Anti-inflammatory role of low intensity pulsed ultrasound in inhibiting LPS-induced M1 polarization of RAW264.7 via Wnt2b/AXIN/β-catenin. Anti-inflammatory role of low intensity pulsed ultrasound in inhibiting LPS-induced M1 polarization of RAW264.7 via Wnt2b/AXIN/β-catenin

Low-intensity pulsed ultrasound (LIPUS) is a special type of low intensity ultrasound. In periodontal disease, LIPUS was applied as an adjuvant and non-invasive therapeutic treatment. While, the specific mechanism of LIPUS in the treatment of periodontal disease is not quite clear.RAW264.7 cells were induced to M1/M2 macrophage-like polarization by LPS/IL4. LIPUS was performed to stimulate RAW264.7 cells at an intensity of 45 mW/cm2, 25 min, interval 24 h, twice. The polyA mRNA sequencing of LPS induced RAW264.7 cells, LPS induced and LIPUS treated RAW264.7 cells were conducted.Our results suggested that LIPUS played an anti-inflammatory role by inhibiting LPS-induced M1 polarization of RAW264.7 cells in an Wnt2b/AXIN/β-catenin dependent way. LIPUS may inhibit inflammation in periodontal diseases by regulating macrophage differentiation, so as to play a therapeutic role in periodontal diseases. Overall design: To investigate the specific mechanism of Low-intensity pulsed ultrasound (LIPUS) in periodontal disease. RAW264.7 cells were induced by 100ng/mL LPS to M1 polarization for 48 hours. LIPUS was performed to stimulate RAW264.7 cells at an intensity of 45 mW/cm2, 25 min, interval 24 hours, twice. RAW264.7 cells induced by LPS, or treated with LPS and LIPUS, were analyzed by RNA sequencing to discover differential gene expression profiling. 3 replicates of each group were analyzed by RNA sequencing.

低强度脉冲超声（Low-intensity pulsed ultrasound, LIPUS）是一类特殊的低强度超声。在牙周疾病中，LIPUS被用作辅助性无创治疗手段，但其用于治疗牙周疾病的具体机制尚不明确。
本研究采用脂多糖（Lipopolysaccharide, LPS）/白细胞介素4（Interleukin-4, IL4）将RAW264.7细胞诱导为M1/M2型巨噬细胞样极化细胞。以45 mW/cm²的强度对RAW264.7细胞进行LIPUS刺激，每次25分钟，间隔24小时，共刺激2次。随后对脂多糖诱导的RAW264.7细胞，以及经脂多糖诱导联合LIPUS处理的RAW264.7细胞开展polyA信使RNA（polyA mRNA）测序。
研究结果表明，LIPUS可通过依赖Wnt2b/AXIN/β-连环蛋白（β-catenin）的通路抑制脂多糖诱导的RAW264.7细胞M1极化，从而发挥抗炎功效。提示LIPUS或可通过调控巨噬细胞分化改善牙周炎症，进而对牙周疾病产生治疗作用。
总体实验设计如下：为探究低强度脉冲超声治疗牙周疾病的具体机制，本研究采用100ng/mL脂多糖诱导RAW264.7细胞向M1型极化48小时。以45 mW/cm²的强度对RAW264.7细胞进行LIPUS刺激，每次25分钟，间隔24小时，共刺激2次。通过RNA测序分析脂多糖诱导组、脂多糖诱导联合LIPUS处理组的RAW264.7细胞，以筛选差异基因表达谱。每组设置3个生物学重复，均进行RNA测序分析。