Central Laboratory, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou, China.
Department of Stomatology, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou, China.
PeerJ. 2024 Nov 13;12:e18448. doi: 10.7717/peerj.18448. eCollection 2024.
Low-intensity pulsed ultrasound (LIPUS) is a special type of low-intensity ultrasound. In periodontal disease, LIPUS is applied as an adjuvant and non-invasive treatment. It has been reported that LIPUS significantly shifts the macrophage phenotype from M1 to M2, but the specific mechanism behind this shift is still unknown.
RAW264.7 cells were induced to M1/M2 polarization with lipopolysaccharide (LPS)/interleukin-4 (IL4). LIPUS was performed for 25 min two times, 24 h apart, at an intensity of 45 mW/cm to stimulate RAW264.7 cells. PolyA mRNA sequencing was conducted of both the LPS-induced RAW264.7 cells and the LPS-induced RAW264.7 cells with LIPUS treatment. The expression of Wnt2b in RAW264.7 cells was downregulated by siRNA. The macrophage surface markers and downstream inflammatory cytokines were detected using flow cytometry. The relative expression of proteins in the Wnt2b/AXIN/β-catenin pathway was assessed using reverse transcription real-time polymerase chain reaction (RT-qPCR) and Western blot.
LIPUS reversed the M1 polarization of RAW264.7 cells, with decreased expression of CD80 and CD86. In addition, LIPUS enhanced the M2 polarization of RAW264.7 cells, with upregulated expression of CD163 and CD206. The polyA mRNA sequencing results indicated that the Wnt signaling pathway participated in the M1 polarization of LIPUS-treated RAW264.7. The results of the RT-qPCR showed a higher expression of Wnt2b in LIPUS-treated and M1- or M2-polarized RAW264.7 cells. Knocking down Wnt2b was shown to reverse the inhibitory effect of LIPUS on M1 polarization and increase the expression of CD80 and CD86. Wnt2b knockdown also regulated downstream AXIN, β-catenin, and inflammatory factors such as tumor necrosis factor alpha (TNFα) and interleukin-6 (IL6).
LIPUS plays an anti-inflammatory role by inhibiting LPS-induced M1 polarization of RAW264.7 cells in a Wnt2b/AXIN/β-catenin-dependent way. LIPUS may play a therapeutic role in periodontal diseases by inhibiting inflammation through the regulation of macrophage differentiation.
低强度脉冲超声（LIPUS）是一种特殊类型的低强度超声。在牙周病中，LIPUS 被用作辅助和非侵入性治疗。据报道，LIPUS 可显著将巨噬细胞表型从 M1 向 M2 转移，但这种转移的具体机制尚不清楚。
用脂多糖（LPS）/白细胞介素-4（IL4）诱导 RAW264.7 细胞向 M1/M2 极化。用 45mW/cm 的强度对 LPS 诱导的 RAW264.7 细胞进行两次 25 分钟的 LIPUS 刺激，两次间隔 24 小时。对 LPS 诱导的 RAW264.7 细胞和 LPS 诱导的并用 LIPUS 处理的 RAW264.7 细胞进行 PolyA mRNA 测序。用 siRNA 下调 RAW264.7 细胞中的 Wnt2b 表达。用流式细胞术检测巨噬细胞表面标志物和下游炎症细胞因子。用逆转录实时聚合酶链反应（RT-qPCR）和 Western blot 评估 Wnt2b/AXIN/β-catenin 通路中蛋白的相对表达。
LIPUS 逆转了 RAW264.7 细胞的 M1 极化，降低了 CD80 和 CD86 的表达。此外，LIPUS 增强了 RAW264.7 细胞的 M2 极化，增加了 CD163 和 CD206 的表达。PolyA mRNA 测序结果表明，Wnt 信号通路参与了 LIPUS 处理的 RAW264.7 的 M1 极化。RT-qPCR 结果显示，LIPUS 处理和 M1 或 M2 极化的 RAW264.7 细胞中 Wnt2b 的表达较高。敲低 Wnt2b 可逆转 LIPUS 对 M1 极化的抑制作用，并增加 CD80 和 CD86 的表达。Wnt2b 敲低还调节下游 AXIN、β-catenin 和肿瘤坏死因子-α（TNFα）和白细胞介素-6（IL6）等炎症因子。
LIPUS 通过 Wnt2b/AXIN/β-catenin 依赖性方式抑制 LPS 诱导的 RAW264.7 细胞的 M1 极化发挥抗炎作用。LIPUS 可能通过调节巨噬细胞分化抑制炎症在牙周病中发挥治疗作用。
