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We have previously shown that low-intensity ultrasound (LIUS) can modulate mitochondrial complex I activity and the generation of mitochondrial reactive oxygen species (mtROS) in PC12 cells. This study investigated the mechanism of LIUS by comparing its effect on mitochondrial dysfunction by three different pathways. LIUS was shown to reverse the effects of rotenone, a Q-site blocker, on the complex I inhibition, mtROS generation, and drop of mitochondrial membrane potential (Δψm). In contrast, common antioxidants, N-acetyl cysteine (NAC), and uric acid (UA) blocked rotenone-induced mtROS generation and Δψm drop without recovering the complex I activity, which suggested that Δψm drop is correlated with mtROS generation rather than complex I inhibition itself. Ionomycin, an ionophore for Ca2+, and L-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, induced mtROS generation and Δψm drop without inhibiting complex I activity via different mechanisms. LIUS showed no effect on ionomycin-induced Δψm drop but showed partial inhibition on the other effects of ionomycin and BSO. These results suggest that LIUS might have redundant mechanisms but acted mainly on the complex I activity thereby modulating mtROS and Δψm levels. LIUS appeared to act on the Q-module of complex I because it showed no inhibitory effect on Zn2+, an inhibitor of the proton transporting P-module of complex I. Interestingly, pretreatment of LIUS for up to an hour in advance blocked the rotenone effect as efficiently as the co-treatment. Further studies are needed to reveal the exact mechanism of LIUS to inhibit complex I activity.

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Ovarian carcinoma is the key cause of cancer death from gynecological malignancy of women. Chemotherapy-resistance, metastasis and relapse contribute to the high mortality in ovarian cancer patients. Cancer stem cells (CSCs) stand for the root of kinds of cancer types such as ovarian cancer, are the key driver of tumor initiation, cancer metastasis, and resistance to conventional chemotherapy as well as genomic targeted therapy. Thus, the approach to eliminate CSCs and uncovering the mechanism will have substantial impact on cancer therapy. However, targeting CSC remains unfeasible in clinical practice in ovarian cancer therapy. In this study, we first found that Low-intensity ultrasound (LIUS) was capable of reducing the CSC populations in the xenograft model with ovarian cancer, with blocking survival, anti-apoptosis, self-renewal, and downregulating the cancer stemness genes in ovarian CSCs. Moreover, LIUS ameliorated IL-6/STAT3 inflammatory pathway via inhibiting IL-6-induced STAT3 phosphorylation, DNA binding activity and, the expressions of its downstream effectors in ovarian CSCs while no explicit effect was found in the corresponding bulk cancer cells. Additional approaches in molecular studies showed that LIUS disrupts CSC features via inhibiting IL-6/STAT3 inflammatory pathway. Collectively, our data for the first time elucidate IL-6/STAT3 inflammatory loop as the key CSC or cancer stemness pathway in ovarian cancer by LIUS treatment, providing a novel and potential therapy and a promising target in ovarian cancer.

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BACKGROUND: Therapeutic potential of low-intensity ultrasound (LIUS) has become evident in various musculoskeletal diseases. We have previously shown that LIUS has an inhibitory effect on local edema in various diseases including the arthritis and brain injury. In this study, we examined whether LIUS can attenuate paw edema formation vis-à-vis vascular permeability and inflammation in rats induced by carrageenan. LIUS with a frequency of 1 MHz and the intensities of 50, 100, or 200 mW/cm2 were exposed on rat paws for 10 min immediately after carrageenan injection. RESULTS: Carrageenan injection induced paw edema which was peaked at 6 h and gradually decreased nearly to the initial baseline value after 72 h. LIUS showed a significant reduction of paw edema formation at 2 and 6 h at all intensities tested. The highest reduction was observed at the intensity of 50 mW/cm2. Histological analyses confirmed that LIUS clearly decreased the carrageenan-induced swelling of interstitial space under the paw skin and infiltration of polymorphonuclear leukocytes. Moreover, Evans Blue extravasation analyses exhibited a significant decreases of vascular permeability by LIUS. Finally, immunohistochemical staining showed that expression of pro-inflammatory proteins, namely, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) induced by carrageenan injection was reduced back to the normal level after LIUS stimulation. CONCLUSIONS: These results provide a new supporting evidence for LIUS as a therapeutic alternative for the treatment of edema in inflammatory diseases such as cellulitis.

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Diabetic retinopathy (DR) is a severe micro-vascular complication of diabetes. High glucose (HG)-evoked nitric oxide (NO) production mediated by increased oxidative stress is a key factor in DR pathogenesis. In this study, we examined whether low-intensity ultrasound (LIUS) stimulation can reduce HG-induced NO generation. We determined that LIUS stimulation decreased the HG-induced NO generation possibly via inhibition of reactive oxygen species (ROS) and subsequently diminished the associated pro-inflammatory pathway involving the induced expression of inducible nitric oxide synthase, cyclooxygenase-2 and vascular endothelial growth factor. In addition, we determined that LIUS stimulation reduced the quantity of NO produced by N-acetylcysteine, which was not mediated by ROS. These results indicate that LIUS can inhibit both ROS-dependent and -independent NO generation processes in ARPE-19 cells. We envision LIUS as a potential therapeutic alternative to treat DR. Further studies are required to understand the underlying mechanism of the LIUS-induced reduction of NO generation for DR therapy.

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OBJECTIVES: Low-intensity ultrasound (LIUS) has been shown to enhance bone and cartilage regeneration from stem cells. The ease of its incorporation makes it an attractive mechanical stimulus for not only osteogenesis and chondrogenesis, but also cardiomyogenesis. However, to date, no study has investigated its effects on cardiomyogenesis from embryonic stem cells. METHODS: In this study, murine embryonic stem cells were differentiated via embryoid body formation and plating, and after 3 days they were subjected to daily 10 minutes of LIUS treatment with various conditions: (1) low-pulsed (21 mW/cm2 , 20% duty cycle), (2) low-continuous, (3) high-pulsed (147 mW/cm2 , 20% duty cycle), and (4) high-continuous LIUS. RESULTS: Low-pulsed and high-continuous LIUS had improved beating rates of contractile areas as well as increased late cardiac gene expressions, such as α- and ß-myosin heavy chain and cardiac troponin T, showing its benefits on cardiomyocyte differentiation. Meanwhile, an early endodermal marker, α-fetoprotein, was significantly attenuated after LIUS treatments. CONCLUSIONS: With these observations, it is demonstrated that LIUS simulation could enhance cardiomyogenesis from embryonic stem cells and increase its selectivity toward cardiomyocytes by reducing spontaneous differentiation.

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Many studies have shown that mitochondrial dysfunction and the subsequent oxidative stress caused by excessive reactive oxygen species (ROS) generation play a central role in the pathogenesis of Parkinson's disease (PD). We have previously shown that low-intensity ultrasound (LIUS) could reduce ROS generation by L-buthionine-(S,R)-sulfoximine (BSO) in retinal pigment epithelial cells. In this study, we studied the effects of LIUS stimulation on the ROS-dependent α-synuclein aggregation in 1-methyl-4-phenylpyridinium ion (MPP+)-treated PC12 cells. We found that LIUS stimulation suppressed the MPP+-induced ROS generation and inhibition of mitochondrial complex I activity in PC12 cells in an intensity-dependent manner at 30, 50, and 100 mW/cm2. Furthermore, LIUS stimulation at 100 mW/cm2 suppressed inhibition of mitochondrial complex activity by MPP+ and actually resulted in a decrease of α-synuclein phosphorylation and aggregation induced by MMP+ treatment in PC12 cells. LIUS stimulation also inhibited expression of casein kinase 2 (CK2) that appears to mediate ROS-dependent α-synuclein aggregation. Finally, LIUS stimulation alleviated the death of PC12 cells by MPP+ treatment in an intensity-dependent manner. We, hence, suggest that LIUS stimulation inhibits ROS generation by MPP+ treatment, thereby suppressing α-synuclein aggregation in PC12 cells.

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AIMS: Brain oedema is a major contributing factor to the morbidity and mortality of a variety of brain disorders. Although there has been considerable progress in our understanding of pathophysiological and molecular mechanisms associated with brain oedema so far, more effective treatment is required and is still awaited. Here we intended to study the effects of low intensity ultrasound (LIUS) on brain oedema. METHODS: We prepared the rat hippocampal slice in vitro and acute water intoxication in vivo models of brain oedema. We applied LIUS stimulation in these models and studied the molecular mechanisms of LIUS action on brain oedema. RESULTS: We found that LIUS stimulation markedly inhibited the oedema formation in both of these models. LIUS stimulation significantly reduced brain water content and intracranial pressure resulting in increased survival of the rats. Here, we showed that the AQP4 localization was increased in the astrocytic foot processes in the oedematous hippocampal slices, while it was significantly reduced in the LIUS-stimulated hippocampal slices. In the in vivo model too, AQP4 expression was markedly increased in the microvessels of the cerebral cortex and hippocampus after water intoxication but was reduced in the LIUS-stimulated rats. CONCLUSIONS: These data show that LIUS has an inhibitory effect on cytotoxic brain oedema and suggest its therapeutic potential to treat brain oedema. We propose that LIUS reduces the AQP4 localization around the astrocytic foot processes thereby decreasing water permeability into the brain tissue.

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Mesenchymal stem cells (MSCs) have a capacity to differentiate into the chondrogenic lineage and are a valuable allogenic source for cartilage tissue engineering. However, they still have critical limitations of relatively inefficient chondrogenic differentiation in vitro and of dedifferentiation and/or hypertrophic changes at late stages of differentiation. Numerous approaches using biochemical and mechanical factors have been tried but have so far failed to overcome these problems. Recent studies by other groups and ours have shown that low-intensity ultrasound (LIUS) is an efficient tool for promoting the chondrogenic differentiation of MSCs both in vitro and in vivo. A series of our experiments suggests that LIUS not only induces chondrogenic differentiation of MSCs but also has diverse additional activities that enhance the viability of MSCs, increase possibly the integrity of the differentiated tissues and delays hypertrophic changes during differentiation. Therefore, LIUS could be an innovative and versatile tool for chondrogenic differentiation of MSCs and for cartilage tissue engineering.