TFEB-NF-κB inflammatory signaling axis: a novel therapeutic pathway of Dihydrotanshinone I in doxorubicin-induced cardiotoxicity

Abstract

Background: Doxorubicin is effective in a variety of solid and hematological malignancies. Unfortunately, clinical application of doxorubicin is limited due to a cumulative dose-dependent cardiotoxicity. Dihydrotanshinone I (DHT) is a natural product from Salvia miltiorrhiza Bunge with multiple anti-tumor activity and anti-inflammation effects. However, its anti-doxorubicin-induced cardiotoxicity (DIC) effect, either in vivo or in vitro, has not been elucidated yet. This study aims to explore the anti-inflammation effects of DHT against DIC, and to elucidate the potential regulatory mechanism.

Methods: Effects of DHT on DIC were assessed in zebrafish, C57BL/6 mice and H9C2 cardiomyocytes. Echocardiography, histological examination, flow cytometry, immunochemistry and immunofluorescence were utilized to evaluate cardio-protective effects and anti-inflammation effects. mTOR agonist and lentivirus vector carrying GFP-TFEB were applied to explore the regulatory signaling pathway.

Results: DHT improved cardiac function via inhibiting the activation of M1 macrophages and the excessive release of pro-inflammatory cytokines both in vivo and in vitro. The activation and nuclear localization of NF-κB were suppressed by DHT, and the effect was abolished by mTOR agonist with concomitant reduced expression of nuclear TFEB. Furthermore, reduced expression of nuclear TFEB is accompanied by up-regulated phosphorylation of IKKα/β and NF-κB, while TFEB overexpression reversed these changes. Intriguingly, DHT could upregulate nuclear expression of TFEB and reduce expressions of p-IKKα/β and p-NF-κB.

Conclusions: Our results demonstrated that DHT can be applied as a novel cardioprotective compound in the anti-inflammation management of DIC via mTOR-TFEB-NF-κB signaling pathway. The current study implicates TFEB-IKK-NF-κB signaling axis as a previously undescribed, druggable pathway for DIC.

Keywords: Cardiotoxicity; Dihydrotanshinone I; Doxorubicin; Inflammation; TFEB-NF-κB.

Fig. 1

DHT improved cardiac function and protected against pathological injury in zebrafish and in mice. a Microscopic examination showed that DHT preserved the heart atrium, protected against extensive pericardial edema and blood accumulation in tail, and preserved the heart atrium. By using a high-speed camera, results and analysis showed that DHT increased FS values (b) and blood flow (d). DHT could improve heart rate (c) and survival rate (e). f M-mode echocardiography was assessed to detect cardiac function in each group. g Echocardiography data showed that DHT increased EF% and FS%, and decreased LVEDD and LVESD. h HE staining showed that DHT protected against the structural damage caused by DOX, scale bar = 20 μm. i Quantification of inflammatory cell infiltration (%) showed that DHT decreased the inflammatory cell rate. j Quantification of SOD and MDA levels in plasma. N ≥ 10 per group in zebrafish; N ≥ 5 per group in mice. All data were presented as means ± SD in triplicate. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 is significantly different as indicated, for values in the DOX group

Fig 2

DHT suppressed the activation of M1 macrophages in mice and in RAW264.7 cells. (A) Flow cytometry assay showed that DHT or Rapamycin reduced accumulation of macrophages and activation of M1 macrophages in mice. In (a), X-axis represents PE-Cy7 anti-CD11b and Y-axis represents APC anti-F4/80; in (b), X-axis represents PE anti-CD86 and Y-axis represents FITC anti-CD206. N ≥ 3 per group. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 is significantly different as indicated, for values in the DOX group. b Immunofluorescence assay showed that DHT suppressed the protein expressions of CD86 and F4/80 in mice, scale bar = 20 μm. N = 3 per group. c DHT treatment for 24 h had no cytotoxic effect on RAW264.7 cells at the dosages of 10, 50 and 100 nM. N = 6 per group. d RAW 264.7 cells were stimulated with LPS (1 μg/mL) in the absence or presence of DHT (10, 50 and 100 nM) for 24 h and the releases of TNF-α and IL-1β in cell supernatants were detected by Elisa assay. N ≥ 5 per group. ^*p < 0.05, ^***p < 0.001 is significantly different as indicated, for values in the LPS group. e The protein expression of CD86 was detected by immunofluorescence assay. DHT suppressed the expression of CD86 in RAW264.7 cells, scale bar = 100 μm. N = 12 per group. f DHT reduced activation of M1 macrophages in LPS-stimulated RAW264.7 cells. N ≥ 3 per group. ^***p < 0.001 is significantly different as indicated, for values in the LPS group. g DHT suppressed the nuclear localization of p-NF-κB in LPS-stimulated RAW264.7 cells, scale bar = 50 μm. N = 20 per group. All data were presented as means ± SD in triplicate.

Fig. 3

DHT inhibited the production of pro-inflammatory cytokines mediated by NF-κB in mice and in cardiomyocytes. a Western blotting showed that DHT downregulated the expressions of activated NF-κB, TNF-α, COX2 and IL-8 in mice. N = 5 per group. All data were presented as means ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 is significantly different as indicated, for values in the DOX group. b Quantification of TNF-α level in heart tissue. N = 5 per group. c IHC and quantitative results showed that DHT or Rapamycin suppressed the expression of TNF-α, scale bar = 20 μm. N = 5 per group. CCK-8 assay results showed the non-toxic range of concentration (d) and the protective concentrations (e). N = 12 per group. f Western blotting showed that DHT downregulated the expressions of activated NF-κB, TNF-α, COX2 in cardiomyocytes. N = 5 per group. All data were presented as means ± SD in triplicate. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 is significantly different as indicated, for values in the DOX group

Fig. 4

DHT regulated mTOR-TFEB-NF-κB signaling pathway to ameliorate inflammation in DOX-stimulated cardiomyocytes. a Western blotting showed that DHT or rapamycin downregulated the expressions of phosphorylated mTOR and S6K in mice. N = 5 per group. All data were presented as means ± SD in triplicate. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 is significantly different as indicated, for values in the DOX group. b Western blotting showed that with MHY1485 co-treatment, the effect of DHT on inflammation-related proteins including TNF-α and COX2 was abrogated in cardiomyocytes. N = 5 per group. All data were presented as means ± SD in triplicate. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 is significantly different as indicated, for values in the DOX group. ^##p < 0.01, ^###p < 0.001 is significantly different as indicated, for values in the DOX + DHT group. c Cardiomyocytes were transfected with GFP-TFEB and results showed that co-treatment with MHY1485 abolished the effects DHT on the nuclear localization of TFEB, scale bar = 50 μm. N = 20 per group

Fig. 5

DHT inhibited NF-κB transcriptional activity via TFEB-IKK signaling pathway. a Cardiomyocytes were transfected with Lentiviral vector (Lv) carrying GFP-TFEB to monitor the location of TFEB. Lv-GFP transfected cells were set as a negative control. Representative GFP fluorescence and immunofluorescence staining of p-NF-κB were shown under different treatment, scale bar = 100 μm. N = 20 per group. b, c, d Western blotting showed that DOX reduced the nuclear expression of TFEB and increased phosphorylated levels of IKKα/β and NF-κB, while DHT up-regulated the nuclear expression of TFEB. In particular, TFEB overexpression or DHT treatment down-regulated phosphorylated levels of IKKα/β and NF-κB. N = 3 per group. e Representative GFP-TFEB fluorescence and immunofluorescence staining of p-NF-κB under different treatment, scale bar = 50 μm. N = 20 per group. All data were presented as means ± SD in triplicate. *^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 6

DHT inhibited apoptosis to facilitate the anti-inflammation effect. a The representative photomicrographs of a left ventricle section of the heart showed the expression of Cleaved caspase-3 under different treatment, scale bar = 20 μm. N = 5 per group. b Western blots detected the expressions of Bax and Bcl-2 proteins in heart tissues. N = 5 per group. c The results of Hoechst staining under different treatment, scale bar = 40 μm. N = 12 per group. d Western blots assessed the expressions of Bax and Bcl-2 proteins in H9C2 cells. N = 5 per group. All data were presented as means ± SD in triplicate. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 is significantly different as indicated, for values in the DOX group. ^#p < 0.05, ^##p < 0.01 is significantly different as indicated, for values in the DOX + DHT group