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Basic nutritional investigation Nutritional compounds in uence tissue factor expression and in ammationof chronic kidney disease patients in vitro Cecilia M. Shing Ph.D. a , * , Murray J. Adams Ph.D. a , Robert G. Fassett M.D., Ph.D. b , c , Jeff S. Coombes Ph.D. c a School of Human Life Sciences, University of Tasmania, Launceston, Australia b Renal Medicine, Royal Brisbane and Women ’ s Hospital Brisbane, Australia; School of Medicine, University of Queensland, Brisbane, Australia c School of Human Movement Studies, University of Queensland, Brisbane, Australia a r t i c l e i n f o Article history: Received 17 May 2010Accepted 6 October 2010 Keywords: Fish oilColostrum, CurcuminKidney diseaseCytokinesProcoagulant activity a b s t r a c t Objective: Nutritional compounds that potentially limit in ammation and tissue factor expressionmay decrease the progression of chronic kidney disease (CKD) and associated cardiovasculardisease. This project aimed to determine the effect of curcumin, bovine colostrum, and sh oil onin ammatory cytokine and tissue factor procoagulant activity of peripheral blood mononuclearcells (PBMCs) from patients with CKD before dialysis. Methods: Peripheral blood mononuclear cells from patients with CKD before dialysis ( n ¼ 13) andage- and sex-matched healthy controls ( n ¼ 12) were cultured alone and with low and high dosesof the nutritional compounds for 24 h. Cells were cultured with and without lipopolysaccharide.Supernatants were analyzed for tumor necrosis factor- a , interleukin (IL)-6, IL-8, monocyte che-moattractant protein-1, IL-1 b , C-reactive protein, and tissue factor procoagulant activity. Results: The production of C-reactive protein, monocyte chemoattractant protein-1, IL-6, and IL-1 b by PBMCs was inhibited by low- and high-dose sh oil in the CKD group ( P < 0.05). Curcumindecreased secretion of IL-6 ( P ¼ 0.015) and IL-1 b ( P ¼ 0.016). Curcumin was more effectivethan colostrum at decreasing the procoagulant activity of PBMCs in the CKD and control groups( P < 0.019). Conclusion: Fish oil decreased in ammatory cytokine secretion from CKD PBMCs. In addition, thebene cial effects of curcumin were demonstrated in decreasing in ammation in vitro, often toa similar magnitude as sh oil. 2011 Elsevier Inc. All rights reserved. Introduction Increased levels of in ammation and increased expression of tissue factor (TF) are associated with increasing severity of chronic kidney disease (CKD) [1,2]. There is growing evidencethat the elevated levels in ammation and TF that characteriseCKD contribute to the accelerated progression of cardiovasculardisease in patients with CKD [3 – 6]. Nutritional compounds withthe potential to limit in ammation and TF expression may makea signi cant contribution to the health of patients with CKD andlimit the progression of associated cardiovascular disease.Therehasbeenagrowingnumberofstudiesinvestigatingthebene tsof shoiloritsconstituentsfordecreasingin ammationin clinical populations. Fish oil supplementation for a period of 26 wk decreased the expression of proin ammatory andatherogenic genes of peripheral blood mononuclear cells(PBMCs) [7], and eicosapentaenoic acid has been demonstratedto decrease the production of in ammatory cytokines in hyper-cholesterolemic subjects [8]. Bovine colostrum has also beenshownto modulate cytokine production in healthyadultsinvivo[9] and in vitro [10]. This modulation may be attributable to an inhibition of the nuclear factor- k B (NF- k B) pathway, which isinvolvedintheupregulationofin ammatorycytokinesinPBMCs[11] and is upregulated in atherosclerotic tissue. Bovine colos-trum has recently been shown to inhibit NF- k B signaling inintestinal epithelial cells [12] and inhibition of the NF- k Bpathway has been associated with a decrease in TF, the major invivo trigger of blood coagulation [13]. Curcumin (derived from This work was supported by the Clifford Craig Medical Research Trust, Laun-ceston, Tasmania. * Corresponding author. Tel.: þ 61-3-6324-5484; fax: þ 61-3-6324-3658. E-mail address:
[email protected] (C. M. Shing).0899-9007/$ - see front matter 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.nut.2010.10.014 Contents lists available at ScienceDirect Nutrition journal homepage: www.nutritionjrnl.com Nutrition 27 (2011) 967 – 972 tumeric)hasalsobeenshowntodecreasein ammatorycytokineproductioninthediabeticrat[14]andinhibittheNF- k Bpathway[15,16].Thecomparisonoftheeffectivenessofnutritionalcompoundsassigned anti-in ammatory properties to decrease markers of in ammationandTFexpressionwithinthesamemodelis,toourknowledge,yettobeinvestigated.Thisstudyaimedtodetermineand compare the effectiveness of nutritional compounds todecrease markers associated with the progression of CKD andassociated cardiovascular disease. Speci cally, we aimed todetermine the effectiveness of curcumin, bovine colostrum, and sh oil to decrease in ammation and TF expression of PBMCsisolated from patients with CKD. We also aimed to determinehowsolublein ammatorymarkersandTFexpressioninpatientswith CKD were in uenced by lipopolysaccharide (LPS), whichfurther stimulates the in ammatory process. We hypothesizedthat these compounds would decrease the production of proin ammatorycytokines, associatedmarkersof in ammation,and the expression of TF. Moreover, this experimental modelenabled us to directly compare the effectiveness of nutritionalcompounds at concentrations re ective of plasma levels after invivo supplementation in decreasing in ammatory markers andTF procoagulant activity in vitro. Materials and methods Subjects Fasted and rested blood samples were donated by patients with CKD beforedialysis who were non-diabetic but hypertensive, with a median glomerular ltration rate of 43 mL/min per 1.73 m 2 (range 29 – 58 mL/min per 1.73 m 2 ; n ¼ 9women and 4 men, 55 y old, range 44 – 63 2 y) and age- and sex-matchedhealthy controls ( n ¼ 8 women and 4 men, 55 y old, range 44 – 64). Subjectswere excluded if they had malignant disease, were receiving immunosuppres-sants, had a cardiac event in the previous 6 mo, had an acute illness (e.g.,infection, virus), or had taken nutritional supplements in the previous 2 mo. Allpatients with CKD and healthy controls completed a medical history question-naire and gave their written informed consent. Healthy controls were free fromknown disease and not currently taking any medication. This study was con-ducted according to the guidelines laid down in the Declaration of Helsinki andall procedures involving human subjects/patients were approved by the Tas-manian statewide human research ethics committee. Written informed consentwas obtained from all subjects/patients. Whole blood collection and PBMC separation Twenty milliliters of whole blood from each subject was collected intoVacutainer tubes containing ethylenediaminetetra-acetic acid (BD Diagnostics,Franklin Lakes, NJ, USA). PBMCs were separated from whole blood byFicoll-Hypaque 1077 (Sigma, St. Louis, MO, USA) and then resuspended in RPMI-1640 medium supplemented with 10% sterile fetal calf serum and 100 U of penicillin-streptomycin-glutamine (Gibco, Invitrogen, Camarillo, CA, USA).PBMCs (1 10 6 /mL) were added to a 24-well culture plate before the addition of each nutritional compound. Cell incubation Peripheral blood mononuclear cells were cultured alone (control) and withlow and high doses of bovine colostrum, curcumin, and sh oil (3:2, eicosa-pentaenoic acid:docosahexaeonic acid). The doses added to each culture re ec-ted previously reported plasma concentrations after supplementation with eachcompound [17 – 21]. Final concentrations of nutritional compounds in the culturemedium for bovine colostrum (Intact, Numico Research Australia, Pasadena, SA,Australia) were 64.42 and 191.2 m g/mL, low-dose sh oil concentrations of eicosapentaenoic acid and docosahexaenoic acid were 0.367 and 0.258 mmol/L,and high-dose sh oil concentrations were 0.734and 0.515 mmol/L, respectively.Final concentrations of curcumin (C7727, Sigma) were 1 and 3 m M.Cells were cultured in the presence of these compounds with and withoutLPS stimulation. LPS (3 m g/mL) from Escherichia coli (K 235, Sigma) was added tocell cultures as a potent enhancer of monocytes and macrophage cytokinerelease, representing a model of systemic stress [22]. Cytokine production byPBMCreachespeakconcentrationsatorbefore24h[23],andthereforecytokinesin this study were sampled after 24 h of incubation. Cells were incubated at 37 Cwith 5% CO 2 . Cell supernatants were collected at 24 h of culture, centrifuged at900 g for 5 min to remove extraneous cells, and frozen at 80 C until analysis. Cytokine determination Quanti cation of cytokine production was chosen in preference to intracel-lularcytokinestaining todeterminetheconcentrationofcytokinessecreted withthepotentialtoalterthelocalenvironment.Supernatantconcentrationsoftumornecrosis factor- a (TNF- a ), interleukin (IL)-6, IL-8, monocyte chemoattractantprotein-1 (MCP-1), and IL-1 b were quantitatively determined using a HumanCytokine Lincoplex Kit (HCYTO-60K, LINCO Research, St. Charles, MO, USA). Theassay was performed according to the manufacturer ’ s instructions and analyzedon a Luminex 100 (Luminex Corporation, Austin, TX, USA). The lower limits of detection for individual cytokines were as follows: TNF- a , 0.66 pg/mL; IL-6,1.29pg/mL; IL-8, 2.6 pg/mL; MCP-1, 1.29 pg/ mL, and IL-1 b , 0.86 pg/mL. All samplesfrom each subject were assayed on the same day and in the same plate. Theintra-assaycoef cients of variation for each cytokinewere 10.9% for TNF- a ,12.7%for IL-6,11% for IL-8, 6.5% for MCP-1, and 13.3% for IL-1 b . TF procoagulant activity Tissue factor procoagulant activity was determined by the ability of PBMCculture supernatants to shorten the clotting time of pooled normal plasma usinga modi ed one-stage clotting assay. Fifty microliters of supernatant was incu-bated with 50 m L of pooled normal plasma for 2 min at 37 C. Fifty microliters of 25 mM CaCl 2 was then added totrigger the clotting reaction.Clotting times weredetermined in duplicate using the STarT4 coagulation analyzer (DiagnosticaStago, Parsippany, NJ, USA). Ten-fold serial dilutions (10 1 to 10 6 ) of recombi-nant thromboplastin (Innovin, Dade-Behring, Marburg, Germany) were used togenerate a dose-dependent standard curve. One unit per milliliter of TF pro-coagulantactivitywasarbitrarilyusedtogenerateaclottingtimeof100s.ACaCl 2 control (no TF) generated clotting times of > 300 s. The intra-assay coef cient of variation was 3.0%. C-reactive protein analysis Supernatant C-reactive protein (CRP) was measured using an enzyme-linkedimmunosorbent assay kit (DCRP00, R&D Systems, Inc., Minneapolis, MN, USA).The intra-assay coef cient of variation was 9.8%. Data analysis All statistical analyses were performed using SPSS 16.0 for Windows (SPSS,Inc., Chicago, IL, USA). Data were non-normally distributed and, hence, non-parametric statistical analyses were performed. Data were analyzed using theMann-Whitney test to determine differences between groups. Within-groupdifferences were determined using the Wilcoxon signed-rank test. Statisticalsigni cance was set at P < 0.05. Data are presented as median (quartiles 1 to 3). Results Cytokine secretionTumor necrosis factor- a Low-dose curcumin decreased the secretion of TNF- a intothesupernatant in the CKD group compared with the control group( P ¼ 0.047). TNF- a concentration from unstimulated CKD PBMCswas decreased after coincubation with low- and high-dose shoil ( P < 0.024; Fig.1). Bovine colostrum stimulated TNF- a releasefrom CKD PBMCs in a dose-dependent manner ( P ¼ 0.007) andlow-dose sh oil signi cantly decreased TNF- a concentrationscompared with low-dose bovine colostrum. Fish oil, curcumin,andbovinecolostrumdidnotin uencethereleaseofTNF- a fromPBMCs of healthy controls ( P < 0.05).When CKD PBMCs were stimulated with LPS, bovine colos-trum and high-dose curcumin promoted the release of TNF- a .High-dosebovinecolostrumalsostimulatedTNF- a releaseinthecontrol group ( P < 0.05). Interleukin-6 Unstimulated IL-6 secretion from CKD PBMCs was decreasedwith the addition of low-dose sh oil ( P ¼ 0.016) and low-dose C. M. Shing et al. / Nutrition 27 (2011) 967 – 972 968 bovine colostrum ( P ¼ 0.027) compared with control PBMCs.CKD PBMCsstimulatedwithLPS andculturedwithlow-dose shoil secreted lower concentrations of IL-6 ( P ¼ 0.049) comparedwith control cells for the same condition.Within the CKD group low-dose sh oil and low- and high-dose curcumin signi cantly decreased IL-6 secretion( P < 0.015), whereas LPS-stimulated PBMCs cocultured withhigh-dose curcumin promoted IL-6 release ( P ¼ 0.028; Fig. 2).Control PBMCs stimulated with LPS secreted more IL-6 whencoculturedwithlow-dose sh oiland low-and high-dose bovinecolostrum and curcumin ( P < 0.034).Curcumin decreased IL-6 secretion from CKD PBMCs ina dose-dependent manner ( P < 0.012). Fish oil was superior tobovine colostrum in decreasing IL-6 secretion in the CKD group. Interleukin-8 There was no signi cant group effect for IL-8 secretion( P > 0.05); however, within the CKD group high-dose sh oildecreased IL-8 secretion from unstimulated PBMCs ( P ¼ 0.02),whereas high-dose bovine colostrum stimulated IL-8 secretion( P ¼ 0.009; Table 1). For control PBMCs high- and low-dosebovine colostrum and low-dose curcumin stimulated IL-8secretion ( P < 0.021), whereas high-dose sh oil decreased IL-8concentrations ( P ¼ 0.004).Fish oil decreased IL-8 secretion to a greater extent thanbovine colostrum and curcumin in the CKD group ( P < 0.023),whereas sh oil was superior to bovine colostrum in the controlgroup. Monocyte chemoattractant protein-1 There was no signi cant group effect for MCP-1 secretion( P > 0.05); however, low- and high-dose sh oil decreased thesecretion of MCP-1 from unstimulated CKD PBMCs ( P ¼ 0.002;Table 1). Only high-dose sh oil decreased MCP-1 secretion inthe control group ( P ¼ 0.01). Low-dose bovine colostrumsigni cantly increased the secretion of MCP-1 ( P ¼ 0.034) in theCKD group. Bovine colostrum stimulated MCP-1 release ina dose-dependent manner, with low-dose bovine colostrumstimulatingMCP-1productiontoagreaterextentthanhigh-dosebovine colostrum ( P ¼ 0.028).When cells were stimulated with LPS, high-dose sh oilinhibited MCP-1 secretion in the CKD and control groups( P < 0.011). In the control group low-dose sh oil also decreasedLPS-stimulated MCP-1 secretion, whereas low-dose curcuminstimulated MCP-1 release.Fish oil decreased MCP-1 secretion to a greater extent thanbovine colostrum and curcumin in the CKD and control groups( P < 0.05). Interleukin-1 b Unstimulated PBMC production of IL-1 b alone or whenincubated with sh oil, bovine colostrum, or curcumin was notsigni cantly different between groups ( P > 0.05). When stimu-lated with LPS and incubated with low-dose sh oil CKD PBMCssecreted lower concentrations of IL-1 b ( P ¼ 0.01) than controlcells (Table 1). Low- and high-dose sh oil and high-dose cur-cumin inhibited IL-1 b secretion from unstimulated CKD PBMCs( P < 0.004), whereas high-dose bovine colostrum potentiatedthe release of IL-1 b for the CKD ( P ¼ 0.023) and control( P ¼ 0.016) groups.Fish oil decreased, whereas high-dose bovine colostrum andlow-dose curcumin stimulated, LPS-induced IL-1 b secretion inthe control group. CKD PBMCs stimulated with LPS secreted lessIL-1 b when cocultured with low- and high-dose sh oil (up toa 98% decrease; P < 0.005).High-dose bovine colostrum stimulated the secretion of IL-1 b from PBMCs signi cantly more than low-dose bovine colostrum( P < 0.016) in both groups. Fish oil decreased unstimulated andstimulated IL-1 b secretion to a greater extent than bovinecolostrum in the CKD group ( P < 0.029). AB Fig. 1. TNF- a concentrations from (A) chronic kidney disease PBMCs and (B)healthy control PBMCs. * Signi cantly different from PBMCs; # signi cantlydifferent from PBMCs þ LPS. H, high-dose; L, low-dose; LPS, lipopolysaccharide;PBMC, peripheral blood mononuclear cell; TNF- a , tumor necrosis factor- a . Fig. 2. IL-6 concentrations from (A) chronic kidney disease PBMCs and (B) healthycontrol PBMCs. * Signi cantly different from PBMCs; # signi cantly different fromPBMCs þ LPS. H, high-dose; L, low-dose; IL-6, interleukin-6; LPS, lipopolysaccha-ride; PBMC, peripheral blood mononuclear cell. C. M. Shing et al. / Nutrition 27 (2011) 967 – 972 969 C-reactive protein Production of CRP by unstimulated PBMCs was greater in theCKD group compared with healthy controls ( P ¼ 0.04). CRPproduction was also greater for the CKD group when PBMCswere incubated with low-dose sh oil ( P ¼ 0.008) and low- andhigh-dose bovine colostrum ( P ¼ 0.013 and P ¼ 0.04, respec-tively). PBMCs cultured with low-dose bovine colostrum andhigh-dose curcumin and stimulated with LPS were also associ-ated with higher CRP concentrations in the CKD group than inthe control group ( P ¼ 0.028).The production of CRP by PBMCs was inhibited by low- andhigh-dose sh oil and stimulated with low-dose curcumin in theCKD group ( P < 0.029; Fig. 3). In the healthy controls only low-dose sh oil decreased CRP production ( P ¼ 0.043), whereashigh-dose curcumin stimulated CRP release ( P ¼ 0.043). Stimu-lated PBMCs from the CKD group produced less CRP whenincubated with low and high doses of sh oil ( P < 0.038),whereas low and high doses of curcumin and high-dose bovinecolostrum potentiated CRP release ( P < 0.017). For the controlgroupthe addition of low-dose sh oil toPBMCs stimulatedwithLPS decreased CRP secretion ( P ¼ 0.043).Low-dose sh oil inhibited unstimulated CRP release toa greater extent than low doses of bovine colostrum and curcu-min in the CKD ( P < 0.029) and control ( P < 0.044) groups.LPS-stimulated PBMCs produced less CRP when cocultured withlow-andhigh-dose shoilintheCKDgroup( P < 0.023)andlow-dose sh oil in the control group ( P ¼ 0.043) compared withbovine colostrum and curcumin. TF procoagulant activity Therewasnosigni cantgroupeffectforTFactivity( P > 0.05);however, within-group differencesweredemonstrated(Table1).Low-dose curcumin was more effective at decreasing TF pro-coagulant activity of PBMCs for the CKD and control groups( P < 0.019)comparedwithlow-dosebovinecolostrum.Low-andhigh-dose bovine colostrum stimulated TF procoagulant activityin the control group ( P < 0.009), whereas only high-dose bovinecolostrum signi cantly stimulated TF procoagulant activity fromCKD PBMCs ( P ¼ 0.028). In contrast to CKD PBMCs, controlPBMCscoculturedwith LPSand bovineand curcumin stimulatedTFprocoagulantactivity( P < 0.017).Low-dose shoilstimulatedLPS-induced TF activity in both groups ( P < 0.035). Discussion This study investigated the effects of nutritional compoundswith anti-in ammatory properties to decrease the production of in ammatory markers and decrease TF procoagulant activity of PBMCs from patients with CKD. Fish oil was superior to bovine Fig. 3. CRP concentrations from (A) chronic kidney disease PBMCs and (B) healthycontrol PBMCs. * Signi cantly different from PBMCs; # signi cantly different fromPBMCs þ LPS. CRP, C-reactive protein; H, high-dose; L, low-dose; LPS, lipopoly-saccharide; PBMC, peripheral blood mononuclear cell. Table 1 Cytokine concentrations and TF activity in unstimulated and stimulated PBMCs exposed to low and high concentrations of sh oil, bovine colostrum, and curcumin * IL-1 b (pg/mL) MCP-1 (pg/mL) IL-8 (pg/mL) TF activityCKD Control CKD Control CKD Control CKD ControlUnstimulated PBMC 29.4 (4 – 149) 3.8 (0.6 – 28.3) 1276 (1057 – 4012) 1554 (723 – 3424) 3480 (1706 – 50 854) 5439 (3131 – 24 981) 0 (0 – 0.01) 0 (0 – 0) L sh 0 y (0 – 17.7) 4.1 (0 – 58.9) 40 y (15 – 117) 28 (20 – 309) 2069 y (1017 – 4132) 2955 (1330 – 37 104) 0 (0 – 0) 0 (0 – 0.1) H sh 0.6 (0 – 21.3) 0.6 (0 – 4.7) 40 y (10 – 108) 11 y (0 – 24) 1160 y (659 – 2386) 1133 y (469 – 2280) 0 (0 – 0) 0 (0 – 0.4) L bovine 82 (17 – 143) 102 (89 – 195) 4428 y (1527 – 6160) 2764 (1483 – 8295) 44 747 (21 714 – 85 238) 94 411 y (30 922 – 170 533) 0.01 (0 – 0.89) 0.13 y (0 – 1.2) H bovine 206 y (126 – 368) 236 (137 – 585) 2468 (757 – 5218) 1975 (1292 – 3236) 74 391 y (43 335 – 108 181) 98 136 y (43 990 – 200 006) 0.06 y (0 – 10.1) 2.7 y (0.4 – 19.9) L curcumin 4.4 (0 – 35.7) 8.3 (2.7 – 189) 2919 (793 – 5232) 2492 (1250 – 3724) 3417 (1857 – 8527) 12 466 y (4291 – 40 063) 0 (0 – 0) 0 (0 – 0) H curcumin 4.4 y (0 – 26.2) 0.6 y (0 – 27.7) 1545 (846 – 4433) 900 (473 – 5787) 5522 (2182 – 25 098) 5710 (1851 – 12 521) 0 (0 – 0) 0 (0 – 0)Stimulated PBMC 414 (186 – 487) 476 (197 – 862) 1909 (793 – 3016) 1611 (545 – 2868) 69 596 (4579 – 94 512) 26 718 (9937 – 143 229) 0.2 (0.0 – 27) 1.0 (0.0 – 15.4) L sh 62 z (25.6 – 119) 268 z (139 – 543) 562 (103 – 1623) 968 z (122 – 1827) 53 438 (8730 – 150 538) 72 906 (29 313 – 123 820) 8.6 z (1.1 – 42.5) 13.9 z (0.9 – 50.3) H sh 34.1 z (8.7 – 53.5) 102 z (5.6 – 281) 56 z (20 – 138) 50 z (8 – 762) 25 906 (10 822 – 36 703) 36 861 (7681 – 50 912) 1.5 (0 – 18.1) 3.9 (0.0 – 18.2) L bovine 358 (76.8 – 504) 639 (191 – 1089) 1357 (663 – 2657) 1828 (287 – 2981) 54 146 (30 120 – 103 964) 81 278 (37 114 – 177 955) 0.7 (0.0 – 4.3) 4.05 z (18 – 48.7) H bovine 510 (271 – 782) 961 z (373 – 1206) 1123 (648 – 1968) 1157 (1023 – 2550) 80 758 (34 566 – 122 947) 111 174 (40 082 – 118 864) 0.5 (0.1 – 27.4) 8.5 z (2.3 – 130) L curcumin 417 (296 – 730) 846 z (456 – 1521) 1481 (765 – 2404) 1223 z (1111 – 2055) 81 771 (46 261 – 118 219) 85 219 (43 157 – 153 810) 0.2 (0.0 – 16.3) 13.8 z (0.2 – 95.1) H curcumin 356 (238 – 782) 805 (285 – 1389) 1759 (955 – 2760) 1488 (1041 – 2434) 85 828 (40 823 – 122 591) 90 172 (39 932 – 143 050) 0.2 (0.0 – 27.1) 9.8 z (0.5 – 42.2)CKD, chronic kidney disease; H, high-dose; IL-1 b , interleukin-1 b ; IL-8, interleukin-8; L, low-dose; MCP-1, monocyte chemoattractant protein-1; PBMC, peripheral blood mononuclear cells; TF,tissue factor * Data are presented as median (quartiles 1 to 3). y Signi cantly different from unstimulated PBMCs. z Signi cantly different from stimulated PBMCs. C. M. Shing et al. / Nutrition 27 (2011) 967 – 972 970 colostrum and curcumin in decreasing unstimulated in amma-tory cytokine and CRP production from cultured PBMCs.Furthermore, sh oil decreased LPS-stimulated TF procoagulantactivity, whereas bovine colostrum potentiated TF procoagulantactivity and the secretion of TNF- a , IL-6, IL-1 b , and MCP-1 inPBMCs from the CKD group. Our results provide further supportfor the ef cacy of sh oil as a bene cial compound for CKD andare the rst to demonstrate the bene cial effects of curcumin indecreasingIL-6andIL-1 b ,oftentoasimilarmagnitudeas shoil.Our experimental model enabled us to directly compare theeffectiveness of nutritional compounds in decreasing in am-matory markers and TF procoagulant activity in vitro atconcentrations re ective of plasma levels after in vivo supple-mentation. Curcumin is absorbed into the bloodstream withoutchanges in chemical composition, and despite poor bioavail-ability, concentrations of curcumin chosen for the present studyare re ective of plasma levels after ingestion [19]. Fish oil andbovine colostrum concentrations in vitro are also re ective of plasma concentrations after supplementation [17,18,20,21]. Alimitation of the present work is that the effect of the nutritionalcompounds was not determined after an in vivo supplementa-tion period; however, the experimental model allows for novelcomparisons between compounds because the concentrationschosen are typical of plasma concentrations after ingestion.Work by Bouwens et al. [7] showed that sh oil decreased PBMCmRNA expression of different in ammatory markers (includingIL-6) after in vivo supplementation. In the present study weshowedthat shoildecreasedIL-6secretion,suggestingthat shoil in vitro may be re ective of in vivo actions.In ammation is characteristic of CKD, with patients display-ing increased levels of proin ammatory TNF- a [24] and IL-6 andIL-1 b [1] in combination with other markers of in ammation.Curcumin [25] and eicosapentaenoic acid have been shown toinhibit the activation of NF- k B in cancer cells; more recently,bovine colostrum has been shown to inhibit the transcriptionfactor in intestinal epithelial cells [12]. NF- k B may be stimulatedby TNF- a and its expression is increased in vessels prone toatherosclerosis [26]. Decreasing TNF- a secretion could havepotentially bene cial effects for CKD and associated cardiovas-cular disease. In the present study the suppression of TNF- a andIL-6 production from PBMCs was greater in the CKD groupcompared with healthy controls when coincubated with low-dose curcumin and low-dose sh oil, respectively. Receptorexpression of toll-like receptor-4 [27] is decreased and advancedglycation endproduct receptor is increased [28] in uremicpatients. Differences in responsiveness of CKD PBMCs to nutri-tional compounds compared with healthy controls may beattributable to differences in receptor expression, in uencingsensitivity to these compounds.The in ammatorycytokine MCP-1 is believed to playa roleinmonocyte recruitment of early atherosclerotic lesions [29].Plasma levels of MCP-1 are increased with increasing severity of CKD [30] and are associated with plaque formation and desta-bilization [31]. Recent work by Bouwens et al. [7] showed that sh oil supplementation decreased in ammatory and athero-genic gene expression of PBMCs. The dose-dependent inhibitionof MCP-1, IL-1 b , TNF- a , and IL-6 by sh oil in the present studycould prove bene cial in decreasing the progression of cardio-vascular disease in patients with CKD.In combination with in ammatory cytokines, increased CRPlevels are a predictor of morbidity and cardiovascular events inpatients with CKD [32]. Findings on decreased systemic CRPaftera period of sh oil supplementation are equivocal [33,34].However, in the present study low- and high-dose sh oilsigni cantly decreased the production of CRP from unstimulatedand LPS-stimulated PBMCs. In contrast to sh oil, curcuminpotentiated the release of CRP from PBMCs. Curcumin inhibitsNF- k Bincancercelllinesandwehypothesizedthatthecompoundwould decrease CRP production, particularly in stimulatedcultures, because inhibitors of NF- k B have been shown todecreased LPS-induced expression of CRP from PBMCs [35].Although curcumin did not decrease the secretion of CRP fromPBMCs, it may provide a protective effect after monocyte differ-entiation.Macrophagelipoproteinlipaselevelsareupregulatedinatheroscleroticlesionsandcurcuminhasbeen shownto decreasethe expression of CRP-induced lipoprotein lipase from macro-phages [36].Although curcumin did not suppress the secretion of CRP, itwas effective at decreasing the cytokine secretion of IL-6 and IL-1 b , often to a similar magnitude as sh oil. Long-term increasesin IL-6 are associated with increased cardiovascular risk [37],possibly mediated by signal transducer and activator of tran-scription3phosphorylation,whichisinhibitedbycurcumin[16].Curcuminhasalsorecentlybeenshowntoamelioraterenalfailurein nephrectomized rats, which was associated with a decrease inserum TNF- a concentration and an increase in peroxisome pro-liferator-activatedreceptor- g [38],aregulatorofin ammation[39].TNF- a concentrations were decreased in the present studyafter incubation with curcumin, but this was not signi cant.Tissue factor is well described as the physiologic trigger of invivo blood coagulation. Indeed, high levels of TF are associatedwith an increased risk of developing blood clots [40] and havebeen linked to atherosclerosis [2,41]. Furthermore, TF plays animportantroleinlinkingcoagulationandin ammationbyactingas an important modulator of in ammation [42]. The effects of nutritional compoundson in ammation and TFactivityhavenotbeen extensively investigated, although a recent study hasdemonstrated that curcumin decreases LPS-induced TF activityat a concentration of 40 m M but increases TF activity ata concentration of 10 m M in a human monocytic leukemia cellline [43]. Interestingly, our results also suggest that curcumin ata concentration lower than 10 m M stimulates TF procoagulantactivity in healthy controls, although the same effect was notdemonstrated in the CKD group, with curcumin, sh oil, andlow-dose colostrum not signi cantly increasing TF procoagulantactivity with LPS stimulation.In our study, LPS-stimulated TF procoagulant activity of control PBMCs was increased when cells were cocultured withlow- and high-dose bovine colostrum and curcumin and bylow-dose sh oil, indicating that these compounds increase ratherthan decrease the expression of LPS-stimulated TF activity inhealthy controls. LPS-induced TF expression is correlated withtoll-like receptor-4 expression [44] and CKD has been associatedwith a decrease in toll-like receptor-4. This decreased receptorexpression may potentially explain differences in sensitivity toparticular nutritional compounds between the CKD and healthycontrol groups in the present study, but this remains to becon rmed. Conclusion Fish oil was superior to bovine colostrum and curcumin indecreasing unstimulated in ammatory cytokine and CRPproductionfromculturedPBMCs.Thedose-dependentinhibitionofMCP-1andotherin ammatorymarkersby shoilcouldprovebene cialindecreasingtheprogressionofcardiovasculardiseasein patients with CKD. C. M. Shing et al. / Nutrition 27 (2011) 967 – 972 971
