Translational Medical Innovation Center, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, China.
Department of Orthopedics, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, China.
Cell Prolif. 2022 Oct;55(10):e13291. doi: 10.1111/cpr.13291. Epub 2022 Jun 16.
The main target of current drugs for alleviating bone loss is osteoclasts. However, the long-term application of such drugs will also cause side effects. Therefore, it is of great need to develop new and safer therapeutics for osteoporosis. In recent years, drug development based on gut microbiota has gradually attracted attention. This manuscript investigates the inhibitory effect of urolithin B (UB) on osteoclastogenesis and differentiation in vitro and in ovariectomized (OVX) mice.
CCK-8 was used to analyse the cytotoxicity of UB; BMMs cells were differentiated into osteoclasts by RANKL, and respectively treated with 1, 5, and 25 μmol/L UB during this process. After one week of intervention, tartrate-resistant acid phosphatase (TRAP) staining was used to analyse the number and average area of osteoclasts. F-actin staining and immunofluorescence staining were conducted to evaluate the morphology and function of osteoclasts. Bone resorption function of osteoclasts was detected by Pit Formation Assay. The expression of osteoclast-related protein genes in RAW264.7 cells were investigated via western blot and RT-PCR assays. Western blot analysis of RANKL-mediated activation of MAPK/NF-κB pathway after 0, 5, 15, 30, 60 min of intervention. For in vivo experiments, OVX mice received intraperitoneal injection of 10, 50 mg/kg every two days, 8 weeks later, the femurs of mice were taken for morphological analysis, and the serum content of CTX-1, a bone metabolism index, was analysed.
UB could inhibit the osteoclast differentiation of rankl-induced bone marrow macrophages (BMMs) and RAW264.7 cells in vitro, suppress the uptake activity of hydroxyapatite and expression of osteoclast-related gene MMP9, CTSK, NFATc1 and c-fos. Furthermore, UB repressed the rankl-induced phosphorylation and degradation of IκB and the phosphorylation of P65 in the NF-κB pathway of RAW264.7 cells, and also down-regulated the phosphorylation level of ERK in the MAPK pathway. For in vivo studies, UB-treated OVX mice showed more significant improved various parameters of distal femur compared with the control group, with fewer NFATc1, MMP9 and TRAP-positive osteoclasts in bone tissues, and less serum content of CTX-1.
Urolithin B attenuated bone loss in OVX mice by inhibiting the formation and activation of osteoclasts via down-regulation of the ERK/NF-κB signalling pathway.
目前用于缓解骨质流失的药物主要针对破骨细胞。然而，此类药物的长期应用也会引起副作用。因此，开发新的、更安全的骨质疏松症治疗方法非常有必要。近年来，基于肠道微生物群的药物开发逐渐受到关注。本文研究了尿石素 B（UB）在体外和去卵巢（OVX）小鼠中对破骨细胞形成和分化的抑制作用。
用 CCK-8 法分析 UB 的细胞毒性；用 RANKL 将骨髓单核细胞（BMM）细胞分化为破骨细胞，在此过程中分别用 1、5 和 25 μmol/L 的 UB 进行干预。干预 1 周后，采用抗酒石酸酸性磷酸酶（TRAP）染色分析破骨细胞的数量和平均面积。用 F-肌动蛋白染色和免疫荧光染色评估破骨细胞的形态和功能。通过 Pit 形成分析检测破骨细胞的骨吸收功能。用 Western blot 和 RT-PCR 检测 RAW264.7 细胞中破骨细胞相关蛋白基因的表达。用 Western blot 分析 RANKL 介导的 MAPK/NF-κB 通路激活后 0、5、15、30、60 min 的情况。在体内实验中，OVX 小鼠腹腔注射 10、50 mg/kg，每两天一次，8 周后取小鼠股骨进行形态学分析，分析骨代谢指标 CTX-1 的血清含量。
UB 可抑制体外 RANKL 诱导的骨髓巨噬细胞（BMM）和 RAW264.7 细胞的破骨细胞分化，抑制羟磷灰石摄取活性和破骨细胞相关基因 MMP9、CTSK、NFATc1 和 c-fos 的表达。此外，UB 抑制了 RANKL 诱导的 RAW264.7 细胞中 IκB 的磷酸化和降解以及 P65 在 NF-κB 通路中的磷酸化，并下调了 MAPK 通路中 ERK 的磷酸化水平。在体内研究中，与对照组相比，UB 处理的 OVX 小鼠的远端股骨的各种参数有更显著的改善，骨组织中 NFATc1、MMP9 和 TRAP 阳性破骨细胞较少，CTX-1 的血清含量较少。
UB 通过下调 ERK/NF-κB 信号通路，抑制破骨细胞的形成和激活，减轻 OVX 小鼠的骨质流失。
