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Background/aims: Pseudohypericin (P-HY) and its congener hypericin are the major hydroxylated phenanthroperylenediones present in Hypericum species. Our previous study indicated that hypericin was able to function as a sonosensitizer. The potential use of P-HY as a sonosensitizer for sonodynamic therapy (SDT) requires further exploration. Thus, this study aimed to investigate the effects of P-HY-SDT on THP-1 macrophages., Methods: THP-1 macrophages were incubated with P-HY, and cell viability was measured using a CCK-8 assay. Fluorescence microscopy assessed the intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm ) and mitochondrial permeability transition pore (mPTP) opening. Apoptotic and necrotic cell levels were measured by the flow cytometry analysis. Western blots were employed to assay BAX, Cytochrome C expression and apoptosis-related proteins., Results: P-HY-SDT induced THP-1 macrophage apoptosis. The levels of ROS were significantly increased in the SDT group, resulting in both mPTP opening and ΔΨm loss, which led to apoptosis. In addition, the translocation of BAX, release of Cytochrome C and the upregulated expression of apoptosis-related proteins after P-HY-SDT were observed, all of which were reversed by N-acetyl cysteine (NAC)., Conclusion: P-HY-SDT induced THP-1 macrophage apoptosis through the mitochondria-caspase pathway via ROS generation, the translocation of BAX and the release of Cytochrome C to regulate the mPTP opening., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Atherosclerosis (AS) is the most vital cardiovascular disease, which poses a great threat to human health. Macrophages play an important role in the progression of AS. Photodynamic therapy (PDT) has emerged as a useful therapeutic modality not only in the treatment of cancer but also in the treatment of AS. The purpose of this study was to determine the molecular mechanisms underlying the activity of PDT, using mesoporous-silica-coated upconversion fluorescent nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) in the induction of apoptosis in THP-1 macrophages. Here, we investigated the ability of UCNPs-Ce6-mediated PDT to induce THP-1 macrophage apoptosis by facilitating the induction of reactive oxygen species (ROS) and regulation of mitochondrial permeability transition pore (MPTP) to depolarize mitochondrial membrane potential (MMP). Both Bax translocation and the release of cytochrome C were examined using immunofluorescence and Western blotting. Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis. In addition, immunofluorescence and Western blotting revealed that PDT induced both Bax translocation and the release of cytochrome C, as well as upregulation of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase. Therefore, we demonstrated that UCNPs-Ce6-mediated PDT induces apoptosis in THP-1 macrophages via ROS bursts. The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release. This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

Purpose: To investigate the sonoactivity of hypericin (HY), together with its sonodynamic effect on THP-1 macrophages and the underlying mechanism., Materials and Methods: CCK-8 was used to examine cell viability. Confocal laser scanning microscopy was performed to assess the localization of HY in cells, reactive oxygen species (ROS) generation, and opening of the mitochondrial permeability transition pore (mPTP) after different treatments. Apoptosis was analyzed using Hoechst-propidium iodide and transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) collapse was detected via fluorescence microscopy. Lipoprotein oxidation was determined in malondialdehyde (MDA) assays. Western blotting was conducted to determine the translocation of BAX and cytochrome C and the expression of apoptosis-related proteins., Results: HY was sublocalized among the nuclei and the mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosome in the cytosol of THP-1 macrophages. Under low-intensity ultrasound irradiation, HY significantly decreased cell viability and induced apoptosis. Furthermore, greater ROS generation, higher MDA levels, and greater ΔΨm loss were observed in the sonodynamic therapy (SDT) group. Both ROS generation and MDA levels were significantly reduced by the ROS scavenger N-acetyl cysteine (NAC) and the singlet oxygen scavenger sodium azide. Most of the loss of ΔΨm was inhibited by pretreatment with NAC, sodium azide, and the mPTP inhibitor cyclosporin A (CsA). mPTP opening was induced upon SDT but was reduced by pretreatment with bongkrekic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium, CsA, and NAC. Western blot analyses revealed translocation of BAX and cytochrome C, downregulated expression of Bcl-2, and upregulated expression of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase in the SDT group, which were reversed by NAC., Conclusion: HY mediated SDT-induced apoptosis in THP-1 macrophages via ROS generation. Then, the proapoptotic factor BAX translocated from the cytosol to the mitochondria, increasing the ratio of BAX/Bcl-2, and the mPTP opened to release cytochrome C. This study demonstrated the great potential of HY-mediated SDT for treating atherosclerosis.

Background/Aims: Sonodynamic therapy (SDT) is considered a new approach for the treatment of atherosclerosis. We previously confirmed that hydroxyl acetylated curcumin (HAC) was a sonosensitizer. In this study, we investigated the mechanism of THP-1 macrophage apoptosis and autophagy induced by HAC mediated SDT (HAC-SDT). Methods: Cell viability was measured using a CCK-8 assay. Laser scanning confocal microscopy was used to measure the levels of intracellular reactive oxygen species (ROS), sub-cellular HAC localization, BAX and cytochrome C translocation, LC3 expression, monodansylcadaverine staining and Dillabeled oxidized low density lipoprotein (Dil-ox-LDL) uptake. Flow cytometry was used to analyze apoptosis and autophagy via Annexin V/propidium iodide and acridine orange staining, respectively. The expression levels of apoptosis- and autophagy-related proteins were detected by Western blot. Oil red O was used to measure intracellular lipid accumulation. Results: We identified HAC (5.0 μg/mL) located in lysosomes, endoplasmic reticulum, Golgi apparatus and mitochondria after 4 h of incubation. Compared with other sonosensitizers (e.g., curcumin and emodin), HAC had a more obvious sonodynamic effect on macrophages. Furthermore, the mitochondrial-caspase pathway was confirmed to play a crucial role in the HAC-SDT-induced apoptosis; BAX translocated from the cytosol to the mitochondria during HAC-SDT. Subsequently, mitochondrial cytochrome C was released into the cytosol, activating the caspase cascade in a time-dependent manner. Furthermore, HAC-SDT could induce PI3K/ AKT/mTOR pathway dependent autophagy, accompanied by a decrease in the lipid uptake of THP-1 macrophages. This mechanism was demonstrated by the formation of acidic vesicular organelles, the conversion of LC3 I to LC3 II, the expression of related proteins, and the attenuation of both Dil-ox-LDL and oil red O staining. Moreover, pre-treatment with the autophagy inhibitor 3-methyladenine enhanced the HAC-SDT-induced apoptosis. Additionally, HAC-SDT-induced autophagy and apoptosis were both blocked by ROS scavenger N-acetyll-cysteine. Conclusion: The results suggested that autophagy not only played an inhibitory role in the process of apoptosis but also could effectively attenuate lipid aggregation in THP-1 macrophages during HAC-SDT. As important intracellular mediators, the ROS generated by HAC-SDT also played a crucial role in initiating apoptosis and autophagy. [ABSTRACT FROM AUTHOR]