Hematoporphyrin monomethyl ether pretreatment could substantially enhance the growth inhibition of ultrasound on osteosarcoma, which suggests that HMME is an efficient sonosensitizer, and ultrasound radiation with HMME could be developed as a new modality for treating osteosarcoma.

Objective: The present study aims to investigate the possible mechanisms of hematoporphyrin monomethyl ether (HMME) enhancing the cytotoxicity of ultrasound in osteosarcoma cells. Methods: Osteosarcoma cell line UMR-106 was treated by HMME and ultrasound radiation, with the HMME concentration kept at 20 mg/mL and ultrasound radiation for 10 seconds at the intensity of 0.5 W/cm 2 . Cell proliferation was investigated at 12, 24, 36, and 48 hours using MTT assay after ultrasound and HMME treatment. Ultrastructural morphology was observed using transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) was measured using a flow cytometry with DCFH-DA staining and intracellular free calcium ion (Ca 2+ ) with Fluo-3-AM staining. Results: The UMR-106 cells proliferated rapidly in the sham radiation and HMME treatment alone group, but ultrasound-treated cells and HMME-ultrasound-treated cells proliferated slowly. There was a significant difference between HMME-ultrasound treatment and the controls, including ultrasound radiation, HMME treatment alone, and sham radiation (P < .05). TEM showed endoplasmic reticulum and mitochondrial swelling in the ultrasound-treated cells, and more cells presented apoptosis and necrosis after treatment with ultrasound and HMME together. Intracellular ROS and Ca 2+ in the cells increased more significantly after both ultrasound and HMME treatment than after ultrasound treatment alone. Conclusions: HMME could effectively enhance the inhibition effect of ultrasound on osteosarcoma cells. Intracellular ROS and Ca 2+ in the UMR-106 cells increased more significantly after the treatment of HMME and ultrasound together, indicating that the enhancement of HMME on ultrasound cytotoxicity to osteosarcoma cells possibly involves both intracellular ROS and Ca 2+ elevation.

Following traumatic insult and associated pathogen infection, innate immunity is activated during the perioperative period, especially the NLRP3 inflammasome in macrophages. The neuroendocrine response is also rapidly activated to regulate excessive inflammation; however, the molecular mechanisms are still not completely clear. This study is aimed at investigating the modulation of NLRP3 inflammasome priming by endogenous glucocorticoids (corticosterone, CORT) and its relationship with xanthine oxidase (XO). RAW264.7 murine macrophages were stimulated with LPS (1 μg/ml). LPS-induced NLRP3 expression was pretreated by CORT at different concentrations (0-900 ng/ml). Then, the effect of higher concentrations of CORT (700 ng/ml) on LPS-induced NLRP3 expression and the effect of allopurinol (250 μg/ml) were observed. Finally, the effects of a CORT antagonist (RU486) on XO expression and activity and NLRP3 expression in macrophages were further analyzed. Supernatant levels IL-1β and IL-18 were measured. The results showed that LPS-induced NLRP3 expression was upregulated further by pretreatment with CORT (300 ng/ml) (P<0.05); however, higher concentrations of CORT (greater than 700 ng/ml) downregulated NLRP3 expression (P<0.01) and the expression and activity of XO (P<0.05 and P<0.01, respectively). Allopurinol significantly inhibited NLRP3 expression. However, XO expression and activity, NLRP3 expression, and supernatant IL-1β and IL-18 levels were significantly increased in the RU486 group compared with the CORT group. In conclusion, our results suggested that CORT inhibits LPS-induced NLRP3 inflammasome priming in macrophages. The underlying mechanism is related to the modulation of XO expression and activity, which may be involved in priming and activating the NLRP3 inflammasome.

High-intensity focused ultrasound (HIFU) is a representative non-invasive method of cancer therapy, but its low therapeutic efficacy and risk of damage to surrounding normal tissue hinder its further clinical development and application. Sonodynamic therapy (SDT) kills tumor cells through reactive oxygen molecules produced by sonosensitizers during ultrasound treatment. SDT can enhance HIFU efficacy like microbubbles. In this work, we developed nanoscale N2O microbubbles (N2O-mbs) by an improved mechanical oscillation method. These microbubbles showed good biocompatibility and tumor cell binding. The sonosensitivity of the N2O-mbs was detected both extracellularly and intracellularly through the detection of reactive oxygen species generation. The toxic effects of these sonodynamic microbubbles on tumor cells and the synergistic effect on HIFU treatment were evaluated. Significant apoptosis was caused by reactive oxygen species produced by N2O-mbs under ultrasound irradiation. N2O-mbs combined with HIFU increased tumor cell necrosis and apoptosis in vitro and the coagulative necrotic volume and echo intensity in the bovine liver target area ex vivo. These sonodynamic microbubbles have been also demonstrated to efficiently inhibit tumor growth in vivo. N2O-mbs have a significant impact on the treatment and ablation effect of HIFU due to the advantages of microbubble and extraordinary sonosensitivity. This finding suggests that N2O-mbs may be a novel auxiliary agent for ultrasound that can be used to promote HIFU tumor thermal ablation.

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The arachidonic acid pathway metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemia/reperfusion brain injury. Inhibition of 20-HETE formation can protect the developing brain from global ischemia. In previous studies, we have found that treatment with the 20-HETE synthesis inhibitor N-hydroxy-N-4-butyl-2-methylphenylformamidine (HET0016) can protect the immature brain from traumatic brain injury (TBI), but its hydrophobic nature limits its full potential. We designed a reactive oxygen species-responsive HET0016 prodrug, which consists of a thioketal link between HET0016 and stearyl alcohol (HET-TK-SA), and used the nanoprodrug strategy to successfully synthesize liposomes HET0016 prodrug liposomes (HPLs) to facilitate the application of HET0016 in protection from TBI. HPLs demonstrated spherical shape, size of about 127.8 nm, a zeta potential of −28.8 mv, a narrow particle size distribution and good stability. Male rats at postnatal day 16–17 underwent controlled cortical impact (CCI) followed by intravenous injection with vehicle or HET0016 (1 mg/kg, 2 h post-injury, once/day for 3 days). The results of the in vivo demonstrated that HPLs has good biosafety and can pass through the blood-brain barrier. Not only that compared with HET0016, HPLs better-inhibited inflammation and improved neuronal degeneration, which further led to lesion volume reduction, upgraded behavioral task performance, and ameliorated the degree of TBI impairment. Our results demonstrated HPLs could be a new strategy for juvenile TBI therapy.