Guo Wen, Li Haijun, Lou Yan, Zhang Yue, Wang Jia, Qian Ming, Wei Haifeng, Xiao Jianru, Xu Youjia
Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, China.
Department of Orthopedics, Taizhou People's Hospital, Taizhou, 225300, Jiangsu, China.
J Orthop Translat. 2021 Apr 10;28:148-158. doi: 10.1016/j.jot.2021.01.005. eCollection 2021 May.
Tyloxapol is a non-ionic surfactant with diverse pharmacological effects including anti-inflammatory, anti-malignant tumor and antioxidant activities. However, the effect of tyloxapol on osteoclastogenesis has not been elucidated. In this study, we intended to clarify the effect of tyloxapol on RANKL-stimulated osteoclastogenesis and the molecular mechanism both ex vivo and in vivo.
In vitro osteoclastogenesis assay was performed in BMMs and Raw 264.7 cells. The mature osteoclasts were visualized by TRAP staining. The osteoblsats were visualized by alkaline phosphatase (ALP) staining and Von Kossa staining. To assess whether tyloxapol inhibited the function of mature osteoclasts, F-actin belts and pit formation assays were carried out in BMMs. To evaluate the effect of tyloxapol on post-menopausal osteoporosis, the OVX mouse model were utilized. The bone tissue TRAP staining was used to evaluate the osteoclast activity in vivo. The von kossa staining and micro computed tomography were used to evaluate the histomorphometric parameters. The Goldner's staining was used to evaluate the osteoblast activity. The expression of osteoclastogenesis-associated markers were evaluated by Real-time PCR. The NF-κB and NFATc1 transcriptional activities were illustrated utilizing the assay of luciferase reporter. The effect of tyloxapol pretreatment on IκBa degradation and p65 phosphorylation was evaluated using Western bloting assay. The effect of tyloxapol pretreatment on p65 nuclear translocation was evaluated utilizing immunofluorescence. The effect of tyloxapol pretreatment on the phosphorylatio of ERK, p38 and JNK was examined utilizing Western bloting assay.
In our research, we found that tyloxapol suppresses RANKL-stimulated osteoclastogenesis in a dose dependent manner and in the initial stage of osteoclastogenesis. Through F-actin belts and pit formation assays, we found that tyloxapol had the ability to inhibit the function of mature osteoclasts in vitro. The results of animal experiments demonstrated that tyloxapol inhibits OVX-induced bone mass loss by inhibiting the activity of osteoclasts but had a limited effect on osteoblastic differentiation and mineralization. Molecularly, we found that tyloxapol suppresses RANKL-stimulated NF-κB activation through suppressing degradation of IκBα, phosphorylation and nuclear translocation of p65. At last, MAPK signaling pathway was also suppressed by tyloxapol in dose and time-dependent manners.
Our research illustrated that tyloxapol was able to suppress osteoclastogenesis in vitro and ovariectomized-induced bone loss in vivo by restraining NF-κB and MAPK activation. This is pioneer research could pave the way for the development of tyloxapol as a potential therapeutic treatment for osteoporosis.
This study explores that tyloxapol, also known as Triton WR-1339, may be a drug candidate for osteoclastogenic sicknesses like osteoporosis. Our study may also extend the clinical therapeutic spectrum of tyloxapol.
泰洛沙泊是一种具有多种药理作用的非离子表面活性剂，包括抗炎、抗肿瘤和抗氧化活性。然而，泰洛沙泊对破骨细胞生成的影响尚未阐明。在本研究中，我们旨在阐明泰洛沙泊对RANKL刺激的破骨细胞生成的影响以及体内外的分子机制。
在骨髓巨噬细胞（BMMs）和Raw 264.7细胞中进行体外破骨细胞生成试验。通过抗酒石酸酸性磷酸酶（TRAP）染色观察成熟破骨细胞。通过碱性磷酸酶（ALP）染色和冯科萨（Von Kossa）染色观察成骨细胞。为了评估泰洛沙泊是否抑制成熟破骨细胞的功能，在BMMs中进行F-肌动蛋白环和骨吸收陷窝形成试验。为了评估泰洛沙泊对绝经后骨质疏松症的影响，使用去卵巢（OVX）小鼠模型。骨组织TRAP染色用于评估体内破骨细胞活性。冯科萨染色和显微计算机断层扫描用于评估组织形态计量学参数。戈德纳（Goldner）染色用于评估成骨细胞活性。通过实时聚合酶链反应（Real-time PCR）评估破骨细胞生成相关标志物的表达。利用荧光素酶报告基因检测法说明核因子κB（NF-κB）和活化T细胞核因子c1（NFATc1）的转录活性。使用蛋白质免疫印迹法评估泰洛沙泊预处理对IκBα降解和p65磷酸化的影响。利用免疫荧光评估泰洛沙泊预处理对p65核转位的影响。使用蛋白质免疫印迹法检测泰洛沙泊预处理对细胞外信号调节激酶（ERK）、p38丝裂原活化蛋白激酶（p38）和应激活化蛋白激酶（JNK）磷酸化的影响。
在我们的研究中，我们发现泰洛沙泊在破骨细胞生成的初始阶段以剂量依赖性方式抑制RANKL刺激的破骨细胞生成。通过F-肌动蛋白环和骨吸收陷窝形成试验，我们发现泰洛沙泊在体外具有抑制成熟破骨细胞功能的能力。动物实验结果表明，泰洛沙泊通过抑制破骨细胞活性抑制OVX诱导的骨量丢失，但对成骨细胞分化和矿化的影响有限。在分子水平上，我们发现泰洛沙泊通过抑制IκBα降解、p65磷酸化和核转位来抑制RANKL刺激的NF-κB活化。最后，泰洛沙泊还以剂量和时间依赖性方式抑制丝裂原活化蛋白激酶（MAPK）信号通路。
我们的研究表明，泰洛沙泊能够通过抑制NF-κB和MAPK活化在体外抑制破骨细胞生成，并在体内抑制去卵巢诱导的骨质流失。这项开创性研究可为泰洛沙泊作为骨质疏松症潜在治疗药物的开发铺平道路。
本研究探索了泰洛沙泊（也称为聚氧乙烯月桂醚）可能是骨质疏松症等破骨细胞相关疾病的候选药物。我们的研究还可能扩展泰洛沙泊的临床治疗谱。
