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

Abstract

Background: 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.

Methods: 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/cm² 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.

Results: 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).

Conclusions: 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.

Keywords: Anti-inflammatory; Low-intensity pulsed ultrasound; M1 polarization; Periodontal disease; WNT2b.

Figure 1. Low-intensity pulsed ultrasound inhibited the M1 polarization of RAW264.7 cells.

(A) Expression of CD80 was detected by flow cytometry. (B) Expression of CD86 was detected by flow cytometry. (C) Relative expression of CD80 and CD86 referring to β-actin detected by RT-qPCR. RAW indicates RAW264.7 cells used as control. RAW + LPS indicates RAW264.7 cells treated with 100 ng/mL LPS to induce macrophage-like M1 polarization. RAW + LPS + LIPUS indicates RAW264.7 cells treated with 100 ng/mL LPS and low-intensity pulsed ultrasound. RAW + LIPUS indicates RAW264.7 cells treated with low-intensity pulsed ultrasound. *P < 0.05, ****P < 0.0001.

Figure 2. Low-intensity pulsed ultrasound promoted the M2 polarization of RAW264.7 cells.

(A) Expression of CD163 was detected by flow cytometry. (B) Expression of CD206 was detected by flow cytometry. (C) Relative expression of CD163 and CD206 referring to β-actin detected by RT-qPCR. RAW indicates RAW264.7 cells used as control. RAW+IL4 indicates RAW264.7 cells treated with 20 ng/mL IL4 to induce macrophage-like M2 polarization. RAW+IL4+LIPUS indicates RAW264.7 cells treated with 20 ng/mL LIPUS and low-intensity pulsed ultrasound. RAW+LIPUS indicates RAW264.7 cells treated with low-intensity pulsed ultrasound. *P < 0.05, ****P < 0.0001.

Figure 3. PolyA mRNA sequencing results of LPS-induced RAW264.7 cells and LPS-induced RAW264.7 cells treated with low-intensity pulsed ultrasound.

(A) Heatmap and (B) Volcano plot of the sequencing results show that there were 335 upregulated and 288 downregulated genes. (C) Principal component analysis (PCA) indicated that the six samples were split into two groups: LPS and LIPUS-LPS. (D) GO and (E) KEGG pathway analyses indicated that the Wnt signaling pathway and HIPPO signaling pathway participated in the inhibition of M1 polarization of RAW264.7 cells by low-intensity pulsed ultrasound. LPS indicates RAW264.7 cells treated with 100 ng/mL LPS. LIPUS-LPS indicates RAW264.7 cells treated with 100 ng/mL LPS and low-intensity pulsed ultrasound.

Figure 4. Low-intensity pulsed ultrasound promoted the relative expression of Wnt2b.

The relative expression of Wnt2b, one of the key proteins in the Wnt signaling pathway, was detected by RT-qPCR in RAW264.7 cells, during treatment with (A) LIPUS and LPS or (B) LIPUS and IL4. RAW indicates RAW264.7 cells used as control. RAW-LPS indicates RAW264.7 cells treated with 100 ng/mL LPS to induce macrophage-like M1 polarization. RAW+LPS+LIPUS indicates RAW264.7 cells treated with 100 ng/mL LPS and low-intensity pulsed ultrasound. RAW+LIPUS indicates RAW264.7 cells treated with low-intensity pulsed ultrasound. RAW+IL4 indicates RAW264.7 cells treated with 20 ng/mL IL4 to induce macrophage-like M2 polarization. RAW+IL4+LIPUS indicates RAW264.7 cells treated with 20 ng/mL IL4 and low-intensity pulsed ultrasound. *P < 0.05, **P < 0.005, ****P < 0.0001.

Figure 5. Knocking down Wnt2b could reduce the inhibitory effect of low-intensity pulsed ultrasound on M1 polarization of RAW264.7 cells.

(A) CD80 and (B) CD86 were detected by flow cytometry during M1 polarization of RAW264.7 cells. RAW+Wnt2bsiNC indicates RAW264.7 cells transfected with negative control of Wnt2b siRNA, used as control. RAW+LPS+Wnt2bsiNC indicates RAW264.7 cells transfected with negative control of Wnt2b siRNA and treated with 100 ng/mL LPS to induce macrophage-like M1 polarization. RAW+LPS+LIPUS+Wnt2bsiNC indicates RAW264.7 cells transfected with negative control of Wnt2b siRNA and treated with 100 ng/mL LPS and low-intensity pulsed ultrasound. RAW+LPS+LIPUS+Wnt2bsi1/2/3 indicates RAW264.7 cells transfected with Wnt2b siRNA and treated with low-intensity pulsed ultrasound. ***P < 0.0005, ****P < 0.0001.

Figure 6. Low-intensity pulsed ultrasound inhibited the M1 polarization of RAW264.7 cells in a Wnt2b/AXIN/β-catenin-dependent way.

Relative expression of (A) Wnt2b, (B) AXIN, and (C) β-catenin detected by Western blot. RAW-Wnt2bsiNC indicates RAW264.7 cells transfected with negative control of Wnt2b siRNA, used as control. RAW+LPS+Wnt2bsiNC indicates RAW264.7 cells transfected with negative control of Wnt2b siRNA and treated with 100 ng/mL LPS to induce macrophage-like M1 polarization. RAW+LPS+LIPUS+Wnt2bsiNC indicates RAW264.7 cells transfected with negative control of Wnt2b siRNA and treated with 100 ng/mL LPS and low-intensity pulsed ultrasound. RAW+LPS+LIPUS+Wnt2bsi1/2/3 indicates RAW264.7 cells transfected with Wnt2b siRNA and treated with 100 ng/mL LPS and low-intensity pulsed ultrasound. ****P < 0.0001.

Figure 7. Downregulation of Wnt2b reduced the effect of low-intensity pulsed ultrasound on inhibiting the expression of inflammatory factors IL6 and TNFα in M1 polarization of RAW264.7 cells.

(A) The expression of IL2, IL4, IL6, IL10, TNFα, IFNγ, and IL17A were detected by flow cytometry. (B) The expression of IL6 and TNFα was increased by the downregulation of Wnt2b during LIPUS and LPS treatment. RAW+Wnt2bsiNC indicates RAW264.7 cells transfected with negative control of Wnt2b siRNA, used as control. RAW+LPS+Wnt2bsiNC indicates RAW264.7 cells transfected with negative control of Wnt2b siRNA and treated with 100 ng/mL LPS to induce macrophage-like M1 polarization. RAW+LPS+LIPUS+Wnt2bsiNC indicates RAW264.7 cells transfected with negative control of Wnt2b siRNA and treated with 100 ng/mL LPS and low-intensity pulsed ultrasound. RAW+LPS+LIPUS+Wnt2bsi1/2/3 indicates RAW264.7 cells transfected with Wnt2b siRNA and treated with 100 ng/mL LPS and low-intensity pulsed ultrasound. *P < 0.05, ****P < 0.0001.