Low-intensity pulsed ultrasound suppresses proliferation and promotes apoptosis via p38 MAPK signaling in rat visceral preadipocytes

Abstract

Low-intensity pulsed ultrasound (LIPUS) has been used widely in clinical therapy for bone fracture and soft tissue injury. However, whether LIPUS regulates primary preadipocyte function and adipogenesis remains unknown. In this study, we investigated the potential role of LIPUS in regulating visceral preadipocyte function. Resuspended rat visceral preadipocytes were treated with LIPUS (0.5 MHz, 109.44 mW/cm2) for 1 min and then cultured for an additional 48 hours. Cell proliferation was examined using the CCK-8 assay, and the early apoptosis rate was determined by flow cytometry. In addition, we evaluated the related signaling pathway via examination of proliferating cell nuclear antigen (PCNA), peroxisome proliferator-activated receptor gamma (PPARγ), Bcl2, Bax, cleaved caspase 3 (C-C3), and mitogen-activated protein kinase (MAPK) member protein levels using western blot or quantitative real-time PCR (qRT-PCR). LIPUS inhibited preadipocyte proliferation and induced cell apoptosis. The protein expression of proliferation markers decreased, while expression of the apoptosis-related modulators increased following LIPUS treatment. LIPUS treatment decreased extracellular signal-regulated kinase (ERK) phosphorylation and increased p38 MAPK phosphorylation. Inhibition of p38 MAPK rescued the LIPUS-induced proliferation inhibition and apoptosis induction. Thus, treatment of rat visceral preadipocytes with 0.5 MHz LIPUS suppresses proliferation and promotes apoptosis via activation of p38 MAPK signaling.

Keywords: LIPUS; apoptosis; p38 MAPK; proliferation; rat visceral preadipocytes.

Conflict of interest statement

None.

Figures

Figure 1

LIPUS inhibits preadipocyte proliferation and increases apoptosis. Primary cultured preadipocytes were treated with different doses of ultrasound (500, 800, 1100, 1400, 1700, and 2000 cycles) and different duration (0.5, 1, and 1.5 min). (A) Preadipocyte proliferation was evaluated with the CCK-8 assay. Ultrasound doses reaching 1700 cycles (average sound intensity 109.44 mW/cm2) led to a remarkable reduction of preadipocyte proliferation. (B) Effects of LIPUS on preadipocytes proliferation were evaluated at different points in time and the 1-min duration was determined to be the optimal duration. (C, D) The early apoptosis rate was analyzed by flow cytometry using Annexin-V-FITC staining. LIPUS treatment increased the number of early apoptotic preadipocytes compared to control treatment. All values are expressed as the mean ± SEM of three independent trials. For (A and B), data were analyzed with one-way ANOVA analysis, for (C), data were analyzed with independent t test. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

LIPUS affects expression of proteins associated with proliferation and apoptosis in preadipocytes. The expression levels of key proliferation- and apoptosis-related molecules (PCNA, PPARγ, Bcl2, Bax, and C-C3) were examined by western blotting in primary preadipocytes with or without LIPUS (1700 cycles, 1 min) treatment. A, B. The protein levels of PCNA protein decreased and the levels of PPARγ increased in LIPUS treated cells compared to untreated cells. C, D. LIPUS treatment downregulated the protein expression of Bcl2 and the Bcl2/Bax ratio, and upregulated the protein expression of C-C3. All values are expressed as the mean ± SEM of three independent trials. Data were analyzed with independent t test. *P<0.05, **P<0.01.

Figure 3

LIPUS does not promote transcription of PPAR family members during the early stage. mRNA levels of PPAR and C/EBP family members were assessed by qRT-PCR in LIPUS treated and untreated cells. β-tubulin was utilized as the internal standard for normalization. A. LIPUS did not affect the mRNA levels of PPARα, PPARγ, and PPARδ of rat visceral preadipocytes. B. LIPUS treatment downregulated the C/EBPδ mRNA level but did not affect the C/EBPα and C/EBPβ mRNA levels of rat visceral preadipocytes. All values are expressed as the mean ± SEM of three independent trials. Data were analyzed with independent t test. **P<0.01.

Figure 4

LIPUS induces phosphorylation of p38-MAPK. The expression and modification of MAPK members were assessed by western blotting in primary preadipocytes with or without LIPUS treatment. A. LIPUS treatment significantly promoted the phosphorylation level of p38 and inhibited the phosphorylation of ERK, and no change of the expression level of p-JNK were observed. B. Quantification of p-38, p-p38, ERK, p-ERK, JNK, and p-JNK protein levels were. Data were analyzed with independent t test. All values are expressed as the mean ± SEM of three independent trials. *P<0.05.

Figure 5

Inhibition of p38 phosphorylation rescues the effects of LIPUS. In order to explore whether the biological effects of LIPUS on preadipocytes are associated with p-p38, cell suspension exposed to LIPUS was treated with or without p38 MAPK inhibitor SB203580. A, B. p38 inhibition rescued the LIPUS-induced apoptosis as shown by flow cytometric analysis. The percentage of early apoptotic preadipocytes (%) was analyzed with one-way ANOVA analysis. C. CCK-8 assay showed that inhibition of p38 activation reversed the LIPUS-induced proliferation inhibition in primary preadipocytes. D. p-p38, p-ERK, C-C3, PCNA and Bcl2 protein levels were assessed by western blotting with or without p38 inhibitor treatment. Relative protein levels were analyzed with one-way ANOVA analysis. All values are expressed as the mean ± SEM of three independent trials. *P<0.05.