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American Journal of Translational Research logoLink to American Journal of Translational Research
. 2019 Jan 15;11(1):418–429.

p38 MAPK signaling is a key mediator for low-intensity pulsed ultrasound (LIPUS) in cultured human omental adipose-derived mesenchymal stem cells

Yaqing Wang 1,*, Li Jiang 1,*, Tianhua Xu 1, Zhongping Su 1, Xiasheng Guo 2, Juan Tu 2, Dong Zhang 2, Wei Sun 1, Xiangqing Kong 1
PMCID: PMC6357340  PMID: 30787998

Abstract

Visceral obesity is an independent risk factor for cardiovascular disorders and lacks effective, non-drug based clinical therapy. The use of low-intensity pulsed ultrasound (LIPUS) to treat chronic pain and bone fracture is well-known, but its application for visceral obesity treatment has not been studied. Here, we evaluated the therapeutic potential of LIPUS by studying its effects, at varying doses, on human omental adipose-derived mesenchymal stem cells (hAMSCs). LIPUS stimulation was applied for 1 min at intensities between 70 and 210 mW/cm2. Cell viability was measured using the Cell Counting Kit-8 assay. Cell apoptosis was quantified by flow cytometry and immunoblotting of apoptosis marker proteins. We found that a high dose of LIPUS (210 mW/cm2) promoted apoptosis in hAMSCs, while a low dose (70 mW/cm2) increased hAMSC viability. Phosphorylation of p38, a mitogen-activated protein kinase (MAPK), increased with high dose LIPUS treatment, but markedly decreased with a low dose. Inhibition of p38 phosphorylation by SB203580, an inhibitor of p38 MAPK activity, rescued the apoptotic effects of high dose LIPUS. Our results showed the dose-dependent, opposing effects of LIPUS on hAMSCs and suggested that p38 plays a key role in mediating the effects of LIPUS on hAMSCs.

Keywords: LIPUS, apoptosis, cell viability, p38 MAPK, human omental adipose-derived mesenchymal stem cells

Introduction

Obesity is a risk factor for medical conditions such as diabetes mellitus, respiratory disorders, osteoarthritis, cancer, chronic kidney disease, and cardiovascular disease [1-3]. It is a growing health threat worldwide. Although body mass index (BMI) levels are widely used as a general guide to evaluate obesity, several studies have reported that waist circumference is a better predictor of risks associated with obesity [4,5] because it indicates abdominal fat levels. The accumulation of visceral fat (fat surrounding internal organs) is more dangerous than subcutaneous fat (thin layer of fat under the skin and all around the body) [6]. Obesity therapy mainly includes rigorous lifestyle modifications, pharmacotherapy, use of medical devices, and bariatric surgery [7]. Omentectomy (surgical removal of the omentum, which is a thin layer of fatty tissue surrounding the stomach, large intestine, and other abdominal organs) prevents metabolic syndrome in obese rats [8]. Our unpublished data showed that women who underwent omentectomy had lower systemic blood pressure after the procedure.

Low-intensity pulsed ultrasound (LIPUS) is characterized by ultrasound intensities less than 5 W/cm2. This technique has been used for medical therapy since the 1950s [9]. LIPUS enhances the healing of bone fractures and chronic fracture nonunions [10-12]. It has been suggested that cells translate the mechanical signal of LIPUS in tissues to a biochemical response in bone via integrin-mediated mechano-receptors. This leads to the production of cyclo-oxygenase 2, which stimulates fracture repair [11]. LIPUS also facilitates soft-tissue healing by promoting cell proliferation [12]. LIPUS has shown promise for promoting cell apoptosis of hepatocellular carcinoma cells (ultrasound intensity: 500 mW/cm2) [13] and osteoclasts (ultrasound intensity: 30 mW/cm2) [14]. LIPUS has also been reported to inhibit proliferation of osteosarcoma cells (ultrasound intensity: 30 mW/cm2) [15]. Cong et al. showed that LIPUS (27.37 mW/cm2) promotes Schwann cell viability and proliferation [16]. A similar dose of LIPUS (30 mW/cm2) enhances migration and proliferation of MG63s cells [17]. It is interesting that despite variable effects, most of these studies reported a similar dose of LIPUS.

Several studies have also reported that LIPUS influences differentiation of adipose-derived stem cells [18-20]. We inferred that LIPUS could also affect adipose cell function and help treat obesity. Indeed, the beneficial effects of exercise on obesity are enhanced by LIPUS [21]. However, the efficacy and mechanism of LIPUS-mediated therapy for obesity remain unknown. Our previous study showed that a specific dose of LIPUS (109.44 mW/cm2) inhibits rat visceral preadipocyte proliferation and promotes apoptosis via the p38 pathway [22]. In this study, we showed that different LIPUS intensities differentially affected human omental adipose-derived mesenchymal stem cells (hAMSCs). Specifically, a high intensity of LIPUS (210 mW/cm2) promoted apoptosis in hAMSCs via the mitogen-activated protein kinase (MAPK) pathway, while a low intensity (70 mW/cm2) increased cell viability but did not affect proliferation of hAMSCs.

Materials and methods

Human omental adipose-derived mesenchymal stem cell isolation and culture

Omental adipose tissue was obtained from men (BMI > 25 kg/m2) who underwent radical gastrectomy for gastric cancer. This study was approved by the Ethical Committee of the First Affiliated Hospital of Nanjing Medical University and written informed consent was obtained from each patient. hAMSCs were isolated by collagenase digestion as described previously [23] and cultured in mesenchymal stem cell medium (MSCM; Cat. 7501, ScienCell). Normal cells from passages 3-5 were used for future experiments.

Low-intensity ultrasound stimulation

The LIPUS apparatus consisted of an ultrasonic generator (Verasonics, Inc., USA), a wideband power amplifier (Verasonics, Inc), a planar transducer (0.5 MHz; Haifu, China), and a degassing pump to obtain de-aerated water. The ultrasound transmitter included a signal generator (33250A, Agilent Technologies, Santa Clara, CA, USA), a broadband power amplifier, and a transducer. The receiving end consisted of a needle-shaped hydrophone (HNA-0400, ONDA, Sunnyvale, CA, USA) with an effective diameter of 0.4 mm to measure the acoustic pressure amplitude. The 3D motion system, which was controlled by LabVIEW software (controller model: XPS-C8, Newport, Irvine, CA, USA), scanned the acoustic field distribution of the XY plane. The collected data were digitized and denoised by a digital oscilloscope. Finally, the data were processed using Matlab to generate an acoustic field distribution. A 6-cm dish, seeded with 1 × 106 cells, was placed on top of the transducer with de-aerated water in between. LIPUS stimulation was applied for 1 min at a frequency of 0.5 MHz and a voltage of 44 V in 10-ms pulse bursts. The number of cycles was 1,000-3,000. The acoustic pressure in the 6-cm dish was measured (Supplementary Figure 1). A temperature test paper (TMCHallcrest, USA) was adhered to the inner surface of a 6-cm dish to test the temperature. The temperature of the cell culture media in the dishes was maintained below 37°C during the ultrasound procedures (Supplementary Table 1).

CCK-8 assay

Cell viability was determined using a Cell Counting Kit-8 (CCK-8; Dojindo, Japan) according to the manufacturer’s protocol. LIPUS-treated hAMSCs and untreated control cells were seeded at a density of 8 × 103 cells/well in 96-well plates and cultured in MSCM (Cat. 7501, ScienCell) for 24 h at 37°C in a CO2 incubator. The culture medium was replaced with Dulbecco’s Modified Eagle Medium/Nutrient mixture F12 (DMEM/F12) and incubated for another 24 h. Cells were then cultured in 100 µl of fresh DMEM/F12 supplemented with 10 µl of CCK-8 solution and incubated for an additional 4 h at 37°C. Absorbance at 450 nm was measured using a Synergy™ 2 Microplate Reader (BioTek, USA).

Flow cytometry

LIPUS-treated hAMSCs and control cells were seeded in 12-well plates at a density of 4 × 105 cells/well and then harvested after 24 h. The cells were then incubated with phycoerythrin (PE)-conjugated propidium iodide (PI) and allophycocyanin (APC)-conjugated Annexin-V (Fcmacs Biotech Co. Ltd., China) in binding buffer for 15 min at room temperature, according to the manufacturer’s instructions. Cells were washed and sorted using a FACS-Calibur (BD Biosciences, Germany) within 1 h of staining. To test the role of the p38 pathway in LIPUS-induced cell apoptosis, ultrasound-stimulated hAMSCs were treated with the p38 MAPK inhibitor SB203580 (2 mM/per well), for 24 h and then the extent of cell apoptosis was assayed as above.

TUNEL assay

TUNEL staining was performed to observe DNA fragmentation. Briefly, LIPUS-treated hAMSCs (1.5 × 105 cells/well) were plated in 24-well plates. After 24 h, the TUNEL assay (Cat. 12156792910, Roche) was performed and the percentage of TUNEL-positive cells in a viewing field was quantified. At least five viewing fields containing at least 150 cells each were quantified under 20X magnification to obtain each data point.

Real-time analysis of cell growth

LIPUS-treated and control hAMSCs were plated in 16-well E-plates at a concentration of 2 × 103 cells/well and impedance-based growth was monitored continuously for 48 h using the xCELLigence RTCA TP System (ACEA Biosciences Inc., USA).

EdU (5-Ethynyl-2’-deoxyuridine) cell proliferation assay

After LIPUS treatment, the EdU assay (Ribobio) was used to measure cell proliferation according to manufacturer’s instructions. Cells were labeled with EdU (10 μmol/L) for 12 h. After fixation in 4% paraformaldehyde for 30 min, cells were permeabilized in 0.5% Triton X-100 for 15 min. Next, the cells were incubated with Apollo® reaction solution and then nuclei were stained with Hoechst33342. After rinsing three times, cells were observed using an inverted fluorescence microscope (Zeiss) with five random fields of view.

Western blot

The hAMSCs were lysed and centrifuged for extraction of whole cell proteins. For western blotting, 30 ng of total protein per sample was loaded in each lane and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE; gel percentage of 10-15%), transferred onto polyvinylidene difluoride membranes (PVDF; Millipore, USA) and blocked with bovine serum albumin. Membranes were incubated with primary antibodies against total p38 (1:1,000; Cat. 8690, Cell Signaling Technology, USA), phosphorylated p38 (p-p38; 1:1,000; Cat. 4511, Cell Signaling Technology), total extracellular signal-regulated kinase (ERK; 1:1,000; Cat. 4695, Cell Signaling Technology), phosphorylated ERK (p-ERK; 1:1,000; Cat. 4370, Cell Signaling Technology), total c-Jun N-terminal kinase (JNK; 1:1,000; Cat. 9252, Cell Signaling Technology), phosphorylated JNK (p-JNK; 1:1,000; Cat. 4668, Cell Signaling Technology), B-cell lymphoma 2 (Bcl2; 1:1,000; Cat. 2876, Cell Signaling Technology), Bcl-2 associated X (Bax; 1:1,000; Cat. 2772, Cell Signaling Technology), cleaved caspase-3 (CC3; 1:1,000; Cat. 9661, Cell Signaling Technology), phosphorylated elF2α (p-elF2α; 1:1,000; Cat. 3597, Cell Signaling Technology), activating transcription factor 4 (ATF-4; 1:1,000; Cat. 11815, Cell Signaling Technology), phosphorylated transforming growth factor beta-activated kinase 1 (p-TAK1; 1:1,000; Cat. 9339, Cell Signaling Technology), and total TAK1 (1:1,000; Cat. 5206, Cell Signaling Technology) at 4°C overnight. The following day, membranes were incubated with secondary antibodies and then developed using an enhanced chemiluminescence (ECL) reagent (ThermoFisher). Blots were scanned on a ChemiDoc MP imager (Bio-Rad) and protein levels were quantified by Image J software. Original western images for all relevant western blots were presented in Supplementary Figures 2 and 3.

Statistical analysis

All data were expressed as the mean ± standard error of the mean (SEM) of three independent experiments. Treatment group values were compared with control values using GraphPad Prism 6.0 software. The Student’s t-test was used to independently determine statistical differences between two groups. One-way analysis of variance (ANOVA) and Bonferroni’s multiple comparison tests were used to compare three groups. P-values < 0.05 were considered significant.

Results

High dose of LIPUS promotes apoptosis in hAMSCs

Our previous study showed that a specific dose of LIPUS induces p38-mediated apoptosis in rat visceral pre-adipocytes [15]. To determine the minimum dose of LIPUS that induces apoptosis in hAMSCs, we tested different doses of ultrasound stimulation, with varying power and intensity, on suspensions of hAMSCs (Supplementary Table 1 and Supplementary Figure 1). An increase in cell apoptosis rate was observed at an ultrasound intensity of 210 mW/cm2 (Figure 1A and 1B). The CCK-8 assay also showed a marked decrease in cell viability at this ultrasound intensity, suggesting that this high dose of LIPUS prevented growth of hAMSCs (Figure 1C and 1D). The proapoptotic effects of LIPUS on hAMSCs were also assessed by the TUNEL assay. The total number of cells were visualized by Hoechest staining and apoptotic cells were visualized by TUNEL staining (Figure 1E and 1F). Ultrasound intensities as low as 140 mW/cm2 promoted apoptosis compared to the control treatment. Caspase-3 is a protein responsible for proteolysis during apoptosis, and the detection of its cleaved form, CC3, is a reliable marker for cells that undergoing apoptosis [24]. Immunoblotting analysis of CC3 expression confirmed the increase in hAMSCs apoptosis after LIPUS treatment (210 mW/cm2 intensity; Figure 1G). Bcl-2 and Bax belong to the Bcl-2 family of proteins that regulate mitochondrion-mediated apoptotic cell death. The ratio of Bax/Bcl-2 is a measure of a cell’s susceptibility to apoptosis [24]. Our results showed that levels of Bax were slightly increased in cells treated with LIPUS at an intensity of 210 mW/cm2 compared with untreated control cells (Figure 1H). No significant differences in the ratio of Bax/Bcl-2 were found between LIPUS-treated and control cells (Figure 1H). We also measured the temperature following 70-210 mW/cm2 doses of LIPUS and found that at the 70 mW/cm2 dose, the temperature reached 30°C, while doses of 140 and 210 mW/cm2 increased the temperature to 35°C (Supplementary Table 1).

Figure 1.

Figure 1

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LIPUS promotes apoptosis in hAMSCs. A. Flow cytometric analysis of apoptosis in hAMSCs. Different doses of ultrasound intensities (70, 140, 210 mW/cm2) were applied. The lower right quadrant shows early apoptotic cells. B. Apoptosis rate of hAMSCs at 24 h post-LIPUS treatment. Data are mean ± SEM. **P < 0.01 versus control, one-way ANOVA. C. CCK-8 analysis of hAMSC cell viability at different ultrasound intensities (70, 140, 210 mW/cm2) 24 h after LIPUS treatment. Data are mean ± SEM. ***P < 0.001 versus control, one-way ANOVA. D. CCK-8 analysis of hAMSC cell viability at different ultrasound intensities (70, 140, 210 mW/cm2) 48 h after LIPUS treatment. Data are mean ± SEM. ns P > 0.05, ***P < 0.001 versus control, one-way ANOVA. E. Apoptosis rate was quantified by the TUNEL assay. Scale bar = 50 μm. F. Quantification of TUNEL staining showed that 140 and 210 mW/cm2 LIPUS doses increased the number of TUNEL-positive cells (apoptosis) compared with control treatment. Data are mean ± SEM. ns P > 0.05, **P < 0.01, ***P < 0.001 versus control, one-way ANOVA. G. Western blot analysis of cleaved caspase-3 and GAPDH expression in hAMSCs treated with an ultrasound intensity of 210 mW/cm2. Cleaved caspase-3 levels were quantified relative to GAPDH levels. Data are mean ± SEM. **P < 0.01 versus control, unpaired t-test. H. Western blot analysis of Bax and Bcl-2 expression in hAMSCs treated with an ultrasound intensity of 210 mW/cm2. Bax/Bcl-2 ratio was calculated. Data are mean ± SEM. **P < 0.01 versus control, unpaired t-test.

Low dose of LIPUS enhances viability of hAMSCs, but does not affect cell proliferation

It has been shown that lower doses of LIPUS considerably enhance fracture healing compared with high doses [11]. Therefore, we tested how different doses of LIPUS affected the viability and proliferation of hAMSCs. The cells were stimulated with ultrasound intensities ranging from 70-210 mW/cm2 and then cultured for 24 h. Cell viability was compared between the differently treatments. We found that cells treated with an ultrasound intensity of 70 mW/cm2 showed significantly enhanced cell viability compared with the control cells (Figure 1C). Cell viability decreased in a dose-dependent manner in cells treated with LIPUS intensities of 140 and 210 mW/cm2 (Figure 1C). We then tested whether the low dose treatment of LIPUS promoted cell proliferation in addition to cell viability in hAMSCs. CCK-8 assays were performed on hAMSCs that were grown in serum-deprived medium to synchronize cell division. No differences in viability were seen between control cells and cells treated with a LIPUS intensity of 140 mW/cm2 (Figure 1D). However, increased cell viability was seen with an ultrasound intensity of 70 mW/cm2, while the 210 mW/cm2 dose decreased cell viability (Figure 1D), similar to the results shown in Figure 1C. We performed real-time cell analysis (RTCA) of LIPUS-treated synchronized hAMSCs to study differences in cell proliferation rates under different ultrasound intensities. We found no significant differences in proliferation rates between control cells and cells treated with an ultrasound intensity of 70 mW/cm2 (Figure 2A). Furthermore, ultrasound intensities of 140 mW/cm2 and 210 mW/cm2 significantly inhibited cell proliferation compared with the control treatment (Figure 2A). To further confirm the effect of LIPUS on cell proliferation, the cells were then incubated with EdU to label DNA synthesis and cell proliferation. As expected, the 70 mW/cm2 dose of LIPUS did not affect cell proliferation, but 140 mW/cm2 and 210 mW/cm2 LIPUS doses decreased cell proliferation (Figure 2B and 2C). Our results indicated that a low dose LIPUS treatment enhanced cell viability but not proliferation in hAMSCs, while high dose LIPUS treatment inhibited cell viability and proliferation.

Figure 2.

Figure 2

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LIPUS does not affect proliferation. A. RTCA analysis of hAMSC proliferation at different ultrasonic doses (70, 140, 210 mW/cm2). Cell Index was plotted for cells that were starved for 9 h in serum-free medium. Proliferation rate was quantified. Data are mean ± SEM. ns P > 0.05, 70 mW/cm2 versus control. **P < 0.01, ***P < 0.001 140 mW/cm2 versus control. #P < 0.01, ###P < 0.001, 210 mW/cm2 versus control, one-way ANOVA. B. Proliferation was quantified by EdU assay. Scale bar = 50 μm. C. Quantification of EdU staining showed that 140 and 210 mW/cm2 LIPUS doses decreased the number of EdU-positive cells (proliferation) compared with control treatment. Data are mean ± SEM. ns P > 0.05, ***P < 0.001 versus control, one-way ANOVA.

Dose-dependent opposing effects of LIPUS on cell viability are mediated by p38 phosphorylation

Our previous study showed that a specific intensity of LIPUS (109.44 mW/cm2) promoted cell apoptosis via the p38 MAPK pathway [22]. To test if the apoptotic effects of high dose LIPUS treatment on hAMSCs were also mediated by the p38 pathway, we measured levels of MAPK pathway proteins in cells treated with an ultrasound intensity of 210 mW/cm2. LIPUS treatment increased the levels of phosphorylated p38 and JNK, and decreased the level of the p-ERK (Figure 3A-D). Furthermore, LIPUS treatment increased the level of p-TAK1, which regulates p38 activation in cell apoptosis (Figure 3A and 3E). To determine the downstream targets of p38 in LIPUS induced cell apoptosis, we also quantified p-elF2α, the key protein involved in endoplasmic stress, and its downstream effector ATF-4. We found that LIPUS treatment significantly increased the levels of both p-elF2α and ATF-4 (Figure 3F-H). We then examined whether p38 modulated this signaling transduction from TAK1 to p-elF2α via p38 inhibition. Importantly exposure of high dose LIPUS-treated hAMSCs to the p38 inhibitor, SB203580, significantly decreased cell apoptosis (Figure 4A, 4B). Furthermore, p38 inhibition (Figure 4C) significantly attenuated the LIPUS-enhanced expression of p-TAK1 and p-elF2α, but not ATF-4 (Figure 4D-F). In contrast, LIPUS stimulation of hAMSCs with an intensity of 70 mW/cm2 significantly decreased the levels of p-p38 and increased the levels of p-ERK (Figure 5A-C). A slight elevation in the levels of p-JNK was also observed (Figure 5A and 5D). Our results showed that the dose-dependent LIPUS-mediated effects on cell apoptosis occurred via the p38/MAPK-mediated signaling pathway.

Figure 3.

Figure 3

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High dose of LIPUS promotes apoptosis via phosphorylation of p38. A. Western blot analysis of p-p38, total p38, p-ERK, total ERK, p-JNK, total JNK, p-TAK1 and total TAK1 expression in hAMSCs treated with an ultrasound intensity of 210 mW/cm2. B. Quantification of p-p38 levels relative to total p38. Data are mean ± SEM. ***P < 0.001 versus control, unpaired t-test. C. Quantification of p-ERK levels relative to total ERK. Data are mean ± SEM. **P < 0.01 versus control, unpaired t-test. D. Quantification of p-JNK levels relative to total JNK. Data are mean ± SEM. *P < 0.05 versus control, unpaired t-test. E. Quantification of p-TAK1 levels relative to total TAK1. Data are mean ± SEM. **P < 0.01 versus control, unpaired t-test. F. Western blot analysis of p-elF2α, ATF-4 and GAPDH expression in hAMSCs treated with an ultrasound intensity of 210 mW/cm2. G. Quantification of p-elF2α levels relative to GAPDH. Data are mean ± SEM. ***P < 0.001 versus control, unpaired t-test. H. Quantification of ATF-4 levels relative to GAPDH. Data are mean ± SEM. *P < 0.05 versus control, unpaired t-test.

Figure 4.

Figure 4

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Inhibition of p38 abolishes LIPUS-induced cell apoptosis. A. Flow cytometric analysis of apoptosis rates in hAMSCs at an ultrasound intensity of 210 mW/cm2 and treated with the MAPK p38 inhibitor, SB203580 (2 mM), for 24 h. The lower right quadrant shows early apoptotic cells. B. Apoptosis rate of hAMSCs 24 h after LIPUS treatment and treatment with the MAPK p38 inhibitor, SB203580 (2 mM). Data are mean ± SEM. ## P < 0.01 versus 210 mW/cm2, one-way ANOVA. C. Western blot analysis of p-p38 and p38. Quantification of p-p38 levels relative to total p38. p38 inhibition by SB203580 was verified. Data are mean ± SEM. ***P < 0.001, one-way ANOVA. D. Western blot analysis of p-TAK1 and total TAK1. Quantification of p-TAK1 levels relative to total TAK1. p38 inhibition slightly reversed the enhanced expression of p-TAK1. Data are mean ± SEM. *P < 0.05, **P < 0.01. one-way ANOVA. E. Western blot analysis of p-elF2α, ATF-4, and GAPDH expression in hAMSCs treated with SB203580 after treatment with an ultrasound intensity of 210 mW/cm2. F. Quantification of the indicated proteins relative to GAPDH. Data are mean ± SEM. ns P > 0.05, *P < 0.05, ***P < 0.001, one-way ANOVA.

Figure 5.

Figure 5

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Low dose of LIPUS decreases phosphorylation of p38. A. Western blot analysis of p-p38, total p38, p-ERK, total ERK, p-JNK, and total JNK expression in hAMSCs treated with an ultrasound intensity of 70 mW/cm2. B. Quantification of p-p38 levels relative to total p38. Data are mean ± SEM. ***P < 0.001 versus control, unpaired t-test. C. Quantification of p-ERK levels relative to total ERK. Data are mean ± SEM. *P < 0.05 versus control, unpaired t-test. D. Quantification of p-JNK levels relative to total JNK. Data are mean ± SEM. *P < 0.05 versus control, unpaired t-test.

Discussion

Visceral obesity is a major reason for serious health complications such as diabetes and cardiovascular disease [3]. It has been shown that 500 cm3 of visceral adipose tissue above baseline levels results in a 1.3-fold increase in the risk of developing hypertension and a 2.58-fold increase in the risk of developing metabolic syndrome in women [1,2]. Omentectomy prevents metabolic syndrome by reducing appetite and body weight in diet-induced obesity rat models [8]. The widespread global prevalence of obesity and its associated health issues require the need for urgent therapeutic measures. Bariatric surgery is one option to treat severe obesity, but its invasive approach limits its clinical applications. The use of medications for reducing obesity carries risks of side effects. Lifestyle modification is the safest way to tackle obesity. However, a long-term commitment to exercise and diet control is challenging for many people. Also, some patients fail to respond to lifestyle changes because of certain pre-existing genetic disorders [25]. Therefore, an alternative means of therapy is necessary for the control of visceral obesity.

Adipose tissue contains a population of mesenchymal stem cells (MSCs) located in the stromal vascular fraction [26,27]. The adipogenic differentiation ability of MSCs enables them to spontaneously repair adipose tissue after injury. The removal of adipose tissue (such as liposuction) is not a long-term remedy for obesity because it results in a compensatory increase in visceral adipose tissue [28]. The removal of fat tissue likely sends a false signal to MSCs to activate this repair mechanism. Obesity also results in impaired MSC function and remodeling of adipose tissue, which worsens the condition [29]. Treatments targeting adipose MSCs may provide a potential therapeutic strategy for visceral obesity.

LIPUS is emerging as a safe and non-invasive therapy for treating nonunion fractures [30]. Several studies have shown that LIPUS improves endothelial function [25] and protects against endothelial-mesenchymal transition [31] and spinal fusion [17]. LIPUS has also been reported to alter cell apoptosis, viability and proliferation [13-16]. Several studies have reported that LIPUS influences differentiation of adipose-derived stem cells [18-20]. It is clear that the mechanical effects of LIPUS are translated into various cellular effects. The cellular effects of LIPUS on adipose-derived MSCs have not been previously studied. Here, we showed that different intensities of LIPUS induced specific cellular effects in hAMSCs. We also explored its potential therapeutic value in obesity control.

We found that a high dose of LIPUS (210 mW/cm2) promoted apoptosis in hAMSCs, while a low dose (70 mW/cm2) enhanced cell viability without affecting proliferation. Phosphorylation of p38 significantly increased in cells treated with high dose LIPUS, but dramatically decreased in low dose-treated cells. Meanwhile, LIPUS also increased the levels of p-TAK1, which activates p38 during stress-induced cell apoptosis. These data suggest that a high dose of LIPUS promoted cell apoptosis via activation of the TAK1-p38 axis. Alternatively, high dose LIPUS also increased the expression of p-elF2α and its downstream effector ATF-4. These results revealed that LIPUS-induced cell apoptosis involves endoplasmic reticulum stress. Furthermore, our data showed that inhibition of p38 phosphorylation by SB203580 rescued both the apoptotic effects of LIPUS and the associated changes in protein levels of p-elF2α, but not ATF4. This suggests that LIPUS-induced endoplasmic reticulum stress is partially p-p38 dependent. Interestingly, p38 inhibition did not affect LIPUS-induced ATF-4 expression, indicating that there is some endogenous compensation. Our findings indicate that the p38 MAPK pathway plays a key role in mediating the differential effects of LIPUS on hAMSCs.

In our previous study [22], LIPUS-treated rat primary visceral preadipocytes showed increased rates of apoptosis at a dosage intensity of 109.44 mW/cm2. In this study, a dosage intensity of 140 mW/cm2 was not sufficient to induce apoptosis in hAMSCs. Increased rates of apoptosis were only observed when hAMSCs were treated with LIPUS at a high dosage intensity of 210 mW/cm2. These results suggest that hAMSCs needed larger amounts of ultrasound energy to undergo cellular apoptosis compared with rat primary visceral preadipocytes. The difference in species and cellular origins of the two cell types analyzed in the two studies may contribute to their different energy requirements to undergo apoptosis. The primary visceral adipocytes from rats used in our previous study were derived from epididymal adipose tissue [22], while the hAMSCs used in the current study were derived from human omental adipose tissue. Omental adipose tissue acts as a shock absorber that provides padding to protect inner organs from physical injury. This type of adipose tissue is enriched in fibrillar proteins compared with subcutaneous adipose tissue, which contributes to its mechanical properties [32]. Subcutaneous adipose tissue has a lower energy dissipation density and slower stress relaxation properties than omental adipose tissue, which suggests that these two tissue types have different biomechanical properties [32]. Our results reflected these differences in tissue types derived from rat and human cells. Thus LIPUS parameters obtained from animal studies may not be extrapolated to human cells. The clinical development of LIPUS therapy for abdominal obesity requires better analysis of effective ultrasound parameters.

It has been shown that a low dose of LIPUS treatment, but not a high dose, enhances fracture healing [11,33]. Low-density bone volume fractions and woven bone percentages were compared between cells treated with 30 mW/cm2 and 150 mW/cm2 doses of LIPUS. Cells that were treated with an ultrasound dose intensity of 30 mW/cm2 showed significantly enhanced fracture healing compared with cells treated with an ultrasound dose intensity of 150 mW/cm2 [27]. A low dose of LIPUS (30 mW/cm2) has been shown to suppress adipogenic differentiation and promote osteogenic differentiation in 3T3-L1 and ST2 cell lines by inducing ERK phosphorylation [28]. In contrast, a high dose of LIPUS (100 mW/cm2) promotes adipogenic differentiation of adipose-derived stem cells in mice [29]. These studies suggest a key role for ERK phosphorylation in adipogenic differentiation of AMSCs. In our study, we showed that different doses of LIPUS had different cellular effects on hAMSCs. Specifically, a high dose of LIPUS (210 mW/cm2) promoted apoptosis, while a low dose (70 mW/cm2) enhanced cell viability. ERK phosphorylation was increased in cells treated with a high dose of LIPUS, but decreased in cells treated with a low dose. It is possible that ERK phosphorylation is also involved in adipogenic differentiation of hAMSCs; however, further studies are needed to test this premise.

To determine the main force in LIPUS treatment, we tested temperature upon 70-210 mW/cm2 doses of LIPUS and found that upon 70 mW/cm2, the temperature reached 30°C, while upon 140 and 210 mW/cm2, the temperature reached 35°C. Because the 210 mW/cm2 dose of LIPUS promoted cell apoptosis, while 140 mW/cm2 did not, we speculate that the pro-apoptotic effects of LIPUS may be caused by the mechanical rather than thermal changes.

In summary, we found that a high dose of LIPUS (210 mW/cm2) promoted apoptosis, while a low dose (70 mW/cm2) enhanced cell viability in hAMSCs. Phosphorylation of p38 was a potential key mechanism mediating the differential effects of LIPUS. Further in vivo studies are needed to determine the optimal doses of ultrasound intensities that reduce visceral adipose volume without causing side effects. Our study showed the potential use of LIPUS as a safe and non-invasive therapy to control visceral obesity and attenuate related health complications.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 81627802, No. 81570247) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD2014-2016). Dr. Wei Sun is an assistant Fellow at the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Dr. Xiangqing Kong is a Fellow at the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine.

Disclosure of conflict of interest

None.

Supporting Information

ajtr0011-0418-f6.pdf (764.3KB, pdf)

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