Soy protein supports cardiovascular health by downregulating
hydroxymethylglutaryl–coenzyme A reductase and sterol regulatory
element-binding protein–2 and increasing antioxidant enzyme activity in
rats with dextran sodium sulfate–induced mild systemic inflammation
Tanya G. Marsh, Rachel K. Straub, Fatima Villalobos, Mee Young Hong⁎
School of Exercise and Nutritional Sciences, San Diego State University, San Diego, CA 92182, USA
Received 11 May 2011; revised 23 September 2011; accepted 30 September 2011
Animal and human studies have indicated that the presence of soy in the diet improves
cardiovascular health. Inflammation plays a pivotal role in the progression of cardiovascular disease
(CVD). However, little is known about how dextran sodium sulfate (DSS)–induced systemic
inflammation impacts overall heart health and, correspondingly, how soy protein modulates risk of
CVD development in DSS-induced systemic inflammation. We hypothesized that soy protein–fed
rats would have a lower risk of CVD by beneficial alteration of gene expression involving lipid
metabolism and antioxidant capacity in DSS-induced systemic inflammation. Forty Sprague-Dawley
rats were divided into 4 groups: casein, casein + DSS, soy protein, and soy protein + DSS. After 26
days, inflammation was induced in one group from each diet by incorporating 3% DSS in drinking
water for 48 hours. Soy protein–fed rats had lower final body weights (P = .010), epididymal fat
weights (P = .049), total cholesterol (P b .001), and low-density lipoprotein cholesterol (P b .001). In
regard to gene expression, soy protein–fed rats had lower sterol regulatory element-binding protein–
2 (P = .032) and hydroxymethylglutaryl–coenzyme A reductase (P = .028) levels and higher low-
density lipoprotein receptor levels (P = .036). Antioxidant enzyme activity of superoxide dismutase
and catalase was higher among the soy protein groups (P = .037 and P = .002, respectively). These
results suggest that soy protein positively influences cardiovascular health by regulating serum lipids
through modified expression of sterol regulatory element-binding protein–2 and its downstream
genes (ie, hydroxymethylglutaryl–coenzyme A reductase and low-density lipoprotein receptor) and
by promoting the antioxidant enzyme activity of superoxide dismutase and catalase.
© 2011 Elsevier Inc. All rights reserved.
Keywords: Soy proteins; Cardiovascular diseases; DSS-induced systemic inflammation; Rat; SREBP-2; HMGCR;
CAT, catalase; CVD, cardiovascular disease; DSS, dextran sodium sulfate; FAS, fatty acid synthase; GPx,
glutathione peroxidase; GST, glutathione S-transferase; HDL, high-density lipoprotein; HMGCR, hydroxy-
methylglutaryl–coenzyme A reductase; IL-6, interleukin-6; LDL, low-density lipoprotein; LDLR, low-density
lipoprotein receptor; ROS, reactive oxygen species; RT, reverse transcription; SOD, superoxide dismutase;
SREBPs, sterol regulatory element-binding proteins; TG, triglyceride.
Cardiovascular disease (CVD) is the leading cause of
death in the United States . The lipid profile — plasma
triglyceride (TG), total cholesterol, low-density lipoprotein
Available online at www.sciencedirect.com
Nutrition Research 31 (2011) 922–928
⁎Corresponding author. School of Exercise and Nutritional Sciences,
San Diego State University, San Diego CA 92182-7251, USA. Tel.: +1 619
594 2392; fax: +1 619 594 6553.
E-mail address: firstname.lastname@example.org (M.Y. Hong).
0271-5317/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
(LDL) cholesterol, and high-density lipoprotein (HDL)
cholesterol — is a strong indicator of CVD risk .
Pharmaceutical means can be used to improve TG and
cholesterol levels  but are often associated with adverse
effectssuchas cognitive loss, neuropathy,and pancreatic and
hepatic dysfunction . Generally safe and cost-effective,
dietary changes can also improve markers of CVD [4,5].
Epidemiological studies have indicated that Asian
populations have reduced risk and incidence of heart disease,
which can be at least partly attributed to the high soy intake
common among these cultures [5,6]. Animal and human
studies have indicated that the presence of soy in the diet
improves cardiovascular health [7-12] and has particularly
beneficial effects for those with elevated LDL  and
Lipid balance is regulated within the liver by sterol
regulatory element-binding proteins (SREBPs), a group of
transcription factors . They control genes involved in the
synthesis and uptake of cholesterol, fatty acids, and TGs
. Three types of SREPBs exist in mammals: SREBP-1a,
SREBP-1c, and SREBP-2 . All 3 proteins contribute to
lipid balance, but SREPB-2 preferentially activates choles-
terol synthesis . The presence of SREBP-2 within the
liver correlates with the expression of hydroxymethylglu-
taryl–coenzyme A reductase (HMGCR), fatty acid synthase
(FAS), and LDL receptor (LDLR) . Soy and its
corresponding isoflavones may influence lipid metabolism
transcription factors, yet the process remains unclear .
Systemic inflammation, in addition to lipid balance, plays
a pivotal role in the progression of cardiovascular disease
[17,18]. Proinflammatory cytokines, such as interleukin-6
(IL-6), are indicative of systematic inflammation  and
have been shown to correlate with cardiovascular-associated
death . Dextran sodium sulfate (DSS) is an inflamma-
tion-inducing agent that elevates IL-6 .
Oxidative stress has also been found to be a major
contributor in the development of cardiovascular disease
[22,23]. Functioning to remove reactive oxygen species
(ROS) from the body, higher antioxidant enzymatic activity
may reduce oxidative stress . Superoxide dismutase
(SOD), glutathione peroxidase (GPx), catalase (CAT), and
glutathione S-transferase (GST) are key antioxidants playing
a role in the removal of ROS. Soy appears to activate
antioxidant defense genes ; and genistein, a major
isoflavone of soy, was observed to elevate GPx activity .
Dextran sodium sulfate induces oxidative stress  and
causes systemic inflammation  as well as colonic
inflammation (inflammatory bowel disease) . However,
little is known about how DSS-induced inflammation
impacts overall heart health. We evaluated the influence of
DSS-induced inflammation and the effects of soy protein on
markers of CVD development in rats with DSS-induced
systemic inflammation and mild colonic inflammation.
Presently, there is no study that has examined the effects
of soy protein on prevention of heart disease with DSS-
induced systemic inflammation. Therefore, the purpose of
the study was to investigate the mechanism of soy protein on
the risk factors of CVD in DSS-induced systemic inflam-
mation. Because lipid metabolism and antioxidant capacity
play an important role in CVD prevention, we hypothesized
that a soy protein diet would lower the risk of cardiovascular
disease by beneficial alteration of gene expression involving
lipid metabolism (SREBP-2, HMGCR, FAS, and LDL-R)
and by enhancing antioxidant enzyme activity in rats with
DSS-induced systemic inflammation.
2. Methods and materials
2.1. Animals and diets
Forty 28-day-old maleSprague-Dawley rats(120 ± 2.26 g)
purchased from Harlan Laboratories (Springfield, Ind, USA)
were housed individually in wire cages within a vivarium
maintained at 20°C to 24°C, 40% to 45% humidity, and 12-
hour light/dark cycle. Food and water were given ad libitum.
The present study was approved by the Institutional Animal
Care and Use Committee at San Diego State University.
Diets were mixed in our laboratory and varied only in
protein source, casein or soy protein. The soy protein was
provided by Supro Soy Protein Isolate from NutraBio.com
(Middlesex, NJ, USA); all other components of the diet were
obtained from Dyets, Inc (Bethlehem, Pa, USA) (Table 1).
2.2. Experimental design and procedures
After acclimation, the rats were divided into 4 experi-
mental groups (n = 10 per group): casein (Cas), soy protein
Ingredient composition of the experimental diets
Ingredient (g/100 g diet)Cas/Cas-DSS Soy/Soy-DSS
Soy protein isolate
Genistein (mg/g SPI)a
Daidzein (mg/g SPI)
Glycitein (mg/g SPI)
AIN-93 salt mixture
AIN-93 vitamin mixture
Protein (% energy)
Carbohydrate (% energy)
Fat (% energy)
SPI indicates soy protein isolate.
aSoy isoflavones were analyzed using high-performance liquid
chromatography in the Food Sciences Laboratory in Iowa State University
(Ames, Iowa, USA).
923 T.G. Marsh et al. / Nutrition Research 31 (2011) 922–928
(Soy), casein with DSS-treatment (Cas-DSS), and soy
protein with DSS-treatment (Soy-DSS). On day 27, rats in
the DSS-treated groups received 3% DSS (MP Biochemi-
cals, Solon, Ohio, USA) in their drinking water for 48 hours
to induce inflammation. Our preliminary data and the study
by Naito et al  showed that DSS induces systemic
inflammation via elevation of proinflammatory cytokines.
Rats were fasted for 5 hours before euthanasia by an
overdose of carbon dioxide gas, and organ and blood
samples were collected. The blood samples sat at ambient
temperature for coagulation for exactly 10 minutes and then
were centrifuged for 15 minutes at 1200 rpm at 4°C. Serum
samples were stored at −80°C before further experiments.
Food and water intake, along with body weight, was
measured throughout the duration of the study.
2.3. Biochemical analysis of serum
Serum IL-6 was determined using an enzyme-linked
immunosorbent assay (Invitrogen, Carlsbad, Calif, USA)
according to the manufacturer's protocol. Serum TG, total
cholesterol, and HDL cholesterol were analyzed using
commercial enzyme kits (Stanbio Laboratory, Boerne, Tx,
USA). Serum LDL cholesterol was calculated by subtracting
HDL cholesterol and (TG/5) from the total cholesterol .
Superoxide dismutase, CAT, GPx, and GST were
analyzed using diagnostic kits (Cayman Chemical Company,
Ann Arbor, Mich, USA). Trolox equivalent total antioxidant
capacity was analyzed using a commercial kit from Sigma-
Aldrich (St Louis, Mo, USA). During the antioxidant
enzyme assay and total antioxidant capacity assay, serum
samples were covered with aluminum foil and placed on ice
to prevent potential oxidative stress.
2.4. Gene expression of liver
Total RNA of the liver was extracted using TRIzol
method, and reverse transcription (RT) was performed using
oligo(dT)12-18 primers with SuperScript III Reverse
Transcriptase (Invitrogen) . Quantitative real-time poly-
merase chain reaction (ABI 7500; Applied Biosystems,
Foster City, Calif, USA) was used to investigate the liver
mRNA level of HMGCR, SREBP-2, FAS, and LDLR.
TaqMan probes (Applied Biosystems) for the target genes
were used, and relative quantitation values were calculated
using comparative CT system including ΔΔCT numerical
quantity. The transcription levels of target genes were
normalized to r18S expression. To confirm the reproduc-
ibility of the assay, every other sample had the RT reaction
on a separate occasion, followed by polymerase chain
reaction and quantitation. To confirm the absence of
contamination or an anomaly, every set of RT reactions
contained a minus RT negative control .
2.5. Statistical analyses
All data were analyzed using SPSS Version 17.0 (IBM,
Somers, NY, USA). Power analysis indicated that 10 rats
were needed to detect differences with 80% power and an α
of P b .05 on lipid profiles. A 2-way analysis of variance was
used to determine the effect of diet and treatment on the
dependent variables (ie, serum lipids, body weight, antiox-
idant enzyme activity, gene expressions, and serum IL-6).
Post hoc multiple comparisons were conducted using
Student-Newman-Keuls test to follow up significant differ-
ences. P values b .05 were considered significant. Results
are presented as means ± SE.
3.1. Body weight, weight gain, fat weight, food intake, and
Initial body weights (120 ± 2.26 g) were not different
among groups (data not shown). The final body weights of
rats given the soy protein diet were lower than those given
casein before (P = .037) and after the DSS treatment (P =
.010) (Table 2). Body weight gain before the DSS treatment
was lower in soy protein groups compared with casein
groups (P = .002). During the DSS treatment, body weight
gain was much lower in the DSS-treated groups regardless
of protein source (P b .001). There was no difference in
body weight gain between diets during the treatment. Food
intake between diet groups did not differ before DSS
treatment. However, during treatment, DSS-treated rats had
significantly lower food and water intakes (P b .001) (Table
Effects of diets on body weight (grams), weight gain (grams), food intake (grams per day), water intake (grams per day), and epididymal fat weight (grams) on
rats fed casein or soy protein diets with or without DSS treatment
Cas Cas-DSS SoySoy-DSS
Before treatment: body weight
After treatment: body weight
Before treatment: body weight gain
During treatment: body weight gain
Before treatment: food intake
During treatment: food intake
During treatment: water intake
255.5 ± 4.20a
270.2 ± 4.48a
135.5 ± 3.30a
14.69 ± 0.64a
18.9 ± 0.70
18.7 ± 0.57a
25.7 ± 1.36a
2.58 ± 0.18a
254.3 ± 5.08a
259.6 ± 5.69a
134.2 ± 3.50a
5.30 ± 1.15b
17.8 ± 0.50
14.7 ± 0.47b
16.5 ± 0.88b
2.62 ± 0.14a
245.4 ± 5.16b
253.5 ± 5.33b
122.4 ± 4.10b
12.12 ± 0.73a
17.9 ± 0.40
18.6 ± 0.51a
27.4 ± 1.52a
2.45 ± 0.11b
243.8 ± 4.58b
248.2 ± 5.09b
123.7 ± 3.18b
4.48 ± 1.38b
18.4 ± 0.56
15.1 ± 0.46b
17.1 ± 0.72b
2.23 ± 0.16b
Values represent the means ± SE; values in the same row with different superscripts differ significantly at P b .05; N = 40; 10 per group.
924 T.G. Marsh et al. / Nutrition Research 31 (2011) 922–928
2); and this might contribute to the lower body weight gain
in DSS-treated rats. The weight of epididymal fat was
significantly lower in soy protein–fed rats than in those
given casein (P = .049) (Table 2).
3.2. Serum lipids and IL-6
Soy protein–fed rats had significantly lower serum total
cholesterol (P b .001) and LDL cholesterol (P b .001) than
casein-fed rats (Table 3). However, diet type had no
significant effects on TG or HDL cholesterol serum
concentration. Dextran sodium sulfate treatment had no
effect on serum total cholesterol, LDL cholesterol, HDL
cholesterol, and TG.
Serum IL-6 is an inflammatory cytokine that is relevant to
many diseases processes. Dextran sodium sulfate–treated
rats had significantly higher IL-6 levels than nontreated rats
(P = .050) (Fig. 1). This confirms that DSS induces systemic
inflammation. However, there were no differences in IL-6
levels between diet groups (Fig. 1).
3.3. Gene expression in the liver
Hepatic enzymes involving lipid metabolism were
determined as a function of soy protein diet at transcriptional
levels. Fatty acid synthase gene expression (Table 4) was
downregulated in the soy protein diet groups compared with
the casein groups (P = .054). Soy protein–fed rats had
significantly lower expression of HMGCR (P = .028) and
SREBP-2 (P = .032) in the liver compared with casein-fed
rats (Table 4). Low-density lipoprotein receptor expression
was up-regulated 1.5-fold in the soy protein groups
compared with casein groups (P = .048). There were no
significant differences with DSS treatment in mRNA levels
of the hepatic enzyme.
3.4. Effects on antioxidant enzyme activities and
Soy protein–fed rats had significantly higher SOD (P =
.037) and CAT (P = .002) antioxidant enzyme activities than
not significantly different between diet groups, although there
was a trend of soy protein groups showing numerically higher
GPx activity. There were no statistical differences with DSS
administration on SOD,CAT,and GPx activities.Glutathione
S-transferase activity was significantly lower in the DSS-
treated groups compared with those untreated (P = .035),
although no differences were observed among diet types.
Treatment type, but not diet type, affected total
antioxidant capacity (Fig. 2). Levels of total antioxidant
capacity were significantly lower in groups treated with DSS
administration (P = .002).
In the present study, we have demonstrated that soy
protein may reduce CVD risk by decreasing body and
epididymal fat weights. Despite no differences in food intake
between diet types, the total body weights and epididymal fat
of the soy protein–fed rats weighed 6% and 5%, respective-
ly, less than the casein-fed rats after 4 weeks of feeding.
Torre-Villalvazo et al  and Akahoshi et al  reported
similar results. Torre-Villalvazo et al  proposed that a
soy protein diet increases the expression of uncoupling
Effects of diets on serum lipid concentration in rats
Lipid (mmol/L)CasCas-DSSSoy Soy-DSS
Total cholesterol 3.42 ± 0.16a
HDL cholesterol 0.72 ± 0.04
1.01 ± 0.13 0.86 ± 0.098 0.86 ± 0.17
3.23 ± 0.19a
0.75 ± 0.07
2.09 ± 0.21a
0.96 ± 0.12
2.08 ± 0.07b
0.65 ± 0.03
2.05 ± 0.12b
0.81 ± 0.13
0.97 ± 0.14b0.94 ± 0.09b
2.24 ± 0.16a
Values represent the means ± SE; values in the same row with different
superscripts differ significantly at P b .05; N = 40; 10 per group.
Fig. 1. Effects of diets on serum IL-6. Values represent the means ± SE.
Means without a common letter are significantly different (P b .05). N = 40;
10 per group.
Effects of diets on gene expression relevant to lipid metabolism (arbitrary
unit) in the liver
Cas Cas-DSSSoy Soy-DSS
1.4 ± 0.49a
1.1 ± 0.20a
5.0 ± 1.22a
2.3 ± 0.85a
2.5 ± 0.51a
1.3 ± 0.21a
4.5 ± 1.29a
2.2 ± 0.85a
0.91 ± 0.27b
0.78 ± 0.20b
1.7 ± 1.22b
4.2 ± 0.90b
0.98 ± 0.26b
0.80 ± 0.23b
2.9 ± 1.22b
3.8 ± 0.90b
Values represent the means ± SE; values in the same row with different
superscripts differ significantly at P ≤ .05; N = 40; 10 per group.
Effects of diets on antioxidant enzyme activity
2.3 ± 0.78a
38 ± 5.30a
8.3 ± 0.78
33 ± 2.58a
1.2 ± 0.66a
41 ± 5.68a
8.9 ± 0.78
25 ± 2.58b
4.2 ± 1.75b
57 ± 3.08b
9.0 ± 0.78
32 ± 2.58a
3.9 ± 1.62b
53 ± 4.00b
9.6 ± 0.78
28 ± 2.89b
Values represent the means ± SE; values in the same row with different
superscriptsa,bdiffer significantly at P b .05; N = 40; 10 per group.
925T.G. Marsh et al. / Nutrition Research 31 (2011) 922–928
protein 1, which increases energy expenditure through
thermogenesis and hence expedites weight loss. Soy protein
may also lower body weight by positively influencing
insulin activity, as the specific amino acid pattern of soy and
its isoflavones has been found to reduce insulin secretion as
well as increase insulin sensitivity . Furthermore, soy
protein may influence body weight by decreasing fatty acid
synthesis, as FAS showed trends of lower expression in the
soy protein–fed groups.
This study also indicates that soy protein in the diet
positively affects the lipid profile. The soy protein–fed rats
had lower serum concentrations of total cholesterol and LDL
cholesterol compared with the casein-fed rats, although no
differences in TG or HDL cholesterol concentrations were
detected. Other studies report parallel results [34-36]. The
observed changes in the soy protein groups' lipid profile may
be explained by positive changes in gene expression,
specifically SREBP-2 and its downstream genes (eg,
HMGCR and LDLR). In our study, the soy protein–fed
groups exhibited a reduced expression in SREBP-2 and
HMGCR, and an increased expression in LDLR. The LDLR
expression was increased by 1.5-fold in the soy protein
group compared with the casein group. The results of Shukla
et al  were similar to ours in regard to SREBP-2 and
HMGCR, although no increase in LDLR was found with a
soy protein diet. However, the findings of Lovati et al 
support the theory that soy protein causes an upregulation of
LDLR activity. Fatty acid synthase expression was down-
regulated in the soy protein groups in our study. Tovar et al
 reported significantly lower levels of FAS in obese rats
when fed soy protein. Combined findings of the current
study and previous research suggest that a soy protein diet
contributes to a healthier lipid profile through the up-
regulation of SREBP-2 and the associated expression of its
down-stream genes (ie, HMGCR, FAS, and LDLR) that alter
cholesterol synthesis and degradation.
Down-regulation of HMGCR, SREBP-2, and FAS gene
expression explains the observed lower cholesterol and TG
levels in the soy protein diet groups. Because high serum
lipids including cholesterol and TG are major sources of
oxidation, the lowered lipid levels may have presumably
lead to reduction of oxidation sources that results in lower
Reactive oxygen species contribute to processes resulting
in tissue injury. Mammalian species combat the effects of
ROS through antioxidants. We measured the antioxidant
activity of SOD, CAT, and GPx, all of which work in a series
of enzymatic reactions to convert O2
SOD and CAT in rats fed soy protein was increased, but no
differences in GPx were noted. In addition, antioxidant
activity of GST, which works as a detoxifying enzyme to
make xenobiotics more water soluble , was measured.
Glutathione S-transferase activity was not found to increase
in animals given soy protein diet. These findings parallel the
results of Yousef et al , who found no changes in GST
activity in rabbits given isoflavones. However, Tachibana
et al  found a 1.82 increase in the liver gene expression of
GST in rats fed soy protein compared with those fed casein.
Therefore, more research is needed to determine the
influence of soy protein on antioxidant activities, particularly
GST. These results support the notion that soy protein may
reduce oxidative stress , particularly through the
increased enzymatic activity of SOD and CAT.
Trolox equivalent total antioxidant capacity was also
measured to determine total antioxidant capacity. Even
though soy protein groups increased SOD and CAT
enzyme activities, total antioxidant capacity was not
significantly different among groups. There was a trend
of higher total antioxidant capacities in the soy protein
groups compared with the control, although it was not
statistically significant. The increased SOD and CAT
activities in the soy protein groups might not have been
large enough to change total antioxidant capacity. In
addition, total antioxidant activity represents not only
antioxidant enzymes but also other antioxidant molecules.
Alternation of potential antioxidant molecules as a function
of soy protein remains to be further studied.
We measured activity of SOD and CAT, but we have not
determined expression (mRNA and/or protein) of the
antioxidant genes to correlate with observed changes in
their activities. The current study is the early stage of soy
protein study on inflammation and antioxidant capacity.
Effects of soy on the antioxidant capacity will be further
studied at the transcriptional and translational levels. In
addition, oxidized and reduced glutathione levels in the liver
will be determined in future experiments.
Systematic inflammation was measured via IL-6 levels.
Inflammation was successfully induced with DSS treatment;
however, no differences between diet types on IL-6 levels
were found. The lack of differences within diet types caused
by DSS treatment is inconclusive in regard to soy protein
protecting against the effects of inflammation. Even though
inflammation was induced with treatment, the degree of
inflammation was very mild. A diet effect may have been
observed if either the duration of exposure or the dosage of
DSS had been higher. Nevertheless, the correlation between
−to H2O. The activity of
Fig. 2. Effects of diets on antioxidant activity. Values represent the means ±
SE. Means without a common letter are significantly different (P b .05). N =
40; 10 per group.
926 T.G. Marsh et al. / Nutrition Research 31 (2011) 922–928
IL-6 and death related to cardiovascular disease ; and the
implication of this study and others regarding the protective
effects of soy and soy protein on cardiovascular health
suggests that the relationship between soy protein and
inflammation warrants further investigation.
Equol is a bioactive metabolite of the soy isoflavone
daidzein. Although most studies have examined the effect of
dietary equol on reproduction tissues and bone health, one
study demonstrated that daidzein metabolite equol rapidly
activates endothelial nitric oxide synthase via Akt signaling,
which may be associated with modulation of blood pressure
. Sierens et al  showed that equol inhibits oxidative
stress, which would be beneficial in heart health. Further
study is needed to examine serum equol levels and their roles
on the antioxidant capacity.
In conclusion, this study supports soy protein as an
effective dietary component for sustaining cardiovascular
health. Through modifications in SREBP-2 expression and
its downstream genes HMGCR and LDLR, soy protein
promoted healthier total and LDL cholesterol levels as we
hypothesized. In addition to the beneficial effects on the lipid
profile, soy protein limited weight gain and enhanced the
antioxidant activity of SOD and CAT.
The authors thank the students of N302L Advanced
Nutrition Laboratory at San Diego State University for their
help on sample collection. This study was funded by San
Diego State University Grant Program.
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