4-辛基衣康酸抑制脂多糖诱导的小鼠颅骨炎性骨吸收的效果研究

doi:10.12439/kqhm.1005-4979.2023.06.003

摘要/Abstract

摘要：

目的：探讨4-辛基衣康酸(4-octyl itaconate，4-OI)对脂多糖（lipopolysaccharide，LPS）诱导的小鼠颅骨溶解的影响。方法：使用CCK-8法检测不同浓度4-OI对RAW 264.7巨噬细胞及小鼠原代骨髓来源巨噬细胞（bone marrow-derived macrophages，BMMs）的细胞活性影响；使用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）、细胞免疫荧光和细胞内活性氧（reactive oxygen species，ROS）检测试剂盒（DCFH-DA）探究不同浓度（12.5 µmmol/L和50 µmmol/L）4-OI对巨噬细胞炎症反应的影响；使用抗酒石酸酸性磷酸酶（tartrate-resistant acid phosphatase，TRAP）染色探究4-OI对破骨细胞分化的作用；构建LPS诱导的小鼠颅骨溶解模型，通过micro-CT和组织学切片评价4-OI对炎性骨溶解的治疗效果。结果：CCK-8结果显示，浓度在50 µmmol/L以下时，4-OI对RAW 264.7巨噬细胞及小鼠BMMs无明显细胞毒性；RT-qPCR及免疫荧光结果显示，4-OI可以浓度依赖性地抑制促炎因子的表达；TRAP染色结果显示，4-OI可有效抑制核因子κB受体活化因子配体（receptor activator of nuclear factorκB ligand，RANKL）诱导的破骨细胞分化；micro-CT和组织学切片结果显示，4-OI可有效缓解颅骨溶解周围的炎症反应及骨吸收程度。结论：4-OI可有效抑制炎性骨溶解，在口腔颌面部骨溶解疾病的治疗方面具有极大的应用前景。

关键词: 4-辛基衣康酸, 炎性骨溶解, 巨噬细胞, 脂多糖

Abstract:

Objective: To investigate the effect of 4-octyl itaconate (4-OI) on lipopolysaccharide (LPS)-induced calvarial osteolysis in mice. Methods: CCK-8 assay was used to evaluate the effect of different concentrations of 4-OI on the cell viability of RAW 264.7 macrophages and murine primary bone marrow-derived macrophages (BMMs). Real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence staining, and intracellular reactive oxygen species (ROS) detection kit (DCFH-DA) were used to explore the effect of 4-OI at different concentrations (12.5 µmmol/L and 50 µmmol/L) on the inflammatory response of macrophages. Tartrate-resistant acid phosphatase (TRAP) staining was used to explore the effect of 4-OI on osteoclast differentiation. The murine LPS-induced mouse calvarial osteolysis model was established, and the therapeutic effect of 4-OI on inflammatory osteolysis was evaluated by micro-CT and histological staining. Results: The results of CCK-8 showed that 4-OI below a concentration of 50 µmmol/L had no obvious cytotoxicity to RAW 264.7 macrophages and murine BMMs. RT-qPCR and immunofluorescence results showed that 4-OI was concentration-dependent, and inhibited the expression of pro-inflammatory cytokines. TRAP staining showed that 4-OI effectively inhibited the osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL). Micro-CT and histological staining demonstrated that 4-OI effectively alleviated the inflammatory response and bone resorption around the osteolysis site. Conclusion: 4-OI can effectively inhibit inflammatory osteolysis, and it has great potential application in the treatment of osteolysis related diseases in oral and maxillofacial region.

Key words: 4-octyl itaconate, inflammatory osteolysis, macrophage, lipopolysaccharide

中图分类号:

R782

温从鹏, 童无忧, 陈旭卓, 赖林锋. 4-辛基衣康酸抑制脂多糖诱导的小鼠颅骨炎性骨吸收的效果研究[J]. 《口腔颌面外科杂志》, 2023, 33(6): 370-378.

WEN Congpeng, TONG Wuyou, CHEN Xuzhuo, LAI Linfeng. The effect of 4-octyl itaconate on lipopolysaccharide-induced murine calvarial osteolysis[J]. 《Journal of Oral and Maxillofacial Surgery》, 2023, 33(6): 370-378.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2023/V33/I6/370

图/表 7

表1 炎症基因引物序列

Table 1 Primers for inflammatory genes and their sequences

基因	上游引物序列(5'-3')	下游引物序列(5'-3')
β-actin	GTCCCTCACCCTCCCAAAAG	GCTGCCTCAACACCTCAACCC
TNF-α	GGCGGTGCCTATGTCTCA	GGCAGCCTTGTCCCTTGA
IL-1β	AGCTTCAGGCAGGCAGTATCAC	CCAGCAGGTTATCATCATCATCC
IL-6	TGCCTTCTTGGGACTGAT	CTGGCTTTGTCTTTCTTGTT
iNOS	ATGGAACAGTATAAGGCAAACACC	GTTTCTGGTCGATGTCATGAGCAAAGG

图1 4-OI对RAW 264.7巨噬细胞及小鼠原代BMMs的细胞毒性检测 A. 4-OI的化学结构式（上图）与分子结构（下图），分子式为C₁₃H₂₂O₄，分子量为242.31 g/mol；B. 不同浓度4-OI对RAW 264.7巨噬细胞细胞活力的影响（上图为培养24 h时，下图为培养48 h时）；C. 不同浓度4-OI对小鼠BMMs细胞活力的影响（上图为培养48 h时，下图为培养96 h时），***表示P <0.001，与0 µmmol/L组比较。

Figure 1 The effect of 4-OI on cell viability of RAW 264.7 macrophages and murine primary BMMs

图2 4-OI对LPS诱导的巨噬细胞炎症的影响 A. RT-qPCR检测4-OI对RAW 264.7巨噬细胞促炎相关基因mRNA表达的影响；B. 免疫荧光检测4-OI对RAW 264.7巨噬细胞内iNOS表达的影响（×400）；C. iNOS阳性细胞比例统计（%）。*表示P <0.05，***表示P <0.001，与LPS组比较。

Figure 2 The effect of 4-OI on LPS-induced inflammation of macrophage

图3 4-OI对LPS诱导的炎症巨噬细胞内ROS水平的影响 A. DCFH-DA探针检测4-OI对RAW 264.7巨噬细胞内ROS水平的影响（×400）；B. DCFH-DA相对荧光强度统计（%），**表示P <0.01，***表示P <0.001，与LPS组比较。

Figure 3 The effect of 4-OI on ROS level in inflammatory macrophages

图4 4-OI对RANKL诱导的破骨分化的影响 A. RANKL刺激第5天时小鼠BMMs的TRAP染色图（×100）；B. 破骨细胞数量统计；C. 破骨细胞面积统计。**表示P <0.01，***表示P <0.001，与RANKL组比较。

Figure 4 The effect of 4-OI on RANKL-induced osteoclastogenesis

图5 Micro-CT检测4-OI对LPS诱导的小鼠颅骨溶解的治疗效果 A. 三维重建；B. BV/TV统计；C. 孔隙率统计。*表示P <0.05，**表示P <0.01，***表示P <0.001，与LPS组比较。

Figure 5 Micro-CT detection of the effect of LPS-induced murine calvarial osteolysis treated by 4-OI

图6 组织学检测4-OI对LPS诱导的小鼠颅骨溶解的治疗效果 A. HE染色（×100）、iNOS免疫荧光染色（×200）与TRAP染色（×200）；B. iNOS阳性细胞百分比；C. TRAP染色阳性细胞数量。**表示P <0.01，***表示P <0.001，与LPS组比较。

Figure 6 Histology detection of the effect of LPS-induced murine calvarial osteolysis treated by 4-OI

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