NPLC0393 attenuated CCl₄- and BDL-induced collagen expressions <i>in vivo</i>.

<p>(A) Collagen deposition in livers was evaluated by Masson staining and determined by image quantification. Scale bar, 100 µm. (B) Collagen mRNA expression was examined by real-time PCR analysis. ***P ≤ 0.001, n = 3 for control (CTL), model (MOD) and 2.5 mg/kg NPLC0393-treated (NPLC0393) mice. (C) Hydroxyproline content in liver was also measured. Significant difference versus model group, *P<0.05. n = 9 for control (CTL), model (MOD) and 2.5 mg/kg NPLC0393-treated (NPLC0393) mice.</p

NPLC0393 induced cell cycle arrest in HSCs.

<p>(A,B) LX-2 cells and the isolated primary rat HSCs were treated with increasing concentrations of NPLC0393 for the indicated time points. MTT assay was performed to assess the effects of NPLC0393 on cell viability. The values were indicated as relative units normalized to the control. *P<0.05; **P<0.01; ***P ≤ 0.001 compared with control group at the indicated time point. (C, D) LX-2 cells were exposed to increasing concentrations of NPLC0393 for 48 h. Then cells were harvested and the cell-cycle distribution was analyzed by Flow cytometry analysis. (E) LX-2 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014230#pone-0014230-g003" target="_blank">Figure 3C</a>. Effect of NPLC0393 on Cdk2 phosphorylation was assessed by western blotting. For the PDGF-induced Cdk2 phosphorylation, LX-2 cells were cultured to confluence and growth-arrested for 24 h in DMEM with 10% FBS, and then for an additional 24 h treatment with NPLC0393 and PDGF (10 ng/ml) in DMEM plus with 0.2% FBS. (F) Control and shPP2Cα cells were treated with increasing concentrations of NPLC0393 for 24 h. Effects of NPLC0393 on cell viability in shPP2Cα cells and control cells were assessed by MTT assay. Significant difference of the reduction on cell viability by NPLC0393 in shPP2Cα cells versus that in control cells at indicated dose, *P<0.05, **P<0.01. (G) Cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014230#pone-0014230-g003" target="_blank">Figure 3F</a> and harvested for Western blotting.</p

Magnolol as a biased agonist on PPRE transcription.

<p>(<b>A–B</b>) Magnolol could not activate the transcription of RXRE mediated by RXRα:RXRα homodimer (<b>A</b>), while activating the transcription of PPRE mediated by RXRα:PPARγ heterodimer in a dose-dependent manner (<b>B</b>). RXRα agonist 9<i>c</i>RA, RXRα antagonist HX531, PPARγ agonist Rosiglitazone, and PPARγ antagonist GW9662 were used as controls. (<b>C</b>) Activating curves of magnolol and Rosiglitazone on PPRE transcription indicated that magnolol was a PPARγ full agonist, although magnolol exhibited lower activities in their lower concentrations.</p

Crystal structures of RXRαLBD-magnolol-SRC1 and PPARγLBD-magnolol.

<p>(<b>A</b>) Electron density of magnolol bound into RXRα ligand-binding pocket in stereo view (contoured at 1.0σ level). (<b>B</b>) Comparison of receptor-ligand interactions between 9<i>c</i>RA-bound and magnolol-bound RXRαLBDs. 9<i>c</i>RA (in blue sticks) formed hydrogen bonds with Arg316 (in magenta sticks) in the C-terminus of helix 5 (in magenta ribbon), while magnolol (in yellow sticks) formed hydrogen bonds with Asn306 (in cyan sticks) in the N-terminus of helix 5. Density map around Asn306 was shown in the right to indicate its conformational changes. All other hydrophobic residues involving 9<i>c</i>RA (in magenta lines) or magnolol interactions were the same (shown in cyan lines). (<b>C</b>) Electron density map of magnolol bound into PPARγ ligand-binding pocket in stereo view (contoured at 1.0σ level). (<b>D</b>) The two magnolol molecules formed hydrogen bonds with Ser342 in β-sheet, Tyr473 in AF-2 motif, and Ser289 in helix 3 of PPARγ, as well as water-mediated hydrogen bonds.</p

NPLC0393 decreased TGFβ-induced α1(I) collagen expression in HSCs.

<p>(A) LX-2 cells and the isolated primary rat HSCs were treated with increasing concentrations of NPLC0393 for 48 h. Cells were harvested for real-time PCR experiment. ***P<0.001 compared with vehicle group. (B) LX-2 cells were treated with NPLC0393 and TGFβ for 48 h, cells were then harvested and the total RNA was extracted. ^#P<0.001 compared with non-TGFβ-treated group; **P<0.01; ***P<0.001 compared with TGFβ-treated group with vehicle treatment. (C) Characterization of stable LX-2 cell line expressing shPP2Cα by western blot analysis (upper panel). Control and shPP2Cα cells were treated with increasing concentrations of NPLC0393 for 24 h and harvested for Western blotting (lower panel). (D) Control and shPP2Cα cells were treated with NPLC0393 and TGFβ for 48 h and harvested for western and real-time PCR analysis. Significant difference of the reduction on α1(I) procollagen mRNA by NPLC0393 in shPP2Cα cells versus that in control cells, *P<0.05.</p

NPLC0393 attenuated CCl₄- and BDL-induced α-SMA expressions <i>in vivo</i>.

<p>(A) Expression of α-SMA in CCl₄- and BDL-intoxicated mice was evaluated by immunohistochemical staining, and quantified by counting five random chosen high-power fields. Scale bar, 50 µm. n = 9 for control (CTL), model (MOD) and 2.5 mg/kg NPLC0393-treated (NPLC0393) mice. (B) α-SMA expression was also assessed by western blotting and quantified from three independent experiments, *P<0.05, **P<0.01. n = 3 for control (CTL), model (MOD) and 2.5mg/kg NPLC0393-treated (NPLC0393) mice.</p

Activation of PP2Cα inhibited both TGFβ-Smad3 and TGFβ-p38 signaling pathways and induced cell cycle arrest in HSCs.

<p>(A) Flag-hPP2Cα, shPP2Cα494 and control vectors were electransfected into LX-2 cells. At 48 h post-transfection, 2 ng/ml of TGFβ was used to stimulate the cells for 1 h. Cells were harvested and proteins were immunoblotted with the indicated antibodies. (B) At 24 h post-transfection with hPP2Cα and shPP2Cα494, LX-2 cells were stimulated with TGFβ (2ng/ml) for another 24 h. Cells were harvested for real-time PCR experiment. ^#P<0.001 compared with non-TGFβ-treated group; *P<0.05, **P<0.01 compared with TGFβ-treated group tranfected with control vector. (C) Cell viability was assessed by MTT assay at 48 h post-transfection with increasing concentrations of hPP2Cα (upper panel). Increasing expressions of PP2Cα were verified by western blotting (lower panel). (D) Cells were harvested at 48 h post-transfection with hPP2Cα and shPP2Cα494 and total cell extracts were analyzed by western blotting.</p

NPLC0393 reduced TGFβ-Smad3 and TGFβ-p38 phosphorylations in HSCs.

<p>(A,B) LX-2 cells and the isolated primary rat HSCs were treated with increasing concentrations of NPLC0393 for indicated time points. Cells were harvested and the total cell extracts were analyzed by western blotting. (C) LX-2 cells were treated with NPLC0393 for 48 h followed by TGFβ (1ng/ml) stimulation for another 1 h. Effects of NPLC0393 on the nuclear translocation of P-Smad3 were assessed by Immunofluorescence experiment. Images were taken by IN Cell Analyzer 1000 and quantified by counting six random chosen fields in each well. Each treatment was performed in three wells. ^#P<0.001 compared with non-TGFβ-treated group; **P<0.01; ***P<0.001 compared with TGFβ-treated group with vehicle treatment.</p

Magnolol as a dual agonist of RXRα and PPARγ.

<p>(<b>A</b>) Chemical structure of magnolol. (<b>B–C</b>) Magnolol dose-dependently activated the transcription of GAL4DBD-RXRαLBD (<b>B</b>) and GAL4DBD-PPARγLBD (<b>C</b>) in HEK-293T cells, which could be suppressed by RXRα antagonist HX531 and PPARγ antagonist GW9662, respectively. RXRα agonist 9-<i>cis</i>-retinoic acid (9<i>c</i>RA) and PPARγ agonist Rosiglitazone were used as positive controls. (<b>D–E</b>) Magnolol dose-dependently bound to RXRαLBD (<b>D</b>) and PPARγLBD (<b>E</b>) in SPR technology based assays. (<b>F–G</b>) Magnolol dose-dependently enhanced SRC1 recruitment to RXRαLBD (<b>F</b>), instead of PPARγLBD (<b>G</b>) in SPR technology based assays.</p

Identification of NPLC0393 as a small molecular PP2Cα activator.

<p>(A) Chemical structure of NPLC0393. (B, C) NPLC0393 activated the recombinant human PP2Cα activity using pNPP (B) and phosphopeptide FLRTpSCG (C) as substrates. Data are expressed as the mean ± S.D. of three independent experiments. (D) Binding affinity of NPLC0393 to PP2Cα as evaluated by Biacore 3000. Sensorgrams obtained from NPLC0393 injection over the immobilized PP2Cα surface. NPLC0393 was injected for 60s, and dissociation was monitored for more than 120s. (E) ITC analysis of NPLC0393/PP2Cα interaction.</p