低强度脉冲式超声波在脂多糖诱导的RAW264.7巨噬细胞分化中的抗炎和抗氧化作用

The mechanism of anti-inflammatory and antioxidant effects of low-intensity pulsed ultrasound on lipopolysaccharide-induced RAW264.7 macrophage differentiation

目的研究低强度脉冲式超声波(LIPUS)对RAW264.7巨噬细胞极化的影响及相关分子机制。方法100 ng/mL脂多糖(LPS)和10 ng/mL白细胞介素(IL)-4诱导巨噬细胞RAW264.7分别向M1和M2型极化,45 mW/cm2强度LIPUS对巨噬细胞处理25 min。采用流式细胞术检测巨噬细胞氧化应激活性氧(ROS)水平、M1分化标志物CD80和CD11b,以及M2分化标志物CD163的表达水平。采用反转录聚合酶链反应(RT-PCR)技术检测CD80、CD11b、核转录因子κB(NF-κB)p65、肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β和IL-6的mRNA水平。采用Western blot技术检测细胞p65、p-p65、TNF-α、IL-1β和IL-6表达水平。采用流式细胞术检测细胞培养上清TNF和IL-6表达水平。结果LIPUS可明显减少LPS诱导的RAW264.7细胞ROS水平。LPS诱导后,RAW264.7细胞M1分化标志物CD80和CD11b表达和转录水平均上调;LIPUS可抑制LPS对RAW264.7细胞向M1的诱导,差异具有统计学意义。并且,LIPUS可促进IL-4诱导的巨噬细胞M2分化标志物CD163表达。LPS诱导的RAW264.7细胞炎症因子NF-κB p65、TNF-α、IL-1β和IL-6 mRNA水平上调,LIPUS可下调这些炎症因子的mRNA水平,差异均具有统计学意义。LIPUS抑制了LPS对RAW264.7细胞因子蛋白p65和p-p65、IL-1β、TNF-α和IL-6蛋白表达上调的作用。胰酶可以通过激活ROS-NF-κB通路,回复LIPUS促进巨噬细胞M1分化的作用。结论LIPUS可通过ROS-NF-κB抑制RAW264.7向巨噬细胞M1型分化,促进RAW264.7向巨噬细胞M2型分化。氧化应激和炎症因子表达水平被抑制。LIPUS可能在牙周疾病中起到抑制氧化和炎症的作用,从而发挥对牙周疾病的治疗功能。

Objective To study the effects of low-intensity pulsed ultrasound(LIPUS)on the polarization of RAW264.7 macrophages and related molecular mechanisms.Methods Lipopolysaccharide(LPS,100 ng/mL)or IL-4(10 ng/mL)was used to induce M1 or M2 polarization of macrophages RAW264.7.The macrophages were treated with 45 mW/cm2 LIPUS for 25 min.Flow cytometry was used to detect the reactive oxygen species(ROS)level of macrophages and the expression levels of CD80 and CD11b,or CD163,which were markers of M1 or M2.The mRNA levels of CD80,CD11b,NF-κB p65,TNF-α,IL-1βand IL-6 were detected by RT-PCR.The expression levels of p65,p-p65,TNF-α,IL-1βand IL-6 were detected by Western blot.The expression levels of TNF and IL-6 in the supernatant of cell culture were detected by flow cytometry.Results LIPUS could significantly reduce the ROS level of LPS-induced RAW264.7 cells.After LPS induction,the expression and transcription levels of CD80 and CD11b,markers of M1 differentiation,were up-regulated in RAW 264.7 cells.LIPUS inhibited the differentiation induction of LPS on RAW264.7 cells to M1,and the difference was statistically significant.Moreover,LIPUS promoted the expression of CD163,a marker of M2.The mRNA levels of NF-κB p65,TNF-α,IL-1βand IL-6 in LPS-induced RAW264.7 cells were up-regulated,and LIPUS down-regulated the mRNA levels of these inflammatory factors,with statistically significant differences.LIPUS inhibited the up-regulation of RAW264.7 cytokine proteins p65 and p-p65,IL-1β,TNF-αand IL-6 by LPS.Trypsin restored the role of LIPUS in promoting macrophage M1 differentiation by activating the ROS-NF-κB pathway.Conclusions LIPUS inhibited the LPS-induced differentiation of RAW264.7 into macrophages M1 type through ROS-NF-κB,oxidative stress and the expression levels of inflammatory factors of RAW264.7.LIPUS promoted the M2 polarization of RAW264.7.LIPUS may play a therapeutic role in periodontal diseases by inhibiting M1 differentiation of macrophages,which reduced oxidative stress and inflammation.