Low-frequency low energy ultrasound combined with microbubbles induces distinct apoptosis of A7r5 cells

The present study aimed to investigate whether low frequency low energy ultrasound combined with microbubbles induces apoptotic cell death of A7r5 rat aortic vascular smooth muscle cells, and to identify the possible mechanisms underlying this effect. Ultrasonic waves (45 kHz with 0.3 Wcm(2) of intensity for 0, 10, 20 and 30 sec) were used together with different dosages of SonoVue (TM) microbubbles (0, 14, 28,42 and 56 mu l), respectively. The cell viability and apoptotic rate were determined by trypan blue staining immediately following treatment and flow cytometry 24 h thereafter. The treatment conditions resulting in the lowest amount of necrosis, highest apoptotic rate and lowest microbubble dosage was selected for the US+MB group, which was treated with ultrasound combined with Microbubbles. The cell proliferation 24 h following treatment was determined and western blot analysis was applied to examine the expression of apoptosis-associated proteins, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). The harmonic acoustic pressure amplitude was measured to obtain the cavitation intensity. The combination of 20 sec ultrasound irradiation and 14 mu l SonoVue (TM) was selected as the treatment conditions for the US+MB group. The results demonstrated that both ultrasound alone (the US group) and in combination with microbubbles significantly inhibited the proliferation of A7r5 cells compared with that of the control (P<0.01), and the suppression in the US+MB group was significantly greater (P<0.01). The apoptotic rate in A7r5 cells induced by this combination treatment (16.62 +/- 0.93%) was significantly higher than that in the control (3.93 +/- 0.39%; P<0.01) and US (6.88 +/- 1.87%; P<0.01) groups. Treatment with ultrasound combined with microbubbles increased the expression of Bax and decreased the ratio of Bcl-2/Bax compared with those in the control and US groups. The cavitation induced by ultrasound combined with microbubble treatment was more intense than that by ultrasound alone. The results demonstrated that the cell death and apoptosis of A7r5 cells were associated with ultrasound duration and microbubble dosage. Low frequency ultrasound combined with microbubbles induced apoptosis in A7r5 cells through the upregulation of Bax and the downregulation of the Bcl-2/Bax ratio, where the cavitation effect may have an important role.