Colchicum

luteum hydroalcoholic extract (CLHE) in completeFreund's adjuvant (CFA)-induced arthritis in rats [4].Along with decrease in joint swelling, a decrease in serumand synovial expression of proinflammatory cytokines wasalso demonstrated in CLHE-treated animals as comparedto control. However, in the pathophysiology of CFA-induced arthritis, along with macrophages, there is also theinvolvement of fibroblasts and leukocytes like neutrophilsand lymphocytes [3, 5]. Also, colchicine, which is theprinciple anti-inflammatory alkaloid present in C. luteum,has been shown to exert an inhibitory activity on a largenumber of proinflammatory cells [6]. As a result, it couldonly be concluded that macrophage inhibition wasprobably one of the underlying mechanism responsiblefor the observed antiarthritic activity.

Therefore, the present study was carried out tospecifically evaluate the inhibitory activity of CLHE onmacrophages in a model of subcutaneous cotton pelletimplantation-induced granuloma, as this model is moredepictive of circulating macrophage activation andaggregation [7, 8]. In order to evaluate the effect oftreatment on global effect of inflammation, we estimatedthe serum levels of proinflammatory cytokines (TNF-alpha, IL-6, and IL-1beta). Additionally, we also deter-mined the expression of TNF-R1 protein on CFA-

1 Department of Pharmacology, All India Institute of MedicalSciences (AIIMS), Ansari Nagar, New Delhi 110029, India

2 To whom correspondence should be addressed at Department ofPharmacology, All India Institute of Medical Sciences (AIIMS),Ansari Nagar, New Delhi 110029, India. E-mail: [email protected]

0360-3997/12/0300-0881/0 # 2011 Springer Science+Business Media, LLC

Inflammation, Vol. 35, No. 3, June 2012 (# 2011)DOI: 10.1007/s10753-011-9389-2

881

stimulated peritoneal macrophage as this receptor has beenimplicate in most of the pathophysiological actions ofTNF-alpha [9, 10].

MATERIALS AND METHODS

Animals

Adult male albino rats (150–180 g) of the Wistarstrain from our institutional breeding stock were used inthe study. Animals were housed at 25±2°C in cleanpolypropylene cages in batches of three with access tofood and water ad libitum. The experimental protocolwas approved by the Institutional Animal Ethics Com-mittee, All India Institute of Medical Sciences, NewDelhi, and all experiments were carried out in accord-ance with the “Guidelines for care and use of animals inscientific research (Indian National Science Academy1998, Revised 2000)”.

Test Drug

Standardized C. luteum Baker hydroalcoholic (50%methanol) extract which was previously evaluated by usfor its antiarthritic activity was used for the present study[4]. The extract was golden brown in color, resinous inconsistency, and contained alkaloids, tannins, and sugars asthe principle secondary metabolites. The total alkaloidcontent of CL was found to be 0.90% w/w, and the totalcolchicine content in CL was found to be 0.19% w/w. Theoral LD50 of CLHE was found to be >2,000 mg/kg, andoral administration of the extract at a dose of 340 mg/kg for28 days did not produce any significant physiological/biochemical changes in the tested animals [4].

Carrageenan Administration-Induced Paw Edemain Rats

Overnight fasted animals were divided into fivegroups (n=6). Dose selection for the test drug was doneon the basis of our previously published report [4].Group I received vehicle (1% gum acacia perorally) andserved as the control; group II received indomethacin(3 mg/kg perorally); and groups III, IV, and V receivedCLHE at a dose of 17, 34, and 68 mg/kg respectively perorally. Sixty minutes after administration of the drug/vehicle, paw edema was induced by subcutaneousadministration of 0.1 mL of 1% λ-carrageenan (con-stituted in normal saline) into the left hind paw of theanimal [11]. Paw edema was measured using a digital

plethysmometer (Ugo Basile, Italy) at 1, 3, and 6 h post-carrageenan administration.

Cotton Pellet Implantation-Induced GranulomaFormation

Animals were divided into five groups (n=6).Group I received vehicle (1% gum acacia perorally)and served as the control; group II received indometha-cin (3 mg/kg perorally); and groups III, IV, and Vreceived CLHE at a dose of 17, 34, and 68 mg/kg,respectively, perorally. Thirty minutes after administra-tion of drug/vehicle, the animals were anaesthetized withdiethyl ether and a sterile cotton pellet (made frombleached cotton weighing 30±1 mg), saturated withnormal saline was implanted subcutaneously bilaterallybelow the axilla. This was considered to be day 0.Animals were kept under aseptic conditions for theentire duration of the study. Drug/vehicle treatment wascontinued for the duration of 6 more days. On day 7,terminal blood collection was carried out and the cottonpellets were excised. Serum was separated by centrifug-ing at 3,000 rpm and used for cytokine estimation. Thepellets were dried overnight at 60°C till a constantweight was recorded at two consecutive recordings. Thedifference between the initial and postimplantationweight was considered to be the dry weight of thegranuloma tissue [7]. Estimation of circulating cytokineswas only carried out in the serum of control, indomethacin,and CLHE- (68 mg/kg) treated animals.

ELISA for Estimation of Serum TNF-alpha Levels

TNF-alpha estimation was carried out by usingcommercial ELISA kit (U-CyTech Biosciences, TheNetherlands) according to the manufactures instructions.

Dot-Blot Analysis for Estimation of Serum IL-6and IL-1beta Levels

Dot-blot analysis for the estimation of IL-6 and IL-1beta protein was only carried out in the serum of control,indomethacin, and highest dose CLHE- (68 mg/kg) treatedanimals. Three microliters of serum from each sample wasdiluted with 7 μL of 10 mM PBS (pH 7.2) and spotted onto nitrocellulose membrane and dried at room temperature.Nonspecific sites were blocked with blocking buffer [5%Blot-Quick blocking power (Genotech) in 10 mM PBScontaining 0.05% Tween-20] for 2 h at room temperatureon a shaker. The membrane was washed thrice in washingbuffer [10 mMPBS–0.1%Tween-20] for 10min each. The

882 Nair, Kumar, Singh, and Gupta

membrane was then incubated with either anti-IL-6polyclonal primary antibody (Santa Cruz Biotechnology,Inc., Santa Cruz, USA) or anti-IL-1beta polyclonal primaryantibody (Santa Cruz Biotechnology, Inc.) overnightat 4°C. The wash was repeated and, correspondingHRP-conjugated secondary antibody (BangaloreGenei, India) was added and kept for 2 h at roomtemperature on a shaker. This was followed by threewashes in washing buffer. The blots were thendeveloped with nickel-enhanced diaminobenzidinesubstrate kit (Vector Labs, USA). Protein expressionwas evaluated by determining the intensity of darkness ofdots by a densitometer (Alpha Imager ECGel Doc System,CA, USA) using Alpha-View imaging software.

CFA-Induced Stimulation of Peritoneal Macrophages

Three groups (n=3) of male Wistar albino rats wereused in the study. After overnight fasting, group Ireceived vehicle (1% gum acacia perorally) and servedas the control, group II received indomethacin (3 mg/kgperorally), and group III received CLHE (68 mL/kgperorally). Thirty minutes after administration of drug/vehicle, 0.1 mL of CFA (0.05% w/v dead Mycobacteriumbutyricum in mineral oil) was injected into the peritonealcavity using a 26-gauge needle [12, 13]. This wasconsidered as day 0. Drug/vehicle treatment was continuedfor the duration of 6 more days.

Isolation and Culture of Rat Peritoneal Macrophages

On day 7, animals were sacrificed by an overdoseof anaesthetic ether and sterile RPMI 1640 media(10 mL) was injected into the peritoneal cavity by usinga 21-gauge needle. After gently massaging the abdomenfor 5 min, the injected media were aspirated under sterileconditions and transferred into a 15-mL centrifuge tube.The cellular content was pelleted out by centrifuging at1,000×g for 10 min at room temperature.

The pellet was resuspended and cultured asepticallyin 60-mm culture dishes at a cell number of approx-imately 106 cells/mL in RPMI 1640 medium with L-glutamine (GibcoBRL), containing NaHCO3 (2 g/L),10% FCS (v/v), penicillin (100 U/mL), streptomycin(100 μg/mL), and amphotericin B (250 ng/mL). Thecells were maintained at 37°C in an atmosphere of 95%air and 5% CO2 for 2 h. The supernatant was thendiscarded, and the adherent cells were washed twicewith ice-cold 10 mM PBS. The cells were scraped andsuspended in 1-mL Hank's balanced salt solution, andculture purity was assessed by assay for acid α-naphthyl

acetate esterase activity as described by Ennist and Jones[14] using a hemocytometer. Macrophage concentrationin the suspension obtained by the above protocol wasfound to be ≥90%. Remaining suspension wascentrifuged at 1,000×g for 10 min, and the pellet wasused for preparation of lysate for western blot analysis.

Preparation of Cell Lysate

The macrophage pellets were lysed by strongvortexing in 100 μL of lysis buffer containing 50 mMTris–HCl (pH 7.4), 300 mM NaCl, 0.5% (v/v) Triton X-100, 5 mM EDTA with 2 mM PMSF, 10 μg/mLleupeptin, and 10 U/mL aprotinin. The lysed suspensionwas kept on ice for 30 min and then centrifuged at10,000×g for 15 min at 4°C. The supernatant wascollected, and protein content was determined by usingthe Bradford method [15] with BSA as the standard.

SDS-PAGE and Western Blotting

Seventy-five micrograms of protein was separatedin 12% polyacrylamide gels (1.0 mm thick), overlaidwith a 4% stacking gel in SDS according to the standardprocedure of Laemmli [16]. The proteins were trans-ferred onto nitrocellulose membrane under an electricpotential [17]. The membrane was incubated with block-ing buffer [5% Blot-Quick blocking power (Genotech) in10 mM PBS containing 0.05% Tween-20] for 2 h at roomtemperature on a shaker. The membrane was then washedthrice in 10 mM PBS–0.1% Tween-20, for 10 min each.Anti-TNF-R1 or anti-β-actin polyclonal primary antibody(Santa Cruz Biotechnology, Inc.) was added, and themembrane was incubated overnight at 4°C. The wash wasrepeated, and corresponding HRP-conjugated polyclonalsecondary antibody (Bangalore Genei, India) was addedand kept for 2 h at room temperature on a shaker.Following three washes in PBS–Tween buffer, the blotswere developed with femtoLUCENT detection kit (SantaCruz, CA). Protein expression was evaluated by determin-ing the intensity of darkness of protein bands by adensitometer (Alpha Imager EC Gel Doc System, CA,USA) using Alpha-View imaging software. Protein con-centration of TNF-R1 was expressed as percentage ofintegrated density value (%IDV) after normalizationagainst β-actin.

Statistical Analysis

All data are expressed as mean±SE. Differencebetween groups was analyzed by one-way ANOVA

883Anti-inflammatory and Antigranuloma Activity of Colchicum luteum

followed by Dunnett's multiple comparison (GraphPadInStat; Version 3.05). P<0.05 was considered significant.

RESULTS

Anti-inflammatory Activity of CLHE

Administration of carrageenan produced a time-dependent increase in paw edema in all the testedanimals (Fig. 1). The standard drug indomethacinproduced significant reduction in paw edema at 3 and6 h post-carrageenan administration. A dose-depend-ent decrease in paw edema was observed in theCLHE-treated groups. However, unlike indomethacinwhich did not affect the first phase of inflammation,CLHE in the higher two doses (34 and 68 mg/kg)produced a significant reduction in paw edema

throughout the observation period (1, 3, and 6 hpost-carrageenan administration). Maximum reductionin paw edema was observed in the highest doseCLHE- (68 mg/kg) treated group throughout theobservation period.

Antigranuloma Activity of CLHE

Subcutaneous cotton pellet implantation led to theformation of granuloma tissue in all the tested animals(Fig. 2). CLHE produced a dose-dependent antigranu-loma activity as seen by the decrease in dry granulomaweight with increasing doses. Even though there was adecrease in granuloma weight in all CLHE-treatedanimals as compared to the control, the decrease wassignificant only in the highest dose CLHE- (68 mg/kg)treated group. Indomethacin (3 mg/kg) treatment alsosignificantly decreased granuloma formation as com-

Fig. 1. Effect of CLHE treatment on carrageenan-induced paw edema in rats. Difference between initial paw volume and paw volume at observationpoints post-carrageenan administration was considered to be the increase in paw volume (depictive of paw edema). Each bar represents the mean±SEof six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *p<0.05, **p<0.01.

Fig. 2. Effect of CLHE treatment on subcutaneous cotton pellet implantation-induced granuloma formation in Wistar rats. Difference between initialweight (30±1 mg) and weight after overnight drying was considered to be the dry weight of the granuloma tissue. Each bar represents the mean±SEof six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *p<0.05, **p<0.01.


pared to the control. CLHE (68 mg/kg) demonstratedgreater antigranuloma activity as compared to 3 mg/kgindomethacin in this model.

Effect of CLHE Treatment on Serum Cytokines

Serum TNF-alpha levels in normal animals werebelow the detectable range of the kit that was used in thestudy (U-CyTech Biosciences, The Netherlands). Cottonpellet implantation produced an increase in the serumTNF-alpha levels in all the tested animals (Fig. 3). Anapproximately twofold increase in serum TNF-alphalevels was observed in the indomethacin-treated animalsas compared to control. A similar increase in serumTNF-alpha levels was also seen in the lowest doseCLHE- (17 mg/kg) treated group. However, at the highertwo doses of CLHE, there was a decrease in circulating

TNF-alpha levels as compared to control and thisdecrease was significant at the highest dose tested.

Immunoblot analysis for estimation of IL-6 and IL-1beta levels also demonstrated a significant decrease incirculating proinflammatory cytokine levels in the CLHE-(68 mg/kg) treated group as compared to the control(Figs. 4 and 5). Indomethacin treatment only decreased IL-6 levels significantly as compared to the control. Eventhough there was a slight increase in IL-1beta levels in theindomethacin-treated animals as compared to control, thisincrease was not statistically significant.

Effect of CLHE Treatment on TNF-R1 Expressionin Peritoneal Macrophage

Initially, the percentage of IDV was calculatedseparately for TNF-R1 and β-actin. Thereafter, normal-

Fig. 3. Effect of CLHE treatment on serum TNF-alpha levels after subcutaneous cotton pellet implantation in Wistar rats. Each bar represents themean±SE of six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *p<0.05, **p<0.01.

Fig. 4. Effect of CLHE treatment on serum IL-6 levels after subcutaneous cotton pellet implantation in Wistar rats. Each bar represents the mean±SEof six animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. **p<0.01.


ization of TNF-R1 expression was carried out bycalculating the amount of TNF-R1 expressed per unitof β-actin [(%IDV−TNF-R1/%IDV−β-actin)×100].Comparison of groups demonstrated that treatmentwith indomethacin and CLHE (68 mg/kg) produced asignificant reduction in macrophage expression ofTNF-R1 protein as compared to the control (Fig. 6).A greater inhibition of TNF-R1 protein expressionwas observed in the CLHE-treated group as com-pared to indomethacin.

DISCUSSION

C. luteum Baker, an annual herb which is widelydistributed in the Western Himalayas, has been used bypractitioners of traditional medicine for the treatment ofgout, diseases of liver and spleen, rheumatoid arthritis,and other inflammatory and granulomatous disorders[18, 19]. CL methanolic extract has been demonstratedto possess potent lipoxygenase inhibitory activity inexperimental studies [20]. Additionally, the principal

Fig. 5. Effect of CLHE treatment on serum IL-1beta levels after subcutaneous cotton pellet implantation inWistar rats. Each bar represents the mean±SE ofsix animals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. **p<0.01.

Fig. 6. Effect of CLHE treatment on TNF-R1 expression in CFA-stimulated rat peritoneal macrophages. Each bar represents the mean±SE of threeanimals. Statistical analysis by one-way ANOVA followed by Dunnett's multiple comparison. *p<0.05, **p<0.01.


alkaloid of CL, colchicine, has been demonstrated toprevent polymerization of microtubules in proinflamma-tory cells and thus inhibit their chemotaxis [6]. In thepresent study, we have evaluated the anti-inflammatoryand antigranuloma activity of CLHE in experimentalmodels using the carrageenan-induced paw edemamodel, cotton pellet-induced granuloma model andadjuvant-induced stimulation of peritoneal macrophages.

Carrageenan-induced paw edema model is widelyemployed for evaluating the effect of drugs on theautacoid system [11]. This model depicts three distinctphases of autacoid involvement, the first phase (1–2 h aftercarrageenan administration) being primarily attributed tohistamine, the second phase (2–3 h after carrageenanadministration) being attributed to serotonin and kinins,and the third phase (3 h onwards) primarily attributed toprostaglandins and leukotrienes. In our study, the standarddrug indomethacin produced a significant reduction in pawedema at 3 and 6 h post-carrageenan administration, thusdepicting its primary activity against prostaglandin syn-thesis. On the other hand, CLHE demonstrated anti-inflammatory activity at all observation points, thusdemonstrating its activity against multiple inflammatorymediators involved in carrageenan-induced paw edema.

Foreign body implantation-induced granuloma for-mation is considered to be a reliable in vivo model forstudying chronic inflammation and its effect on macro-phage function. Efficacy in this model is depictive ofinhibitory activity on macrophage activation, infiltration,and aggregation [7, 8, 21]. Effect on macrophageactivation was further studied by using the CFA-inducedstimulation of peritoneal macrophages model. In ourstudy, CLHE demonstrated a dose-dependent reductionin dry granuloma weight as compared to control animalsand its efficacy was comparable to indomethacin.Further, CLHE treatment also produced a significantreduction in serum levels of TNF-alpha, IL-6, and IL-1beta in the treated animals as compared to the control.TNF-R1 receptor expression on CFA-stimulated perito-neal macrophages was also reduced in the CLHE-treatedgroup as compared to the control. Even though bothindomethacin and CLHE treatment produced a reductionin granuloma formation, the effect of these drugs on theserum levels of proinflammatory cytokines was differ-ent. Indomethacin treatment increased circulating TNF-alpha levels and decreased circulating IL-6 levels, whilehaving no effect on circulating IL-1beta levels ascompared to the control. These effects of indomethacintreatment on serum proinflammatory cytokine levels aresimilar to earlier reports [22–24]. On the other hand,

CLHE treatment produced a decrease in the circulatinglevels of all the three proinflammatory cytokines, thusdemonstrating that the antigranuloma activity is notpredominantly dependent on cyclooxygenase inhibitionas is in the case of indomethacin [25]. These effects couldhave been primarily produced by colchicine, which wasfound to be present in a concentration of 0.19% w/w in thetest drug [4] and is known to have an inhibitory effect onactivation and migration of proinflammatory cells [6].Though highly effective, its use in therapeutics is reservedowing to the severity of its adverse effects. However,although the active principle in the test drug is colchicine,toxicity studies have demonstrated its safety on acute andchronic administration in rats [4]. This absence of adverseeffects despite the presence of colchicine cannot beexplained at this moment but can only be attributed to thepresence of other phytochemicals in the test drug, whichcould have contributed towards mitigation of the adverseeffects of colchicine.

Results of the present study thus demonstrate the anti-inflammatory and antigranulomatous activity of CLHE, asit decreased both granuloma formation and the expressionof proinflammatory cytokines/cytokine receptor. Aschronic inflammation and macrophage activation/aggrega-tion has been implicated in the pathogenesis of RA andother granulomatous disease [3], we believe that ourfindings validate the clinical application of CLHE in themanagement of granulomatous disorders by practitionersof complementary and alternative medicine.

ACKNOWLEDGMENT

The study was funded by an extramural researchgrant from the Indian Council of Medical Research, NewDelhi, India. The funding agency did not have any rolein the collection, analysis, interpretation of data, writingof the report, or the decision to submit the paper forpublication.

Conflict of Interest. None to declare.

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