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Vol.3, No.1, 19-25 (2014) Modern Research in Inflammation
http://dx.doi.org/10.4236/mri.2014.31003
Copyright © 2014 SciRes. OPEN ACCESS
Reduction of pro-inflammatory cytokines in rats
following 7-day oral supplementation with a
proprietary eggshell membrane-derived product
Kevin J. Ruff1*, Dale P. DeVore2
1ESM Technologies, LLC, Carthage, USA; *Corresponding Author: kruff@esmingredients.com
2Membrell, LLC, Carthage, USA
Received 21 January 2014; revised 14 February 2014; accepted 20 February 2014
Copyright © 2014 Kevin J. Ruff, Dale P. D eVore. This is an open access article distributed under the Creative Commons Attribution
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ABSTRACT
NEM® brand eggshell membrane is a novel die-
tary supplement that has been clinically shown
to alleviate arthritis joint pain and stiffness;
however the mechanism of action is not well
understood. Preliminary evidence from an in
vitro study of NEM® indicated that the mechan-
ism of action may be based on the reduction of
pro-inflammatory cytokines. In vivo studies were
therefore initiated to evaluate the effects of
NEM® on pro-inflammatory and anti-inflamma-
tory cytokines following oral administration in
rats. NEM® was administered daily at doses of
6.13 mg/kg bw/day (Study 1), 10.0 mg/kg bw/day
(Study 2), or at doses of 0 (control), 26.0 or 52.0
mg/kg bw/day (Study 3) by oral gavage for 7
consecutive days. Inflammation was induced in
the Study 3 rats by intraperitoneal injection of
lipopolysaccharide. Changes in plasma cytokine
levels from baseline following 7 days of oral
supplementation with NEM® at 6.13 mg/kg bw/
day (Study 1) were statistically significant at Day
8 for IL-2, TIMP-1 and VEGF, at Day 21 for IL-10,
and at Day 35 for MCP-1, MCP-3 and TIMP-1, and
at 10.0 mg/kg bw/day (Study 2) were statistically
significant at Day 8 for VEGF, at Day 21 for
MIP-1β, MIP-2 and VEGF, and at Day 35 for
MCP-3, MIP-1β, MIP-2 and VEGF. Changes in
serum cytokine levels versus control at 26.0
mg/kg bw/day (Study 3) were statistically sig-
nificant at all time-points for IL-1β and at 1.5
hours for IL-10, and at 52.0 mg/kg bw/day (Study
3) were statistically significant at 1.5 hours for
IFN-γ, IL-1β and IL-10, and at 3 hours for IL-1β,
and at 24 hours for IL-10. Taken together, these
studies demonstrate that oral supplementation
with NEM® can influence both early-phase pro-
inflammatory cytokines like IL-1β and TNF-α
(Study 3), as well as later-phase cytokines like
MCP-1, MIP-1α & β, RANTES and VEGF (Study 1
& 2). These studies provide a possible basis for
the mechanism of action of NEM® in vivo.
KEYWORDS
Pro-Inflammatory Cytokines; Eggshell
Membrane
1. INTRODUCTION
Many human diseases are characterized by chronic in-
flammation which ultimately leads to tissue destruction.
Inflammatory arthritides like rheumatoid arthritis (RA)
and osteoarthritis (OA) are classic examples of such dis-
eases and the roles that inflammatory chemokines and
cytokines play in the pathogenesis of these diseases are
fairly well accepted [1-6]. Corticosteroids, non-steroidal
anti-inflammatory drugs (NSAIDs) (e.g. ibuprofen, dic-
lofenac, celecoxib), and inflammatory cytokine-specific
biologics (e.g. etanercept, infliximab, adalimumab) are
commonly prescribed to address the underlying inflam-
mation of these debilitating conditions. While some of
these treatments have demonstrated good efficacy in
randomized controlled clinical trials (RCTs), they are
also known to have significant and sometimes severe
side effects [7-10]. NEM® brand eggshell membrane has
previously demonstrated good efficacy in relieving pain
and stiffness associated with OA of the knee in an RCT
[11] and has shown similar efficacy in limited trials for
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
20
other affected joints [12,13] with no reports of any sig-
nificant side effects during these trials.
Eggshell membrane is primarily composed of fibrous
proteins such as Collagen Type I [14]. However, egg-
shell membranes have also been shown to contain other
bioactive components, namely glycosaminoglycans (e.g.
dermatan sulfate [15], chondroitin sulfate [15], hyalu-
ronic acid [16], etc.). ESM Technologies, LLC (Carthage,
MO USA) has developed methods to efficiently and ef-
fectively separate eggshell membrane from eggshells on
a commercial metric-ton scale. The isolated membrane is
then partially hydrolyzed using a proprietary process and
dry-blended to produce NEM® brand eggshell membrane.
Compositional analysis of NEM® conducted by ESM has
identified a high content of protein and moderate quanti-
ties of glucosamine (up to 1% by dry weight), chondroi-
tin sulfate (up to 1%), hyaluronic acid (up to 2%), and
collagen (Type I, up to 5%).
Although NEM® has been clinically shown to alleviate
joint pain and stiffness from arthritis, the mechanism of
action of this eggshell membrane preparation is not well
understood. Preliminary evidence from an in vitro study
of NEM® indicated that the basis for the mechanism of
action may be via the reduction of pro-inflammatory
cytokines [17]. In vivo studies were therefore initiated to
evaluate the effects of NEM® on pro-inflammatory and
anti-inflammatory cytokines following oral administra-
tion in rats. The results of these preliminary studies are
reported herein.
2. MATERIALS AND METHODS
2.1. Animals, Care and Diet
Studies were conducted utilizing the facilities and staff
of Charles River Laboratories (Spencerville, OH) (Study
1 & 2) and Ricerca Biosciences (Taipei, Taiwan) (Study
3). Animals were housed and cared for in general accor-
dance with the “Guide for the Care and Use of Labora-
tory Animals” (National Academies Press, Washington,
D.C. USA, 1996). Male Sprague Dawley rats were ob-
tained from Harlan Sprague Dawley, Inc., Indianapolis,
IN USA (Study 1 & 2) and BioLASCO Taiwan Co., Ltd.
(a Charles River licensee), Taipei, Taiwan (Study 3) at
approximately 7 - 10 weeks of age. Upon receipt, tags
with unique identification numbers were used to indivi-
dually identify the animals. Cage cards displaying the
study number, animal number, and sex were affixed to
each cage. The rats were acclimatized for 5 days prior to
study commencement and were observed daily for overt
physical or behavioral abnormalities, general health/ mo-
ribundity, and mortality. Healthy rats weighing 300 ± 20
g were randomized into groups of three and were housed
in cages under standard experimental conditions (22˚C ±
3˚C; 30% - 70% humidity; 12-hour light/dark cycle;
minimum 10 room air changes per hour) and had access
to standard rat chow [PMI Certified Rodent Chow #5002
(PMI Nutrition International, St. Louis, MO USA)
(Study 1 & 2) or Laboratory Rodent Diet MF-18 (Orien-
tal Yeast Co., Ltd., Tokyo, Japan)(Study 3)] and water
ad libitum.
2.2. Test Article Preparation and Dosing
The test article was prepared by suspending NEM®
powder (ESM Technologies, LLC, Carthage, MO USA)
in 0.5% methylcellulose (Sigma-Aldrich, St. Louis, MO
USA) in distilled water at a concentration of 0.613
mg/mL (Study 1), 1.0 mg/mL (Study 2), or 2.6 mg/mL
and 5.2 mg/mL (Study 3), corresponding to a dose vo-
lume of 10 mL/kg. The test article was stored at ap-
proximately 4˚C with constant stirring between daily
uses. The NEM® suspension was administered daily to
groups of 3 rats (Study 1 & 2) or groups of 8 rats (Study
3) at doses of 6.13 mg/kg bw/day (Study 1), 10.0 mg/kg
bw/day (Study 2), or at doses of 0 (control, vehicle only),
26.0 mg/kg bw/day, or 52.0 mg/kg bw/day (Study 3) by
oral gavage for 7 consecutive days. The rats were ob-
served twice daily following administration of the test
article for mortality and clinical abnormalities during the
study period.
2.3. Induction of Inflammation (Study 3)
Inflammation was induced in the Study 3 rats by
intraperitoneal (i.p.) injection (2.5 mg/kg) of a solution
of lipopolysaccharide (LPS) (E. coli serotype 055:B5,
Sigma-Aldrich, St. Louis, MO USA) in pyrogen free
saline (Taiwan Biotech Co., Ltd., Taoyuan, Taiwan).
Control rats received an i.p. injection of saline only.
2.4. Blood Collection and Cytokine
Measurement
Blood samples (~0.5 mL) were collected via jugular
vein (Study 1 & 2) or tail vein (Study 3) pre-dose (Day 0)
(all studies) and on Days 8, 21 and 35 (Study 1 & 2) or
1.5, 3 and 24 hours post LPS injection (Study 3). Blood
samples were processed at the time of collection into
plasma samples (Study 1 & 2) or serum samples (Study 3)
and were stored at −70˚C until cytokine determination
could be performed.
Cytokine determination for Study 1 & 2 was accom-
plished utilizing the facilities and services of Rules
Based Medicine, Inc., Austin, TX USA using their Ro-
dent MAP® multi-analyte profile platform following the
manufacturer’s instructions. In this instance, the levels of
42 different biomarkers, chemokines and cytokines were
evaluated, however only 16 of these which are related to
inflammation [Granulocyte Chemotactic Protein 2 (GCP-
2), Interferon gamma (IFN-γ), Interleukin 1 beta (IL-1β),
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
21
IL-2, IL-4, IL-6, IL-10, Monocyte Chemotactic Protein 1
(MCP-1), MCP-3, Macrophage Inflammatory Protein 1
alpha (MIP-1α), MIP-1β, MIP-2, Regulated upon Acti-
vation Normal T-cell Expressed and Secreted (RANTES),
Tissue Inhibitor of Matalloproteinase Type 1 (TIMP-1),
Tumor Necrosis Factor alpha (TNF-α), and Vascular
Endothelial Growth Factor (VEGF)] are reported herein.
Cytokine determination for Study 3 was performed by
Ricerca Biosciences, LLC, Bothell, WA USA using the
Luminex xMAP® bead-based multiplex platform (Austin,
TX USA) following the manufacturer’s instructions. In
this instance, the levels of 5 chemokines/cytokines re-
lated to inflammation (IFN-γ, IL-1
β
, IL-6, IL-10, TNF-α)
were evaluated and are reported herein.
2.5. Statistical Analysis
Comparisons of baseline data between groups (Study 3)
were made with a Kruskal-Wallis test for multiple inde-
pendent samples to validate randomization. Statistical
significance was accepted at an α value of < 0.05. Post-
baseline statistical analyses were done as repeated meas-
ures univariate Analysis of Variance (rm-ANOVA) ver-
sus baseline (Study 1 & 2) or versus control (Study 3).
Items found to have statistical significance with
rm-ANOVA were then compared using a post hoc test
for repeated measures. Statistical significance was ac-
cepted at an α value of < 0.05 for both determinations. In
cases where post-baseline cytokine values were below
the Limit of Quantitation (LOQ), a value of ½LOQ (well
above the Limit of Detection for the assays) was incor-
porated for statistical calculations as opposed to incor-
porating zero values (Study 1 & 2). This substitution
approach was developed in consultation with the assay
manufacturer and cases where this approach was used are
denoted in the data tables. Additionally, data points were
excluded in cases where there was >35% variance be-
tween replicates (Study 3). This occurred at a rate of
<4% in the overall dataset and appeared to be randomly
distributed throughout. SYSTAT software (version 13)
was used for all statistical analyses [18].
3. RESULTS
Changes in plasma cytokine levels from baseline fol-
lowing 7 days of oral supplementation with NEM® at
6.13 mg/kg bw/day (Study 1, Table 1) were statistically
significant at Day 8 for IL-2 (153% increase, p = 0.033),
TIMP-1 (11.2% reduction, p = 0.002) and VEGF (27.8%
reduction, p = 0.022), at Day 21 for IL-10 (65.1% reduc-
tion, p = 0.033), and at Day 35 for MCP-1 (30.0% reduc-
tion, p = 0.034), MCP-3 (26.6% reduction, p = 0.007)
and TIMP-1 (14.6% reduction, p = 0.032). There were
non-detectable levels of MIP-1
β
at Day 21 and MIP-2
and TNF-α at Day 35.
Table 1. Change in plasma cytokine levels from baseline in
healthy rats following 7 days of oral supplementation with
NEM® at 6.13 mg/kg bw/day.
Baseline
(Day 0) NEM
(Day 8) NEM
(Day 21) NEM
(Day 35)
n = 3 n = 3 n = 3 n = 3
GCP-2a 0.07 ± 0.02 0.22 ± 0.10 0.24 ± 0.07† 0.06 ± 0.02
IFN-γb < LOQ < LOQ < LOQ < LOQ
IL-1
β
a 1.29 ± 0.99 0.72 ± 0.34 0.62 ± 0.22 0.30 ± 0.12c
IL-2b 11.2 ± 6.2 28.3 ± 9.0* 33.9 ± 23.6 26.0 ± 13.0
IL-4b < LOQ < LOQ < LOQ < LOQ
IL-6b < LOQ < LOQ < LOQ < LOQ
IL-10b 616 ± 98 503 ± 85 215 ± 105* 477 ± 62
MCP-1b 457 ± 65 449 ± 91 553 ± 144 320 ± 101*
MCP-3b 222 ± 51 207 ± 39 256 ± 70 163 ± 42*
MIP-1αa 0.26 ± 0.05 0.17 ± 0.08 0.15 ± 0.03 0.24 ± 0.06
MIP-1
β
b 28.7 ± 11.5 44.2 ± 9.1 39.0 ± 0.0c 27.4 ± 17.0
MIP-2b 3.0 ± 0.7 3.3 ± 0.7 3.6 ± 1.2 3.6 ± 0.0c
RANTESb 86.2 ± 36.6 152 ± 92 319 ± 70† 62.0 ± 22.5
TIMP-1a 8.9 ± 1.4 7.9 ± 1.3* 8.3 ± 0.6 7.6 ± 1.1*
TNF-αa 0.05 ± 0.03 0.05 ± 0.04c 0.05 ± 0.04c 0.07 ± 0.0c
VEGFb 227 ± 38 164 ± 40* 196 ± 55 208 ± 30
Values represent means ± standard deviation, a = ng/mL, b = pg/mL, <LOQ
= below limit of quantitation, P-values determined by repeated measures
ANOVA versus baseline, *P < 0.05, †P < 0.10, c = contained cases where
values measured were below the Limit of Quantitation (LOQ) wherein
values of ½LOQ were incorporated for statistical calculations.
Changes in plasma cytokine levels versus baseline
following 7 days of oral supplementation with NEM® at
10.0 mg/kg bw/day (Study 2, Table 2) were statistically
significant at Day 8 for VEGF (50.1% reduction, p =
0.038), at Day 21 for MIP-1
β
(84.8% reduction, p =
0.022), MIP-2 (77.1% reduction, p = 0.005) and VEGF
(61.5% reduction, p = 0.014), and at Day 35 for MCP-3
(67.2% reduction, p = 0.047), MIP-1
β
(88.4% reduction,
p = 0.002), MIP-2 (76.5% reduction, p = 0.006) and
VEGF (66.4% reduction, p = 0.002). There were trends
toward significance at Day 8 for MIP-2 (64.0% reduction,
p = 0.063), at Day 21 for MCP-3 (61.9% reduction, p =
0.081) and TNF-α (70.0% reduction, p = 0.097), and at
Day 35 for GCP-2 (53.8% reduction, p = 0.075), IL-2
(69.0% reduction, p = 0.098) and MCP-1 (67.3% reduc-
tion, p = 0.079). There were non-detectable levels of
IL-2, MIP-2 and TIMP-1 at Day 35.
There were no differences in serum cytokine levels
between groups (control, 26.0 mg/kg bw/day or 52.0
mg/kg bw/day) at baseline for any of the five cytokines
evaluated (IFN-γ, IL-1
β
, IL-6, IL-10, TNF-α)(Study 3,
Table 3). Changes in serum cytokine levels versus con-
trol following 7 days of oral supplementation with
NEM® at 26.0 mg/kg bw/day (Study 3, Table 4) with
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
22
Table 2. Change in plasma cytokine levels from baseline in
healthy rats following 7 days of oral supplementation with
NEM® at 10.0 mg/kg bw/day.
Baseline
(Day 0) NEM
(Day 8) NEM
(Day 21) NEM
(Day 35)
n = 3 n = 3 n = 3 n = 3
GCP-2a 0.13 ± 0.05 0.06 ± 0.04 0.02 ± 0.03 0.06 ± 0.02†
IFN-γb < LOQ < LOQ < LOQ <LOQ
IL-1
β
a < LOQ < LOQ < LOQ <LOQ
IL-2b 108 ± 44 31.3 ± 21.3 25.3 ± 14.1c 33.5 ± 0.0c†
IL-4b < LOQ < LOQ < LOQ <LOQ
IL-6b < LOQ < LOQ < LOQ <LOQ
IL-10b 460 ± 265 305 ± 15 314 ± 25 294 ± 83
MCP-1b 721 ± 233 439 ± 113 250 ± 122 236 ± 28†
MCP-3b 354 ± 93 226 ± 72 135 ± 44† 116 ± 6*
MIP-1αa < LOQ < LOQ < LOQ <LOQ
MIP-1
β
b 250 ± 31 81 ± 103 38 ± 46* 29 ± 16*
MIP-2b 15.3 ± 1.5 5.5 ± 3.4c† 3.5 ± 0.2c* 3.6 ± 0.0c*
RANTESb 195 ± 118 93 ± 100 23 ± 20 20 ± 4
TIMP-1a 0.28 ± 0.16 0.09 ± 0.06c
0.08 ± 0.03c 0.09 ± 0.00c
TNF-αa 0.20 ± 0.09 0.06 ± 0.06 0.06 ± 0.03c† 0.04 ± 0.03c
VEGFb 429 ± 42 214 ± 75* 165 ± 25* 144 ± 33*
Values represent means ± standard deviation, a = ng/mL, b = pg/mL, < LOQ
= below limit of quantitation, P-values determined by repeated measures
ANOVA versus baseline, *P < 0.05, †P < 0.10, c = contained cases where
values measured were below the Limit of Quantitation (LOQ) wherein
values of ½LOQ were incorporated for statistical calculations.
Table 3. Mean serum cytokine concentrations (pg/mL) in
NEM-supplemented and control groups at baseline.
Control
(0 mg/kg) NEM
(26 mg/kg) NEM
(52 mg/kg)
n = 8 n = 8 n = 8
IFN-γ 2.49 ± 0.16 2.40 ± 0.00 2.49 ± 0.27
IL-1
β
15.8 ± 10.0 13.0 ± 5.5 18.6 ± 13.2
TNF-α 11.1 ± 2.0 10.2 ± 0.7 11.2 ± 2.9
IL-6 9.80 ± 0.00 9.96 ± 1.14 10.2 ± 1.1
IL-10 10.2 ± 1.0 9.80 ± 0.00 10.0 ± 0.6
Values represent means ± standard deviation, P-values determined by
Kruskal-Wallis test for multiple independent samples, *P < 0.05, †P < 0.10.
subsequent inflammatory challenge (LPS, i.p.) were sta-
tistically significant at 1.5 hours (43.7% reduction, p =
0.013), 3 hours (28.8% reduction, p = 0.034) and 24
hours (20.8% reduction, p = 0.006) for IL-1
β
and at 1.5
hours (27.6% reduction, p = 0.028) for IL-10. There was
a trend toward significance at 24 hours for IL-10 (74.6%
increase, p = 0.097). No other changes in serum cytokine
levels were statistically significant at this dose level.
Changes in serum cytokine levels versus control fol-
lowing 7 days of oral supplementation with NEM® at
52.0 mg/kg bw/day (Study 3, Table 5) with subsequent
inflammatory challenge (LPS, i.p.) were statistically sig-
nificant at 1.5 hours for IFN-γ (33.5% reduction, p =
0.047), IL-1
β
(39.4% reduction, p = 0.003) and IL-10
(29.8% reduction, p = 0.015), and at 3 hours for IL-1
β
(23.9% reduction, p = 0.044), and at 24 hours for IL-10
(57.5% increase, p = 0.021). There was a trend toward
significance at 24 hours for IL-1
β
(9.3% reduction, p =
0.093). No other changes in serum cytokine levels were
statistically significant at this dose level.
4. DISCUSSION
Although OA has not traditionally been considered an
inflammatory arthropathy, the scientific understanding of
the pathophysiological progression of the disease has
been gradually trending towards that of a disease involv-
ing the “whole joint” with significant localized inflam-
mation [19]. Evidence of an inflammatory process in OA
is reflected in many of the clinical symptoms of the pro-
gressive disease, including swelling of affected joints,
Table 4. Change in mean serum cytokine concentrations
(pg/mL) in 7-day NEM-supplemented (26 mg/kg bw/day) and
control groups from baseline at 1.5, 3, and 24 hours post LPS
treatment.
Hours post-
treatment
Control NEM % Difference
(NEM-vs-ctrl)
(0 mg/kg)
n = 8 (26 mg/kg)
n = 8
IFN-γ Baseline 2.49 ± 0.16 2.40 ± 0.00 −3.5
1.5 6.35 ± 2.48 4.88 ± 2.60 −23.2
3 135 ± 43 144 ± 85 6.2
24 3.55 ± 2.18 2.49 ± 0.14 −29.8
IL-1
β
Baseline 15.8 ± 10.0 13.0 ± 5.5 −17.9
1.5 62.4 ± 17.1 35.1 ± 16.7* −43.7*
3 87.9 ± 23.5 62.6 ± 15.9* −28.8*
24 12.6 ± 2.1 9.96 ± 0.42* −20.8*
TNF-α Baseline 11.1 ± 2.0 10.2 ± 0.7 −7.6
1.5 1157 ± 828 934 ± 332 −19.3
3 84.5 ± 46.7 75.5 ± 28.7 −10.7
24 9.80 ± 0.00 9.80 ± 0.00 0.0
IL-6 Baseline 9.80 ± 0.00 9.96 ± 1.14 1.7
1.5 321 ± 175 385 ± 182 20.0
3 386 ± 172 329 ± 133 −14.8
24 10.0 ± 0.6 10.7 ± 2.3 6.6
IL-10 Baseline 10.2 ± 1.0 9.80 ± 0.00 −3.8
1.5 42.7 ± 12.3 30.9 ± 8.3* −27.6*
3 18.7 ± 5.6 19.3 ± 7.4 3.1
24 14.2 ± 3.2 24.9 ± 11.5† 74.6†
Values represent means ± standard deviation. P-values determined by re-
peated measures ANOVA, *p < 0.05, †p < 0.10. ctrl = control.
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
23
Table 5. Mean serum cytokine concentrations (pg/mL) in 7-day
NEM-supplemented (52 mg/kg bw/day) and control groups at
baseline and 1.5, 3, and 24 hours post LPS treatment.
Hours post-
treatment
Control NEM % Difference
(NEM-vs-ctrl)
(0 mg/kg)
n = 8 (52 mg/kg)
n = 8
IFN-γ Baseline 2.49 ± 0.16 2.49 ± 0.27 0.3
1.5 6.35 ± 2.48 4.22 ± 0.76* −33.5*
3 135 ± 43 130 ± 34 −4.3
24 3.55 ± 2.18 2.82 ± 0.75 −20.7
IL-1
β
Baseline 15.8 ± 10.0 18.6 ± 13.2 17.3
1.5 62.4 ± 17.1 37.8 ± 9.7* −39.4*
3 87.9 ± 23.5 66.9 ± 15.8* −23.9*
24 12.6 ± 2.1 11.4 ± 1.2 −9.3†
TNF-α Baseline 11.1 ± 2.0 11.2 ± 2.9 1.5
1.5 1157 ± 828 786 ± 161 −32.1
3 84.5 ± 46.7 70.0 ± 15.0 −17.2
24 9.80 ± 0.00 9.80 ± 0.00 0.0
IL-6 Baseline 9.80 ± 0.00 10.2 ± 1.1 4.0
1.5 321 ± 175 256 ± 83 −20.1
3 386 ± 172 306 ± 64 −20.6
24 10.0 ± 0.6 10.3 ± 1.4 3.0
IL-10 Baseline 10.2 ± 1.0 10.0 ± 0.6 −1.8
1.5 42.7 ± 12.3 30.0 ± 8.1* −29.8*
3 18.7 ± 5.6 18.1 ± 3.4 −3.6
24 14.2 ± 3.2 22.4 ± 6.1* 57.5*
Values represent means ± standard deviation. P-values determined by re-
peated measures ANOVA, *p < 0.05, †p < 0.10. ctrl = control.
synovial effusion, and joint stiffness [20]. This clinical
evidence is supported by immunochemical and histolog-
ical data from numerous studies showing infiltration of
the joint synovium by immune cells, primarily macro-
phages and mononuclear lymphocytes such as T-cells
[20-22] accompanied by subsequent inflammatory cyto-
kine expression [23] and synovial fibroblast activation
[24].
The two primary mediators of arthritis inflammation
are IL-1β and TNF-α. These cytokines have been identi-
fied as targets for OA treatment [25,26] and there are
multiple FDA-approved biologic drugs (etanercept, in-
fliximab, adalimumab, etc.) for this indication (mostly
RA). These cytokines, in an autocrine/paracrine manner,
auto-amplify their own expression and induce chondro-
cytes to produce matrix metalloproteinases (MMPs),
chemokines (IL-8, MCP-1, MIP-1α, MIP-1β, RANTES,
etc.), nitric oxide, and prostaglandins [19,26]. This leads
to localized tissue destruction, immune cell infiltration,
inhibition of cartilage matrix synthesis, and increased
pain sensitivity, among others.
The eggshell membrane derived product NEM® has
previously been shown in vitro to reduce a number of
pro-inflammatory cytokines in human immune cells fol-
lowing inflammatory challenge (with phyto-mitogens),
with this effect being most pronounced for IFN-γ and
TNF-α [17]. In this paper, we reported in vivo support for
the reduction of circulating pro-inflammatory cytokines
following oral supplementation with NEM® in both
healthy rats (Study 1 & 2) and inflammatory-challenged
rats (Study 3).
While not statistically significant, NEM® appeared to
demonstrate trends toward reduction in healthy rats for
both IL-1β (6.13 mg/kg bw/day)(Study 1) and TNF-α
(10.0 mg/kg bw/day)(Study 2). Interestingly, there were
statistically significant effects at both dose levels for
nearly all of the chemokines (MCP-1, MIP-1α, MIP-1β,
RANTES, VEGF) currently understood to be key players
in OA/RA inflammation and pathogenesis. MCP-1 and
RANTES have been shown to induce expression of
MMP-3 in both normal and OA chondrocytes [27] and
RANTES has been reported to stimulate MMP-1 release
in chondrocytes as effectively as did IL-1β [28]. These
enzymes are known to degrade chondrocyte extracellular
matrix (ECM) which leads to cartilage destruction.
MCP-1, RANTES, MIP-1α and MIP-1β have all been
shown to inhibit proteoglycan synthesis in chondrocytes
[27,29], a key component of cartilage needed for repair.
VEGF expression is absent in adult healthy cartilage but
is significantly expressed in OA chondrocytes and may
play a role in osteophyte formation [30].
Also interesting is the overall lack of effect from oral
supplementation with NEM® on anti-inflammatory cyto-
kines and chemokines (IL-4, IL-6, IL-10, and TIMP-1) in
healthy rats. While IL-4 and IL-6 were below LOQ at
baseline, there was only a mild downward trend in IL-10
and TIMP-1 levels—consistent with restoring immune
homeostasis following the reductions seen in pro-in-
flammatory cytokines and chemokines. IL-10 is known
to inhibit the production of IL-1β and TNF-α and is
overexpressed in OA chondrocytes compared to normal,
which is likely the body’s attempt to counteract the de-
trimental effects from these pro-inflammatory cytokines
[31]. MMPs are strictly controlled by TIMPs under nor-
mal conditions and an imbalance toward MMPs is be-
lieved to be the basis for cartilage destruction via ECM
degradation in arthritis [32].
NEM® has been shown in clinical trials to have an ef-
fective dose of 500 mg per day. We initially chose to
evaluate doses of 6.13 mg/kg bw/day (Study 1) and 10.0
mg/kg bw/day (Study 2) which, following allometric
conversion [33], equate to a human equivalent dose
(HED) of 59 mg/day and 97 mg/day, respectively, for a
60 kg person. The number of animals (n = 3) was also
small in the preliminary evaluations of inflammatory
K. J. Ruff, D. P. DeVore / Modern Research in Inflammation 3 (2014) 19-25
Copyright © 2014 SciRes. OPEN ACCESS
24
cytokines. These facts, combined with low basal cyto-
kine levels in healthy rats, made it challenging to obtain
statistically significant changes following oral supple-
mentation with NEM®. In a number of instances (IL-1β
in Study 1 and IL-10 & RANTES in Study 2), there ap-
peared to be substantial percent reductions in mean cyto-
kine levels that nevertheless failed to reach statistical
significance. We therefore set out to employ a rat model
in which inflammation was induced (Study 3) to increase
the likelihood of observing clearer effects from NEM®
supplementation. We also increased the number of ani-
mals (n = 8), narrowed the number of cytokines eva-
luated to five, and increased the doses evaluated to 26.0
mg/kg bw/day and 52.0 mg/kg bw/day (HED: 252 mg/
day & 503 mg/day, respectively).
There was a substantial (39% - 44%) and lasting
(through 24 hours) reduction in IL-1β in this inflamma-
tory-challenge model (Study 3) at both doses evaluated.
And, although not statistically significant, there also ap-
peared to be a substantial (19% - 32%) downward trend
in TNF-α levels for both doses, as well. These effects on
the key mediators of arthritis inflammation provide fur-
ther supportive evidence to the observed clinical efficacy
of NEM®. The ability to influence IL-1β and TNF-α in
vivo likely also explains at least some of the effects ob-
served in the downstream chemokines (MCP-1, MIP-1α,
MIP-1β, RANTES, VEGF) in the initial studies. Interes-
tingly, there was a sinusoidal response for the anti-in-
flammatory cytokine IL-10 over the time-course of this
study, in which there was an initial substantial reduction
(28% - 30%) at 1.5 hours leading to a substantial in-
crease (58% - 75%) by 24 hours when compared to con-
trols. This is particularly interesting in the context that
nearly all of the cytokines evaluated had returned to near
baseline levels by the 24-hour study endpoint in the con-
trol animals. The reason for this divergence isn’t com-
pletely clear, but it may be a result of the delayed time-
course of anti-inflammatory cytokines compared to the
rapid time-course of pro-inflammatory cytokines, espe-
cially in this particular animal model.
Taken together, these studies demonstrate that oral
supplementation with NEM® can influence both early-
phase pro-inflammatory cytokines like IL-1β and TNF-α
(Study 3), as well as later-phase pro-inflammatory cyto-
kines like MCP-1, MIP-1α & β, RANTES and VEGF
(Study 1 & 2). There was also a mild effect on the an-
ti-inflammatory cytokine IL-10 in all three studies. A
natural treatment, such as NEM®, which is suitable for
chronic inflammatory diseases like arthritis that could
potentially avoid the unfortunate side effects of currently
available pharmaceutical treatments is of obvious benefit.
These studies provide a possible basis for the mechanism
of action of NEM® in vivo and serve as an important step
in explaining its observed clinical efficacy seen in mul-
tiple human studies.
ACKNOWLEDGEMENTS
KJR is currently employed by the sponsor of the studies. DPD has
served as a paid consultant to the sponsor of the studies. All three stu-
dies were sponsored by ESM Technologies, LLC.
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