• View in gallery
    Figure 1—

    Mean ± SEM PGE2 concentration in samples of tissue culture medium (mPGE2) from cartilage explants conditioned with BLsim (simulated digestion fluid only), BOsim (from oil derived from BO seeds) or a DNsim (0.06 [A and B] or 0.18 [C and D] mg/mL), or INDOsim (0.02 mg/mL). Explants were stimulated with IL-1 (10 ng/mL [A and C]) or unstimulated (B and D). In panels A and C, data from unstimulated BLsim-treated explants are shown for comparison. Explants were conditioned for 48 hours with BLsim, INDOsim,DNsim,or BOsim and then treated with IL-1 (0 [ie, no stimulation] or 10 ng/mL) for the final 48-hour period of culture; the data were obtained during the final 48 hours. a–dFor a given time point, different superscripted letters denote significant (P < 0.05) differences in values among treatments. *Within a given treatment, value is significantly (P < 0.05) different from the 0-hour value.

  • View in gallery
    Figure 2—

    Mean ± SEM GAG concentration in samples of tissue culture medium (mGAG) from cartilage explants conditioned with BLsim, BOsim or a DNsim (0.06 [A and B] or 0.18 [C and D] mg/mL), or INDOsim (0.02 mg/mL). Explants were stimulated with IL-1 (10 ng/mL [A and C]) or unstimulated (B and D). In panels A and C, data from unstimulated BLsim-treated explants are shown for comparison. Explants were conditioned for 48 hours with BLsim, INDOsim, DNsim, or BOsim and then treated with IL-1 (0 [ie, no stimulation] or 10 ng/mL) for the final 48-hour period of culture; the data were obtained during the final 48 hours. See Figure 1 for key.

  • View in gallery
    Figure 3—

    Mean ± SEM NO concentration in samples of tissue culture medium (mNO) from cartilage explants conditioned with BLsim, BOsim oraDNsim (0.06 [A and B] or 0.18 [C and D] mg/mL), or INDOsim (0.02 mg/mL). Explants were stimulated with IL-1 (10 ng/mL [A and C]) or unstimulated (B and D). In panels A and C, data from unstimulated BLsim-treated explants are shown for comparison. Explants were conditioned for 48 hours with BLsim, INDOsim,DNsim,orBOsim and then treated with IL-1 (0 [ie, no stimulation] or 10 ng/mL) for the final 48-hour period of culture; the data were obtained during the final 48 hours. See Figure 1 for key.

  • View in gallery
    Figure 4—

    Ratio of C-AM to EthD-1 fluorescence (mean ± SEM values) in cartilage explants conditioned for 96 hours with BLsim (simulated digestion fluid only), BOsim or a DNsim (0.06 or 0.18 mg/mL), or INDOsim (0.02 mg/mL). Explants were conditioned for 48 hours with BLsim, INDOsim, DNsim, or BOsim and then treated with IL-1 (10 [A] or 0 [no stimulation; B] ng/mL) for the final 48-hour period of culture. Explants were collected at the end of the experiment and immediately stained with C-AM and EthD-1 for determination of cell viability. *Value is significantly (P < 0.05) different from the control BLsim value.

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Effects of simulated digests of Biota orientalis and a dietary nutraceutical on interleukin-1– induced inflammatory responses in cartilage explants

Wendy PearsonDepartment of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada

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Michael W. OrthDepartment of Animal Science, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI 48824

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Niel A. KarrowDepartment of Animal and Poultry Science, Ontario Agriculture College, University of Guelph, Guelph, ON N1G 2W1, Canada

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Michael I. LindingerDepartment of Human Health and Nutritional Sciences, College of Biological Science, University of Guelph, Guelph, ON N1G 2W1, Canada

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Abstract

Objective—To test the hypothesis that simulated digests of Biota orientalis (BO) and a dietary nutraceutical (DN; composed of mussel, shark cartilage, abalone, and BO seed lipid extract) inhibit prostaglandin E2 (PGE2), nitric oxide (NO), and glycosaminoglycan (GAG) production in interleukin (IL)-1–stimulated cartilage explants.

Sample Population—Cartilage tissue from 12 pigs.

Procedures—Articular cartilage explants were conditioned with a simulated digest of BO (BOsim) or DN (DNsim) at concentrations of 0, 0.06, or 0.18 mg/mL or indomethacin (INDOsim; 0 or 0.02 mg/mL) for 72 hours. Control explants received digest vehicle only. Explants were or were not stimulated with recombinant human-IL-1β (10 or 0 ng/mL) during the final 48 hours of culture. Concentrations of PGE2, GAG, and NO in media samples (mPGE2,mGAG, and mNO concentrations, respectively) were analyzed, and explant tissue was stained fluorochromatically to determine chondrocyte viability. Treatment effects during the final 48-hour culture period were analyzed.

Results—IL-1 increased mPGE2, mGAG, and mNO concentrations in control explants without adversely affecting cell viability. Treatment with INDOsim blocked PGE2 production and increased mNO concentration in IL-1–stimulated and unstimulated explants and increased mGAG concentration in unstimulated explants. Treatment with DNsim (0.06 and 0.18 mg/mL) reduced mPGE2 concentration in IL-1–stimulated and unstimulated explants, reduced mNO concentration in IL-1–stimulated explants, and increased mNO concentration in unstimulated explants. Treatment with 0.18 mg of DNsim/mL increased cell viability in the presence of IL-1. In IL-1–stimulated explants, BOsim (0.06 and 0.18 mg/mL) reduced mPGE2 concentration, but 0.18 mg of BOsim/mL increased cell viability.

Conclusions and Clinical Relevance—Effects of IL-1 on cartilage explants in vitro were modulated by DNsim and BOsim.

Abstract

Objective—To test the hypothesis that simulated digests of Biota orientalis (BO) and a dietary nutraceutical (DN; composed of mussel, shark cartilage, abalone, and BO seed lipid extract) inhibit prostaglandin E2 (PGE2), nitric oxide (NO), and glycosaminoglycan (GAG) production in interleukin (IL)-1–stimulated cartilage explants.

Sample Population—Cartilage tissue from 12 pigs.

Procedures—Articular cartilage explants were conditioned with a simulated digest of BO (BOsim) or DN (DNsim) at concentrations of 0, 0.06, or 0.18 mg/mL or indomethacin (INDOsim; 0 or 0.02 mg/mL) for 72 hours. Control explants received digest vehicle only. Explants were or were not stimulated with recombinant human-IL-1β (10 or 0 ng/mL) during the final 48 hours of culture. Concentrations of PGE2, GAG, and NO in media samples (mPGE2,mGAG, and mNO concentrations, respectively) were analyzed, and explant tissue was stained fluorochromatically to determine chondrocyte viability. Treatment effects during the final 48-hour culture period were analyzed.

Results—IL-1 increased mPGE2, mGAG, and mNO concentrations in control explants without adversely affecting cell viability. Treatment with INDOsim blocked PGE2 production and increased mNO concentration in IL-1–stimulated and unstimulated explants and increased mGAG concentration in unstimulated explants. Treatment with DNsim (0.06 and 0.18 mg/mL) reduced mPGE2 concentration in IL-1–stimulated and unstimulated explants, reduced mNO concentration in IL-1–stimulated explants, and increased mNO concentration in unstimulated explants. Treatment with 0.18 mg of DNsim/mL increased cell viability in the presence of IL-1. In IL-1–stimulated explants, BOsim (0.06 and 0.18 mg/mL) reduced mPGE2 concentration, but 0.18 mg of BOsim/mL increased cell viability.

Conclusions and Clinical Relevance—Effects of IL-1 on cartilage explants in vitro were modulated by DNsim and BOsim.

Dietary nutraceuticals are commonly used as a putative prophylactic for joint health in horses,1 dogs,2 and humans.3 One such DNa is composed of NZGLM, SKC, abalone, and lipid extract of BO seeds. In a recent study,4 we demonstrated that simulated digests of NZGLM, SKC, and abalone partially inhibit PGE2 (SKC or NZGLM), GAG (abalone or NZGLM), and NO (abalone) release in cartilage explants stimulated with IL-1 as a model of articular inflammation. Furthermore, the ratio of live cells to dead cells was increased in IL-1–stimulated explants treated with SKC or NZGLM.4 To our knowledge, there are no data available regarding the effects of the fourth constituent (BO) or of the heterogeneous blend of all 4 ingredients (the DN) in cartilage explants stimulated with IL-1.

Biota orientalis is an herb that is native to western China and North Korea and is known by a number of other names, such as Thuja orientalis, Platycladus stricta, and Platycladus orientalis. It is greatly valued in traditional Chinese medicine as 1 of the 50 fundamental herbs.5 In vitro, oil from the seeds of BO reduces arachidonic acid release from phosphatidylcholine in the liver6; thus, a potential application of the oil is reduction of PGE2 production because PGE2 is liberated via oxidation of arachidonic acid. The seeds of BO are a source of α-pinene (approx 24% of the essential oil fraction).7 In vitro, α-pinene (at concentrations of 1, 10, and 100 mg/L) is an effective inhibitor of nuclear factor-κB translocation in human monocytes stimulated with lipopolysaccharide.8 In vivo, α-pinene (at a concentration of 500 mg/kg) inhibits writhing in mice following intraperitoneal injection of p-benzoquinone and reduces development of edema in mice following foot-pad injection of carrageenan.9

The purpose of the study reported here was to test the hypothesis that the BOsim and DNsim inhibit PGE2, NO, and GAG production in IL-1–stimulated cartilage explants without adversely affecting cell viability. It was also hypothesized that the inhibitory effects of the DNsim would be greater than those of any individual ingredient alone at the same concentration.

Materials and Methods

The DN powder was prepared by combining NZGLM (Perna canaliculus), abalone (Haliotis sp), SKC (Galorhinus galeus), BO, and honey and flavoring (< 5%) according to a proprietary formulation.b Biota orientalis was stored at 4°C and warmed to room temperature (approx 21°C) before preparation of the DN powder and BOsim.

Simulated digestion—The DNsim, BOsim, and INDOsim (indomethacin used as a reference anti-inflammatory drug) were prepared as previously described.4 Briefly, DN (0.85 g), BO (2.5 mL; equivalent to 0.85 g of BO), and indomethacin (0.71 g) were agitated individually for 2 hours at 37°C in 35 mL of simulated gastric fluid (37mM NaCl, 0.03 N HCl, and pepsin [3.2 mg/mL]). Acidity was neutralized with NaOH (1.5 mL; concentration of NaOH was 2.2 N), and the suspension was added to 36.15 mL of simulated intestinal fluid (30mM K2HPO4, 160mM NaH2PO4, and pancreatin [20 mg/mL]; pH adjusted to 7.4) and agitated at 37°C for a further 2 hours. The final suspension was centrifuged at 3,000 × g for 25 minutes at 4°C to remove particulates. The supernatant was filtered (0.22 μm) then fractioned by use of a size-exclusion (50-kd) ultrafiltration centrifuge unit.c A BLsim (no product included) was prepared simultaneously by the same method. This BLsim was the vehicle in which the DN, BO, and indomethacin were suspended and was used to control for effects of the simulated digestion protocol, which are independent of the effects of DN, BO, or indomethacin.

Preparation of explants—Thoracic limbs of market-weight pigs were obtained from a local abattoir that had Canadian Food Inspection Agency authorization to process pigs. Full-thickness cartilage explants were aseptically prepared with a sterile 4-mm disposable biopsy tool from grossly normal cartilage in the weight-bearing region of intercarpal joints, as previously described.4 Beginning at culture time 0 hours, the cartilage was cultured in Dulbecco modified Eagle medium with amino acids, sodium selenite, manganese sulfate, NaHCO3, and ascorbic acid in 24-well tissue culture plates (Appendix). The total culture period was 120 hours.

Cartilage explants were 4 mm in diameter and approximately 0.5 mm in thickness; mean ± SEM weight of the explants was 17.9 ± 0.5 mg. Explants were stabilized in this unconditioned medium for the initial 24 hours, after which time 10 μL of BLsim (n = 12 pigs), DNsim (0.06 or 0.18 mg/mL; 12 pigs each), BOsim (0.06 or 0.18 mg/mL; 9 pigs each), or INDOsim (0.02 mg/mL; 12 pigs) was added to fresh medium (990 μL) every 24 hours for the duration of the experiment. Explants from 9 pigs were used in all treatment groups; explants from 3 additional pigs were used in each of the BLsim, DNsim, and INDOsim experiments. Recombinant human IL-1βd (0 [ie, no stimulation] or 10 ng/mL) was added to the conditioned media for the final 48-hour culture period (culture time, 96 to 120 hours).

Every 24 hours beginning immediately after explantation, the entire volume of conditioned medium (ie, 1,000 μL) from each experimental treatment was collected into sterile microcentrifuge tubes containing 10 μL of indomethacin (10 μg of indomethacin/mL of dimethyl sulfoxide, which was added to inhibit postcollection formation of PGE2) and replaced with 990 μL of fresh, unconditioned medium with 10 μL of DN, BO, or indomethacin. Media collected from explants after 24 and 48 hours of culture were discarded; samples collected at the 72-hour culture time point were considered as prestimulation baseline samples (ie, samples in which the accumulations of PGE2, GAG, or NO over the 24-hour period [culture time, 48 to 72 hours] immediately preceding stimulation with IL-1 [0 or 10 ng/mL] were assessed). These prestimulation baseline concentrations reflected the effect of conditioning (BLsim, INDOsim, DNsim, or BOsim) for 48 hours prior to stimulation with IL-1 (0 or 10 ng/mL). Collected samples were immediately frozen at −80°C until analyzed. Following commencement of stimulation with IL-1 (0 or 10 ng/mL), samples of media were collected at 24 hours (culture time, 96 hours) and 48 hours (culture time, 120 hours). At the end of the experiment, explant tissue was collected into sterile microcentrifuge tubes containing sterile PBS solution and stained for determination of cell viability.

Sample analysis—Frozen samples of media were thawed at room temperature and analyzed for PGE2, GAG, and NO concentrations; these concentrations were designated as mPGE2, mGAG, and mNO concentrations. Each sample underwent a single freeze-thaw cycle.

Assessment of mPGE2 concentration—The mPGE2 concentrations in 50-μL volumes of each sample were determined by use of a PGE2-specific ELISA kite; assays were run on coated 96-well microtiter plates according to the manufacturer's protocol. Forty samples in duplicate were run on each plate. Plates were read by use of a microplate readerf with absorbance set at 450 nm. A best-fit, third-order, polynomial standard curve was developed for each plate (R2 ≥ 0.99), and those equations were used to calculate mPGE2 concentrations for samples from each plate.

Assessment of mGAG concentration—The mGAG concentration in samples was determined by use of a spectrophotometric assay.4 For each sample, 75-μL volumes of media samples were added to 96-well plates at 50% dilution and serially diluted 1:2 to a final dilution of 1:64. Guanidine hydrochloride (275 mg/mL) was added to each well followed immediately by addition of 150 μL of dimethylmethylene blue reagent. Plates were incubated in the dark for 10 minutes, and absorbance at 530 nm was measured. Sample absorbance was compared with that of a bovine chondroitin sulfate standardg (range, 2.12 to 11.43 μg/mL). Each 96-well plate contained a standard curve in duplicate and 6 samples in duplicate. A best-fit, linear standard curve was developed for each plate (R2 ≥ 0.99), and these equations were used to calculate mGAG concentrations for samples on each plate. Each sample concentration was compared with the standard curve developed from the plate on which it was analyzed.

Assessment of mNO concentration—Nitrite, a stable oxidation product of NO, was analyzed by use of the Griess reaction.4 Volumes (75 μL each) of undiluted samples were added to 96-well plates. Sulfanilamide (0.01 g/mL) and N-(1)-napthylethylene diamine hydrochloride (1 mg/mL) dissolved in phosphoric acid (0.085 g/L) were added to all wells, and absorbance at 530 nm was measured within 5 minutes. Sample absorbance was compared with that of a sodium nitrite standard (range, 0.06 to 7.75 μg/mL). Each 96-well plate contained a standard curve in duplicate and 39 samples in duplicate. A best-fit, linear standard curve was developed for each plate (R2 ≥ 0.99), and these equations were used to calculate nitrite concentrations for samples from each plate. Each sample concentration was compared with the standard curve developed from the plate on which it was analyzed.

Cell viability—Stock viability stain was prepared by combining C-AMh (a stain for cells with biologically active intracellular esterases) and EthD-1i (a stain for nucleic acids in dead or damaged cells) in sterile PBS solution to concentrations of 4 and 8μM, respectively.4 Each explant was washed 3 times in 500 μL of sterile PBS solution, placed aseptically into a sterile 96-well microtiter plate, and incubated in 200 μL of stock viability stain in the dark at room temperature for 40 minutes. Care was taken to place an explant in the center of the well prior to detection of fluorescence. The microplate reader was set to scan each well, beginning at the bottom, with 10 horizontal steps at each of 3 vertical displacements set 0.1 mm apart. This was done to generate measurements of fluorescence at 30 points at 3 depths across the explant. The C-AM fluorescence of live cells and EthD-1 fluorescence of dead or damaged cells in explants were measured with excitation and emission filters of 485 and 530 nm and 530 and 685 nm, respectively. Mean C-AM fluorescence and EthD-1 fluorescence were determined for each individual explant, and a ratio of C-AM fluorescence to EthD-1 fluorescence was determined as a measure of relative viability. These ratios comprised the individual datum points for each explant. Mean ratios were determined for explants from each pig, and these data were statistically analyzed.

Data analysis—Data were reported as mean ± SEM. Means of replicates of each treatment and animal (mPGE2, mGAG, and mNO concentrations) after the final 48 hours of culture were analyzed by use of a 2-way ANOVA to compare each treatment over time. Viability data were reported as a unitless fluorescence ratio of C-AM (live cells) to EthD-1 (dead cells). Viability data were analyzed by use of a 1-way ANOVA to compare treatments in stimulated and unstimulated explants. When a significant F ratio was obtained, the Holm-Sidak post hoc test was used to identify significantly (P < 0.05) different mean values.

Results

For purposes of the study, samples collected at the 72-hour culture time point were considered as prestimulation baseline samples. Treatment of explants with IL-1 was commenced, and samples of media were collected at 24 hours (culture time, 96 hours) and 48 hours (culture time, 120 hours) for assessment; these time points were referred to as 24 and 48 hours after stimulation, regardless of whether samples were treated with IL-1 at a concentration of 10 ng/mL or 0 ng/mL (ie, no stimulation).

mPGE2 concentration—Compared with findings in unstimulated control samples, stimulation with IL-1 (10 ng/mL) significantly increased mPGE2 concentration in explants treated with the BLsim after 24 hours (419.4 ± 68.1 pg/mL vs 844.2 ± 102.9 pg/mL;) and after 48 hours (363.0 ± 43.5 pg/mL vs 600.4 ± 36.8 pg/mL; Figure 1). There was no effect of BOsim on mPGE2 concentration in unstimulated explants. In IL-1–stimulated explants, BOsim (0.06 and 0.18 mg/mL) reduced mPGE2 concentration at 24 hours (398.8 ± 207.8 pg/mL and 427.5 ± 91.5 pg/mL, respectively), compared with stimulated control explants.

Figure 1—
Figure 1—

Mean ± SEM PGE2 concentration in samples of tissue culture medium (mPGE2) from cartilage explants conditioned with BLsim (simulated digestion fluid only), BOsim (from oil derived from BO seeds) or a DNsim (0.06 [A and B] or 0.18 [C and D] mg/mL), or INDOsim (0.02 mg/mL). Explants were stimulated with IL-1 (10 ng/mL [A and C]) or unstimulated (B and D). In panels A and C, data from unstimulated BLsim-treated explants are shown for comparison. Explants were conditioned for 48 hours with BLsim, INDOsim,DNsim,or BOsim and then treated with IL-1 (0 [ie, no stimulation] or 10 ng/mL) for the final 48-hour period of culture; the data were obtained during the final 48 hours. a–dFor a given time point, different superscripted letters denote significant (P < 0.05) differences in values among treatments. *Within a given treatment, value is significantly (P < 0.05) different from the 0-hour value.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1560

Conditioning of explants with INDOsim significantly inhibited PGE2 release at all time points in both stimulated and unstimulated explants. Treatment with the DNsim (0.06 and 0.18 mg/mL) significantly inhibited IL-1–induced PGE2 release at all time points; mPGE2 concentration was also significantly lower in explants conditioned with DNsim (0.06 and 0.18 mg/mL) in unstimulated explants. In IL-1–treated explants at baseline and at 24 and 48 hours after stimulation, inhibition of PGE2 release by DNsim was comparable with that associated with INDOsim. The mPGE2 concentration in unstimulated explants conditioned with 0.06 mg of DNsim/mL was not different from the concentration in explants conditioned with INDOsim at the 24- and 48-hour time points; the mPGE2 concentration in unstimulated explants conditioned with 0.18 mg of DNsim/mL was not different from the concentration in explants conditioned with INDOsim at all time points.

mGAG concentration—Prestimulation baseline mGAG concentrations (at 72 hours of culture) in unstimulated and stimulated control samples (ie, those conditioned with BLsim) were not different (126.3 ± 9.3 μg/mL vs 142.1 ± 15.8 μg/mL). Compared with findings in unstimulated explants treated with BLsim, stimulation with IL-1 (10 ng/mL) significantly increased mGAG concentration at 24 hours (75.5 ± 6.5 μg/mL vs 169.9 ± 18.6 μg/mL) and 48 hours (55.65 ± 2.9 μg/mL vs 120.1 ± 9.8 μg/mL; Figure 2). Conditioning of IL-1–stimulated explants with BOsim and DNsim (0.06 mg/mL) resulted in a significant increase in mGAG concentration, compared with the respective prestimulation baseline values (an increase that was not seen in stimulated control explants); however, mGAG concentration in these 2 treatment groups was not different from that in the stimulated control explants at any time point. There was no effect of BOsim, INDOsim, or treatment with 0.18 mg of DNsim/mL on mGAG concentration from IL-1–stimulated explants.

Figure 2—
Figure 2—

Mean ± SEM GAG concentration in samples of tissue culture medium (mGAG) from cartilage explants conditioned with BLsim, BOsim or a DNsim (0.06 [A and B] or 0.18 [C and D] mg/mL), or INDOsim (0.02 mg/mL). Explants were stimulated with IL-1 (10 ng/mL [A and C]) or unstimulated (B and D). In panels A and C, data from unstimulated BLsim-treated explants are shown for comparison. Explants were conditioned for 48 hours with BLsim, INDOsim, DNsim, or BOsim and then treated with IL-1 (0 [ie, no stimulation] or 10 ng/mL) for the final 48-hour period of culture; the data were obtained during the final 48 hours. See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1560

Conditioning of unstimulated explants with INDOsim resulted in significantly higher mGAG concentration, compared with findings in unstimulated control explants, at 24 hours (175.7 ± 15.5 μg/mL) and 48 hours (133.1 ± 18.1 μg/mL) after commencement of the stimulation period (Figure 2). There was no effect of BOsim or DNsim (0.06 and 0.18 mg/mL) on mGAG concentration in unstimulated explants.

mNO concentration—Compared with findings in unstimulated control explants, mNO concentration was significantly increased in control explants after stimulation with IL-1 (10 ng/mL) for 24 hours (0.28 ± 0.04 μg/mL vs 1.26 ± 0.13 μg/mL) and for 48 hours (0.19 ± 0.04 μg/mL vs 1.04 ± 0.08 μg/mL; Figure 3).

Figure 3—
Figure 3—

Mean ± SEM NO concentration in samples of tissue culture medium (mNO) from cartilage explants conditioned with BLsim, BOsim oraDNsim (0.06 [A and B] or 0.18 [C and D] mg/mL), or INDOsim (0.02 mg/mL). Explants were stimulated with IL-1 (10 ng/mL [A and C]) or unstimulated (B and D). In panels A and C, data from unstimulated BLsim-treated explants are shown for comparison. Explants were conditioned for 48 hours with BLsim, INDOsim,DNsim,orBOsim and then treated with IL-1 (0 [ie, no stimulation] or 10 ng/mL) for the final 48-hour period of culture; the data were obtained during the final 48 hours. See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1560

Conditioning of explants with INDOsim resulted in significant increases in mNO concentration at baseline and 24 hours after commencement of the stimulation period, compared with findings in control explants, in both stimulated and unstimulated conditions. Compared with stimulated control explants, DNsim significantly reduced mNO concentrations in IL-1–stimulated explants at 24 hours (0.91 ± 0.11 μg/mL and 0.92 ± 0.10 μg/mL for 0.06 and 0.18 mg of DNsim/mL, respectively) and 48 hours (0.61 ± 0.10 μg/mL and 0.58 ± 0.09 μg/mL for 0.06 and 0.18 mg of DNsim/mL, respectively). Conditioning of unstimulated explants with DNsim significantly increased mNO concentrations at the 24-hour stimulation time point (0.66 ± 0.18 μg/mL and 0.67 ± 0.14 μg/mL for 0.06 and 0.18 mg of DNsim/mL, respectively), compared with findings in unstimulated controls. Conditioning with BOsim (0.06 or 0.18 mg/mL) had no effect on NO production by IL-1–stimulated or unstimulated explants.

Cell viability—There was no significant effect of IL-1 stimulation (10 ng/mL) on cell viability in the presence or absence of INDOsim (Figure 4). At a concentration of 0.18 mg/mL, DNsim and BOsim each resulted in a significant increase in cell viability in IL-1–stimulated explants, compared with their effects in stimulated control explants. At a concentration of 0.06 mg/mL, there was no effect of DNsim or BOsim on cell viability.

Figure 4—
Figure 4—

Ratio of C-AM to EthD-1 fluorescence (mean ± SEM values) in cartilage explants conditioned for 96 hours with BLsim (simulated digestion fluid only), BOsim or a DNsim (0.06 or 0.18 mg/mL), or INDOsim (0.02 mg/mL). Explants were conditioned for 48 hours with BLsim, INDOsim, DNsim, or BOsim and then treated with IL-1 (10 [A] or 0 [no stimulation; B] ng/mL) for the final 48-hour period of culture. Explants were collected at the end of the experiment and immediately stained with C-AM and EthD-1 for determination of cell viability. *Value is significantly (P < 0.05) different from the control BLsim value.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1560

Discussion

Results of the present study supported the hypotheses that BOsim and DNsim modulate response of articular cartilage explants to exogenous IL-1 without adversely affecting viability or metabolism of chondrocytes. The use of indomethacin as a reference anti-inflammatory drug provided an allopathic benchmark against which the effects of these 2 nutraceuticals could be compared. As expected, INDOsim caused pronounced inhibition of PGE2 release in both stimulated and unstimulated explants. However, the apparent adverse effects of INDOsim on cartilage metabolism, as evidenced by INDOsim-related increases in mGAG and mNO concentrations in our study and by INDOsim-related inhibition of proteoglycan synthesis in other studies,10,11 suggest that it is an unsuitable treatment for articular inflammation.

Simulated digests of NZGLM, SKC, and abalone (the 3 constituents of the DN other than BO) are known to have anti-inflammatory or cartilage-sparing effects in IL-1–stimulated cartilage explants.4 The data obtained in the present study indicated that the oil from the seed of BO also partially inhibits IL-1–dependent PGE2 production by cartilage explants. There was considerable variability in mean PGE2 concentration from explants conditioned with BOsim, which resulted in a conservative estimation of the PGE2-inhibiting potential of BOsim. This may result, in part, from the difficulty in emulsifying BO in the simulated gastric and intestinal fluids. Pancreatic enzymes (including α-amylase, trypsin, lipases, ribonucleases, and proteases) were added to the simulated intestinal fluid, and although emulsification was improved, miscibility was still incomplete. In the experience of one of the authors (WP), the inclusion of bile acids and Tween 20 in the simulated digest vehicle stimulate PGE2 production in cartilage explants; thus, bile acids and Tween 20 are not appropriate emulsification agents.j Investigation of other possible emulsifiers or processes to improve miscibility of the BO seed oil in this type of experiment should be undertaken; this may include sonicating the oil with RBC membranes with or without plasma proteins prior to simulated digestion, as is used to improve emulsification of silicone oil.12

The heterogeneous composition of the DN (ie, NZGLM, SKC, abalone,4 and BO) resulted in significantly improved NO- and PGE2-inhibiting properties, compared with the effects of the individual ingredients alone at the same dose. This synergistic effect may result from each ingredient influencing divergent PGE2- or NO-synthetic pathways. In the DN, the primary putative bioactive constituents from NZGLM, SKC, and abalone are hexosamine-GAGs, omega-3 fatty acids, and proteins.4,13,14 Several hypotheses have been proposed for the mechanisms by which exogenous hexosamine and GAG salts exert anti-inflammatory effects, including specific inhibition of COX-2 via interference with post translational glycosylation,15 provision of substrate for assembly of new proteoglycan through the hexosamine pathway,16 and interference with IL-1 signal-transduction–associated cascades.17 Specific COX-2 inhibition combined with omega-3 fatty acids results in a synergistic reduction of lipopolysaccharide-dependent tumor necrosis factor-α production in murine macrophages,18 a mechanism that may have contributed to the synergistic reduction in mNO and mPGE2 concentrations detected in the present study.19 Indeed, if such a synergistic decrease in tumor necrosis factor-α production occurred through provision of the DN, this may have contributed to the increase in cell viability in DNsim- and BOsim-conditioned explants.20

If the effects of BOsim and DNsim on IL-1–induced PGE2 release were mediated through a mechanism similar to that of indomethacin, we would predict that BOsim and DNsim, like INDOsim, would not protect cartilage against IL-1–induced NO release and would augment GAG release in cartilage explants. And indeed, in unstimulated explants, conditioning with both doses of DNsim resulted in increases in mNO concentration. However, in explants conditioned with DNsim, IL-1–induced NO release was significantly reduced. This apparent dichotomy may result from action of the DN via an IL-1–independent pathway; however, it may also be explained on the basis of the associative regulation of iNOS and COX, the enzymes that catalyze formation of PGE2 and NO, respectively. Cellular expressions of iNOS and COX-2 are differentially regulated through activation of at least 2 mitogen-activated protein kinases.21 Net expressions of iNOS and COX-2 are at least partially dependent on the relative amounts of pericellular NO and PGE2.22 Thus, products that increase pericellular NO concentration can effectively down-regulate expression of COX-2 and vice versa.22,23 This may provide some explanation as to why the DNsim had a significant inhibitory effect on NO release in IL-1–stimulated explants but caused an increase in mNO concentration in unstimulated explants and also supports a mechanism of COX-2 inhibition by the DN. Further research should investigate cell signaling pathways, particularly p42/44 and p38,21 that may be involved in the effect of the DN on production of NO and PGE2 protein.

Because at least 2 possible cell signaling pathways implicated in IL-1 stimulation of COX-2 expression are immediately associated with expression of catabolic matrix-degrading enzymes,24,25 and because compounds capable of inhibiting COX-2 are associated with decreased expression of matrix mettalloproteinases,25,26 we predicted that the DN would reduce mGAG concentration (an effect similar to that associated with NZGLM or abalone4), thereby providing evidence for a cartilage-sparing effect. However, results of the present study did not indicate any significant effect of BOsim or DNsim on mGAG concentration. Because NZGLM and abalone both inhibit mGAG production in IL-1–stimulated explants,4 further research endeavors should investigate the net flux of cartilage proteoglycan in the presence and absence of DNsim to determine whether the apparent protective effect of NZGLM or abalone is lost when applied in an NZGLM-SKC-abalone-BO preparation or whether the DN is able to stimulate proteoglycan synthesis in cartilage explants.

In the present study, cell viability subsequent to IL-1 challenge was significantly increased by BOsim and DNsim. These increases were not a result of reduced apoptosis, as might have been predicted from the NO inhibitory properties of the DN, because the increases in viability resulted from an increase in C-AM staining (ie, staining of live cells) but not a decrease in EthD-1 staining (ie, staining of dead cells); cell viability in stimulated BOsim-treated and DNsim-treated explants was greater than the corresponding findings in unstimulated control explants. The primary mechanism for chondrocyte growth in cartilage has been attributed to the binding of PGE2 to 1 of its 4 cell-surface receptors (ie, EP1, EP2, EP3, or EP4), and specifically to EP127,28 or EP2.29 The DNsim, perhaps through provision of BOsim (which had almost equal effect on cell viability), may produce an agonist effect on the prostanoid receptor involved in chondrocyte proliferation, resulting in an increase in live-cell staining. This would also provide a basis for the concurrent inhibition of PGE2 release by DNsim. Substances that act as agonists for PGE2 receptors produce an effect similar to that associated with increasing the amount of PGE2 in the pericellular environment. Prostaglandin E2 production from IL-1–stimulated cells is inhibited by increasing extracellular concentrations of PGE2 through a negative feedback mechanism.30 Further research is warranted to investigate preferential inhibition of the 4 prostanoid receptors in the presence of DNsim to determine whether this mechanism is associated with the bioactivity of DNsim in vitro.

Although the data obtained from experiments involving simulated digests of nutraceuticals and cartilage explants stimulated with IL-1 as a model of articular inflammation may more accurately represent the in vivo situation than data obtained from experiments involving traditional cartilage explants alone, the former should still be viewed with some caution from a clinical perspective until in vivo studies to assess safety and efficacy of the test materials are carried out in a target species.

In addition to investigations of other emulsifiers as alternatives to the simulated digestion process, there are several factors that could be examined to further increase the in vivo applicability of data derived from experiments involving the method used in the present study. For example, the bioavailability of the test material of interest should be investigated. In the procedures used in the present study, the digestible portion of the DN product was assumed to be 100% in a treated animal. Pharmacokinetic information on the candidate bioactive constituents within the test material would be useful in assessing the bioavailability of the product. Species differences in absorption and metabolism of the test material should be examined, as these differences are currently assumed to be of negligible importance within the context of the current model. An interesting possibility for further research would be to extract gastrointestinal juices from the stomach and the proximal portion of the small intestine from other animals, including horses and dogs, for direct digestion of nutraceuticals ex vivo to assess differences in the bioactivity between those simulated digests and the digests used in the present study. Furthermore, the digested and absorbed nutraceutical product is assumed to be evenly distributed throughout the total body water compartment of an animal and not preferentially sequestered into any particular tissue or cell type. Substantial biochemical characterization of the ingredients of a test material, followed by pharmacokinetic studies in the target species, would be required to address this factor.

Another factor not considered in the procedures used in our study is biotransformation of test products in the liver. In the study technique, the active constituents of nutraceuticals are assumed to be substantively unaltered by transformation in the liver. The introduction of an S9 fraction of liver microsomes or intact liver microsomes into the preparation of simulated digests may provide additional information on biotransformation products of a test material.

The experimental protocol used in the present study assumed that there are no interactions between other structures and tissues of the joint or cells within synovial fluid that could impact the bioavailability of the nutraceuticals in different components of a joint. Development of cartilage coculture methods, which may include synovial, subchondral bone, or ligamentous explants with or without neutrophils, would provide interesting new information on this aspect of nutraceutical bioactivity.

Another factor of interest is the effect of dynamic compression on cartilage matrix. In the experimental method used in the present study, it was assumed that dynamic compression does not affect the movement of nutraceutical constituents into the cartilage matrix. Further research may integrate the simulated digestion-ultrafiltration protocol with a compression model of cartilage damage,31 which may alter the pharmaco-dynamics of the nutraceutical constituents. Additionally, there may be differences in explant cellularity. The viability assay developed for the present study cannot account for these small differences in explant cellularity resulting from random sampling across the surface of the intercarpal joint, from differences in explant thickness, or from orientation of the explant within the microtiter plate. Thus, viability data may be vulnerable to random sampling bias or to orientation of the explant within the well. The impact of both of these factors could be reduced by increasing the number of individual measurements across a single explant to capture both the articulating surface of the explant and the cartilage zone that interfaces with calcified cartilage or by incorporating a larger number of animals into a study of this nature.

A final limitation of the present study is that it was assumed that there are no constituents in the 50-kd fraction of a digest would undergo extensive physiologic regulation in vivo (eg, blood glucose), resulting in rapid removal of those constituents from the extra-cellular fluids. The impact of this on bioactivity of a nutraceutical product will require in vivo studies to elucidate.

ABBREVIATIONS

BO

Biota orientalis

BLsim

Blank simulated digest

BOsim

Simulated digest of Biota orientalis

C-AM

Calcein acetoxymethyl ester

COX

Cyclooxygenase

DN

Dietary Biota orientalis–nutraceutical

DNsim

Simulated digest of the dietary Biota orientalis–nutraceutical

EthD-1

Ethidium homodimer-1

GAG

Glycosaminoglycan

IL

Interleukin

INDOsim

Simulated digest of indomethacin

iNOS

Inducible nitric oxide synthase

NO

Nitric oxide

NZGLM

New Zealand green-lipped mussel

PGE2

Prostaglandin E2

SKC

Shark cartilage

a.

Sasha's EQ, Interpath Pty Ltd, Ballarat West, VIC, Australia.

b.

Property of Interpath Pty Ltd, Ballarat West, VIC, Australia.

c.

AmiconUltra, Millipore, Etobicoke, ON, Canada.

d.

Medicorp, Montreal, QC, Canada.

e.

Amersham Prostaglandin E2 Biotrak Enzymeimmunoassay System, Amersham, Baie D'Urfé, QC, Canada.

f.

Victor 3 microplate reader, Perkin Elmer, Woodbridge, ON, Canada.

g.

Sigma, Oakville, ON, Canada.

h.

Calcein acetoxymethyl ester, Live/dead viability/cytotoxicity assay, Molecular Probes, Burlington, ON, Canada.

i.

Ethidium homodimer-1, Live/dead viability/cytotoxicity assay, Molecular Probes, Burlington, ON, Canada.

j.

Pearson W. In vitro and in vivo methods to evaluate putative anti-inflammatory nutraceuticals. PhD Thesis, Department of Biomedical Science, Ontario Veterinary College, University of Guelph, Guelph, ON Canada, 2007.

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Appendix

Composition of tissue culture medium used in cultures of porcine cartilage explants.

Medium was composed of 10 g of Dulbecco modified Eagle medium to which additives were included as follows:
Amino acids (mg/L)Methionine, 12.76Tyrosine, 48.01
Glycine, 11.25Phenylalanine, 30.52
Valine, 40.75Histidine, 10.52
Proline, 17.28Alanine, 4.45
Isoleucine, 50.33Serine, 15.75
Arginine, 63.20Leucine, 45.75
Threonine, 41.75Asparagine, 7.51
Lysine, 55.25Tryptophan, 6.98
Aspartic acid, 6.66Glutamine, 0.219 (added immediately before use)
NaHCO3(g/L)3.89
Lactalbumin hydrolysate (mg/L)2
Sodium selenite (pg/mL)1
Penicillin-streptomycin (100 units:100 mg/mL) combination (mL/L)10
Dexamethasone (mg/L)100
Manganese sulfate (mg/L)0.169
Volume was adjusted to 900 mL with double-distilled H20. The pH of the preparation as adjusted to 7.4, and the final volume was adjusted to 1 L with double-distilled H20. Ascorbic acid (50 μg/mL) was added immediately before use.

Contributor Notes

Dr. Pearson's present address is the Department of Plant Agriculture, Ontario Agriculture College, University of Guelph, Guelph, ON N1G 2W1, Canada.

Supported by Interpath Pty Ltd, Australia.

Dr. Pearson was supported by a scholarship from the Natural Sciences and Engineering Research Council (NSERC).

The study sponsor did not participate in the study design; collection, analysis, or interpretation of data; writing of the manuscript; or decision to submit the manuscript for publication.

Address correspondence to Dr. Pearson.