中药龟鹿二仙胶通过体外 Mc3t3 衍生细胞外囊泡抑制破骨细胞的形成和活性。

Chinese Herbal Medicine Guilu Erxian Glue Inhibits Osteoclast Formation and Activity via Mc3t3-Derived Extracellular Vesicles in vitro.

出版信息

Altern Ther Health Med. 2023 Sep;29(6):400-407.

Abstract

BACKGROUND

Osteoporosis is a systemic bone disease characterized by decreased bone density and quality, destruction of bone microstructure, and increased bone fragility. Extracellular vesicles are lipid bilayer nanoparticles that participate in intercellular communication. Extracellular vesicles are becoming popular in the study of osteoporosis and the bone cell microenvironment. Extracellular vesicles can transmit cell signals and regulate bone homeostasis. Our previous studies revealed that the Chinese herbal medicine Guilu Erxian Glue promotes type I collagen synthesis and osteoprotegerin secretion by osteoblasts in rats, reverses the imbalance of bone homeostasis, and alleviates osteoporosis.

OBJECTIVE

We investigated how osteoblast-derived extracellular vesicles treated with Guilu Erxian Glue affected osteoclasts in vitro.

METHODS

We quantified osteoclast differentiation of RAW 264.7 using TRAP staining, cell apoptosis using flow cytometry, extracellular vesicle uptake by fluorescence tracing, bone absorption functions by bone resorption lacuna , and transcription of key genes by quantitative real-time PCR.

RESULTS

Fluorescently labeled mouse preosteoblastic MC3T3-E1 cells secreted nanoscale substances less than 1 μm in diameter. Mouse macrophage RAW 264.7 cells adsorbed these nanoparticles and PKH26-labeled extracellular vesicles derived from MC3T3-E1 cells on the cell membrane surface. Extracellular vesicles from MC3T3-E1 cells treated with Guilu Erxian Glue inhibited the differentiation of osteoclasts induced by receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor and reduced the number of lacunae formed by osteoclasts in vitro compared with controls. Extracellular vesicles from MC3T3-E1 cells treated with Guilu Erxian Glue downregulated the relative messenger RNA expression of c-Fos, cathepsin K, nuclear factor of activated T cells 1, and tartrate-resistant acid phosphatase in osteoclasts, which may be part of the mechanism by which they regulate osteoclasts.

CONCLUSIONS

Our results demonstrate that extracellular vesicles are essential for signal exchange between osteoblasts and osteoclasts. Although we do not know how Guilu Erxian Glue affects the signaling molecules carried by extracellular vesicles, we have shown for the first time, to our knowledge, that Guilu Erxian Glue can inhibit osteoclast differentiation and function via osteoblast-derived extracellular vesicles. Our findings are conducive to providing a new target for the development of osteoporosis drugs.

摘要

背景

骨质疏松症是一种以骨密度和质量降低、骨微观结构破坏以及骨脆性增加为特征的系统性骨骼疾病。细胞外囊泡是参与细胞间通讯的双层脂质纳米颗粒。细胞外囊泡在骨质疏松症和骨细胞微环境的研究中越来越受到关注。细胞外囊泡可以传递细胞信号，调节骨内稳态。我们之前的研究表明，中药龟鹿二仙胶可促进大鼠成骨细胞中 I 型胶原的合成和护骨素的分泌，逆转骨内稳态失衡，缓解骨质疏松症。

目的

我们研究了龟鹿二仙胶处理的成骨细胞衍生的细胞外囊泡对体外破骨细胞的影响。

方法

通过 TRAP 染色定量检测 RAW 264.7 细胞的破骨细胞分化，通过流式细胞术检测细胞凋亡，通过荧光示踪检测细胞外囊泡摄取，通过骨吸收陷窝检测骨吸收功能，通过实时定量 PCR 检测关键基因的转录。

结果

荧光标记的小鼠前成骨细胞 MC3T3-E1 分泌的纳米级物质直径小于 1μm。小鼠巨噬细胞 RAW 264.7 细胞吸附这些纳米颗粒和 PKH26 标记的 MC3T3-E1 细胞来源的细胞外囊泡在细胞膜表面。与对照组相比，龟鹿二仙胶处理的 MC3T3-E1 细胞来源的细胞外囊泡抑制了核因子-κB 配体和巨噬细胞集落刺激因子诱导的破骨细胞分化，并减少了体外形成的破骨细胞陷窝数量。龟鹿二仙胶处理的 MC3T3-E1 细胞来源的细胞外囊泡下调了破骨细胞中 c-Fos、组织蛋白酶 K、激活 T 细胞核因子 1 和抗酒石酸酸性磷酸酶的相对信使 RNA 表达，这可能是其调节破骨细胞的部分机制。