Fig 2: TBM prevented DOX-evoked cellular damage by upregulating SIRT3 in H9c2 cells. (a, b) si-SIRT3 inhibited the expression of SIRT3 as determined by western blotting (n = 4). (c–h) The expression of SIRT3, P-P65, T-P65, IL-6, IL-1ß, and TNF-a was estimated by western blotting in H9c2 cells (n = 3). (i–m) Western blotting was used to detect the expression of Nrf2, HO-1, and NQO1 in vitro (n = 3). (n, p) si-Sirt3 partially reversed the inhibitory roles of TBM in DOX-induced ROS production (n = 4). (o, q) Flow cytometry analysis demonstrated that SIRT3 inhibition blunted the beneficial effects of TBM on DOX-evoked apoptosis (n = 6). (r–v) Western blotting was used to test the expression of apoptosis related proteins (BAX, BCL-2, and C-Caspase3) in H9c2 cells after si-Sirt3 treatment (n = 3). Values indicate the mean ± SD. *P < 0.05 versus the CON group and the TBM group; #P < 0.05 versus the DOX group; ns (not significant) versus the CON group; &P < 0.05 versus the DOX + TBM + siSIRT3 group.

Fig 3: Inhibition of P. gingivalis-medicated ROS production inhibits NF-?B activation, proinflammatory cytokine expression, cell apoptosis, and cell death in bEnd.3 cells. Cells are treated with various concentrations (0, 5, 10 or 20 mM) of NAC for 2 h and then infected with P. gingivalis (200 MOI) for 90 min; the ROS production is determined by DCF fluorescence using a flow cytometer (A). Total protein is collected, and the expression of IL-1ß (precursor), IL-1ß (mature), and TNF-a are determined by Western blotting (D). Morphological alterations are observed under a light microscope (E). The cell survival rate is evaluated by MTT assay (F) and Trypan Blue exclusion test (G). The nuclear condensation is performed by DAPI staining and viewed with a fluorescence microscope (H). Apoptotic cells are determined by Annexin V/PI staining and quantitated by a flow cytometer (I). Cells were treated with NAC (10 mM) and infected with P. gingivalis, the nuclear NF-?B p65 and PARP protein expression are determined by Western blot analysis (B), and transcriptional factor binding activity is determined by NF-?B p65 DNA-binding activity (C). Data are presented as mean values ± SEM (n = 4). Significant differences of the control group and groups with or without NAC treatment are expressed as *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, p > 0.05. Scale bars are 200 µm in (E) and 100 µm in (H).

Fig 4: Suppression of NF-?B activation by THSG in P. gingivalis-infected bEnd.3 cells. THSG (0, 30, 100, or 200 µM) was added to cells 2 h before 90 min P. gingivalis infection (heat-killed MOI 500 or live MOI 200). Cell lysates were collected after 15 min of additional incubation with a fresh medium. (A) I?Ba protein from whole-cell lysate and NF-?B p65 protein from nuclear and cytoplasmic fraction were analyzed by Western blot analysis. The quantitative results are shown in (B,C), respectively. (D) DNA binding activity of NF-?B p65 was determined by the transcription-factor assay and calculated in the fold of control. Data in bar graph are expressed as mean values ± SEM (n = 4). Significant difference of the control and THSG 0 µM group are presented as *, p < 0.05; ***, and p < 0.001. NS: not significant.

Fig 5: The AP-1 and NF-?B pathways mediate nesfatin-1-induced stimulation of IL-1ß. (A–C) OASFs were treated with an AP-1 inhibitor (tanshinone IIA) or NF-?B inhibitor (PDTC and TPCK), or transfected with c-Jun or p65 siRNAs, then stimulated with nesfatin-1. IL-1ß expression was examined by qPCR and ELISA. (D) Cells were incubated with nesfatin-1 for the indicated time intervals; c-Jun and p65 phosphorylation was examined by Western blot. (E–F) Quantitative data for p-c-jun and p-p65 were shown. (G and H) Cells were treated with indicated inhibitors then stimulated with nesfatin-1, and AP-1 and NF-?B luciferase activity was examined. *p < 0.05 compared with the control group; #p < 0.05 compared with the nesfatin-1-treated group.