Schisandrol B and schisandrin B inhibit TGFβ1-mediated NF-κB activation via a Smad-independent mechanism

Abstract

Aberrant transforming growth factor β1 (TGFβ1) signaling plays a pathogenic role in the development of vascular fibrosis. We have reported that Schisandra chinensis fruit extract (SCE), which has been used as a traditional oriental medicine, suppresses TGFβ1-mediated phenotypes in vascular smooth muscle cells (VSMCs). However, it is still largely unknown about the pharmacologic effects of SCE on various TGFβ1 signaling components. In this study, we found that SCE attenuated TGFβ1-induced NF-κB activation and nuclear translocation in VSMCs. Among the five active ingredients of SCE that were examined, schisandrol B (SolB) and schisandrin B (SchB) most potently suppressed TGFβ1-mediated NF-κB activation. In addition, SolB and SchB effectively inhibited IKKα/β activation and IκBα phosphorylation in TGFβ1-treated VSMCs. The pharmacologic effects of SolB and SchB on NF-κB activation were independent of the Smad-mediated canonical pathway. Therefore, our study demonstrates that SCE and its active constituents SolB and SchB suppress TGFβ1-mediated NF-κB signaling pathway in a Smad-independent mechanism. Our results may help further investigations to develop novel multi-targeted therapeutic strategies that treat or prevent vascular fibrotic diseases.

Keywords: NF-κB; TGFβ1; schisandra chinensis; schisandrin B; schisandrol B.

Figure 1. SCE inhibits TGFβ1-induced NF-κB activation in A7r5 cells

The cells were transfected with 3TP-PAI1-Luc (A) or 3×kB-Luc (B) reporter constructs and then treated with TGFβ1 (1 ng/ml) and/or SCE (100 or 500 mg/ml) for 24 h. The luciferase activity was expressed as a relative value compared to that of the untreated cells which was set to 100%. The data were expressed as the mean ± SEM (n = 3–5). ^***p < 0.005. (C) The heatmap shows SCE-regulated NF-κB target genes in TGFβ-treated cells.

Figure 2. SCE inhibits TGFβ1-induced IKK activation and IκBα degradation in A7r5 cells

The cells were treated with TGFβ1 (1 ng/ml) and/or SCE (100 or 500 mg/ml) for the indicated times (A) or for 1 h (B) prior to western blot analysis. (C) The cells were treated with TGFβ1 (1 ng/ml) and/or SCE (100 or 500 mg/ml) for 1 h prior to confocal microscopy. The subcellular localization of p65 was assessed using anti-p65 antibody and FITC-conjugated IgG antibody. DAPI was used to visualize the nucleus. (D) The nuclear/cytosolic ratio of p65 was measured in at least 15 independent fields (n = 4). The data were expressed as the mean ± SEM. ^***p < 0.005.

Figure 3. SolB and SchB inhibit TGFβ1-induced NF-κB activation in A7r5 cells

The cells were transfected with 3×kB-Luc (A–C) or 3TP-PAI1-Luc (D–F) reporter constructs and then treated with TGFβ1 (1 ng/ml) and/or SolB (2 or 10 µM) or SchB (2 or 10 µM) for 24 h. The luciferase activity was expressed as a relative value compared to that of the untreated cells which was set to 100%. The data were expressed as the mean ± SEM (n = 4). ^**p < 0.01, ^***p < 0.005.

Figure 4. SolB and SchB inhibit TGFβ1-induced IKK activation and IκBα degradation in A7r5 cells

The cells were treated with TGFβ1 (1 ng/ml) and/or SolB (2 or 10 µM) or SchB (2 or 10 µM) for 1 h prior to western blot analysis (A, B) or confocal microscopy (C, D). The nuclear/cytosolic ratio of p65 was measured in at least 15 independent fields (n = 4). The data were expressed as the mean ± SEM. ^***p < 0.005.

Figure 5. SolB and SchB inhibit TGFβ1-induced NF-κB target gene production in A7r5 cells

The cells were stimulated with TGFβ1 (1 ng/ml) and/or SolB (2 or 10 µM) or SchB (2 or 10 µM) for 48 h prior to western blot analysis (A–C). (C) For IL-6 measurement, the medium was collected at 48 h after treatment. TNFα (10 ng/ml) used as a positive control for NF-κB activation. IL-6 level was determined by ELISA assay kit according to the manufacturer’s instruction. The data were expressed as the mean ± SEM (n = 4). ^**p < 0.01, ^***p < 0.005.

Figure 6. Smad activity is irrelevant to NF-κB activity in TGFβ1-treated A7r5 cells

The cells were transfected with 3TP-PAI1-Luc (A, B) or 3×kB-Luc (C, D) reporter constructs. Under the condition, the cells were co-transfected with Smad3-DN (A and C) or siSmad3 (B and D). The transfected cells were further treated with TGFβ1 (1 ng/ml) for 24 h. The luciferase activity was expressed as a relative value compared to that of the untreated cells which was set to 100%. The data were expressed as the mean ± SEM (n = 4). ^***p < 0.005. n.s., not significant. (E, F) The cells were transfected with Smad3-DN or siSMAD3 for 48 h and then treated with TGFβ1 (1 ng/ml) for 1 h prior to western blot analysis. ^#GFP-Smad3.