Toll-like receptor 5 deficiency diminishes doxorubicin-induced acute cardiotoxicity in mice

Abstract

Rationale: Clinical application of doxorubicin (DOX) is limited by its toxic cardiovascular side effects. Our previous study found that toll-like receptor (TLR) 5 deficiency attenuated cardiac fibrosis in mice. However, the role of TLR5 in DOX-induced cardiotoxicity remains unclear. Methods: To further investigate this, TLR5-deficient mice were subjected to a single intraperitoneal injection of DOX to mimic an acute model. Results: Here, we reported that TLR5 expression was markedly increased in response to DOX injection. Moreover, TLR5 deficiency exerted potent protective effects against DOX-related cardiac injury, whereas activation of TLR5 by flagellin exacerbated DOX injection-induced cardiotoxicity. Mechanistically, the effects of TLR5 were largely attributed to direct interaction with spleen tyrosine kinase to activate NADPH oxidase (NOX) 2, increasing the production of superoxide and subsequent activation of p38. The toxic effects of TLR5 activation in DOX-related acute cardiac injury were abolished by NOX2 deficiency in mice. Our further study showed that neutralizing antibody-mediated TLR5 depletion also attenuated DOX-induced acute cardiotoxicity. Conclusion: These findings suggest that TLR5 deficiency attenuates DOX-induced cardiotoxicity in mice, and targeting TLR5 may provide feasible therapies for DOX-induced acute cardiotoxicity.

Keywords: Apoptosis; NOX2; TLR5; cardiotoxicity; doxorubicin.

Figure 1

TLR5 deficiency attenuates the DOX-induced cardiotoxic effects in mice. (A-B) TLR5 mRNA and protein expression in the hearts at three days after DOX injection (n = 6-8). (C) Expression of TLR5 protein in NRCMs received DOX for 24 h (n = 6). (D) Body weight change (n = 8). (E) The ratio of heart weight to tibia length (n = 8). (F-G) Serum concentration of CK-MB and LDH (n = 6). (H) H&E staining. Black line indicates the thickness of the myofibril (n = 8). (I) Ejection fraction in TLR5 KO or WT mice with or without DOX (n = 6). (J) DHE staining (n = 6). (K-L) The levels of 4-HNE and MDA in DOX-treated hearts (n = 6). (M) NADPH oxidase activity (n = 6). (N) Protein expression of NOX isoenzymes (n = 6). (O) Apoptosis measured by TUNEL staining (n = 6). ^*P < 0.05 versus WT Saline; ^#P < 0.05 versus WT DOX group.

Figure 2

TLR5 restoration promotes DOX-induced damage in TLR5-deficient mice. (A) TLR5 protein expression (n = 6). (B) The ratio of heart weight to tibia length (n = 10). (C) Serum concentration of CK-MB (n = 6). (D) Ejection fraction in mice with AAV9-TLR5 (n = 6). (E) The levels of 4-HNE in DOX-treated hearts (n = 6). (F) Bax expression (n = 6). (G) The mRNA levels of inflammatory factors (n = 6). ^*P < 0.05 versus KO Saline; ^#P < 0.05 versus KO DOX group.

Figure 3

FL treatment aggravates the DOX-induced cardiotoxic effects in mice. (A) Experimental protocol. (B) Survival for FL+DOX-treated mice (n = 15). (C) Body weight change at three days after DOX injection (n = 12). (D) The ratio of heart weight to tibia length at three days after DOX injection (n = 12). (E) Ejection fraction at three days after DOX injection (n = 6). (F-G) CK-MB and LDH in DOX+FL-treated mice at three days after DOX injection (n = 6). (H) DHE staining at three days after DOX injection (n = 5). (I) NADPH oxidase activity at three days after DOX injection (n = 6). (J) The levels of 4-HNE in DOX-treated hearts at three days after DOX injection (n = 6). (K) TUNEL staining at three days after DOX injection (n = 6). *P < 0.05 versus NS group; ^#P < 0.05 versus DOX group.

Figure 4

TLR5 promotes DOX-induced toxic effects via activation of NOX2 in vitro. (A) Cell viability after FL treatment (n = 6). (B) DCF-DA staining indicated the production of ROS. (C) ROS production as detected by ELISA (n = 6). (D-E) The production of superoxide (n = 6). (F) The NOX2 protein expression (n = 6). (G) The production of superoxide after knockdown of NOX isoenzymes (n = 6). (H) Superoxide production after NOX2 inhibition (n = 6). (I) Cell viability after NOX2 inhibition (n = 6). (J) TLR5 expression (n = 6). (K) The production of superoxide after TLR5 deficiency (n = 6). (L) MDA production after TLR5 deficiency (n = 6). (M) TUNEL staining. (N) Cell viability after TLR5 deficiency (n = 6). (O) NOX2 expression after TLR5 depletion (n = 6). *P < 0.05 versus matched control. The data are expressed as the mean ± SEM from six independent experiments.

Figure 5

NOX2 deficiency abolishes the toxic effects caused by FL in mice. (A) Experimental protocol. (B) NOX2 protein expression (n = 6). (C) Body weight gain (n = 10). (D) EF in the indicated groups (n = 8). (E) CK-MB levels (n = 6). (F) DHE staining and statistical results (n = 6). (G) MDA level after NOX2 deficiency (n = 6). *P < 0.05 versus matched control.

Figure 6

TLR5 regulates NOX2 through direct physical interaction with Syk. (A) The hearts were lysed and reacted with antibodies targeting TLR5, and then complexes were directly subjected to immunoblot analysis with NOX2 antibody or Syk antibody. (B) Immunoblot of a GST pull-down assay. (C) The p-Syk expression in TLR5-deficient mice (n = 6). (D) The production of superoxide after Syk inhibition (n = 6). (E-F) The PLCγ1 expression (n = 6). (G-H) DOX induced translocation of PKCα and p47phox from cytosol to the plasma membrane (n = 6). *P < 0.05 versus matched control. The data are expressed as the mean ± SEM from six independent experiments.