Department of Rheumatology and Immunology, The Second Affiliated Hospital of Jiaxing University, Jiaxing, China.
J Biochem Mol Toxicol. 2023 Jul;37(7):e23362. doi: 10.1002/jbt.23362. Epub 2023 Mar 29.
This work aimed to explore the therapeutic effect and target of sulforaphene (LF) in mice with rheumatoid arthritis (RA). Lipopolysaccharide (LPS) and IFN-γ were added to induce the M1 polarization of SMG cells, and later cells were pretreated with 5 μM and 15 μM LF. M1 cell proportion was detected by flow cytometry (FCM), inflammatory factors were measured by enzyme-linked immunosorbent assay, and protein levels were analyzed by western blotting (WB) assay. Besides, small molecule-protein docking and pull-down assays were carried out to detect the binding of LF to NLRP3. After the knockdown of NLRP3 in SMG cells, the effect of LF was further detected. The RA mouse model was induced with collagen antibody and LPS, after LF intervention, H&E staining was performed to detect the pathological changes in mouse synovial membrane, whereas safranin O-fast green staining was performed to detect cartilage injury, NLRP3 inflammasome and inflammatory factor levels in tissues. LF suppressed M1 polarization of macrophages, reduced M1 cell proportion and inflammatory factor levels, and suppressed the activation of NLRP3 inflammasome. After NLRP3 knockdown, LF did not further suppress the M1 polarization of macrophages. Pull-down assay suggested that LF bound to NLRP3. As revealed by mouse experimental results, LF inhibited bone injury in mice, decreased M1 cell infiltration and inflammatory response in tissues, and inhibited NLRP3 inflammasome expression in tissues. LF targets NLRP3 to suppress the M1 polarization of macrophages and decrease tissue inflammation in RA.
本研究旨在探讨莱菔硫烷（LF）在类风湿关节炎（RA）小鼠模型中的治疗作用及靶点。采用脂多糖（LPS）和干扰素-γ（IFN-γ）诱导 SMG 细胞 M1 极化，用 5μM 和 15μM LF 预处理细胞。采用流式细胞术（FCM）检测 M1 细胞比例，酶联免疫吸附试验（ELISA）检测炎症因子，Western blot（WB）检测蛋白水平。此外，还进行了小分子-蛋白对接和下拉实验，以检测 LF 与 NLRP3 的结合。在 SMG 细胞中敲低 NLRP3 后，进一步检测 LF 的作用。用胶原抗体和 LPS 诱导 RA 小鼠模型，LF 干预后，进行 H&E 染色检测小鼠滑膜组织的病理变化，番红 O-快绿染色检测软骨损伤，组织中 NLRP3 炎性小体和炎症因子水平。LF 抑制巨噬细胞 M1 极化，降低 M1 细胞比例和炎症因子水平，抑制 NLRP3 炎性小体激活。敲低 NLRP3 后，LF 不能进一步抑制巨噬细胞 M1 极化。下拉实验表明 LF 与 NLRP3 结合。小鼠实验结果表明，LF 抑制小鼠骨损伤，减少组织中 M1 细胞浸润和炎症反应，抑制组织中 NLRP3 炎性小体表达。LF 通过靶向 NLRP3 抑制巨噬细胞 M1 极化，减轻 RA 组织炎症。
