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.

Sodium-glucose cotransporter 2 inhibitor-associated perioperative ketoacidosis: a systematic review of case reports.

Although the recommended preoperative cessation period for sodium-glucose cotransporter 2 inhibitors (SGLT2is) changed in 2020 (from 24 h to 3-4 days preoperatively) to reduce the risk of SGLT2i-associated perioperative ketoacidosis (SAPKA), the validity of the new recommendation has not been verified. Using case reports, we assessed the new recommendation effectiveness and extrapolated precipitating factors for SAPKA. We searched electronic databases up to June 1, 2022 to assess SAPKA (blood pH < 7.3 and blood or urine ketone positivity within 30 days postoperatively in patients taking SGLT2i). We included 76 publications with 99 cases. The preoperative SGLT2i cessation duration was reported for 59 patients (59.6%). In all cases with available cessation periods, the SGLT2is were interrupted < 3 days preoperatively. No SAPKA cases with > 2-day preoperative cessation periods were found. Many case reports lack important information for estimating precipitating factors, including preoperative SGLT2i cessation period, body mass index, baseline hemoglobin A1c level, details of perioperative fluid management, and type of anesthesia. Our study suggested that preoperative SGLT2i cessation for at least 3 days could prevent SAPKA. Large prospective epidemiologic studies are needed to identify risk factors for SAPKA.

Bleeding After Central Venous Catheter Placement in a Patient With Undiagnosed Acquired Hemophilia A: A Case Report.

Acquired hemophilia A is a rare condition caused by autoantibodies against endogenous coagulation factor VIII, which results in spontaneous bleeding. Workup of a patient with difficult hemostasis after removing and placing a central venous catheter led to the diagnosis of acquired hemophilia A. A 64-year-old man was transferred with an intramuscular right thigh mass. Initial biopsy at an outside facility showed degenerated muscle and coagula and he was transferred for incisional biopsy and definitive treatment. The patient had difficult venous access, and a right internal jugular venous catheter was placed. The catheter insertion site showed slow continuous bleeding. Achieving adequate hemostasis after removing the catheter was difficult, and a hematoma formed after the placement of an infraclavicular axillary venous catheter under ultrasound guidance. Coagulation studies revealed a prolonged activated partial thromboplastin time at 96 seconds. The patient was then diagnosed with acquired hemophilia A by enzyme-linked immunosorbent assay using anti-factor VIII antibodies. Even if ultrasound-guided central venous catheterization is performed carefully, bleeding may occur in some patients, suggesting the possibility of coagulopathy. Decision-making for performing central venous catheterization requires extensive knowledge of coagulopathies to understand the causes of bleeding complications.

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.

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.