Journal: Molecules

Article Title: Cationic Vitamin E-TPGS Mixed Micelles of Berberine to Neutralize Doxorubicin-Induced Cardiotoxicity via Amelioration of Mitochondrial Dysfunction and Impeding Apoptosis

doi: 10.3390/molecules29051155

Figure Lengend Snippet: Concentration-dependent recovery of treated rat cardiomyocyte (H9C2, left panels) and mouse aortic medial smooth muscle (A10, right panels) cell models, post-elevated DOX exposure. Microplate-based CellTiter Glo ® cytotoxicity assay in cultured H9C2 ( A ) and A10 ( B ) cells, which were treated with 1.0 µg/mL of Brb-Sol, and equivalent concentrations of empty and Brb-loaded micellar formulations, immediately co-exposed for 8 h to acute DOX dose of 5 µM, 15 µM, and 30 µM, to assess mainly DOX-induced apoptotic cell death; overall cell viability profile assessed after 24 h via Cytotox-ONE cell membrane integrity assay of cultured H9C2 ( C ) and A10 ( D ) cells, which were treated with 1.0 µg/mL of Brb-Sol, and equivalent concentrations of empty and Brb-loaded micellar formulations, immediately co-exposed to extended DOX challenge of doses of 0-to-96 µM, all incubated at 37 °C, 5% CO 2 conditions. ( n = 4–5, mean ± SE, values denoted with unlike symbols (*, †, ‡, #) are statistically different, p ≤ 0.05).

Article Snippet: Both 6- and 12-well tissue culture-treated microplate plates and black 96-well tissue culture plates were purchased from BD-biosciences (San Jose, CA, USA).

Techniques: Concentration Assay, Cytotoxicity Assay, Cell Culture, Membrane, Integrity Assay, Incubation

Journal: Molecules

Article Title: Cationic Vitamin E-TPGS Mixed Micelles of Berberine to Neutralize Doxorubicin-Induced Cardiotoxicity via Amelioration of Mitochondrial Dysfunction and Impeding Apoptosis

doi: 10.3390/molecules29051155

Figure Lengend Snippet: Evaluation of mitochondrial protection against DOX-induced cardiovascular cell apoptosis after micellar berberine treatments. Microplate-based MitoOrange Dye fluorescence assay using cultured H9C2 ( A ) and A10 ( B ) cells, which were exposed to acute DOX challenge at two dose levels of 30 µM and 60 µM, co-treated with 1.0 µg/mL of Brb-Sol, and equivalent concentrations of empty and Brb-loaded micellar formulations, and then fluorescence intensity was measured after 4 h; cellular apoptosis assayed using Annexin V-FITC (panels ( C – I )), following 6 h co-incubation with 30 µM DOX challenge, along with the corresponding 1.0 µg/mL of Brb-treatments (as free Sol, admixtured or encapsulated with OA/TPGS mMic) and equivalent empty OA/TPGS mMic control. Apoptotic cells were detected by flow cytometry. The mean fluorescence intensity of Annexin V/PI staining in both H9C2 myocytes and A10 SMCs was calculated ( C ), following quantitative FACS analysis, as demonstrated in H9C2 myocytes (panels ( D – I )). All cells were cultured in standard 37 °C, 5% CO 2 conditions. ( n = 4, mean ± SD values denoted with unlike symbols (*, #, †, ‡, ¶) are statistically different, p ≤ 0.05).

Article Snippet: Both 6- and 12-well tissue culture-treated microplate plates and black 96-well tissue culture plates were purchased from BD-biosciences (San Jose, CA, USA).

Techniques: Fluorescence, Cell Culture, Incubation, Flow Cytometry, Staining

Journal: Molecules

Article Title: Cationic Vitamin E-TPGS Mixed Micelles of Berberine to Neutralize Doxorubicin-Induced Cardiotoxicity via Amelioration of Mitochondrial Dysfunction and Impeding Apoptosis

doi: 10.3390/molecules29051155

Figure Lengend Snippet: Mitohormetic effects of Brb-mMic activate mitochondrial stress protection against DOX-induced mitochondrial apoptosis in model cardiac cells. Colorimetric microplate Caspase-8 and Caspase-9 activation assays in model H9C2 myocytes ( A ) and A10 SMCs ( B ), where cardiac cells were exposed to acute DOX dose of 15 µM and co-treated with 1.0 µg/mL of Brb-Sol and equivalent concentrations of empty and Brb-loaded micellar formulations, for 4 h at 37 °C in 5% CO 2 conditions. After washing treated cells, they were normalized by protein content, then incubated with either Caspase-8 or Caspase-9 substrate solution, in reaction buffers according to manufacturer’s instructions; the sample color absorbance values were measured and analyzed relative (%) to the corresponding untreated cell control; Western blot analysis for the relative accumulation of proapoptotic Bax protein and antiapoptotic Bcl-2 protein markers of mitochondrial membrane integrity in rat H9C2 cardiomyocytes treated with acute 15 µM DOX challenge, along with Brb-loaded mMic and blank mMic vehicle control, or un-encapsulated drug controls (either free Brb-solution or a simple mix of Brb-solution + empty mMic), at concentration equivalent to 1.0 µg/mL of Brb for 2 h, respectively. A total of 20 µg of protein extracts was loaded per well for H9C2 cells, and β-actin served as a loading control. Nitrocellulose membrane blots were immunoblotted with either anti-BAX or anti-Bcl-2 monoclonal antibodies, as well as an anti-β-actin monoclonal antibody, and finally visualized ( C ), followed by quantification of intensities of the protein bands using ImageJ Gel analysis (ver. 1.53e), comparing Bcl-2 vs. Bax activation ( D ). ( n = 3–4, mean ± SD values denoted with unlike symbols (*, †, ‡, #) are statistically different, p ≤ 0.05).

Article Snippet: Both 6- and 12-well tissue culture-treated microplate plates and black 96-well tissue culture plates were purchased from BD-biosciences (San Jose, CA, USA).

Techniques: Activation Assay, Incubation, Western Blot, Membrane, Concentration Assay