Original Article

Protective Effects of Japanese Soybean Paste (Miso) on
Stroke in Stroke-Prone Spontaneously Hypertensive Rats(SHRSP)
Hiromitsu Watanabe,1,* Megumi Sasatani,2,* Toshiki Doi,3 Takao Masaki,3 Kenichi Satoh,4 andMasao Yoshizumi1
BACKGROUND AND HYPOSESIS
Soybean isoflavones have been shown to reduce the risk of cerebral
infarction in humans according to epidemiological studies. However,whether intake of miso can reduce the incidence of stroke in animalmodels remains unknown. In this study, we investigated the effects ofsoybean paste (miso) in an animal model of stroke.

and the low salt group (P ≤ 0.001). Large hemorrhagic macules were
found in the cerebrum in the high salt group, whereas none were
found in the other 2 groups. There were also fewer histological andimmunohistochemical changes in the brain and kidneys in the misogroup compared to the high salt group.

METHODS
Stroke-prone spontaneously hypertensive rats (SHRSP) were fed a miso
diet (normal diet 90%, miso 10%; final NaCl content 2.8%), a high salt
diet (normal diet and NaCl 2.5%; final NaCl content 2.8%), or a low salt
diet (normal diet; final NaCl content 0.3%).

CONCLUSION
Our results suggest that miso may have protective effects against
stroke despite its high salt content.

RESULTS
Kaplan–Meier survival curves revealed a significantly lower survival
rate in the high salt group compared to the miso group (P = 0.002)

Miso is a traditional Japanese food fermented from soybeans.
It is used regularly as a flavoring in soup and is an essential
ingredient in Japanese-style cooking. We previously reported
that miso can suppress the development of liver tumors, gastrictumors, lung tumors, and aberrant crypt foci and colon tumors
in mice and rats.1 We also reported that miso can prevent theinduction of hypertension in Dahl salt-sensitive hypertensive
rats despite its high salt content.2 These findings were further
supported by similar studies conducted by Yoshinaga et al.3
Kokubo et al.4 reported an inverse association between
isoflavone intake and risk of cerebral and myocardial infarctions in Japanese women. In this context, we thought that
it would be interesting to examine the association between
brain infarction and nutrition; more specifically, whether
administration of miso can reduce the incidence of strokein animal models. Stroke-prone spontaneously hypertensive rats (SHRSP) are generated from spontaneously

Correspondence: Hiromitsu Watanabe (tonko@hiroshima-u.ac.jp).
Initially submitted April 23, 2017; date of first revision June 28, 2017;
accepted for publication July 20, 2017.

Keywords: blood pressure; brain protection; hypertension; miso
(Japanese soy bean paste); SHRSP; stroke.
doi:10.1093/ajh/hpx129

hypertensive rats (SHR),5–11 and excessive salt intake in thismodel increases the rate of fatal strokes. Sepehrdad et al.9
reported that all SHRSP provided with a 1% NaCl drinking
solution died by 16.4 weeks. Camargo et al.10 reported thatsurvival of SHRSP at 12 weeks was 26% in the 4% NaCl dietgroup and Kim-Mitsuyama et al.11 reported that all SHRSP
died by 42 days when provided a 8% NaCl diet. In this study,
we examined the effects of miso intake on the incidence ofstroke in SHRSP.
MATERIALS AND METHODSThis study was carried out in accordance with guidelinesof the Institute of Laboratory Animal Science, Hiroshima
University. The experimental protocols were approved by
the ethics committee on animal experiments of Hiroshima
University (Permission Number: A-14–114).

1Department of Cardiovascular Physiology and Medicine, Instituteof Biomedical and Health Sciences, Hiroshima University, Hiroshima,
Japan; 2Department of Experimental Oncology, Research Institutefor Radiation Biology, Hiroshima University, Hiroshima, Japan;
3Department of Nephrology, Hiroshima University Hospital, Hiroshima
University, Hiroshima, Japan; 4Department of Environmetrics andBiometrics Research Institute for Radiation Biology and Medicine,
Hiroshima University, Hiroshima, Japan.

*These authors contributed equally to this work.

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Watanabe et al.

A total of 36 4-week-old male SHRSP (SHRSP/Izm)
were purchased from Nihon SLC (Hamamatsu, Japan) anddivided into 3 groups: low salt diet (normal diet, Oriental
Yeast, Tokyo, Japan; final NaCl content 0.3%), miso diet
(normal diet 90% and miso 10%; final NaCl content 2.8%),
and high salt diet (normal diet supplemented with 2.5%
NaCl; final NaCl content 2.8%). We used red rice miso,
which was fermented for 180 days and freeze-dried by the
supplier (Miyasaka Jozo, Tokyo, Japan).
Rats were maintained as previously reported.2 Normal
tap water was provided ad libitum. Animals were observed
3 times per day and autopsied under ether anesthesia if they
showed ataxic movements, were motionless, or were moribund. Animals alive at the end of experiment (63 days) were
examined under ether anesthesia. Animal weight and foodand water intake were measured.
Blood pressure was measured at 14, 28, and 42 days after initiation of the specific diets using the tail-cuff method (BP-98E,
Softron, Tokyo, Japan).9 Bodies and major organs were weighed
and fixed in phosphate-buffered 3% formalin. Histologicalevaluation was performed by routine procedures with H&E,
periodic acid-Schiff-Alcian blue, and Azan-Mallory staining.
Using a cross-section containing the cerebral cortex, thalamus, third ventricle, and hippocampus at level II, as describedby Solleld et al.,12 we examined all arteries (A) and veins (V) on
the surface (S) of the brain and referred to them as A-S and V-S,
respectively. Similarly, arteries and veins inside (I) the brain were
examined and referred to as A-I and V-I, respectively. Vessels ineach classification (A-S, V-S, A-I, and V-I) were scored as zero orone. When no thrombus was observed, the score was zero. If one
or more thrombi were identified, a score of one was assigned.

The scores of the 4 classifications were summed to generate a
total score (minimum, 0; maximum, 4), and the mean total score± SD was calculated for each group.
Kidney sections (3-µm thick) were treated for 30 minutes atroom temperature with 2% bovine serum albumin and incubated with primary antibodies against CD68 (diluted 1:200;
Serotec MCA341R), and monoclonal mouse anti-α smoothmuscle antigen (dilute 1:1000, Sigma-Aldrich A2547) antibodies overnight at 4 °C. Serial sections were used for negative controls (no primary antibody). All slides were exposed
to a biotinylated secondary antibody and streptavidin-peroxidase using the Ultra Tech HRP kit (Ultra Tech HRP PN
IM2391). Peroxidase activity was visualized by treatment
with H2O2 and diaminobenzidine for 30 minutes. At the
final step, α smooth muscle antigen-stained sections werecounterstained with periodic acid-Schiff. CD68-positive
cells were counted per 10 sites at 200× magnification, and αsmooth muscle antigen-positive areas were measured using
an image analyzer (Win Roof, Mitani, Fukui, Japan).
Statistical significance was determined with Dunnett’s
method, the χ2-test, fitted linear regression lines with thecommon intercept and different slopes on a scatter plot
(multiple regression model), and Kaplan–Meier survival
curves as assessed by log–rank test.
RESULTS
Body weight

Body weight at 56 days after initiation of the diets did not
differ between the 3 groups (Supplementary Table 1).

Figure 1. Blood pressure and event-free survival of rats. (a) Scatter plot of systolic blood pressure (SBP) and diastolic blood pressure (DBP) in SHRSP
with fitted linear regression lines showing the common intercept and different slopes. Animals were given a low salt diet (normal diet, 0.3% NaCl), miso
diet (2.8% NaCl), or high salt diet (2.8% NaCl) for the indicated durations. (b) Kaplan–Meier survival curves in the low salt (normal diet), miso, and high salt
groups, as assessed by log–rank test. The experimental span (from the first treatment of different diets to when the animal died or was sacrificed) of thehigh salt group was significantly shorter (P < 0.001) compared to the low salt group and the miso group (P = 0.002). There was no significant difference
between the low salt group and the miso group (P = 0.062).

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Protective Effects of Miso on Stroke in SHRSPIntake of water and foodAnimal survival

After initiation of the diets, the amount of drinking waterand food consumed by each group were measured for 56 days
and mean values were calculated as ml/animal/day or gram/animal/day, respectively. Water consumption in the high salt
group (42.6 ± 7.4 ml/animal/day) was significantly higher
than in the miso group (38.3 ± 4.7 ml/animal/day), which
was greater than in the low salt group (29.8 ± 4.4 ml/animal/day). There were no significant differences in amount of foodconsumed between groups (Supplementary Table 2).

Of the 36 total rats, we observed 18 events of paralysis and1 death. Rats were autopsied immediately after these events.
None of the rats in the high salt group survived for 64 days,
while survival was noted for 7 (54%) in the miso group and
10 (83%) in the low salt group (Supplementary Table 3). The
Cox proportional hazards model revealed that event-freetime was significantly shorter in the high salt group compared to the low salt group (P < 0.001) and the miso group(P = 0.002) (Figure 1b). There was no significant difference
between the low salt group and the miso group (P = 0.062).

Blood pressure

The fitted linear regression lines on a scatter plot revealed asignificantly higher systolic blood pressure (SBP) in the highsalt group compared to the low salt group (P < 0.001) and themiso group (P = 0.009). There was no significant differencein systolic blood pressure between the low salt group and themiso group (P = 0.457). Diastolic blood pressure was significantly higher in the high salt group compared to the low salt
group (P = 0.006) and the miso group (P = 0.027). There wasno significant difference in diastolic blood pressure between
the low salt group and the miso group (P = 0.611).

Histological findings in the brain

Macroscopically, large hemorrhagic macules were observedon the surface of the cerebrum in 6 of 12 rats in the high salt
group (Figure 2a, A), whereas no macules were observed in
the miso and low salt groups (P < 0.01, Figure 2b). After macroscopic inspection, brains were sectioned at the caudal border of the mammillary body for microscopic analysis.12 Small
hemorrhagic sites were observed in both the miso and highsalt groups (Figure 2a, B), with no significant difference in thenumber of sites between the 2 groups (Figure 2b).

Figure 2. Macroscopic and microscopic analysis of brain. (a) Bleeding in the brain (macroscopic (A) and microscopic (B)). (b) Incidence of bleeding in
the brain (by macroscopic and microscopic observations). In the high salt group, the number of large hemorrhagic macules in the brain was significantly
higher than that in the miso and low salt groups. (c) Microscopic analysis of arteries and veins on the surface of the brain in the low salt (A), miso (B),and high salt (C) groups. Thrombi in arteries and veins on the surface of brain are indicated by arrows (C). The bar indicates 100 µm. (d) Thrombus scoresfor all vessels in the brain. Dots (●) indicate the score of each animal. At the bottom of the figure, scores for each group are expressed as mean ± SD.
**Significantly different from low salt group (P < 0.01), *Significantly different from miso group (P < 0.05).

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Watanabe et al.

Thrombi were frequently observed in cerebral arteries
and veins in the high salt group (Figure 2c, C). The thrombus score significantly differed between the 3 groups (lowsalt, 1.92 ± 1.00; miso, 2.23 ± 0.90; high salt, 3.25 ± 1.06;
Figure 2d).
Biochemical markers

Supplementary Table 3 summarizes the biochemicalparameters of the 3 groups. Blood glucose and total cholesterol levels in the miso and high salt groups were significantly
increased compared to the low salt group. Conversely, total
protein, albumin, ALP, Na, and Cl levels were lower in the
miso and high salt groups compared to the low salt group. It
is noteworthy that creatinine and BUN in the high salt groupwas significantly higher than in the miso and low salt groups.
Histological findings in kidneys

Protein casts and thickened adventitia of arteries wereincreased in the miso and high salt groups compared to
the low salt group (Figure 3a), while the number of degenerated glomeruli was only increased in the high salt group(Supplementary Table 4). Area of collagen fibers was alsosignificantly larger in the high salt group compared to theother 2 groups. Pale staining of columnar epithelium of renal
tubules was detected in all groups (Figure 3a).
The numbers of CD68-positive cells and α smooth muscle
antigen-positive areas, both of which are markers of kidneydamage, were decreased in the miso group compared to thehigh salt group (Figures 3b and c).
DISCUSSION

In the present study, miso intake reduced the incidence ofstroke in a rat stroke model despite its high salt content, andalso suppressed injuries to the brain and kidneys. Alderman
et al. proposed an interesting paradox that among people
with high sodium consumption, Japanese people have alonger lifespan.13 Anderson et al. showed that, when comparing Japan, the United States, the United Kingdom, andChina, people in Japan had the highest sodium intake, of
which approximately 30% was from fermented foods suchas miso and soy sauce; however, blood pressure was the lowest in Japan.14 We previously reported that miso has potent
antihypertensive effects in Dahl salt-sensitive hypertensive
rats.2 In that study, the difference in blood pressure between
the miso group and high salt group was substantial andreached 35 mm Hg. Here, we found that miso also has antihypertensive effects in a different model of hypertension.
However, the effects of miso were somewhat weaker in thismodel, with a difference in blood pressure of only about
10 mm Hg between the miso and high salt groups.
Yamori et al. reported that inclusion of supplemental proteins such as fish or soybean protein, calcium, potassium,magnesium, and fiber in food can effectively prevent strokein SHRSP.7 Although the antihypertensive effect of miso
was not so remarkable in SHRSP, miso clearly reduced the

Figure 3. Microscopic and immunohistological analysis of kidneys. (a) Histological examination of kidneys in the low salt (A), miso (B), and high salt (C)groups. Pale staining columnar epithelium (arrows), protein cast in renal cortex (P), degeneration of glomeruli (D), and thickening of adventitia of arteries(T). Bar indicates 100 µm, Azan Mallory staining. (b) Number of CD68-positive cells in the kidney cortex. Cell number per one microscopic field under 200×
magnification was counted and expressed as mean ± SD for each group. (c) αSMA-positive areas in the kidney cortex. αSMA-positive areas per field (200×magnification) were measured and expressed as mean ± SD (µm2) for each group. Abbreviation: αSMA, anti-α smooth muscle antigen.

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Protective Effects of Miso on Stroke in SHRSP

incidence of fatal stroke, suggesting that miso components
may have a direct effect on stroke prevention.
Isoflavones found in soybeans are known to have cholesterol-lowering effects.15 Liu et al. reported in a meta-analysisof 11 randomized controlled trials that ingestion of 65–153
mg of soy isoflavones per day lowered blood pressure in
hypertensive subjects.16 Furthermore, Kokubo et al. reported
an inverse association between dietary intake of isoflavones,
miso, and beans and cerebral and myocardial infarctionsin Japanese women without cardiovascular disease.4 Given
that miso contains a lower amount of isoflavones than beans(Nakano K, Central Miso Institute, Tokyo, Japan, personalcommunication, 2010–2017), isoflavones may play a minor
role in the antistroke effects of miso.
We recently found that the levels of 25 substances, including genistein, several antihypertensive substances, antidiabetic
substances, and some antioxidants, increase during the fermentation/maturation of miso (unpublished data). This raises thepossibility that miso may contain compounds that are directly
protective against stroke. Further studies will be needed to identify the effective factors in miso that confer protection against
stroke and to elucidate the mechanisms underlying this effect.
As for the strength of the present study, our findings may
encourage Japanese people—who are accustomed to foods
with salty taste—to use more miso instead of salt for seasoning dishes, given that the Japanese diet is generally high insalt. However, there is a limitation to our study: we used an
animal model. Therefore, further investigation will be necessary to verify our results in humans.
In conclusion, our findings suggest that a miso-containing diet, regardless of its high salt content, may be protective
against stroke.
SUPPLEMENTARY MATERIAL

Supplementary data are available at American Journal ofHypertension online.
ACKNOWLEDGMENTS

This work was supported by a grant-in-aid from theCentral Miso Institute, Tokyo, Japan (2010).
DISCLOSURE

The authors declared no conflict of interest.

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