Chrysophanol protects against doxorubicin-induced cardiotoxicity by suppressing cellular PARylation

Abstract

The clinical application of doxorubicin (DOX) in cancer chemotherapy is limited by its life-threatening cardiotoxic effects. Chrysophanol (CHR), an anthraquinone compound isolated from the rhizome of Rheum palmatum L., is considered to play a broad role in a variety of biological processes. However, the effects of CHR׳s cardioprotection in DOX-induced cardiomyopathy is poorly understood. In this study, we found that the cardiac apoptosis, mitochondrial injury and cellular PARylation levels were significantly increased in H9C2 cells treated by Dox, while these effects were suppressed by CHR. Similar results were observed when PARP1 activity was suppressed by its inhibitors 3-aminobenzamide (3AB) and ABT888. Ectopic expression of PARP1 effectively blocked this CHR׳s cardioprotection against DOX-induced cardiomyocyte injury in H9C2 cells. Furthermore, pre-administration with both CHR and 3AB relieved DOX-induced cardiac apoptosis, mitochondrial impairment and heart dysfunction in Sprague-Dawley rat model. These results revealed that CHR protects against DOX-induced cardiotoxicity by suppressing cellular PARylation and provided critical evidence that PARylation may be a novel target for DOX-induced cardiomyopathy.

Keywords: 3AB, 3-aminobenzamide; ADR, adriamycin; ANOVA, one-way analysis of variance; Apoptosis; CHR, chrysophanol; CMC-Na, sodium carboxymethyl; CO, cardiac output; Cardiotoxicity; Chrysophanol; Cyt c, Cytochrome c; DOX, doxorubicin; Doxorubicin; EF, ejection fraction; FBS, fetal bovine serum; FS, fractional shortening; HE, hematoxylin-eosin; HR, heart rate; IVSd, end-diastolic interventricular septum; IVSs, end-systolic interventricular septum; LV, end-systolic volume; LVEDV, LV end-diastolic volume; LVIDd, LV end-diastolic internal diameter; LVIDs, LV end-systolic internal diameter; LVPWd, LV end-diastolic posterior wall thickness; LVPWs, LV end-systolic posterior wall thickness; Mitochondria; NS, normal saline; PAR, polymers of ADP-ribose; PARP1, poly(ADP-ribose) polymerase 1; PARylated, poly(ADP-ribosyl)ated; PARylation; PARylation, poly(ADP-ribosyl)ation; PBS, phosphate-buffered saline; RCR, respiratory control ratio; ROS, reactive oxygen species; Rh123, rhodamine 123; SD, Sprague–Dawley; TUNEL, TdT-mediated dUTP nick end labeling; VDAC1, voltage dependent anion channel 1.

Graphical abstract

Figure 1

Chrysophanol (CHR) attenuated DOX-induced cardiomyocyte apoptosis and mitochondrial injury in vitro. (A) The chemical structure of CHR (1,8-dihy-droxy-3-methylanthraquinone). (B) H9C2 cells were incubated with the different doses of DOX (0, 1, 5, 25, 50, 100, and 200 μmol/L) for 12 h, and the cell viability were measured by MTS assay. H9C2 cells were pre-cultured with CHR at different doses (1, 10 and 20 μmol/L) for 12 h before stimulation with DOX (1 μmol/L). (C) The cellular morphology was observed by light microscopy. Sclae bar: 200 nm. (D) The nuclear condensation was identified by Hoechst 33342 staining. Sclae bar: 100 nm. (E)–(G) The mitochondrial membrane potential (Δψm) and matrix swelling was determinated using staining with TMRE, Rh123 or Mitotracker. Sclae bar: 100 nm. Representative images of five independent experiments are shown. (H) The cleavage and activation of PARP1 and caspase 3, as well as the BCL-2/BAX ratio were detected by Western blot analysis. (I) The cardiac mitochondria were isolated from H9C2 cells, and the lysed extracts were submitted to Western blot analysis to measure the release of Cyt c from mitochondria to cytoplasm. (J) The cardiac PARylation levels of H9C2 cells were measured by Western blot analysis. The results were normalized to those of VDAC1 or α-tubulin and were presented as the means±SEM. ^*P < 0.05 vs. the control group, ^#P < 0.05 vs. the DOX group, n = 3.

Figure 2

Inhibition of PARP1 alleviated DOX-triggered cardiomyocyte apoptosis and mitochondrial damage in vitro. H9C2 cells were pre-treated with the ABT888 (5 μmol/L) or 3AB (20 μmol/L) for 12 h before stimulation with DOX (1 μmol/L). (A) The cardiac PARylation levels of H9C2 cells were measured by Western blot analysis. (B) The cellular morphology was observed by light microscopy. Sclae bar: 200 nm. (C) The nuclear condensation was identified by Hoechst 33342 staining. Sclae bar: 100 nm. (D)–(F) The mitochondrial membrane potential (Δψm) and matrix swelling was determinated using staining with TMRE, Rh123 or Mitotracker. Sclae bar: 100 nm. Representative images of five independent experiments are shown. (G) The cardiac cleavage and activation of PARP1 and caspase 3, as well as the BCL-2/BAX ratio were detected by Western blot analysis. (H) The lysed extracts were measured by Western blot analysis to measure the release of Cyt c from mitochondria to cytoplasm. The results were normalized to those of α-tubulin/VDAC1 and were presented as the means ± SEM. ^*P < 0.05 vs. the control group, ^#P < 0.05 vs. the DOX group, n = 3.

Figure 3

PARP1 was involved in the protection of CHR on DOX-induced cardiotoxicity in vitro. H9C2 cells were pre-infected with Ad-PARP1 or Ad-GFP followed by CHR (10 and 20 μmol/L) incubation with or without DOX. (A) The cellular morphology was observed by light microscopy. Sclae bar: 200 nm. (B) The nuclear condensation was identified by Hoechst 33342 staining. Sclae bar: 100 nm. (C)–(E) The mitochondrial membrane potential (Δψm) and matrix swelling was determinated using staining with TMRE, Rh123 or Mitotracker. Sclae bar: 100 nm. Representative images of five independent experiments are shown. (F) The cleavage and activation of PARP1 and caspase 3, as well as the BCL-2/BAX ratio were determined by Western blot analysis. (G) The cardiac mitochondria were isolated from H9C2 cells, and the lysed extracts were submitted to Western blot analysis to detect the release of Cyt c from mitochondria to cytoplasm. The results were normalized to those of VDAC1 or α-tubulin and were presented as the means ± SEM. ^*P < 0.05 vs. the control group, ^#P < 0.05 vs. the Ad-GFP+DOX group, ^&P < 0.05 vs. the 20+Ad-GFP+DOX group, n = 3.

Figure 4

Both Chrysophanol (CHR) and 3-aminobenzamide (3AB) protected against DOX-induced heart injury in Sprague–Dawley (SD) rats. SD rats were intragastrically treated with different doses of CHR (5, 20, and 40 mg/kg/day) or intraperitoneally injected with 3AB (40 mg/kg/day) for 7 days followed by DOX intraperitoneal injection (its cumulative doses were 15 mg/kg by three equal injections for 15 days) or an equal volume of sterile normal saline/sodium carboxymethylcellulose (CMC-Na). (A)–(C) Gross hearts, Sirus red and HE-stained transections of the left ventricle. Sclae bar: 100 nm. (D)–(F) The heart weight (HW), the body weight (BW), as well as the heart weight to the tibia length (HW/TL) ratios were calculated. The results were attended as the means ± SEM. ^*P < 0.05 vs. Normal saline group, ^#P < 0.05 vs. the DOX group, n = 10. ns: no statistical difference.

Figure 5

Both Chrysophanol (CHR) and 3-aminobenzamide (3AB) relieved DOX-induced heart dysfunction of Sprague–Dawley (SD) rats. SD rats were intragastrically treated with different doses of CHR (5, 20, and 40 mg/kg/day) or intraperitoneally injected with 3AB (40 mg/kg/day) for 7 days followed by DOX intraperitoneal injection (its cumulative doses were 15 mg/kg by three equal injections for 15 days) or an equal volume of sterile normal saline/sodium carboxymethylcellulose (CMC-Na). (A) and (A׳) The representative echocardiographic graphs are presented. (B)–(I) The echocardiographic parameters were measured, including the ejection fraction (EF), fractional shortening (FS), cardiac output (CO), heart rates (HR), interventricular septum (IVS), left ventricular diameter (LVID), left ventricular volume (LVV) and left ventricular posterior wall thickness (LVPW). The results were attended as the means ± SEM. ^*P < 0.05 vs. Normal saline group, ^#P < 0.05 vs. the DOX group, n = 10. ns: no statistical difference.

Figure 6

Both Chrysophanol (CHR) and 3-aminobenzamide (3AB) relieved DOX-induced cardiac apoptosis of Sprague–Dawley (SD) rats. SD rats were intragastrically treated with different doses of CHR (5, 20, and 40 mg/kg/day) or intraperitoneally injected with 3AB (40 mg/kg/day) for 7 days followed by DOX intraperitoneal injection (its cumulative doses were 15 mg/kg by three equal injections for 15 days) or an equal volume of sterile normal saline/sodium carboxymethylcellulose (CMC-Na). (A) The sections of rat heart were stained with TUNEL labeling, were observed by light microscopy. Representative images of five independent experiments are shown. Sclae bar: 100 nm. (B) The lysed extracts were detected the apoptotic protein levels, such as the protein cleavage and activation of PARP1 or caspase 3, as well as the BCL-2/BAX ratio. The results were presented as the means ± SEM. ^*P < 0.05 vs. Normal saline group, ^#P < 0.05 vs. the DOX group, n = 10. ns: no statistical difference.

Figure 7

Chrysophanol (CHR) and 3-aminobenzamide (3AB) both relieved DOX- induced mitochondrial dysfunction of Sprague–Dawley (SD) rats. SD rats were intragastrically treated with different doses of CHR (5, 20, and 40 mg/kg/day) or intraperitoneally injected with 3AB (40 mg/kg/day) for 7 days followed by DOX intraperitoneal injection (its cumulative doses were 15 mg/kg by three equal injections for 15 days) or an equal volume of sterile normal saline/sodium carboxymethylcellulose (CMC-Na). (A)–(E) The rat heart state III and IV respiration, the heart respiratory control ratio (RCR), ADP/O and ATP content was respectively determined. (F) The heart PARylation levels of SD rats were photographed using EVOS FL Auto. Representative images of five independent experiments are shown. ^*P < 0.05 vs. Normal saline group, ^#P < 0.05 vs. the DOX group, n =10. ns: no statistical difference.