Ritodrine hydrochloride is used for pregnancy prolongation and intrauterine fetal resuscitation. However, its clinical significance in intraamniotic inflammation during preterm labor and intrauterine fetal distress is unclear. We investigated the effects of maternal ritodrine hydrochloride administration (MRA; 200 μg/min for 2 h, followed by 800 μg/min for 2 h after 24 h) on fetal physiological parameters. For this purpose, we used chronically instrumented pregnant sheep at 113-119 d (term = 145 d) of gestation without (Group 1, n = 5) and with (Group 2, n = 5) intraamniotic inflammation induced by lipopolysaccharide injection into the amniotic cavity. The changes in fetal heart rate (FHR) and short-term variability (STV) and long-term variability (LTV) in FHR, fetal blood pressure, and fetal arterial blood gas (FABG) values were measured before and at 1 and 2 h after initiating MRA. Before MRA, all parameters were similar between Groups 1 and 2; however, there was significantly higher STV in Group 2 than in Group 1 before MRA at 800 μg/min, significantly higher partial arterial pressure of carbon dioxide in FABG in Group 2 than in Group 1 before MRA at 200 μg/min, and significantly lower blood glucose (BG) in Group 2 than in Group 1 before MRA at 800 μg/min. One hour after MRA, the FHR, STV, and LTV were significantly higher at 800 μg/min than those at the baseline in Group 1, as determined by the Friedman test; however, no significant difference was observed in Group 2. Additionally, the FABG pH significantly decreased 1 h after MRA at 800 μg/min in Group 2, whereas FABG lactate and BG significantly increased 2 h after MRA at 800 μg/min in Groups 1 and 2. Thus, short-term MRA at 800 μg/min increased the FHR, STV, and LTV significantly; these values were further modified under intraamniotic inflammation.

OBJECTIVE: Postmenopausal women are at increased risk of metabolic diseases such as obesity and diabetes. Therefore, the chemoprevention of postmenopausal changes in health via dietary supplements is important. Syringic acid (SA) is a phenolic compound present in the fruit of the assai palm, Euterpe oleracea, and in the mycelium of the shiitake mushroom, Lentinula edodes. This compound shows no affinity for estrogen receptors and may exert disease-preventive effects. Reportedly, dietary SA ameliorates high-fat diet-induced obesity in mice; however, its effects on estrogen deficiency-induced obesity are still unclear. Therefore, in this study, we investigated whether and how dietary SA affects these factors in ovariectomized (OVX) mice. METHODS: Ten-week-old OVX mice were fed SA-containing diets (100 mg/kg body weight/d) for 12 weeks. Their body weights, food intake, and uterus weights as well as other parameters were measured and comparisons were made with mice in the control group. RESULTS: Dietary SA did not affect the body weight, food intake, or uterus weight of OVX mice over the study period; however, the SA-fed group showed lower fat mass (ie, visceral, subcutaneous, and total fat) than the OVX-control group (11.1 ± 3.3 vs. 8.3 ± 2.4, P < 0.05; 7.9 ± 1.1 vs. 5.9 ± 1.6, P < 0.05; 19.0 ± 4.2 vs. 14.1 ± 3.8, P < 0.05, respectively). Furthermore, blood analysis revealed that SA-treatment resulted in a dose-dependent decrease and increase in serum triglyceride (59.2 ± 8.3 vs. 43.9 ± 12.2 mg/dL P < 0.05) and adiponectin (7.7 ± 0.3 vs. 9.5 ± 0.6 μg/mL, P < 0.05) levels, respectively. CONCLUSIONS: These results suggest that the SA diet improves lipid metabolism without affecting the uterus in OVX mice. Therefore, dietary SA has potential applicability for the prevention of postmenopausal obesity and type 2 diabetes.

In recent years, the number of patients with osteoporosis has increased as population grows older. Therefore, the chemoprevention of osteoporosis by better nutrition is important. White-rot fungi degrades milled wood lignin for growth and development. This degradation results in the formation of phenolic compounds such as syringic acid (SA) and vanillic acid (VA). In the artificial culture of edible mushrooms using a mushroom bed, the disposal of waste beds after mushroom cultivation is an important issue. The present study investigated the presence and amount of both SA and VA in the discarded waste beds after mushroom cultivation. The extracts from waste beds after cultivation of shiitake mushrooms, Lentinula edodes; buna shimeji, Hypsizygus marmoreus; maitake, Grifola frondosa; king trumpet mushrooms, Pleurotus eryngii; and butterscotch mushrooms, Pholiota microspora were analyzed using high performance liquid chromatography. Although the content of SA and VA was considerably different among the mushrooms, SA and VA were present in extracts obtained from all the waste beds. We also demonstrated that SA and VA exert their anti-osteoporotic effect independently of the estrogen receptor-mediated pathway using murine monocytic RAW264.7 cells, ovariectomized mice, and human breast cancer MCF-7 cells. Thus, these results suggest that the extracts are effective sources of SA and VA, which are effective in preventing osteoporosis.

OBJECTIVE: Osteoclastogenic activation of macrophages (OCG) occurs in human abdominal aortic aneurysms (AAAs) and in calcium chloride-induced degenerative AAAs in mice, which have increased matrix metalloproteinase activity. As the activity of OCG in dissecting aneurysms is not clear, we tested the hypothesis that OCG contributes to angiotensin II (Ang II)-induced dissecting aneurysm (Ang II-induced AAA) in apolipoprotein E knockout mice. METHODS: AAAs were produced in apolipoprotein E knockout mice via the administration of Ang II. Additionally, receptor activator of nuclear factor kB ligand (RANKL)-neutralizing antibody (5 mg/kg) was administered to one group of mice 7 days prior to Ang II infusion. Aneurysmal sections were probed for presence of RANKL and tartrate-resistant acid phosphatase via immunohistochemistry and immunofluorescence staining. Mouse aortas were also examined for RANKL and matrix metalloproteinase 9 expression via Western blot. In vitro murine vascular smooth muscle cells (MOVAS) and murine macrophages (RAW 264.7) were analyzed for the expression of osteogenic factors via Western blot, qPCR, and flow cytometry in response to Ang II or RANKL stimulation. The signaling pathway that mediates Ang II-induced RANKL expression in MOVAS cells was also investigated via application of TG101348, a Janus kinase 2 (JAK2) inhibitor, and Western blot analysis. RESULTS: Immunohistochemical staining of Ang II-induced AAA sections revealed OCG as evidenced by increased RANKL and tartrate-resistant acid phosphatase expression compared with control mice. Immunofluorescence staining of AAA sections revealed co-localization of vascular smooth muscle cells and RANKL, revealing vascular smooth muscle cells as one potential source of RANKL. Systemic administration of RANKL-neutralizing antibody suppressed Ang II-induced AAA, with significant reduction of the maximum diameter of the abdominal aorta compared with vehicle controls (1.5 ± 0.4 mm vs 2.2 ± 0.2 mm). Ang II (1 μM) treatment induced a significant increase in RANKL messenger RNA expression levels in MOVAS cells compared with the vehicle control (1.0 ± 0.2 vs 2.8 ± 0.2). The activities of JAK2 and signal transducer and activator of transcription 5 (STAT5) were also significantly increased by Ang II treatment. Inhibition of JAK2/STAT5 suppressed Ang II-induced RANKL expression, suggesting the involvement of the JAK2/STAT5 signaling pathway. CONCLUSIONS: OCG with increased RANKL expression was present in Ang II-induced AAA, and neutralization of RANKL suppressed AAA formation. As neutralization of RANKL has been used clinically to treat osteoporosis and other osteoclast-related diseases, additional study of the effectiveness of RANKL neutralization in AAA is warranted.

本年度は、破骨細胞を活性化した時の腹部大動脈瘤病変を観察すると共に、食品成分による腹部大動脈瘤予防効果の作用機構の解明を試みた。 女性は65歳以上になると腹部大動脈瘤に発症する可能性が高いことが知られているが、これは閉経によるエストロゲン欠乏により破骨細胞が活性化されることが原因である可能性がある。そこで、卵巣摘出マウスと卵巣摘出擬似手術マウスにおいてCaPO4誘発性の腹部大動脈瘤モデルを作成した。その結果、卵巣摘出マウスは卵巣摘出擬似手術マウスと比較し、破骨細胞が活性化され、腹部大動脈瘤の発症を促進させた。これらのデータは、「破骨細胞」は動脈瘤の発症に重要な役割を果たしていることを示唆しており、腹部大動脈瘤の発症機構の解明や予防・治療薬の開発を目指すための重要な知見になると考えられる。 C-グリコシド型イソフラボン・プエラリンは葛 （クズ、Pueraria lobata）に含有するイソフラボンである。申請者らは、プエラリンは破骨細胞の分化を抑制し、腹部大動脈瘤を予防することを報告したが、詳細な作用機構は不明である。そこで、培養細胞（RAW264.7）を用いて、プエラリンによる破骨細胞の分化抑制効果の作用機構の解明を試みた。次世代シーケンサーを用い、RNA-Seqによりプエラリンを添加した破骨細胞において発現変動する遺伝子を網羅的に解析した結果、脂質代謝関連の遺伝子発現に変動がみられた。