title: Combination of Hydroxyl Acetylated Curcumin and Ultrasound Induces Macrophage Autophagy with Anti-Apoptotic and Anti-Lipid Aggregation Effects.
abstract: OBJECTIVE
  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 Dil-labeled 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-acetyl-l-cysteine.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathophysiology, Key Laboratory of Cardiovascular Pathophysiology, Harbin Medical University, Harbin, China.
authors.forename: Longbin
authors.initials: L
authors.lastname: Zheng
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Xuesong
authors.initials: X
authors.lastname: Li
authors.forename: Jiayuan
authors.initials: J
authors.lastname: Kou
authors.forename: Zhaoyu
authors.initials: Z
authors.lastname: Zhong
authors.forename: Yueqing
authors.initials: Y
authors.lastname: Jiang
authors.forename: Zhongni
authors.initials: Z
authors.lastname: Liu
authors.forename: Ye
authors.initials: Y
authors.lastname: Tian
authors.forename: Liming
authors.initials: L
authors.lastname: Yang
mesh_terms: D000107:Acetylation; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics / Q000528:radiation effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D003474:Curcumin / Q000031:analogs & derivatives / Q000494:pharmacology*; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects / Q000648:ultrastructure; D015870:Gene Expression; D006056:Golgi Apparatus / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects / Q000648:ultrastructure; D006801:Humans; D006900:Hydroxylation; D008055:Lipids / Q000737:chemistry; D008077:Lipoproteins, LDL / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects / Q000648:ultrastructure; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects / Q000648:ultrastructure; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D000069453:Ultrasonic Waves; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D003474:Curcumin; D045304:Cytochromes c; D008055:Lipids; D008077:Lipoproteins, LDL; D008869:Microtubule-Associated Proteins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D017382:Reactive Oxygen Species; D058570:TOR Serine-Threonine Kinases; D051028:bcl-2-Associated X Protein; C494826:BAX protein, human; C477012:MAP1LC3A protein, human; C099252:oxidized low density lipoprotein; C546842:MTOR protein, human