Low-Intensity Pulsed Ultrasound Modulates RhoA/ROCK Signaling of Rat Mandibular Bone Marrow Mesenchymal Stem Cells to Rescue Their Damaged Cytoskeletal Organization and Cell Biological Function Induced by Radiation

Abstract

Osteoradionecrosis of the jaw (ORNJ) is an infrequent yet potentially devastating complication of head and neck radiation therapy. Low-intensity pulsed ultrasound (LIPUS) has been widely accepted as a promising method for the successful management of ORNJ, but the mechanism remains unclear. In this study, the effects of LIPUS on cytoskeletal reorganization, cell viability, and osteogenic differentiation capacity of rat mandible-derived bone marrow mesenchymal stem cells (M-BMMSCs) induced by radiation were determined by immunofluorescence staining, CCK-8 cell proliferation assay, quantification of alkaline phosphatase (ALP) activity, alizarin red staining, and real-time RT-PCR, respectively. Moreover, the involvement of the RhoA/ROCK signaling pathway underlying this process was investigated via western blot analysis. We found that radiation induced significant damage to the cytoskeleton, cell viability, and osteogenic differentiation capacity of M-BMMSCs and downregulated their expression of RhoA, ROCK, and vinculin while increasing FAK expression. LIPUS treatment effectively rescued the disordered cytoskeleton and redistributed vinculin. Furthermore, the cell viability and osteogenic differentiation capacity were also significantly recovered. More importantly, it could reverse the aberrant expression of the key molecules induced by radiation. Inhibition of RhoA/ROCK signaling remarkably aggravated the inhibitory effect of radiation and attenuated the therapeutic effect of LIPUS. In the light of these findings, the RhoA/ROCK signaling pathway might be a promising target for modifying the therapeutic effect of LIPUS on osteoradionecrosis.

Figure 1

The isolation and characterization of rat mandible-derived BMMSCs (M-BMMSCs). (a) The stem cells were isolated from rat mandibular tissues. The rats were immersed in 75% ethanol for 5 min after sacrifice. (A1) The mouths were opened and the mandibles with muscles were revealed after disinfection. (A2) The intact mandibular bones were obtained after the remove of muscles. (A3) The teeth were extracted from the mandibles with tweezers in super clean bench. (A4) A 5 ml syringe with DMEM was used to wash the bone marrow cavity repeatedly, and the flushing solution was collected. (b) Observation of M-BMMSCs at P0 and P1 with the inverted phase contrast microscope (magnification, ×100). (c) Flow cytometric analysis of surface marker expression of M-BMMSCs showed negative expression of CD31 and CD45 while positive expression of CD73, CD90, and CD105 (1.88%, 2.19%, 98.7%, 96.2%, and 97.3%), respectively. (d) Alizarin red staining of M-BMMSCs after induction in osteogenic differentiation medium for 21 d. (e) Oil red O staining of M-BMMSCs after induction in adipogenic differentiation medium for 14 d. Both pictures were observed by using an inverted phase contrast microscope. Scale bars 200 μm.

Figure 2

Radiation disrupted the cytoskeleton arrangement and vinculin distribution in M-BMMSCs. The cells were divided into three groups: the control group (a) that received no treatment; the radiation group (b) that was irradiated with 4 Gy of X-ray for 12 h; and the inhibitor group (c) that was subjected to Y-27632 30 min before radiation. Then, the cells were immunostained with F-actin (A1, B1, and C1) and counter stained with TRITC-vinculin (A2, B2, and C2) and DAPI (A3, B3, and C3). Graphs A4, B4, and C4 are the merged images. The white arrowheads represent the change in the cytoskeleton. The yellow arrowheads represent the change in vinculin distribution. Scale bars 50 μm.

Figure 3

Radiation inhibited the expression of key molecules in the RhoA/ROCK signaling pathway. Cells from different groups were treated as previously described. Cell lysates were then analyzed for RhoA (a), ROCK (b), and vinculin (c) using western blot. β-Actin was used as an internal reference. The results are representative of those obtained in three separate experiments. Each sample was repeated in triplicate. ∗p < 0.05.

Figure 4

LIPUS induced cytoskeleton remodeling and vinculin redistribution after irradiation via a RhoA/ROCK signaling pathway. The cells were divided into four groups: the control group (d) that received no treatments; the radiation group (e) that was irradiated with 4 Gy of X-ray for 12 h; the LIPUS group (f) that was exposed to 2 h of LIPUS treatment after irradiation for 12 h; and the inhibitor group (g) that was subjected to Y-27632 30 min before the LIPUS treatment. Then, the cells were immunostained with F-actin (D1, E1, F1, and G1) and counter stained with TRITC-vinculin (D2, E2, F2, and G2) and DAPI (D3, E3, F3, and G3). Graphs D4, E4, F4, and G4 are the merged images. The white arrowheads represent the changes in the cytoskeleton. The yellow arrowheads represent the changes in the vinculin distribution. Scale bars 50 μm.

Figure 5

LIPUS initiated the recovery of cell viability and osteogenic differentiation capacity after radiation exposure via a RhoA/ROCK signaling pathway. In (a), CCK-8 activity at day 3 was analyzed. The optical density was measured at 450 nm using a microplate reader. In (b), group division was conducted as previously described. To assess the osteogenic differentiation activity, ALP activity was measured and scored. Absorbance was read at a wavelength of 450 nm. At the end of the experiment, the ALP levels were normalized to the total protein content. In (c), the osteogenic gene (Runx2, ALP, and OCN) expression of M-BMMSCs after incubation with osteogenic differentiation medium for 7 d was detected by quantitative real-time RT-PCR. In (d) and (e), the mineral formation of M-BMMSCs under osteogenic differentiation medium for 28 d was detected by alizarin red staining. Staining semiquantification was done by dissolving with leaching solution. All of the data represented the average ± SD from three separate experiments. Each sample was repeated in triplicate. ∗p < 0.05.

Figure 6

LIPUS elevated the decrease in the p-FAK/FAK ratio induced by radiation via the RhoA/ROCK signaling pathway. Cells from the different groups were treated as previously described. Cell lysates were then analyzed for FAK (a), and p-FAK (b) using western blot. β-Actin was used as an internal reference. The ratio of p-FAK/FAK was also determined (c). The results are representative of those obtained in three separate experiments. Each sample was repeated in triplicate. ∗p < 0.05.

Figure 7

LIPUS attenuated the inhibitory effects of radiation on the RhoA/ROCK signaling pathway. Cells from the different groups were treated as previously described. The cell lysates were then analyzed for RhoA (a), ROCK (b), and vinculin (c) using western blot. β-Actin was used as an internal reference. The results are representative of those obtained in three separate experiments. Each sample was repeated in triplicate. ∗p < 0.05.