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

doi:10.3877/cma.j.issn.1674-1366.2023.01.004

Abstract

Abstract:

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-1β and IL-6 were detected by RT-PCR. The expression levels of p65, p-p65, TNF-α, IL-1β and 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-1β and 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-α and 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.

Key words: Low-intensity pulsed ultrasound, Reactive oxygen species, Inflammation, Macrophage differentiation, Periodontal diseases

Juan Yin, Xing Yang, Ping Li, Minxin Xu, Yu Bao, Zhipeng Zhang, Hui Xue. The mechanism of anti-inflammatory and antioxidant effects of low-intensity pulsed ultrasound on lipopolysaccharide-induced RAW264.7 macrophage differentiation[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2023, 17(01): 26-36.

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References 24

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基因	引物序列
β-actin	正向：5′-GCAGGAGTACGATGAGTCCG-3′
	反向：5′-ACGCAGCTCAGTAACAGTCC-3′
IL-6	正向：5′-CTGCAAGAGACTTCCATCCAG-3′
	反向：5′-AGTGGTATAGACAGGTCTGTTGG-3′
TNF-α	正向：5′-CCGATGGGTTGTACCTTGTC-3′
	反向：5′-AGATAGCAAATCGGCTGACG-3′
IL-1β	正向：5′-CAGGCAGGCAGTATCACTCA-3′
	反向：5′-AGGTGCTCATGTCCTCATCC-3′
CD11b	正向：5′-CCAGTACTTCGGGCAGTCTC-3′
	反向：5′-GGCTCTGAGCAGCAGAAGAT-3′
CD80	正向：5′-GGAGATGCTCACGTGTCAGA-3′
	反向：5′-CAACGATGACGACGACTGTT-3′
NF-κB p65	正向：5′-TCAATGGCTACACAGGACCA-3′
	反向：5′-GGCAGAGGTCAGCCTCATAG-3′

基因	引物序列
β-actin	正向：5′-GCAGGAGTACGATGAGTCCG-3′
	反向：5′-ACGCAGCTCAGTAACAGTCC-3′
IL-6	正向：5′-CTGCAAGAGACTTCCATCCAG-3′
	反向：5′-AGTGGTATAGACAGGTCTGTTGG-3′
TNF-α	正向：5′-CCGATGGGTTGTACCTTGTC-3′
	反向：5′-AGATAGCAAATCGGCTGACG-3′
IL-1β	正向：5′-CAGGCAGGCAGTATCACTCA-3′
	反向：5′-AGGTGCTCATGTCCTCATCC-3′
CD11b	正向：5′-CCAGTACTTCGGGCAGTCTC-3′
	反向：5′-GGCTCTGAGCAGCAGAAGAT-3′
CD80	正向：5′-GGAGATGCTCACGTGTCAGA-3′
	反向：5′-CAACGATGACGACGACTGTT-3′
NF-κB p65	正向：5′-TCAATGGCTACACAGGACCA-3′
	反向：5′-GGCAGAGGTCAGCCTCATAG-3′

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