Facilitatory effect of low-pulse repetition frequency ultrasound on release of extracellular vesicles from cultured myotubes.

PURPOSE: Extracellular vesicles (EVs) serve as carriers of intracellular factors with therapeutic effects, including tissue regeneration and attenuation of inflammatory responses. The majority of EVs in vivo are derived from skeletal muscle, which is reported to have anti-inflammatory effects. While high-intensity pulsed ultrasound (US) irradiation has been shown to promote EV secretion from myotubes, the impact of pulse repetition frequency, a US parameter affecting pulse length, on EV release remains unclear. This study aimed to investigate the impact of pulse repetition frequency of US on the release of EVs from myotubes. METHODS: C2C12 myoblasts were used in this study. After differentiation into C2C12 myotubes, US was performed for 5 min at an intensity of 3.0 W/cm2, duty cycle of 20%, acoustic frequency of 1 MHz, and different pulse repetition frequencies (100 Hz, 10 Hz, or 1 Hz). After 12 h, EVs and cells were collected for subsequent analyses. RESULTS: US did not cause a reduction in cell viability across all US groups compared to the control. The concentration of EVs was significantly higher in all US groups compared to the control group. In particular, the highest increase was observed in the 1-Hz group on EV concentration as well as intracellular Ca2+ level. CONCLUSION: This study investigated the effect of three different pulse repetition frequencies of US on the release of EVs from cultured myotubes. It is concluded that a low-pulse repetition frequency of 1 Hz is the most effective for enhancing EV release from cultured myotubes with pulsed ultrasound.

Pulsed ultrasound promotes secretion of anti-inflammatory extracellular vesicles from skeletal myotubes via elevation of intracellular calcium level.

The regulation of inflammatory responses is an important intervention in biological function and macrophages play an essential role during inflammation. Skeletal muscle is the largest organ in the human body and releases various factors which mediate anti-inflammatory/immune modulatory effects. Recently, the roles of extracellular vesicles (EVs) from a large variety of cells are reported. In particular, EVs released from skeletal muscle are attracting attention due to their therapeutic effects on dysfunctional organs and tissues. Also, ultrasound (US) promotes release of EVs from skeletal muscle. In this study, we investigated the output parameters and mechanisms of US-induced EV release enhancement and the potential of US-treated skeletal muscle-derived EVs in the regulation of inflammatory responses in macrophages. High-intensity US (3.0 W/cm2) irradiation increased EV secretion from C2C12 murine muscle cells via elevating intracellular Ca2+ level without negative effects. Moreover, US-induced EVs suppressed expression levels of pro-inflammatory factors in macrophages. miRNA sequencing analysis revealed that miR-206-3p and miR-378a-3p were especially abundant in skeletal myotube-derived EVs. In this study we demonstrated that high-intensity US promotes the release of anti-inflammatory EVs from skeletal myotubes and exert anti-inflammatory effects on macrophages.

Electrical stimulation facilitates NADPH production in pentose phosphate pathway and exerts an anti-inflammatory effect in macrophages.

Macrophages play an important role as effector cells in innate immune system. Meanwhile, macrophages activated in a pro-inflammatory direction alter intracellular metabolism and damage intact tissues by increasing reactive oxygen species (ROS). Electrical stimulation (ES), a predominant physical agent to control metabolism in cells and tissues, has been reported to exert anti-inflammatory effect on immune cells. However, the mechanism underlying the anti-inflammatory effects by ES is unknown. This study aimed to investigate the effect of ES on metabolism in glycolytic-tricarboxylic acid cycle (TCA) cycle and inflammatory responses in macrophages. ES was performed on bone marrow-derived macrophages and followed by a stimulation with LPS. The inflammatory cytokine expression levels were analyzed by real-time polymerase chain reaction and ELISA. ROS production was analyzed by CellRox Green Reagent and metabolites by capillary electrophoresis-mass spectrometry. As a result, ES significantly reduced proinflammatory cytokine expression levels and ROS generation compared to the LPS group and increased glucose-1-phosphate, a metabolite of glycogen. ES also increased intermediate metabolites of the pentose phosphate pathway (PPP); ribulose-5-phosphate, rebose-5 phosphate, and nicotinamide adenine dinucleotide phosphate, a key factor of cellular antioxidation systems, as well as α-Ketoglutarate, an anti-oxidative metabolite in the TCA cycle. Our findings imply that ES enhanced NADPH production with enhancement of PPP, and also decreased oxidative stress and inflammatory responses in macrophages.

Association between Patients' Body Mass Index and the Effect of Monophasic Pulsed Microcurrent Stimulation on Pressure Injury Healing.

This secondary analysis study aimed to detect individual variables that influence the efficacy of monophasic pulsed microcurrent on pressure injury healing. Eleven patients with pressure injuries showing delayed healing underwent a microcurrent stimulation period and a placebo period. We analyzed the correlation between the individual variables and the following three outcomes using monophasic pulsed microcurrent: the wound reduction rate in the electrical stimulation period, the reduction rate in the placebo period, and the difference between these two reduction rates. Furthermore, the patients were divided into two groups, one with a wound reduction rate of more than 10% and the other with less than 10%, and the relationship between each variable was compared. As a result, the wound reduction rate in the electrical stimulation period and the difference in the reduction rate between the two periods showed significant positive correlations with patients' body mass index. In addition, a significant difference was observed in the body mass index between subjects with a reduction rate of 10% or higher and those with a reduction rate of less than 10%. This study found a correlation between the effect of monophasic pulsed microcurrent for pressure injury healing and the level of patients' body mass index.

The role of death receptor signaling pathways in mouse Sertoli cell avoidance of apoptosis during LPS- and IL-18-induced inflammatory conditions.

Lipopolysaccharide (LPS) triggers infectious acute inflammation, and interleukin (IL)-18 is an inflammasome-mediated cytokine. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are high. Additionally, high-dose recombinant IL-18 (rIL-18) induced Leydig cell apoptosis. The blood-testis barrier formed by Sertoli cells protects testicular germ cells from both exogenous and endogenous harmful substances. However, the impact of LPS and IL-18 on Sertoli cells remained unclear. We stimulated TM4 cells, a mouse Sertoli cell line, with LPS (200 or 1000 ng/mL) or rIL-18 (0.1-100 ng/mL) at levels that induced Leydig cell apoptosis in our previous study and assessed caspase 3 cleavage and the mRNA expression of inflammatory cytokines and markers of apoptotic pathways (Tnfr1, Fasl, Fas, Fadd) after stimulation. Il6 mRNA was increased by LPS stimulation. Tnfα mRNA was increased by 200 ng/mL LPS but not 1000 ng/mL LPS. Fas was increased, but Fasl was decreased, by LPS. LPS had little influence on Tnfr1 or Fadd mRNA expression and did not induce apoptosis. Il18 mRNA was not increased, and Il18r1 was significantly decreased following LPS treatment. Treatment with rIL-18 increased Il18r1 mRNA and induced inflammation, but decreased Tnfr1 and had little influence on apoptosis, as indicated by Tnfα, Fasl, Fas, Fadd and cleaved caspase 3. These results suggested that Sertoli cells do not easily undergo apoptosis despite strong inflammatory stimuli. Additionally, Sertoli cells may resist inflammation and play a larger role in protecting testicular homeostasis than other component cells of the testis.

Skeletal myotube-derived extracellular vesicles enhance itaconate production and attenuate inflammatory responses of macrophages.

Introduction: Macrophages play an important role in the innate immunity. While macrophage inflammation is necessary for biological defense, it must be appropriately controlled. Extracellular vesicles (EVs) are small vesicles released from all types of cells and play a central role in intercellular communication. Skeletal muscle has been suggested to release anti-inflammatory factors, but the effect of myotube-derived EVs on macrophages is unknown. As an anti-inflammatory mechanism of macrophages, the immune responsive gene 1 (IRG1)-itaconate pathway is essential. In this study, we show that skeletal muscle-derived EVs suppress macrophage inflammatory responses, upregulating the IRG1-itaconate pathway. Methods: C2C12 myoblasts were differentiated into myotubes and EVs were extracted by ultracentrifugation. Skeletal myotube-derived EVs were administered to mouse bone marrow-derived macrophages, then lipopolysaccharide (LPS) stimulation was performed and inflammatory cytokine expression was measured by RT-qPCR. Metabolite abundance in macrophages after addition of EVs was measured by CE/MS, and IRG1 expression was measured by RT-PCR. Furthermore, RNA-seq analysis was performed on macrophages after EV treatment. Results: EVs attenuated the expression of LPS-induced pro-inflammatory factors in macrophages. Itaconate abundance and IRG1 expression were significantly increased in the EV-treated group. RNA-seq analysis revealed activation of the PI3K-Akt and JAK-STAT pathways in macrophages after EV treatment. The most abundant miRNA in myotube EVs was miR-206-3p, followed by miR-378a-3p, miR-30d-5p, and miR-21a-5p. Discussion: Skeletal myotube EVs are supposed to increase the production of itaconate via upregulation of IRG1 expression and exhibited an anti-inflammatory effect in macrophages. This anti-inflammatory effect was suggested to involve the PI3K-Akt and JAK-STAT pathways. The miRNA profiles within EVs implied that miR-206-3p, miR-378a-3p, miR-30d-5p, and miR-21a-5p may be responsible for the anti-inflammatory effects of the EVs. In summary, in this study we showed that myotube-derived EVs prevent macrophage inflammatory responses by activating the IRG1-itaconate pathway.

Effect of Partial Foot Amputation Level on Gait Independence in Patients With Chronic Lower Extremity Wounds: A Retrospective Analysis of a Japanese Multicenter Database.

Partial foot amputation (PFA) is generally planned to minimize the amputation level; nonetheless, the effect of PFA levels on gait independence in amputees remains unclear. This study aimed to investigate the impact of PFA levels of the forefoot on gait independence in patients with chronic lower extremity (LE) wounds. This multicenter retrospective cohort study included 232 hospitalized Japanese patients treated and rehabilitated for chronic LE wounds. A multivariate analysis based on PFA levels was conducted for gait independence at discharge, with age and comorbidities as independent variables. Patients with Lisfranc amputation had significantly less independent gait than patients with more distal amputation and those without amputation (<22% vs >40%; P = .027; Fisher's exact test). Logistic regression analysis revealed that Lisfranc amputation (odds ratio [OR]: 0.257, P = .047), age (OR: 0.559, P = .043), and chronic limb-threatening ischemia (OR: 0.450, P = .010) were independent factors associated with gait independence. Additionally, the regression model confirmed discrimination performance using the C index (0.691, P < .001) with receiver operating characteristic analysis. In patients with chronic LE wounds undergoing PFA, Lisfranc amputation was negatively associated with gait independence.

Acute Effects of Combination Therapy by Triceps Surae Stretching and Electrical Stimulation to the Tibialis Anterior on Medial Forefoot Plantar Pressure During Gait in Patients With Diabetes Mellitus.

High plantar flexor moment and limited ankle mobility are known to cause high plantar pressure under the forefoot. Stretching is an effective physical therapy for the limited ankle range of motion (ROM), and electrical stimulation is used to regulate the activity of antagonistic muscle via the action of reciprocal inhibition. Additionally, stretching paired with electrical stimulation has been reported to improve the limited ROM significantly. This study aims to investigate the influences of stretching on triceps surae (STR), electrical stimulation to tibialis anterior (ES), and the combination (ES+STR) on the ROM, kinematic parameters, and plantar pressure distribution during gait in patients with diabetes mellitus. Planter pressure and other parameters were measured before and after the intervention of ES, STR, ES+STR, or the rest sitting on the bed (CON) for 10 min. Pressure time integral under the medial forefoot decreased in the ES+STR compared to CON (P< .05). Interestingly, ES+STR increased passive and dynamic ROM on ankle dorsiflexion during gait and increased the lateral center of pressure excursion (P < .05). Furthermore, these changes were followed by decreased contact duration under the medial forefoot (P < .05). The combined therapy improves ankle mobility during gait and reduces the contact duration and the plantar pressure under the medial forefoot in patients with diabetes mellitus.

The migration speed of nucleolar precursor bodies in pronuclei affects in vitro fertilization-derived human embryo ploidy status and live birth.

Purpose: To study the relationship between clinical outcomes after assisted reproduction and the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN). Methods: NPB migration speed, embryo ploidy status, and live birth (LB) were retrospectively analyzed in IVF-derived zygotes. The central coordinates of the mPN, fPN, and NPBs were noted at multiple timepoints. The migration distance of NPBs between two sequential images was measured to calculate NPB migration speed. Results: The NPB migration speeds in mPN and fPN were significantly faster in euploid zygotes than in aneuploid zygotes. In multivariate logistic analysis, NPB migration speed in mPN and the female age were associated with euploidy. The NPB migration speeds in mPN and fPN were also significantly faster in zygotes that led to LB than in zygotes that led to no pregnancy. In a receiver operating characteristic curve analysis of LB by NPB migration speed in mPN, the cut-off value was 3.74 µm/h (AUC: 0.825, 95%CI: 0.688-0.963). When the zygotes were categorized by this cut-off value, there were significantly more LBs in zygotes with migration speed ≥ the cut-off (78.9% vs. 21.1%). Conclusions: Zygotes with quickly migrating NPBs demonstrated the developmental potential to become a baby.

Immediate Effects of Weight-Bearing Calf Stretching on Ankle Dorsiflexion Range of Motion and Plantar Pressure During Gait in Patients with Diabetes Mellitus.

High plantar pressure is a risk factor for diabetic foot ulcers, and it is known that restriction of ankle dorsiflexion range of motion (ROM) causes high plantar pressure. Stretching is a non-invasive and general means to improve ROM; however, the effect of stretching on the ROM and plantar pressure has not been clarified in patients with diabetes mellitus. We aimed to study the effects of intermittent weight-bearing stretching on ankle dorsiflexion ROM and plantar pressure during gait in patients with diabetes mellitus. Seven patients with diabetes mellitus participated, and their triceps surae was stretched using weight-bearing stretching with a stretch board. Five minutes of stretching was performed 4 times with a rest interval of 30 s. Ankle dorsiflexion ROM was measured with the knee flexed and extended. Peak pressure and pressure-time integral during gait were measured and calculated for the rearfoot, midfoot, forefoot, and total plantar surface before and after stretching. Ankle dorsiflexion ROM with the knee extended or bent increased significantly after stretching (P < .05). Peak pressure and the pressure-time integral decreased significantly, especially in the forefoot (P < .01), and these also decreased significantly in the total plantar surface (P < .05). The duration of foot-flat decreased after stretching (P < .05). Weight-bearing stretching improved ankle dorsiflexion ROM and reduced plantar pressure during gait. These results suggest that weight-bearing calf stretching may be an effective means to prevent and treat diabetic foot ulcers.

High Concentrations of Nucleotides Prevent Capillary Regression during Hindlimb Unloading by Inhibiting Oxidative Stress and Enhancing Mitochondrial Metabolism of Soleus Muscles in Rats.

Prolonged inactivity in skeletal muscles decreases muscle capillary development because of an imbalance between pro- and antiangiogenic signals, mitochondrial metabolism disorders, and increased oxidative stress. Nucleotides have been shown to exert a dose-dependent effect on disuse-induced muscle atrophy. However, the dose-dependent effect on capillary regression in disused muscles remains unclear. Therefore, this study investigated the dose-dependent effect of nucleotides on capillary regression due to disuse. For this purpose, Wistar rats were divided into five groups as follows: control rats fed nucleotide-free diets (CON), hindlimb-unloaded rats fed nucleotide-free diets (HU), and hindlimb-unloaded rats fed 1.0%, 2.5%, and 5.0% nucleotide diets, (HU + 1.0% NT), (HU + 2.5% NT), and (HU + 5.0% NT), respectively. Unloading increased reactive oxygen species (ROS) production and decreased mitochondrial enzyme activity, thereby decreasing the number of muscle capillaries. In contrast, 5.0% nucleotide-containing diet prevented increases in ROS production and reductions in the expression levels of NAMPT, PGC-1α, and CPT-1b proteins. Moreover, 5.0% nucleotide-containing diet prevented mitochondrial enzyme activity (such as citrate synthase and beta-hydroxy acyl-CoA dehydrogenase activity) via NAMPT or following PGC-1α upregulation, thereby preventing capillary regression. Therefore, 5.0% nucleotide-containing diet is likely to prevent capillary regression by decreasing oxidative stress and increasing mitochondrial metabolism.

Efficacy of Low-frequency Monophasic Pulsed Microcurrent Stimulation Therapy in Undermining Pressure Injury: A Double-blind Crossover-controlled Study.

Objectives: This double-blind crossover-controlled trial aimed to verify the effect of electrical stimulation therapy on pressure injuries with undermining. Methods: : In this trial, we compared the healing rates between a sham period and a treatment period using monophasic pulsed microcurrent therapy. The participants were randomly assigned to the sham or treatment group and received stimulation for 2 weeks. All the participants, physical therapists, and researchers were blinded to the allocation. For the main analysis, data on the effect of the intervention on changes in weekly healing and contraction rates of the wound areas, including undermining, were analyzed based on a two-period crossover study design. The intervention effect was estimated by examining the mean treatment difference for each period using Wilcoxon's signed-rank test. Results: : The reduction of the entire wound area, including the undermining area, resulted in significantly higher healing and contraction rates in the treatment group (overall wound area reduction rate: contraction rate, P=0.008; period healing rate, P=0.002). Conclusions: : Electrical stimulation therapy for pressure injuries, using conditions based on the findings of an in vivo culture study, was effective in reducing the wound area.

Electrical Shunting Prevents the Decline of Galvanotaxis After Monophasic Pulsed Microcurrent Stimulation in Human Dermal Fibroblasts.

Background: Electrical stimulation (ES) therapy is recommended for healing pressure injuries. Monophasic pulsed microcurrent stimulation promotes the migration of human dermal fibroblasts (HDFs) to the cathode, and ES potentially accelerates pressure injury healing. A reverse current is generated after ES in the human body; however, the effects of the electrical shunt in preventing the reverse current from migrating are unclear. Therefore, this study aimed to investigate the effects of an electrical shunt on the migration of HDFs. Methods: In the shunt groups, HDFs were electrically stimulated (0, 200, 400, and 600 µA) for 8 hours, and an electrical shunt was used to remove the electricity after ES. HDFs were observed under time-lapse microscopy for 24 hours. The migration ratio toward the cathode was calculated for each dish. Results: The migration ratio was significantly higher in the 200-µA group than in the other groups. HDFs migrated toward the anode after ES in the non-shunt groups with greater than 400 µA ES; however, HDFs did not migrate toward the anode with electrical shunting. Conclusions: A post-ES electrical shunt is important in preventing a decline in the migration effect of ES.

Pulsed-Ultrasound Irradiation Induces the Production of Itaconate and Attenuates Inflammatory Responses in Macrophages.

Background: Itaconate is a key metabolite in the innate immune system and exerts strong anti-inflammatory effects in macrophages. For the production of itaconate in macrophages, immune-responsive gene 1 (IRG1) is an imperative enzyme, and activating the IRG1-itaconate pathway is reported to alleviate inflammatory diseases by upregulating nuclear factor-erythroid 2-related factor 2 (NRF2). However, there are very few reports on strategies to increase itaconate production. Ultrasound therapy is a widely used intervention for anti-inflammatory and soft-tissue regeneration purposes. Here we show the effect of ultrasound irradiation on the production of itaconate in macrophages. Methods: Murine bone marrow-derived macrophages (BMDMs) were exposed to pulsed ultrasound (3.0 W/cm2) for 5 minutes. Three hours after irradiation, the intracellular levels of metabolites and mRNA expression levels of Irg1 and Nrf2 were measured using CE/MS and qPCR, respectively. To evaluate macrophage inflammation status, 3 h after irradiation, the cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) for 1.5 h and the mRNA expression levels of pro-inflammatory factors (Il-1ß, Il-6, and Tnf-α) were measured. Student's t-test, one-way ANOVA and Tukey's multiple comparison test were used for statistical processing, and the significance level was set to less than 5%. Results: Ultrasound irradiation significantly increased the intracellular itaconate level and the expression levels of Irg1 and Nrf2 in BMDMs. Upregulation of Il-1ß, Il-6, and Tnf-α by LPS was significantly suppressed in BMDMs treated with ultrasound. Ultrasound irradiation did not affect cell viability and apoptosis. Conclusion: Ultrasound irradiation induces the production of itaconate by upregulating Irg1 expression and attenuates inflammatory responses in macrophages via Nrf2. These results suggest that ultrasound is a potentially useful method to increase itaconate production in macrophages.

Prediction of callus and ulcer development in patients with diabetic peripheral neuropathy by isosceles triangle-forming tuning fork.

Objectives: Tuning fork vibration sensation testing is widely used as a diagnostic test to detect diabetic neuropathy. However, evidence-based literature indicates that reliability between examiners is low. Attaching isosceles triangle diagrams on tuning forks lowers the discrepancy between examiners. This study aimed to analyze the relationship between vibration sensation measurement using an improved tuning fork and the presence of callus and wound development in patients with diabetic peripheral neuropathy. Methods: Participants included 56 general older adults and 52 patients with diabetic peripheral neuropathy. The methods included confirmation of the presence or absence of callus, range of motion of the ankle and the first metatarsophalangeal joint, vibratory sensitivity of the medial malleolus and the dorsal aspect of the first distal phalanx using an improved tuning fork, and touch-pressure sensitivity of the plantar aspect of the hallux. Patients with diabetic peripheral neuropathy were followed up for 3 years to check for the presence or absence of wounds. Results: When compared with the general older adults, the patients with diabetic peripheral neuropathy had significantly lower touch-pressure sensitivity (p < 0.01), vibratory sensitivity at the distal phalanx (p < 0.01) and medial malleolus (p < 0.01), ankle dorsiflexion range of motion (p < 0.01), and metatarsophalangeal joint extension range of motion (p < 0.01). The area under the receiver operating characteristic curve with callus formation was 0.93 for the medial malleolus and 0.96 for the distal phalanx, indicating that the accuracy of the distal phalanx was higher (p < 0.01) than the medial malleolus. According to the Cox proportional hazard analysis, the vibratory sensitivity of the distal phalanx was a significant risk factor for ulcer development (p < 0.05). Conclusion: These findings suggest that the vibration sensation test, which we improved via the technique described in this study, is useful for predicting the occurrence of callus and ulcer.

Enhancement of astaxanthin incorporation by pulsed high-intensity ultrasound in LPS-stimulated macrophages.

PURPOSE: Ultrasound (US) has been reported to improve the permeability of cell membranes to pharmaceuticals by causing cavitation. Astaxanthin (AX) potently terminates the induction of inflammation, but it has low oral bioavailability, which limits its incorporation in local cells and organs and its therapeutic potential. In this study, we aimed to investigate the contribution of US to AX incorporation to compensate for the limited incorporation of AX, and regulation of the pro-inflammatory factor interleukin-1ß (IL-1ß) by AX. METHODS: Murine bone marrow-derived macrophages were stimulated by lipopolysaccharide (LPS). After 2 h, cells were treated with 10 µM AX and/or pulsed high-intensity US irradiation. The cells were then incubated for another 3 h and harvested. AX incorporation in cells was measured by absorbance, and the expression of IL-1ß was measured by qPCR. All values are expressed as means ± standard error of the mean. RESULTS: The combination of AX and US significantly increased AX incorporation in cells compared to AX alone (p < 0.05). In addition, this combination further suppressed the expression of IL-1ß compared to AX alone (p < 0.05). CONCLUSION: Pulsed high-intensity US irradiation combined with AX treatment promoted AX incorporation in cells and enhanced the anti-inflammatory effect on macrophages.

Preventive effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against endotoxin-induced muscle atrophy.

BACKGROUND & AIMS: Muscle atrophy is a public health issue and inflammation is a major cause of muscle atrophy. While docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which are typical ω-3 polyunsaturated fatty acids, are reported to have anti-inflammatory effects on endotoxin-induced inflammatory responses, their effects on inflammatory muscle atrophy have not been clarified. In this study, we aimed to investigate the effects of DHA and EPA on inflammatory muscle atrophy. METHODS: DHA or EPA was added to C2C12 myotubes at a concentration of 25, 50, or 100 µM, and 1 h later, lipopolysaccharide (LPS) was added at a concentration of 1 µg/mL. Two hours after the first LPS addition, mRNA expression of atrogin-1 and Murf-1 in C2C12 myotubes was measured. The second LPS addition was performed 24 h after the first LPS addition, and myotube diameter, myofibrillar protein, and cell viability were measured. One-way ANOVA and Tukey's multiple comparison test were used for statistical processing of the results, and the significance level was set to less than 5 %. RESULTS: The LPS-added group significantly decreased the myotube diameter and the myofibrillar protein content compared to the control group. The myotube diameter was significantly higher in the 25 µM, 50 µM DHA and 25 µM EPA-added groups compared to the LPS group. In the 25 µM DHA and EPA-added groups, the myofibrillar protein content was significantly higher than that in the LPS group. The mRNA expression levels of atrogin-1 and murf-1 were significantly suppressed in the 25 µM DHA and EPA-added groups compared to the LPS group. The cell viability did not change by the addition of LPS, DHA, and EPA. CONCLUSIONS: The addition of DHA or EPA suppressed the decrease in myotube diameter and myofibrillar protein content and suppressed the increase in atrogin-1 and murf-1 induced by LPS. This study showed the preventive effect of DHA and EPA on endotoxin-induced muscle atrophy.

Monophasic Pulsed Current Stimulation of Duty Cycle 10% Promotes Differentiation of Human Dermal Fibroblasts into Myofibroblasts.

OBJECTIVE: Many clinical trials have shown the therapeutic effects of electrical stimulation (ES) in various conditions. Our previous studies showed that ES (200 µA and 2 Hz) promotes migration and proliferation of human dermal fibroblasts (HDFs). However, the effective duty cycle and the effect of ES on myofibroblast differentiation are unclear. This study aimed to investigate the relationship between duty cycle and myofibroblast differentiation. METHODS: HDFs were subjected to ES (200 µA and 2 Hz) for 24 h with the duty cycle adapted at 0% (control), 10%, 50%, or 90%. α-smooth muscle actin (SMA) and transforming growth factor (TGF)-ß1 mRNA and α-SMA protein expressions were assessed. Collagen gel contraction was observed for 48 h after ES initiation and the gel area was measured. Cell viability and pH of culture medium were analyzed for cytotoxicity of the ES. RESULTS: Cell viabilities were decreased in the 50% and the 90% groups but ES did not influence on pH of culture media. ES with a duty cycle of 10% significantly promoted the mRNA expression of α-SMA and TGF-ß1. α-SMA protein expression in the 10% group was also significantly higher than that of the control group. Collagen gel subjected to ES with a duty cycle of 10% was contracted. CONCLUSION: Duty cycle can influence on myofibroblast differentiation and ES with a duty cycle 10% is the effective for wound healing.

Pulsed ultrasound prevents lipopolysaccharide-induced muscle atrophy through inhibiting p38 MAPK phosphorylation in C2C12 myotubes.

OBJECTIVE: Inflammation contributes to skeletal muscle atrophy via protein degradation induced by p38 mitogen-activated protein kinase (MAPK) phosphorylation. Meanwhile, pulsed ultrasound irradiation provides the mechanical stimulation to the target tissue, and has been reported to show anti-inflammatory effects. This study investigated the preventive effects of pulsed ultrasound irradiation on muscle atrophy induced by lipopolysaccharide (LPS) in C2C12 myotubes. METHODS: C2C12 myotubes were used in this research. The pulsed ultrasound (a frequency of 3 MHz, duty cycle of 20%, intensity of 0.5 W/cm2) was irradiated to myotube before LPS administration. RESULTS: The LPS increased phosphorylation of p38 MAPK and decreased the myofibril and myosin heavy chain protein (P < 0.05), followed by atrophy in C2C12 myotubes. The pulsed ultrasound irradiation attenuated p38 MAPK phosphorylation and myotube atrophy induced by LPS (P < 0.05). CONCLUSIONS: Pulsed ultrasound irradiation has the preventive effects on inflammation-induced muscle atrophy through inhibiting phosphorylation of p38 MAPK.

Migration speed of nucleolus precursor bodies in human male pronuclei: a novel parameter for predicting live birth.

PURPOSE: To study the relationship between the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN) and human embryo development during assisted reproduction. METHODS: The migration speed of 263 NPBs from 47 zygotes was quantitated, and embryonic development was observed until the blastocyst stage. The central coordinates of mPN, fPN, and NPBs were noted at multiple timepoints. Then, the distance traveled by the NPBs between two sequential images was measured, and migration speed was calculated. Additionally, we investigated the relationship between NPB migration speed and ploidy status (N = 33) or live birth/ongoing pregnancy (LB/OP) (N = 60) after assisted reproduction. RESULTS: The NPB migration speed in both mPN and fPN was significantly faster in the zygotes that developed into blastocysts (N = 25) than that in the zygotes that arrested (N = 22). The timing of blastulation was negatively correlated with NPB migration speed in the mPN. Faster NPB migration was significantly correlated with LB/OP. In multivariate logistic analysis, NPB migration speed in the mPN was the only morphokinetic parameter associated with LB/OP. In a receiver-operating characteristic curve analysis of LB/OP by the NPB migration speed in the mPN, the cut-off value was 4.56 µm/h. When this cut-off value was applied to blastocysts with preimplantation genetic testing for aneuploidy, 100% of the blastocysts faster than or equal to the cut-off value were euploid. CONCLUSION: The NPBs migrated faster in zygotes having the potential to develop into a blastocyst, and eventually into a baby. This predictor could be an attractive marker for non-invasive embryo selection.