Objective

Osteoporosis is affecting the health of postmenopausal women in the world. In case of that, we explored whether FK-506 could ameliorate osteoporosis by inhibiting the activated CaN/NFAT pathway during oxidative stress.

Methods

First, the castrated rat model is constructed through the bilateral ovariectomy. Hologic Discovery (S/N 80347) dual-energy X-ray absorptiometry assessed bone mineral density (BMD) implemented at left femur of rats. Next, hematoxylin–eosin (H&E) staining observed and calculated the changes of bone trabecular, mean trabecular plate separation (Tb.Sp), mean trabecular plate thickness (Tb.Th), and bone volume fraction (BV/TV). Then, CCK-8 assay, TUNEL assay, ALP kit and alizarin red staining detected the viability, apoptosis, alkaline phosphatase (ALP) activity, and capacity of mineralization respectively. At last, commercially available kits detected the levels of ROS and SOD in transfected MC3T3-E1 cells and bone tissues, and Western blot analysis detected proteins related to apoptosis and CaN/NFAT pathway.

Results

FK-506 increased the BMD and changes of bone trabecular in female castrated rats. FK-506 inhibited the oxidative stress and apoptosis by suppressing the activated CaN/NFAT pathway. Low dose of FK-506 improved the viability, ALP activity, and mineralization capacity. What’s more, it suppressed the apoptosis of H₂O₂-induced MC3T3-E1 cells, which was deteriorated by the high dose of FK-506. Briefly, low dose of FK-506 inhibited the oxidative stress by suppressing the activated CaN/NFAT pathway, while high dose of that further inhibited the oxidative stress by suppressing the CaN/NFAT pathway.

Conclusion

FK-506 ameliorates osteoporosis resulted from osteoblastic apoptosis which caused by suppressing the activated CaN/NFAT pathway during oxidative stress.

中文翻译：

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FK506通过抑制氧化应激期间激活的CaN/NFAT通路改善成骨细胞凋亡引起的骨质疏松症

客观的

骨质疏松症正在影响全世界绝经后妇女的健康。在这种情况下，我们探讨了 FK-506 是否可以通过抑制氧化应激期间激活的 CaN/NFAT 途径来改善骨质疏松症。

方法

首先，通过双侧卵巢切除构建去势大鼠模型。Hologic Discovery (S/N 80347) 双能 X 射线吸收测定法评估了大鼠左股骨的骨矿物质密度 (BMD)。接下来，苏木精-伊红（H&E）染色观察并计算骨小梁、平均小梁板间距（Tb.Sp）、平均小梁板厚度（Tb.Th）和骨体积分数（BV/TV）的变化。然后，CCK-8法、TUNEL法、ALP试剂盒和茜素红染色分别检测细胞活力、细胞凋亡、碱性磷酸酶（ALP）活性和矿化能力。最后，市售试剂盒检测转染的MC3T3-E1细胞和骨组织中ROS和SOD的含量，Western印迹分析检测与细胞凋亡和CaN/NFAT通路相关的蛋白。

结果

FK-506 增加了雌性去势大鼠的 BMD 和骨小梁的变化。FK-506 通过抑制激活的 CaN/NFAT 通路来抑制氧化应激和细胞凋亡。低剂量的 FK-506 提高了生存能力、ALP 活性和矿化能力。更重要的是，它抑制了H ₂ O ₂诱导的MC3T3-E1细胞的凋亡，后者因高剂量的FK-506而恶化。简而言之，低剂量的 FK-506 通过抑制激活的 CaN/NFAT 通路来抑制氧化应激，而高剂量的 FK-506 通过抑制 CaN/NFAT 通路进一步抑制氧化应激。

结论

FK-506 可改善成骨细胞凋亡导致的骨质疏松症，该细胞凋亡是由在氧化应激期间抑制激活的 CaN/NFAT 途径引起的。