Cytotoxicity of arenobufagin (Arg) on HeLa cells.

<p>(<b>A</b>) Chemical structure of arenobufagin. (<b>B</b>) Cell viability of HeLa cells treated with arenobufagin at different doses for 24 h, 48 h and 72 h. (<b>C</b>) Representative DNA histograms of HeLa cells obtained by flow cytometry analysis. Accumulation in G₂/M phase was observed in HeLa cells treated with 100 nM arenobufagin for 24 h or 10 nM, 100 nM arenobufagin for 48 h. (<b>D</b>) Morphological change induced by 10 nM or 100 nM arenobufagin in HeLa cells—for 48 h (scale bar = 10 μm). Typical change of apoptotic morphology in arenobufagin-treated cells was observed. (<b>E</b>) Representative flow cytometry result of plasma membrane potential by rhodamine staining assay. Treated with arenobufagin for 3 h, HeLa cells with the potential of disrupted plasma membrane were count. (<b>F</b>) Quantitation in percentage of HeLa cells with the potential of disrupted plasma membrane. Data were statistical results of three independent experiments. Data were expressed as mean ± SD. *Significant difference from the control group at P<0.05.</p

The results of differentially-expressed proteins identified using MALDI-TOF MS/MS.

<p>The results of differentially-expressed proteins identified using MALDI-TOF MS/MS.</p

The molecular structures of Na, K-ATPase α₁β₁γ E2P-arenobufagin complex and the detailed binding mode were shown by computational molecular docking.

<p>(<b>A)</b> The molecular structure of Na, K-ATPase α₁β₁γ E2P and the binding site of arenobufagin in the Na, K-ATPase α1β1γ E2P. (<b>B)</b> The molecular structure of arenobufagin. (<b>C)</b> The binding site of arenobufagin visualized from alpha M2 and M4. (<b>D)</b> The binding site of arenobufagin visualized from the top of alpha M1-6 and the interaction of arenobufagin and the residues of alpha M1-6. Hydrogen bonds were displayed as green dashed lines and amino acid residues interacted with arenobufagin were also shown.</p

The KEGG pathways related to possible target proteins of arenobufagin (Arg) found in proteomic study.

<p>Grey dots were target-related proteins. Full names of these proteins were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159034#pone.0159034.t002" target="_blank">Table 2</a>. The main pathways were shown in red while the branched pathways were shown in blue.</p

Ataxin-1 and translationally-controlled tumor protein might be intermediate proteins between Na, K-ATPase and proteasome.

<p>(<b>A</b>) Protein-protein interaction network constructed to connect α1, α2 or α3 subunits of Na,K-ATPase and proteasomal-related proteins found in the proteomic study. The red dots were α1, α2 or α3 subunits of Na,K-ATPase and the yellow dots were 5 proteasomal-related proteins found in the proteomic study. The intermediate partner proteins except ataxin-1 and translationally-controlled tumor protein were shown in black. Ataxin-1 and translationally-controlled tumor protein (shown in blue) were considered as the most important intermediate partners between Na,K-ATPase and proteasome. (<b>B</b>) The protein expression level of ataxin-1 in control HeLa cells or cells treated with arenobufagin at different doses for 24 h. (<b>C</b>) The protein expression level of translationally-controlled tumor protein in control HeLa cells or cells treated with arenobufagin at different doses for 24 h. Data were statistical results of three independent experiments. Data were expressed as mean ± SD. *Significant difference from the control group at P<0.05.</p

Cellular proteasomal activity was inhibited by arenobufagin, which might be related to the binding of Na, K-ATPase.

<p><b>(A</b>) Inhibition of arenobufagin (Arg) at different doses on cytosolic proteasomal activity for 24h. (<b>B</b>) Three types of cellular proteasome enzyme activities (C-L, T-L and CT-L) in HeLa cells treated with 0.1% DMSO or arenobufagin(Arg) at different concentrations for 24h. (<b>C</b>) Western blotting results of WEE1 and actin in control HeLa cells and cells treated with 10 nM arenobufagin for 24 h and 48 h. Each blot was the representative result of three independent experiments. (<b>D</b>) Inhibition of antibodies against α1 or α3 subunits of Na,K-ATPase or combination of antibodies against α1 or α3 subunits of Na,K-ATPase and 10 nM arenobufagin on cytosolic proteasomal activity for 24h,48h and 72h, respectively. Data were statistical results of three independent experiments. Data were statistical results of three independent experiments. Data were expressed as mean ± SD. *Significant difference from the control group at P<0.05.</p

Summary of differentially-expressed proteins in arenobufagin-treated HeLa cells compared with control.

<p>Summary of differentially-expressed proteins in arenobufagin-treated HeLa cells compared with control.</p

Effect of arenobufagin (Arg) on Na, K-ATPase activity, intracellular Ca(2+) level and ROS level.

<p>(<b>A</b>) Quantitation of Na, K-ATPase activity in HeLa cells treated with arenobufagin(Arg) at different doses for 24h. (B) Western blotting results of Na, K-ATPase α1 and α3 in HeLa cells treated with arenobufagin(Arg) at different doses for 24 h. (<b>C</b>) Quantitation of the intracellular Ca(2+) level in HeLa cells treated with 10 nM arenobufagin for different time periods. (<b>D</b>) Quantitation of the intracellular ROS level in HeLa cells treated with 10 nM arenobufagin for different time periods. (<b>E</b>) Quantitation of the intracellular ROS level in HeLa cells treated with arenobufagin at different doses for 3h. (F) The viability of HeLa cells treated with 10 nM arenobufagin for 72 h in the presence of ROS scavenger NAC (N-acetyl cysteine). Data were statistical results of three independent experiments. Data were expressed as mean ± SD. *Significant difference from the control group at P<0.05.</p

Discriminatory Components Retracing Strategy for Monitoring the Preparation Procedure of Chinese Patent Medicines by Fingerprint and Chemometric Analysis

<div><p>Chinese patent medicines (CPM), generally prepared from several traditional Chinese medicines (TCMs) in accordance with specific process, are the typical delivery form of TCMs in Asia. To date, quality control of CPMs has typically focused on the evaluation of the final products using fingerprint technique and multi-components quantification, but rarely on monitoring the whole preparation process, which was considered to be more important to ensure the quality of CPMs. In this study, a novel and effective strategy labeling “retracing” way based on HPLC fingerprint and chemometric analysis was proposed with Shenkang injection (SKI) serving as an example to achieve the quality control of the whole preparation process. The chemical fingerprints were established initially and then analyzed by similarity, principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) to evaluate the quality and to explore discriminatory components. As a result, the holistic inconsistencies of ninety-three batches of SKIs were identified and five discriminatory components including emodic acid, gallic acid, caffeic acid, chrysophanol-<i>O</i>-glucoside, and <i>p</i>-coumaroyl-<i>O</i>-galloyl-glucose were labeled as the representative targets to explain the retracing strategy. Through analysis of the targets variation in the corresponding semi-products (ninety-three batches), intermediates (thirty-three batches), and the raw materials, successively, the origins of the discriminatory components were determined and some crucial influencing factors were proposed including the raw materials, the coextraction temperature, the sterilizing conditions, and so on. Meanwhile, a reference fingerprint was established and subsequently applied to the guidance of manufacturing. It was suggested that the production process should be standardized by taking the concentration of the discriminatory components as the diagnostic marker to ensure the stable and consistent quality for multi-batches of products. It is believed that the effective and practical strategy would play a critical role in the guidance of manufacturing and help improve the safety of the final products.</p></div

Workflow of the proposed strategy for monitoring the preparation parameters.

<p>RRR, SMRR, AR, and CF are Radix et Rhizoma Rhei, Radix et Rhizoma Salviae Miltiorrhizae, Radix Astragali, and Flos Carthami, respectively.</p