PGC1α activation by pterostilbene ameliorates acute doxorubicin cardiotoxicity by reducing oxidative stress via enhancing AMPK and SIRT1 cascades

Pterostilbene application alleviated DOX-induced H9c2 cell viability inhibition, mitochondrial damage and oxidative stress

As our previous basic research data suggested [4], the dosage of 1 μM DOX was chosen to carry on the present in vitro study. To investigate whether pterostilbene exerts the cardioprotective action against toxicity induced by DOX on H9c2 cells, CCK-8 cell viability assay was conducted to analyze its protective effect. As shown in Figure 1A, pterostilbene treatment (2.5, 5, 7.5 or 10 μM culturing for 24 h) had no effect on H9c2 cell viability, but 1 μM DOX treatment for 24 h significantly inhibited the cell viability. Interestingly, cotreatment of 2.5, 5, 7.5 and 10 μM pterostilbene for 24 h markedly reversed the 1 μM DOX caused decrease of cell viability in a dose-dependent manner. Therefore, this result indicated that pterostilbene exerted cardiac protection against DOX-cardiotoxicity, and 10 μM pterostilbene was chosen to be used for further experimental studies. Generation of ROS thus promoting mitochondrial oxidative stress plays vital actions on the development of cardiac dysfunction. We found 1 μM DOX-exposure for 24 h caused markedly upregulation of ROS level, loss of mitochondrial membrane potential (ΔΨm), and downregulation of ATP content, but pterostilbene cotreatment obviously reversed these DOX-induced mitochondrial oxidative stress by decreasing ROS level and preserving ΔΨm and ATP content (Figure 1B, 1C and Figure 2C). Moreover, the transmission electron microscopic examination on H9c2 cells revealed that 1 μM DOX exposure significantly caused ultrastructural morphology disorder on mitochondria by inducing swelling with cristae disorientation and breakage (Figure 1D). However, pterostilbene cotreatment markedly rescued the myocardial mitochondrion by normalizing the cristae density and architecture (Figure 1D).

Figure 1. Effect of pterostilbene treatment on cell viability, mitochondrial membrane potential, ROS generation, mitochondrial morphologic changes in DOX-treated H9c2 cells (24 h). (A) single pterostilbene (2.5-10 μM) treatment and cotreatment of 1 μM DOX with increasing concentrations of pterostilbene (2.5-10 μM) on H9c2 cell viability (24 h). (B) mitochondrial membrane potential (ΔΨm) was expressed as the ratio of JC-1 polymer/monomer; red fluorescence represents the mitochondrial JC-1 polymer, and green fluorescence represents the monomeric form of JC-1, indicating ΔΨm depolarization. (C) Representative images and ROS level, and the indexes in the control group are defined as 100%. (D) Representative images of the ultrastructural morphology of mitochondria in each group of H9c2 cells are shown. The results are expressed as the mean ± SEM. *P < 0.05 vs. the control group, ^#P < 0.05 vs. the 1 μM DOX-treated group.

Figure 2. Effect of pterostilbene treatment combined with AMPK siRNA on cell viability, ATP content, ROS generation and ΔΨm, and AMPK and PGC1α signaling in DOX-treated H9c2 cells (24 h). (A) Representative western blot results of p-AMPK, AMPK, PGC1α, UCP2 and NRF1 are shown. Membranes were re-probed for β-actin expression to show that similar amounts of protein were loaded in each lane. (B) Cell viability, (C) cellular ATP content, (D) ROS level and (E) ΔΨm are shown, and (B–D) three indexes in the Control group of siCON are defined as 100%. The results are expressed as the mean ± SEM. *P < 0.05 vs. the Control group, ^#P < 0.05 vs. the 1 μM DOX-treated group, ^$P < 0.05 vs. the siCON group with the representative same drug treatments.

Pterostilbene application activated the AMPK, SIRT1 and PGC1α signaling in DOX-treated H9c2 cells

To explore the underlying molecular mechanisms regarding myocardial protective actions of pterostilbene on DOX-cardiotoxicity, we further analyzed the expression of p-AMPK, AMPK, SIRT1, and PGC1α and its downstream signaling proteins (NRF1 and UCP2) in H9c2 cells. Consistent with previously reported studies [4], 1 μM DOX-exposure for 24 h significantly inhibited the AMPK activation (AMPK phosphorylation), and decreased the expression of SIRT1, PGC1α, NRF1 and UCP2 (vs. Control group, P < 0.05, Figure 2A and Figure 3A). Interestingly, cotreatment of 10 μM pterostilbene markedly reversed the above effects caused by DOX treatment compared with DOX group (P < 0.05, Figure 2A and Figure 3A), suggesting activation of AMPK, SIRT1 and PGC1α signaling cascades may be involved in the actions of pterostilbene-exerted protection against DOX-cytotoxicity.

Figure 3. Effect of pterostilbene treatment combined with SIRT1 siRNA on cell viability, ATP content, ROS generation and ΔΨm, and SIRT1 and PGC1α signaling in DOX-treated H9c2 cells (24 h). (A) and (B) Representative western blot results of SIRT1, PGC1α, Ac-PGC1α, UCP2 and NRF1 are shown. Membranes were re-probed for β-actin expression to show that similar amounts of protein were loaded in each lane. IB, immunoblot; IP, immunoprecipitation. (C) Cell viability, (D) cellular ATP content, (E) ROS level and (F) ΔΨm are shown, and (C–E) three indexes in the Control group of siCON are defined as 100%. The results are expressed as the mean ± SEM. *P < 0.05 vs. the Control group, ^#P < 0.05 vs. the 1 μM DOX-treated group, ^$P < 0.05 vs. the siCON group with the representative same drug treatments.

Effect of pterostilbene cotreatment with AMPK siRNA on cell viability, oxidative stress parameters, AMPK and PGC1α signaling in DOX-treated H9c2 cells

Our previous study reported that AMPK was a potential upstream regulator on PGC1α signaling, and activation of AMPK-PGC1α cascades was involved in myocardial protection [4]. To investigate the action of AMPK on the myocardial protection of pterostilbene against DOX-cytotoxicity in vitro, a specific AMPK siRNA was applicated to knockdown the AMPK expression in the H9c2 cells. Western blot analyses showed that knockdown of AMPK hindered pterostilbene application-exerted increase of PGC1α and its downstream signaling (NRF1 and UCP2) protein levels in H9c2 cells with DOX exposure (vs. DOX + PTS group, P < 0.05, Figure 2A). Additionally, compared with the pterostilbene and DOX cotreatment group, AMPK knockdown significantly prevented the pterostilbene cotreatment-exerted rise in H9c2 cell viability, preservation of ΔΨm, cellular ATP content and the decrease of ROS generation with DOX-exposure (P < 0.05, Figure 2B–2E). Therefore, AMPK activation was involved in the pterostilbene-induced heart protection against DOX-cytotoxicity via PGC1α upregulation.

Effect of pterostilbene cotreatment with SIRT1 siRNA on cell viability, oxidative stress parameters, SIRT1 and PGC1α signaling in DOX-treated H9c2 cells

SIRT1 is a well-known deacetylase and involved in cardiac protection and mitochondrial biogenesis through its posttranslational deacetylation of PGC1α protein [16]. The activity of PGC1α is regulated by the post-translational level, and acetylation of PGC1α leads to the decrease of its transcriptional ability [19]. In present study, we found cotreatment of pterostilbene markedly increased the SIRT1 expression and decreased the acetylated PGC1α level (vs. DOX group, P < 0.05, Figure 3A and 3B). To explore the action of SIRT1 on the protective effects of pterostilbene against DOX-induced cardiotoxicity in vitro, the SIRT1 was knockdown after SIRT1 specific siRNA application on the H9c2 cells. Western blot analyses revealed that SIRT1 knockdown markedly promoted PGC1α acetylation without influencing PGC1α expression, and downregulated the UCP2 and NRF1 levels in the DOX + PTS + SIRT1 siRNA group compared with the DOX + PTS group (P < 0.05, Figure 3A and 3B). Moreover, compared with the pterostilbene and DOX cotreatment group, SIRT1 knockdown partially reversed the pterostilbene cotreatment-exhibited upregulation of H9c2 cell viability, ΔΨm and cellular ATP content, and the decrease of ROS generation during DOX-exposure (P < 0.05, Figure 3C–3F). These results suggested that SIRT1 participated in the pterostilbene exerted protection against DOX-induced cardiotoxicity via PGC1α deacetylation (activation).

Effect of cotreatment of pterostilbene with Compound C or EX527 on cardiac toxicity and mitochondrial oxidative stress injury on DOX-impaired mice hearts

The mitochondrial damage and concomitant oxidative stress injury were considered to be an important cause of DOX-cardiomyopathy. After DOX exposure for 6 days, compared with sham group, transmission electron microscopy examination on heart tissues showed the obvious disorders in ultrastructural mitochondrial morphology including mitochondrial swelling with cristae disorientation and breakage in the DOX-treated hearts (Figure 4A). Interestingly, pterostilbene cotreatment alleviated the DOX-induced injury on mitochondria through normalizing cristae density and architecture (Figure 4A). Moreover, pterostilbene cotreatment markedly downregulated the ROS generation and promoted the SOD and GPx activities in the DOX-exhibited hearts (vs. DOX group, P < 0.05, Figure 4B–4D). However, when compared with the DOX + PTS group, cotreatment of compound C (a selective AMPK inhibitor) or EX527 (a selective SIRT1 inhibitor) with pterostilbene clearly mitigated the mitochondrial protective and anti-oxidative effects of pterostilbene on DOX-stimulated hearts (Figure 4A–4D). Pterostilbene application combined with DOX and compound C or EX527 showed severe mitochondrial disorders (Figure 4A), as well as increase of ROS level and decreases in GPx and SOD activities (vs. DOX + PTS group, P < 0.05, Figure 4B–4D).

Figure 4. Effect of pterostilbene treatment combined with Compound C or EX527 on cardiac toxicity and oxidative stress in DOX-exposure mice hearts. (A) Representative images of the ultrastructural morphology of mitochondria from the left ventricular myocardium of experimental mice (magnification: upper panel 9900 ×, lower panel 20,500 ×). (B) ROS level, (C) SOD and (D) GPx activity in the myocardial tissues are showed. The results are expressed as the mean ± SEM. *P < 0.05 vs. the Sham group, ^#P < 0.05 vs. the DOX-treated group, ^$P < 0.05 vs. the DOX+PTS cotreated group.

Effect of cotreatment of pterostilbene with Compound C or EX527 on AMPK, SIRT1 and PGC1α signaling in the myocardial tissue of DOX-stimulated mice

To further confirm the above in vitro results that AMPK and SIRT1 activation was involved in the protective effects of pterostilbene against DOX-cytotoxicity via stimulating PGC1α signaling.

Western blot analyses were applied in the invivo study, and we found pterostilbene treatment markedly upregulated the p-AMPK and SIRT1 levels, and inhibited the PGC1α acetylation and promoted the expression of PGC1α and its downstream signaling (NRF1 and UCP2) proteins in DOX + PTS group (vs. DOX group, P < 0.05, Figure 5). Interestingly, cotreatment of AMPK inhibitor compound C markedly antagonized pterostilbene induced- upregulation of p-AMPK, PGC1α, NRF1 and UCP2 levels in the DOX + PTS + CC group (vs. DOX + PTS group, P < 0.05, Figure 5). Moreover, SIRT1 activity inhibition by EX527 cotreatment significantly reversed pterostilbene promoted PGC1α deacetylation and the increase of NRF1 and UCP2 expression in the DOX + PTS + EX527 group (vs. DOX + PTS group, P < 0.05, Figure 5). Taken together, above results indicated that AMPK activation-induced PGC1α upregulation and SIRT1 activation-promoted PGC1α deacetylation were involved in the pterostilbene-exerted myocardial protection against DOX-cytotoxicity.

Figure 5. Effect of pterostilbene treatment combined with Compound C or EX527 on AMPK, SIRT1 and PGC1α signaling protein levels in DOX-stimulated mice hearts. Representative western blot results of p-AMPK, AMPK, SIRT1, PGC1α, Ac-PGC1α, UCP2 and NRF1 are shown. Membranes were re-probed for Tubulin expression to show that similar amounts of protein were loaded in each lane. IB, immunoblot; IP, immunoprecipitation. The results are expressed as the mean ± SEM. *P < 0.05 vs. the Sham group, ^#P < 0.05 vs. the DOX-treated group, ^$P < 0.05 vs. the DOX+PTS cotreated group.

Cell culture and siRNA transfection

The cardiomyoblast cell line H9c2 (Rattus norvegicus) was purchased from the American Type Culture Collection (ATCC, VA, USA). Cells were cultured in high glucose DMEM medium (Gibco, NY, USA), supplemented with penicillin-streptomycin solution (100 units/ml) (Solarbio, Beijing, China) and 10% fetal bovine serum (FBS, Gibco, USA) according to the providers’ instructions. The commercial rat AMPK siRNA, SIRT1 siRNA and control siRNA were obtained from Santa Cruz Biotechnology (CA, USA). When the H9c2 cells reached 40-60% confluence seeded in the 6-well plates, they were transfected with control or targeted siRNA transfection mixture via using the Lipofectamine 2000 (Invitrogen). Then, cells were cultured in fresh medium after 48 h transfection.

Cell treatments and in vitro experimental design

Pterostilbene, doxorubicin, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pterostilbene and doxorubicin stock solutions were maintained in DMSO and diluted in culture medium at certain concentrations for further experiments. The dosage of DOX was chosen based on previously described [4]. The scramble siRNA, AMPK siRNA or SIRT1 siRNA-treated cells were respectively assigned to 3 groups: (i) Control group; (ii) 1 μM DOX treatment group (DOX); (iii) 1μM DOX and 10 μM pterostilbene cotreatment group (DOX + PTS). After those treatments, cells were harvested for further experiments.

Animals and in vivo experimental design

The animal experiments were performed on healthy adult male C57BL/6 mice at 8 weeks of age; the mice were normally kept in a pathogen-free environment with free access to food and water. All experiments on mice were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (National Institutes of Health Publication No. 85-23, revised 1996). Compound C and EX527 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pterostilbene, doxorubicin, Compound C and EX527 were dissolved in DMSO and then diluted with normal saline. 75 mice were randomly assigned to five groups (n = 15 for each group): (i) sham-operated control group (Sham); (ii) DOX treatment group (DOX); (iii) DOX and pterostilbene cotreatment group (DOX + PTS); (iv) DOX, pterostilbene and Compound C cotreatment group (DOX + PTS + CC); (v) DOX, pterostilbene and EX527 cotreatment group (DOX + PTS + EX527). DOX was intraperitoneally injected at the dose of 10 mg/kg or the same volume of vehicle (saline) was conducted on day 1 and day 4 for a total of 2 times (20 mg/kg cumulative dose of DOX). Pterostilbene (10 mg/kg/day), Compound C (20 mg/kg/day), EX527 (5 mg/kg/day) or the same volume of vehicle was intraperitoneally injected every day for a total of 7 times (1 day before initial DOX treatment). All mice were euthanized 6 days after the initial injection of DOX. The dosages of DOX, pterostilbene, Compound C and EX527 were chosen based on the previous reports [4, 45, 46].

Cell viability analysis

After the different treatments on H9c2 cells for 24 h, the cell viability was detected via using the CCK-8 kit (7Sea, Shanghai, China) according to the previously described [47]. Optical density (OD) values were measured at 450 nm through the microplate reader (SpectraMax 190, Molecular Device, USA). Cell viability was presented as the ratio of OD values compared with the Control group, and representative images were taken by a 600D camera (Canon Company, Japan).

Cell mitochondrial membrane potential analysis

The H9c2 cells’ mitochondrial membrane potential (ΔΨm) was measured by the fluorescent dye JC-1 staining. After different treatments, cells were incubated with JC-1 working solution for 20 min in the dark at 37 °C. Cells exhibiting red fluorescence are in normal ΔΨm state. Green fluorescence represented the monomeric form of JC-1 and suggested ΔΨm depolarization. Images were obtained using the confocal microscope, and the results were analyzed by the SpectraMax 190 microplate reader, and expressed as the proportion of aggregated JC-1 and monomeric JC-1.

Cellular ATP content analysis

Cellular ATP concentrations were assayed with an ATP detection kit (Beyotime Institute of Biotechnology, China). H9c2 cells were lysed through the ATP assay buffer according to the instructions of the manufacturer. The level of ATP was measured by the emitted light of the mixture of the 50 μl cell lysis supernatant and 50 μl luciferase reagent using the microplate reader.

ROS, SOD and GPx determination

To analyze the oxidative stress parameters, the ROS level and the GPx and SOD activities in cultured cells and heart tissues were detected via using commercially available DCFH-DA (Beyotime Institute of Biotechnology, China), SOD and GPx assay kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. The data were analyzed by the SpectraMax 190 microplate reader (Molecular Device, USA).

Transmission electron microscopy

The ultrastructure of mitochondria of H9c2 cells and mouse heart samples were observed via using the transmission electron microscopy. After different treatments, cells were collected and fixed in 2% glutaraldehyde for 24 h at 4 °C, and then cells were diced into 2 mm cubes. 6 days after the initial injection of DOX, the mouse hearts were cut into 2 mm cubes placed in 2% glutaraldehyde at 4 °C for 24 h. Later, both the cell and tissue samples were cut into 75 nm thickness sections, then they were stained with uranium acetate and lead citrate; images were observed using a TECNAI G2 Spirit Biotwin 120 kV electron microscope.

Western blot and immunoprecipitation

After various treatments, the cells and heart samples were collected and lysed by the RIPA buffer; a BCA protein assay was used to analyze the protein concentration (Thermo Scientific, Rockford, IL, USA). As for immunoprecipitation, equal amounts of cell or tissue extracts were incubated with 2 ug anti-PGC1α (Santa Cruz, sc-518025) at 4 °C for 12 h on a rotating incubator, and then the samples were further incubated with the 20 μl Protein A/G Agarose (Beyotime Institute of Biotechnology, China) at 4 °C for 2 h rotation. Immunocomplexes were washed four times with NETN buffer before diluted with SDS-PAGE sample loading buffer. Then, the boiled protein samples of each group were fractionated by SDS-PAGE and transferred to PVDF membranes. Later, After blocked with 5% nonfat milk in TBST for 2 h at room temperature, the membranes were incubated with primary antibodies against AMPK (CST, #5831, 1:1000), p-AMPK Thr¹⁷² (CST, #50081, 1:1000), PGC1α (Abcam, ab54481, 1:1000), Ac-Lys (CST, #9441, 1:1000), NRF1 (Abcam, ab175932, 1:1000), UCP2 (Abcam, ab97931, 1:500), β-actin (Abcam, ab6276, 1:3000) and Tubulin (CST, #2148, 1:3000) for 12 h at 4 °C. Thereafter, the membranes were washed with TBST and exposed to the corresponding secondary antibodies (1:5000, Zhongshan Company, China) at room temperature for 2 h. The fluorescent signal was detected using a BioRad imaging system (BioRad, Hercules, CA, USA), and the signal was quantified using Image Lab Software (BioRad, Hercules, CA, USA).

Statistical analyses

IBM SPSS 23.0 (SPSS Inc., Chicago, USA) software was used for further data analyses. In this study, between-group comparisons, one-way ANOVA followed by Bonferroni post hoc test. Data are presented as the means ± SEM, and the P value < 0.05 was considered as statistically significant.