Journal: Asian Spine Journal

Article Title: Isolation and Characterization of Extracellular Vesicle from Mesenchymal Stem Cells of the Epidural Fat of the Spine

doi: 10.31616/asj.2021.0129

Figure Lengend Snippet: Human epidural fat-derived mesenchymal stem cell (EF-MSC) exosomes isolated using tangential flow filtration system. (A) Representative image of exosomes from EF-MSCs visualized using transmission electron microscopy, size bar=200 nm. (B) Nanoparticle tracking analysis profile of human EF-MSCs exosomes showing size distribution. Isolated particles had an average size of approximately 142.8 nm and the concentration of particles was 1.27×10 10 particles/mL. (C, D) Flow cytometry analysis of exosomes isolated from human EF-MSCs. Exosomes were labeled using the tetraspanins, CD63 (upper panel) and CD81 (below panel), which exhibited 63.4% and 98.4% expression, respectively. (E) Western blots stained against tetraspanins (CD9 and CD81), exosome fusion protein (Flotillin2), and MVB biogenesis protein (TSG101). The positive expression of the cell markers, calnexin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in human EF-MSCs was detected using western blotting.

Article Snippet: The cells were then incubated overnight with primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, NB100-56875; Novus Biologicals), calnexin (NB100-1965; Novus Biologicals), CD9 (NBP1-28363; Novus Biologicals), CD81 (NBP1-44861; Novus Biologicals), flotillin 2 (NBP1-30881; Novus Biologicals), and TSG101 (NB200-112; Novus Biologicals) at 4°C.

Techniques: Derivative Assay, Isolation, Filtration, Transmission Assay, Electron Microscopy, Concentration Assay, Flow Cytometry, Labeling, Expressing, Western Blot, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Atractylenolide-I covalently binds to CYP11B2, selectively inhibits aldosterone synthesis, and improves hyperaldosteronism

doi: 10.1016/j.apsb.2021.09.013

Figure Lengend Snippet: AT-I inhibits the production of ALD in NCI-H295R cells by interacting with CYP11B2. (A) The chemical structure of AT-I, AT-II, and AT-III. (B)‒(E) Inhibitory effect of AT-I and its derivatives on Prog, Cort, Cor, and ALD production. NCI-H295R cells were treated with different concentrations of AT-I and its derivatives (AT-II and AT-III) for 24 h, then Prog, Cort, Cor, and ALD were measured ( n = 4). (F) AT-I/II/III treatment (10 μmol/L) influences the thermal stability of CYP11B2 as assayed by CETSA ( n = 3). (G) AT-I treatment reduces the thermal stability of CYP11B2 in a dose-dependent manner at 55 °C ( n = 3). (H) Evaluation of ALD-inhibiting effects and cell viability of AT-I treatment in NCI-H295R cells ( n = 4). Data are expressed as means ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. Con group that was not drug-treated.

Article Snippet: NCI-H295R cells were transfected with CYP11B2 siRNA or control negative siRNA (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) for 48 h. The transfected cells were used to evaluate the production of ALD.

Techniques:

Journal: Acta Pharmaceutica Sinica. B

Article Title: Atractylenolide-I covalently binds to CYP11B2, selectively inhibits aldosterone synthesis, and improves hyperaldosteronism

doi: 10.1016/j.apsb.2021.09.013

Figure Lengend Snippet: AAT-I molecular probes and siRNA interference test reveal that CYP11B2 is the target of AT-I. (A) The chemical structure of the AAT-I probe and it derivative coumarin-AAT-I fluorescent tracer and AAT-I bead formed by clicking reaction. (B) The ALD release assay with AAT-I probe or AT-I treatment in NCI-H295R cells ( n = 4). (C) Fishing and identifying the potential target protein CYP11B2. Coomassie brilliant blue stain (CBS, left panel) and Western blot analysis (right panel) were used to detect the protein CYP11B2 enriched by AAT-I beads. The marker shows the molecular weight. (D) The co-localization imaging of CYP11B2 protein and AAT-I in NCI-H295R cells with fluorescent confocal microscopy. The AAT-I probe took on a pseudo-green color via the click reaction, and the pseudo-red color represents CYP11B2, which was stained by Alexa Fluor® 594. The yellow merger represents CYP11B2 colocalized with AAT-I. Cells were treated with 1 or 10 μmol/L AT-I, 1 μmol/L AAT-I for 6 h. Scale bar: 7.5 μm. (E) The prevention of CYP11B2 protein expression using a specific CYP11B2 siRNA. Western blot analysis was performed after transfection of NCI-H295R cells with CYP11B2 siRNA for 48 h ( n = 3). (F) ALD release assay of AT-I treatment in NCI-H295R cells, which was induced by Ang II in the absence or presence of CYP11B2 siRNA-transfection ( n = 4). All data are expressed as means ± SD. ### P < 0.001 vs. Con; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. Mod; N.S., no statistical difference.

Article Snippet: NCI-H295R cells were transfected with CYP11B2 siRNA or control negative siRNA (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) for 48 h. The transfected cells were used to evaluate the production of ALD.

Techniques: Release Assay, Staining, Western Blot, Marker, Molecular Weight, Imaging, Confocal Microscopy, Expressing, Transfection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Atractylenolide-I covalently binds to CYP11B2, selectively inhibits aldosterone synthesis, and improves hyperaldosteronism

doi: 10.1016/j.apsb.2021.09.013

Figure Lengend Snippet: AT-I covalently binds with CYP11B2 via an epoxidation nucleophilic addition. (A) UPLC/Q-TOF-MS analysis of the incubation product of AT-I and GSH in NCI-H295R cells. NCI-H295R cells were treated with 1 mmol/L GSH or EDTA, 50 μmol/L AT-I for 24 h, and then cells were harvested for LC‒MS analysis. (B) UPLC/Q-TOF-MS identification of the incubation product of AT-I and CYP11B2 or CYP11B1. (C) In-gel imaging assay of CYP11B2 or CYP11B1. (D) Irreversible binding of AAT-I to CYP11B2. In the co-incubation AT-I group, the recombinant CYP11B2 protein was incubated for 12 h with AAT-I in the absence or presence of AT-I for competitive binding. In the post-incubation group, the recombinant CYP11B2 protein was preincubated with AAT-I for 12 h and then incubated with or without AT-I for 12 h for competitive binding.

Article Snippet: NCI-H295R cells were transfected with CYP11B2 siRNA or control negative siRNA (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) for 48 h. The transfected cells were used to evaluate the production of ALD.

Techniques: Incubation, Imaging, Binding Assay, Recombinant

Journal: Acta Pharmaceutica Sinica. B

Article Title: Atractylenolide-I covalently binds to CYP11B2, selectively inhibits aldosterone synthesis, and improves hyperaldosteronism

doi: 10.1016/j.apsb.2021.09.013

Figure Lengend Snippet: The C8/C9 double bond is considered to be the key pharmacophore for AT-I selective binding to CYP11B2. (A) The in-gel fluorescence competition assay of AT-I and its derivatives. Recombinant CYP11B2 proteins were incubated with AAT-I (100 μmol/L) in the absence or presence of AT-I, AT-II, or AT-III at 4 °C for 12 h, and the products were resolved by the SDS-PAGE for the in-gel imaging assay (green). (B) and (C) The CO difference spectroscopy assay of CYP11B2 or CYP11B1 for AT-I and its derivatives. Recombinant CYP11B2 or CYP11B1 protein (0.1 mg/mL) was incubated with AT-I, AT-II, and AT-III (10 μmol/L) at 4 °C for 12 h, and the products were reduced with sodium dithionite and complexed with CO. The reduced CO complex was scanned between 400 and 780 nm. (D) The FTS assay for CYP11B1 protein against AT-I, AT-II, or AT-III.

Article Snippet: NCI-H295R cells were transfected with CYP11B2 siRNA or control negative siRNA (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) for 48 h. The transfected cells were used to evaluate the production of ALD.

Techniques: Binding Assay, Fluorescence, Competitive Binding Assay, Recombinant, Incubation, SDS Page, Imaging, Spectroscopy

Journal: Acta Pharmaceutica Sinica. B

Article Title: Atractylenolide-I covalently binds to CYP11B2, selectively inhibits aldosterone synthesis, and improves hyperaldosteronism

doi: 10.1016/j.apsb.2021.09.013

Figure Lengend Snippet: AT-I selectively binds with CYP11B2 relying both on Cys450 and Ala320. (A) A representative global view of AT-I with the CYP11B2 protein. Molecular modeling the 8,9 epoxy AT-I covalently bound to the Cys450 of CYP11B2. CYP11B2 protein is shown by the sky-blue ribbon model, heme is shown with the green stick model, and AT-I is shown by the cyan stick model. (B) A sequence comparison of CYP11B2 from different species and different isoforms. (C) In-gel fluorescence imaging assay for CYP11B2 (WT) protein and its mutants (A320V and C450G). (D) and (E) SPR analysis of AT-I binding to CYP11B2 or A320V. (F) The thermal shift assay of CYP11B2 or C450G protein incubated with the AT-I. (G) The CO difference spectra of CYP11B2 with AT-I.

Article Snippet: NCI-H295R cells were transfected with CYP11B2 siRNA or control negative siRNA (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) for 48 h. The transfected cells were used to evaluate the production of ALD.

Techniques: Sequencing, Comparison, Fluorescence, Imaging, Binding Assay, Thermal Shift Assay, Incubation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Atractylenolide-I covalently binds to CYP11B2, selectively inhibits aldosterone synthesis, and improves hyperaldosteronism

doi: 10.1016/j.apsb.2021.09.013

Figure Lengend Snippet: AT-I alleviated Ang II-induced hyperaldosteronism in mice. (A) The in-gel fluorescence imaging and target fishing assay for potential targets in adrenal tissue of mice. (B) The co-localization imaging of CYP11B2 and AAT-I in adrenal tissue of mice with fluorescence confocal microscopy. The pseudo-red color represents CYP11B2, the pseudo-green color represents AAT-I and the yellow merger represents CYP11B2 colocalized with AAT-I. Scale bar: 100 μm. (C)‒(H) The content of Na + , K + , and Cl − in the serum and urine analyzed by an ion detection kit. (I)‒(K) The content of ALD, Cort, and estradiol in the serum was analyzed with an ELISA kit. The mice in the negative control group were fed without treatment (Con). (L)‒(N) The HR, SBP, and DBP of mice with aldosteronism after treatment. Hyperaldosteronism was induced in mice by Ang II (Mod) and treated with Spi (i.p., 20 mg/kg/day) or AT-I (i.p., 10, 20, 40 mg/kg/day) for one week. Data are expressed as means ± SD ( n = 10). ### P < 0.001 vs. Con; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. Mod.

Article Snippet: NCI-H295R cells were transfected with CYP11B2 siRNA or control negative siRNA (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) for 48 h. The transfected cells were used to evaluate the production of ALD.

Techniques: Fluorescence, Imaging, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Negative Control