Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, China.
Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, China.
J Ethnopharmacol. 2022 Mar 1;285:114834. doi: 10.1016/j.jep.2021.114834. Epub 2021 Nov 18.
Semen Cuscutae, called Tu-si-zi in Chinese, is a kind of dried mature seed in the Convolvulaceae family. It mainly distributes in China, Korea, Pakistan, Vietnam, India and Thailand. It is used as a kidney-tonifying drug for treatment of aging related diseases such as osteoporosis in traditional Chinese medicine. However, the exact mechanisms on bone resorption are poorly studied.
The aim of this study was to investigate the potential effect of Semen Cuscutae on ovariectomy (OVX)-induced osteoporosis in mice and clarify the exact mechanisms by which Semen Cuscutae exert the anti-osteoporosis effect.
Qualitative and quantitative analyses of Semen Cuscutae were performed by UPLC-Q-TOF-MS and HPLC-MS/MS, respectively. Changes in bone mineral density (BMD) induced by OVX in mice were measured by dual-energy X-ray absorptiometry and micro-computed tomography (μCT). Tartrate-resistant acid phosphatase (TRAP) staining as well as hematoxylin and eosin (HE) staining were used to observe bone microarchitectural changes. ELISA kits were used to assess the therapeutic effects of Semen Cuscutae on the serum levels of osteoprotegerin (OPG), tartrate-resistant acid phosphatase 5b (TRACP-5b), and receptor activator of nuclear factor-κB (RANKL). The effect of Semen Cuscutae on primary cell viability was assessed using CCK-8 and anti-tartrate phosphatase assays. TRAP staining and actin ring staining were used to observe the effect of Semen Cuscutae on osteoclast differentiation. Western blotting was used to measure the effects of Semen Cuscutae on expressions of NFATC1, c-Src kinase, and c-fos.
Results from UPLC-Q-TOF-MS showed that the main components of Semen Cuscutae were flavonoid compounds that included quercitrin, quercetin, hyperoside, caffeic acid, rutin, chlorogenic acid, luteolin, apigenin, kaempferol, isoquercetin, cryptochlorogenic acid, isorhamnetin-3-O-glucoside, and astragalin. After the Semen Cuscutae extract was orally administered to OVX mice, bone density increased (P < 0.01) and bone microstructure was significantly improved (P < 0.01 or 0.05). Additionally, Semen Cuscutae exhibited a significant descending effect in the levels of serum TRACP-5b and RANKL, while there was a significant increase in OPG in the Semen Cuscutae group compared with the OVX group, especially at high doses. Moreover, we found that increasing of c-fos, c-Src kinase, and NFATC1 protein expressions were reversed by Semen Cuscutae in vitro and in vivo.
Our results showed that Semen Cuscutae exhibited anti-osteoporosis effects through the c-fos/c-Src kinase/NFATC1 signaling pathway.
菟丝子，中文名为菟丝子，是旋花科植物的一种干燥成熟种子。它主要分布在中国、韩国、巴基斯坦、越南、印度和泰国。它在传统中医中被用作补肾药，用于治疗骨质疏松症等与衰老相关的疾病。然而，其对骨吸收的确切机制研究甚少。
本研究旨在探讨菟丝子对去卵巢（OVX）诱导的骨质疏松症小鼠的潜在作用，并阐明菟丝子发挥抗骨质疏松作用的确切机制。
采用 UPLC-Q-TOF-MS 和 HPLC-MS/MS 分别对菟丝子进行定性和定量分析。双能 X 射线吸收仪和微计算机断层扫描（μCT）测量 OVX 诱导的小鼠骨密度（BMD）变化。用抗酒石酸酸性磷酸酶（TRAP）染色和苏木精-伊红（HE）染色观察骨微观结构变化。ELISA 试剂盒用于评估菟丝子对血清骨保护素（OPG）、抗酒石酸酸性磷酸酶 5b（TRACP-5b）和核因子-κB 受体激活剂（RANKL）水平的治疗作用。CCK-8 和抗酒石酸磷酸酶检测法评估菟丝子对原代细胞活力的影响。TRAP 染色和肌动蛋白环染色观察菟丝子对破骨细胞分化的影响。Western blot 用于测量菟丝子对 NFATC1、c-Src 激酶和 c-fos 表达的影响。
UPLC-Q-TOF-MS 结果表明，菟丝子的主要成分是黄酮类化合物，包括槲皮素、槲皮素、金丝桃苷、咖啡酸、芦丁、绿原酸、木犀草素、芹菜素、山奈酚、异槲皮苷、隐绿原酸、异鼠李素-3-O-葡萄糖苷和黄芪苷。菟丝子提取物口服给予去卵巢小鼠后，骨密度增加（P<0.01），骨微结构明显改善（P<0.01 或 0.05）。此外，与 OVX 组相比，菟丝子组血清 TRACP-5b 和 RANKL 水平显著下降，而 OPG 水平显著升高，尤其是高剂量组。此外，我们发现，c-fos、c-Src 激酶和 NFATC1 蛋白表达的增加在体外和体内均被菟丝子逆转。
我们的结果表明，菟丝子通过 c-fos/c-Src 激酶/NFATC1 信号通路发挥抗骨质疏松作用。
