Therapeutic effects of higenamine combined with [6]-gingerol on chronic heart failure induced by doxorubicin via ameliorating mitochondrial function

Abstract

Higenamine (HG) is a natural benzylisoquinoline alkaloid isolated from Aconitum with positive inotropic and chronotropic effects. This study aimed to investigate the possible cardioprotective effects of HG combined with [6]-gingerol (HG/[6]-GR) against DOX-induced chronic heart failure (CHF) by comprehensive approaches. DOX-induced cardiotoxicity model in rats and H9c2 cells was established. Therapeutic effects of HG/[6]-GR on haemodynamics, serum indices and histopathology of cardiac tissue were analysed. Cell mitochondrial energy phenotype and cell mitochondrial fuel flex were measured by a Seahorse XFp analyser. Moreover, UHPLC-Q-TOF/MS was performed to explore the potential metabolites affecting the therapeutic effects and pathological process of CHF. To further investigate the potential mechanism of HG/[6]-GR, mRNA and protein expression levels of RAAS and LKB1/AMPK/Sirt1-related pathways were detected. The present data demonstrated that the therapeutic effects of HG/[6]-GR combination on CHF were presented in ameliorating heart function, down-regulation serum indices and alleviating histological damage of heart tissue. Besides, HG/[6]-GR has an effect on increasing cell viability of H9c2 cells, ameliorating DOX-induced mitochondrial dysfunction and elevating mitochondrial OCR and ECAR value. Metabolomics analyses showed that the therapeutic effect of HG/[6]-GR combination is mainly associated with the regulation of fatty acid metabolites and energy metabolism pathways. Furthermore, HG/[6]-GR has an effect on down-regulating RAAS pathway-related molecules and up-regulating LKB1/AMPKα/Sirt1-related pathway. The present work demonstrates that HG/[6]-GR prevented DOX-induced cardiotoxicity via the cardiotonic effect and promoting myocardial energy metabolism through the LKB1/AMPKα/Sirt1 signalling pathway, which promotes mitochondrial energy metabolism and protects against CHF.

Keywords: Aconiti Lateralis Radix Praeparata; [6]-gingerol; chronic heart failure; doxorubicin; energy metabolism; higenamine.

Figure 1

HG combined with [6]‐GR ameliorates the myocardial function in rats. Haemodynamic parameters, including (A) LVSP; (B) LVEDP; (C) +dp/dtmax; (D) −dp/dtmax, were measured by multichannel physiological signal acquisition system. HG/[6]‐GR reduces serum Renin (E), Ang‐II (F), ALD (G) and ET‐1 (H) levels in CHF rats. (I) Representative photomicrographs for HE staining of heart histological changes. **P < .01, compared with the control group. #P < .05 and ##P < .01, compared with the DOX group. aP < .05, aaP < .01, compared with the HG group. bbP < .01 compared with the [6]‐GR group. (HE stained, 200× magnification)

Figure 2

Metabolic profiles and differentiation of the control, DOX and HG/[6]‐GR groups by multivariate analysis. The score scatter plots of the control, DOX and HG/[6]‐GR‐treated groups from PCA data in the ESI− mode (A) and ESI+ mode (B); OPLS‐DA score scatter plots for the pairwise comparisons between the control and DOX groups in the ESI− mode (C) and ESI+ mode (D); S‐plot of the OPLS‐DA model for the control and DOX groups in the ESI− mode (E) and ESI+ mode (F); OPLS‐DA score scatter plots for the pairwise comparisons between the DOX and HG/[6]‐GR‐treated groups in the ESI− mode (G) and ESI+ mode (H); S‐plot of the OPLS‐DA model for the DOX and HG/[6]‐GR groups in the ESI− mode (I) and ESI+ mode (J)

Figure 3

Potential metabolites changes and related metabolomic pathway involved in the treatment of HG/[6]‐GR on DOX‐induced CHF. (A) The metabolomic pathway involved in the effects of HG/[6]‐GR on DOX‐induced CHF. (B) The cluster heatmap of metabolites both in each group. (C) Signalling networks associated with the differentially expressed metabolic pathways. Potential metabolites changes in DOX‐induced CHF with HG/[6]‐GR treatment in Eicosanoyl‐CoA (D); Pantothenic acid (E); 1,4‐beta‐D‐Glucan (F); Palmitic acid (G); Oleic acid (H); 3‐Methoxy‐4‐hydroxyphenylglycol glucuronide (I); 3‐carboxy‐1‐hydroxypropylthiamine diphosphate (J); Coenzyme A (K). **P < .01, compared with the control group; #P < .05, ##P < .01, compared with the DOX group. aaP < .01 compared with the HG group. bP < .05 and bbP < .01 compared with the [6]‐GR group

Figure 4

HG combined with [6]‐GR mitigates the inhibition effect of DOX on mitochondrial respiration and attenuates H9c2 cells from DOX‐induced decreases in mitochondrial fuel flexibility. Mitochondrial respiration was measured by a Seahorse XFp apparatus to detect OCR and ECAR values. During testing, H9c2 cells were treated with 10 μmol/L oligomycin and 10 μmol/L FCCP. (A) OCR. (B) ECAR. (C) Baseline OCR. (D) Baseline ECAR. (E) Stressed OCR. (F) Stressed ECAR. Mitochondrial substrate analysis was determined. During testing, H9c2 cells were treated with 4 μmol/L etomoxir and 3 μmol/L BPTES/2 μmol/L UK5099 in succession. (G) Effects of HG/[6]‐GR combination on fuel dependency in terms of the fatty acid oxidation pathway. (H) Effects of HG/[6]‐GR combination on fuel capacity in terms of the fatty acid oxidation pathway and oxidation rates of fatty acids expressed in dependency (I), capacity (J) and flexibility (K) to maintain baseline OCR levels determined with the Seahorse XFp respirometer. **P < .01, compared with the control group. #P < .05 and ##P < .01, the compared with DOX group. aP < .05 and aaP < .01 the compared with HG group. bP < .05 and bbP < .01 compared with the [6]‐GR group

Figure 5

Effect of HG/[6]‐GR couple on the relative mRNA expressions in rats with CHF. The relative mRNA expression levels of ACE (A), AT1R (B), NRG1 (C), AngPTL4 (D), CPT‐1 (E), FAS (F), eNOS (G), sGC (H) and PKG1 (I) were analyzed by RT‐PCR. (J) The level of ACE, AGTR1 and NRG1 in heart tissues was measured using immunohistochemical staining. Data are expressed as mean ± SD. **P < .01, compared with the control group; #P < .05, ##P < .01, compared with the DOX group; aP < .05, aaP < .01, compared with the HG group; bP < .05, bbP < .01, compared with the [6]‐GR group. (Immunohistochemical staining, 200× magnification)

Figure 6

Effects of HG/[6]‐GR couple on the relative mRNA and protein expression levels in the heart tissue of rats. (A) Relative LKB1 mRNA level in heart tissue; (B) relative CaMKK2 mRNA level in heart tissue; (C) relative AMPK α1 mRNA level in heart tissue; (D) relative CPT‐1 mRNA level in heart tissue; (E) relative Sirt1 mRNA level in heart tissue; (F) relative PGC‐1α mRNA level in heart tissue; (G) relative p300 mRNA level in heart tissue; (H) schematic for the role of HG/[6]‐GR in LKB1/AMPK α1/Sirt1 signalling pathway in rats; (I) Western blotting images of LKB1, AMPK α1, Sirt1 and PGC‐1α; (J) relative LKB1 protein level in heart tissue; (K) relative AMPK α1 protein level in heart tissue; (L) relative Sirt1 protein level in heart tissue; (M) relative PGC‐1α protein level in heart tissue. ∗∗P < .01 compared with the control group; #P < .05, ##P < .01 compared with the DOX group; aP < .05, aaP < .01 compared with the HG group; bP < .05, bbP < .01 compared with the [6]‐GR group