Administration of Isoflavone Attenuates Ovariectomy-induced Degeneration of Aortic Wall

soy-Abstract: Women are more resistant to vascular diseases; however, the resistance is reduced after menopause. It has been reported that the risk of vascular diseases such as atherosclerosis and abdominal aortic aneurysm is increased in postmenopausal women. Currently, methods to prevent vascular disease in postmenopausal women have not been established. Isoflavones are promising functional food factors that have a chemical structure similar to estrogen. In this study, we investigated the effects of isoflavones on ovariectomized (OVX)-induced degeneration of the aortic wall in mice. Increased destruction of elastic fibers in the thoracic and abdominal aorta was observed in the OVX group, and isoflavones attenuated the destruction of elastic fibers. The positive areas of matrix metalloproteinase (MMP)-2 and MMP-9 in the OVX group were higher than those in the control group. Isoflavones decreased the positive areas of MMP-2 and MMP-9 compared to those in the OVX group. These data suggest that isoflavones have a suppressive effect on OVX-induced degeneration of the aortic wall by inhibiting the increase in MMP-2 and MMP-9.

abdominal aortic aneurysm AAA 3,6 . AAA is characterized by aortic degeneration with progressive dilatation of a portion of the abdominal aorta. Age, smoking history, hypertension, hyperlipidemia, and sex were risk factors for AAA. Many AAA patients are male, and the incidence is approximately four times higher than that of females 7 . However, it has been reported that the rate of progression and rupture of AAA is higher in women than in men 8,9 . It has been reported that sex hormones are involved in these sex differences, but the detailed mechanism is unknown. In addition, most female patients with AAA are postmenopausal 8 , and it has been reported that female patients with early menopause are more likely to have an enlarged mass diameter 10 . Therefore, it is speculated that the decrease in estrogen production associated with menopause may contribute to the increased growth rate of AAA.
Based on the information mentioned above, we focused on isoflavones which have a chemical structure similar to estrogen and exert estrogenic effects on target organs. Isoflavones are polyphenolic nonsteroidal compounds found in the legume family, including food crops such as soy-Abstract: Women are more resistant to vascular diseases; however, the resistance is reduced after menopause. It has been reported that the risk of vascular diseases such as atherosclerosis and abdominal aortic aneurysm is increased in postmenopausal women. Currently, methods to prevent vascular disease in postmenopausal women have not been established. Isoflavones are promising functional food factors that have a chemical structure similar to estrogen. In this study, we investigated the effects of isoflavones on ovariectomized (OVX)-induced degeneration of the aortic wall in mice. Increased destruction of elastic fibers in the thoracic and abdominal aorta was observed in the OVX group, and isoflavones attenuated the destruction of elastic fibers. The positive areas of matrix metalloproteinase (MMP)-2 and MMP-9 in the OVX group were higher than those in the control group. Isoflavones decreased the positive areas of MMP-2 and MMP-9 compared to those in the OVX group. These data suggest that isoflavones have a suppressive effect on OVX-induced degeneration of the aortic wall by inhibiting the increase in MMP-2 and MMP-9.
Key words: ovariectomized, estrogen, abdominal aortic aneurysm, elastic fiber, MMP-2, MMP-9 beans, kidney beans, and solanaceous legumes, and the production of isoflavone-containing foods such as tofu and soy milk is increasing worldwide 11 . Because orally ingested isoflavones could be transported to the blood and increase the blood isoflavone levels 12,13 , consumption of foods containing high amounts of isoflavones has been reported to correlate with a lower incidence of chronic diseases, including coronary heart disease 14 . In addition, isoflavones may contain antioxidants that scavenge free radicals 15,16 , have anti-inflammatory 17,18 and vascular protective 2,14,17 functions, and are said to be effective in preventing diseases associated with low estrogen levels 18 . However, the effect of isoflavones on the degeneration of the aorta associated with menopause remains unknown. In this study, we evaluated the effects of isoflavone administration on the aortic wall of ovariectomized OVX female mice.

Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee and were conducted according to the Kindai University Animal Experimentation Regulations Approval number: KAAG-31-007 . Female 7-week-old mice SHIMIZU Laboratory Supplies Co., Ltd, Kyoto, Japan were fed a free diet and tap water and kept at 25 1 with a 12 h light/dark cycle. The diet composition is shown in Table 1. In this experiment, the mice were divided into three groups: the control group C , OVX group O , and OVX and isoflavone group OI . During the experiment, the C and O groups were administered the control diet, and the OI group was administered an isoflavone diet. One week later, C group underwent sham surgery without ovary removal, and the O and OI groups underwent OVX treatment under anesthesia. Five weeks later, all the mice were sacrificed.
2.2 Serum glucose, triglyceride and total cholesterol levels Five weeks after the sham or OVX treatment, blood was collected from the abdominal vein cava under anesthesia 50 mg/kg pentobarbital, i.p. to determine serum glucose, triglyceride and total cholesterol levels. For serum preparation, blood was centrifuged at 3000 g for 10 min. Then, serum glucose, triglyceride, and total cholesterol were measured using commercial kits Wako Pure Chemical Industries, Osaka, Japan and using the methodology according to the manufacturer s instruction.

OVX and sample collection
After opening the abdomen, the oviducts were isolated from the peri-ovarian fat, and the oviducts were removed with electrocautery. At the end of the experiment, mice were sacrificed, and the abdominal and thoracic aortas were removed and fixed with 4 paraformaldehyde in phosphate-buffered saline PBS for 1 d Nacalai Tesque, Kyoto, Japan . The aorta samples were dehydrated and embedded in paraffin.

Histological analysis
Isolated aortas were serially sectioned into 4 μm thick sections using a microtome PR-50, Yamato Kohki, Japan and stained with hematoxylin and eosin H&E and Elastica van Gieson EVG stains. EVG staining was performed using Sirius red solution instead of fuchsin acid solution with a slight modification to the previously reported method 19 . The deparaffinized tissue sections were soaked in resorcinfuchsin solution Nacalai Tesque, Kyoto, Japan for 30 min. After rinsing in tap water, sections were stained for 10 min with a 1:1 mixture of Weigert s iron hematoxylin solution I 1 hematoxylin in ethanol : solution II 2 ferric chloride in 0.25 HCl . After decolorizing in 1 hydrochloric acid alcohol, the sections were stained with 1 Sirius red solution diluted 1:20 in van Gieson picric acid solution Wako Pure Chemical industries for 15 min. Dehydrated with ethanol, permeated with xylene, and covered with a lipid-soluble mounting medium and glass cover slips. The destruction rate of the wavy configuration of the elastic lamina was calculated by dividing the area of destruction indicated by flattening and fragmentation of the elastic lamina by the entire area of elastic lamina as previously descripted 20 . Collagen fibers were observed using a polarizing microscope, and the positive area of collagen was calculated by binarizing the image into black and red using software National Institutes of Health, Bethesda, MD, USA .

Immunohistochemical staining
The deparaffinized tissue sections were permeabilized Then, to block endogenous peroxidase, it was soaked in 3 hydrogen peroxide in methanol for 8 min. After washing with PBS, the tissue sections were blocked with Blocking One Histo Nacalai Tesque, Kyoto, Japan for 30 min. The tissue sections were incubated with the primary antibody overnight at 4 . The following antibodies were used: rabbit anti-matrix metalloproteinase MMP -2 1:100; GeneTex, Irvine, CA, USA and goat anti-MMP-9 1:50; Santa Cruz Biotechnology, Dallas, TX . The following day, the sections were washed with PBS and incubated with the appropriate secondary antibody conjugated to HRP for 30 min. A DAB kit Vector Laboratories, Burlingame, CA, USA was used to detect the target proteins. The area of positive staining in immunohistochemistry was calculated by binarizing the images into black and white images using ImageJ.

Statistical analyses
The experimental data are expressed as the mean standard error of the mean SEM . Statistical differences were determined using the Fisher PLSD.

Results
3.1 Effects of OVX and iso avones on body weight, food intake, weight of adipose tissue and weight of organs The average body weight was significantly higher in the OI group than in the C group on days 26, 42, and 43 Fig.  1a . No significant differences in food intake were observed among the groups Fig. 1b . Oviduct weight was significantly lower in OVX treated groups O and OI than in the sham treated group Fig. 1c , indicating that OVX treatment was appropriate. There were no significant differences in oviduct peripheral fat weight, brown fat weight, liver weight and kidney weight among all groups. 3.2 Effects of OVX and Iso avone diet on serum parameters Serum glucose and total cholesterol levels were significantly higher in the OI group than in the C group Figs. 2a, 2c . There was no significant difference groups in serum triglyceride levels among the groups Fig. 2b . 3.3 Effects of OVX and Iso avone diet on elastic and collagen bers in the thoracic aortic wall In the thoracic aorta, there was no significant difference in the thickness of the vascular wall between all groups Figs. 3a-3d . The destruction rate of elastin was significantly higher in the O group than the C group, and was significantly lower in the OI group than the O group Figs. 3e-3h . The collagen-positive area was significantly higher in O and OI groups than in C group Figs. 3i-3l .

Effects of OVX and Iso avone diet on elastic and collagen bers in abdominal aortic wall
In the abdominal aorta, there was no significant difference in the thickness of the vascular wall between all groups Figs. 4a-4d . The destruction rate of elastin was significantly higher in O group than in C and OI groups Figs. 4e-4h . There was no significant difference in the collagen-positive area between the groups Figs. 4i-4l .

Effects of OVX and Isoflavone diet on MMP-2 and
MMP-9 in thoracic aorta wall In the MMP-2 positive area, there was no significant difference between all groups in the adventitia and intima Figs. 5a-5d ; however, O group tended to be higher than C group Fig. 5d . In the MMP-9 positive area, there was no significant difference in adventitia Figs. 5e-5h . In the intima-media, the MMP-9 positive area in O group was significantly increased compared to that in C group Figs.
5e-5h .   areas in OI group in intima-media were significantly lower than those in O group Figs. 6a-6h .

Discussion
The average body weight was higher in the OI group than in the C and O groups. It has been reported that OVX treatment increases body weight 21 , and isoflavone has fatburning and anti-obesity effects. In this experiment, body weight increased, but there was no significant difference in fat or organ weight, suggesting that the subjects were not obese or ill.
In serum parameters, blood glucose and total cholesterol levels were higher in the OI group than in the C and O groups. Isoflavone has been reported to lower blood glucose levels in diabetic rats 22 , but the mice used in this experiment were not diabetic and had not fasted before dissection, which may have caused the difference.
OVX treatment has been reported to induce degradation of aortic fibers in the thoracic and abdominal aorta 5,6 . It has also been reported that OVX treatment decreases the levels of smooth muscle cells and elastic fibers in the mouse aortic wall 23 , which is consistent with the results obtained in this study. In addition, the destruction rate of elastin in both the thoracic and abdominal aorta was significantly lower in group OI than in group O Figs. 3h, 4h . These results suggest that the destruction rate of elastic fibers in the thoracic and abdominal aorta induced by OVX treatment may be inhibited by isoflavone administration.
To investigate how the destruction of elastic fibers is induced, immunohistochemical staining was performed to observe MMP-2 and MMP-9, which are involved in the destruction of aortic fiber components. In the thoracic aorta, the MMP-2 positive area in O group tended to be higher than that in C group for both the intima-media and adventitia. In the thoracic aorta, the MMP-9 positive area in the adventitia tended to be lower in OI group than in O group, and in the intima-media was significantly increased in O group compared to C group. In the abdominal aorta, the MMP-2 and MMP-9 positive areas in the intima-media were significantly higher in O group higher in C group, and there was no significant difference between the C group and the OI group. MMP-2 is mainly derived from smooth muscle cells and fibroblasts, while MMP-9 is mainly derived from macrophages 24 . Both MMP-2 and MMP-9 are known to degrade fibrous components of the vascular wall 24 . In O group, both the thoracic aorta and abdominal aorta showed destruction of elastic fibers and increased positive areas of MMP-2 and MMP-9 in the intima-media. However, in the OI group treated with isoflavone, no increase in MMP-2 and MMP-9 positive areas in the intima media of the thoracic and abdominal aorta were observed, suggesting that suppressive mechanisms are associated with a common pathway between MMP-2 and MMP-9.
There are two estrogen receptor ER types, α and β. ERα is expressed in the uterus, ovaries, and hypothalamus, whereas ERβ is specifically expressed in the ovaries. ERα and ERβ play different roles in the immune system, skeletal system, central nervous system, and cardiovascular system 24 26 , and are expressed in the human vascular smooth muscle of the coronary arteries and aorta. ERα and ERβ are expressed in endothelial and smooth muscle cells in the aortic wall 27,28 , and ERs are necessary for estrogenmediated vasoprotection 29 . ERβ expression is more common in females 30 . Both ERα and ERβ are expressed in macrophages 31 . It has been suggested that one of the mechanisms by which estrogen replacement therapy reduces the risk of cardiovascular disease in menopausal women is by improving vascular reactivity 32 . To suppres- sive effect of isoflavones may be involved in ERs. There are several types of the isoflavones, and isoflavone used in this experiment contain more than 50 genistein. It has been suggested that genistein acts through estrogen receptors 33 , and it has been reported that genistein have multiple cellular effects, including the inhibition of MARK pathway 34 and NF-κB activation 35 . It has also been reported to inhibit the expression of MMP-2 and MMP-9 by being involved in the inhibition of MARK pathway and/or NF-κB activity 35,36 . Therefore, we speculate that genistein in the isoflavones suppressed the destruction of elastic fibers by inhibiting the increase of MMP-2 and MMP-9 in the inner tunica media of the aortas through its involvement in the inhibition of the MARK pathway and NF-κB activity. It can be inferred that the inhibition of MMP-2 and MMP-9 in the medial tunica media of the respective aortas suppressed elastic fiber destruction.
In conclusion, we demonstrated the suppressive effect of isoflavones on OVX-induced degeneration of the aortic walls. However, it should be noted weight gain and increased levels of serum glucose and total cholesterol were observed in the OI group. In addition, adverse effects such as excessive estrogen in the body due to excessive estrogen intake have been reported to cause cervical cancer 37 . Further research is needed to determine the optimal conditions that do not adversely affect health and lifespan.