Physical energy-based ultrasound shifts M1 macrophage differentiation towards M2 state

Figure 1 Experimental design for this study in vitro and the method of treating cells. A: At 24 h before lipopolysaccharide (LPS) was added to simulate the inflammatory environment, macrophages at the M1 stage were uniformly subcultured into the six-well plate. Then, The first ultrasound treatment was performed 24 h after the inflammatory environment was maintained. At 48 h, ultrasound was performed on the group requiring a second treatment. The supernatant of culture medium was separated after 24 d of culture (3 d after LPS was added); B: In order to easily adjust the ultrasonic probe to fit the culture holes on the bottom of the six-well plate, we fixed the ultrasonic probe on a sponge pad. Additionally, a box of the same height is used to support the six-hole plate to prevent it from tilting.

Figure 2 Low-Intensity pulsed ultrasound significantly increases the expression of anti-inflammatory cytokine and decreases the expression of pro-inflammatory cytokine. A: Real-time quantitative polymerase chain reaction (qPCR) was used to detect the gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 after being treated by low-intensity pulsed ultrasound (LIPUS); B: ELISA was used to analyze the protein expression of TNF-α, IL-6, and IL-10 after being treated with LIPUS. Data are expressed as the mean ± standard error of the mean. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. Low intensity = 0.25 W/cm², medium intensity = 0.5 W/cm², and high intensity = 0.75 W/cm². TNF-α: Tumor necrosis factor-α; IL: Interleukin.

Figure 3 Low-intensity pulsed ultrasound shifts M1 macrophages towards M2 state. A: The morphology of macrophages was observed by microscopy. Black, red, and yellow arrows represent M0, M1, and M2 macrophages, respectively; B: Quantitative polymerase chain reaction was used to detect the gene expression of cell phenotypes maker iNOS, CD86, CD206, and ARG1 after being treated by low-intensity pulsed ultrasound. Data are expressed as the mean ± standard error of the mean. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. Low intensity = 0.25 W/cm², medium intensity = 0.5 W/cm², and high intensity = 0.75 W/cm².