Dioscin ameliorates doxorubicin‐induced heart failure via inhibiting autophagy and apoptosis by controlling the PDK1‐mediated Akt/mTOR signaling pathway

Abstract

Heart failure (HF) is a disease with high mortality and morbidity rate. Autophagy is critically implicated in HF progression. The current research was designed to investigate the function of Dioscin on oxidative stress, autophagy, and apoptosis in HF. In this study, doxorubicin (Dox) was employed to induce HF model and HL‐1 cell damage model. Echocardiography implied that Dioscin could dramatically relieve heart function in vivo. Western blotting determined that Dioscin treatment reversed the promotive effect of autophagy caused by Dox through modulating levels of key autophagy‐associated molecules, including Atg5 and Beclin1. Dioscin also impaired apoptosis by regulating apoptosis‐related protein, including Bcl‐2 and cleaved caspase‐3 following Dox treatment in vivo and in vitro. Furthermore, the impacts of Dioscin were mediated by upregulation of PDK1‐mediated Akt/mTOR signaling. The mTOR inhibitor (rapamycin) could counteract the therapeutic impact of Dioscin in vitro. Taken together, Dioscin could relieve cardiac function through blocking apoptosis and autophagy by activating the PDK1‐elicited Akt/mTOR pathway.

1. INTRODUCTION

Heart failure (HF) is a global medical problem and complex clinical syndrome, which is the final stage of a wide range of cardiovascular diseases characterized by impaired heart capacity to contract or relax, and the leading cause of cardiac disease‐associated death. ¹ , ² It was demonstrated that HF was closely related to cardiac hypertrophy, dilated cardiomyopathy, hypertrophic cardiomyopathy, age, obesity, diabetes, and hypertension. ³ , ⁴ Even though the availability of effective treatments aimed at ameliorating HF, the 5‐year death rate from the disease is still close to 50%. ⁵ In the pathological process of HF, apoptosis leads to the reduction of cardiomyocytes and the decrease of myocardial contractility, thus boosting the occurrence and development of HF, confirming the vital function of apoptosis in HF. ⁶ Autophagy is a conserved system in eukaryotic cells to recycle damaged organelles and proteins, which plays a crucial function in maintaining cell homeostasis and survival. However, overactivation of autophagy has also been shown to be harmful. For instance, stimulated autophagy by myocardial reperfusion can lead to cardiac injury. ⁷ Similarly, autophagy elicited by doxorubicin (Dox) treatment in myocardium promoted the cardiotoxicity of Dox. ⁸ Thus, autophagy inhibition could be a potential strategy for the treatment of Dox‐triggered cardiomyopathy. Notably, Dox was widely used to produce animal and cellular models of HF. ⁹ , ¹⁰ Therefore, a thorough understanding of the mechanism of cardiomyocyte apoptosis and autophagy in Dox‐induced HF is imperative to develop effective treatment methods and evaluate the prognosis of HF patients.

Phosphoinositide dependent protein kinase‐1 (PDK1) is a serine/threonine kinase belonging to the AGC kinase family. It was reported that PDK1 plays a fundamental function in the PI3K‐PDK1‐Akt pathway. ¹¹ Moreover, PI3K activation resulted in the synthesis of phosphatidylinositol‐3,4,5‐triphosphate. ¹² Afterward, PDK1 and Akt are recruited to the plasma membrane, and then the membrane‐bound PDK1 phosphorylates Akt at Thr308, resulting in activating Akt, which was involved in cardiovascular pathological processes such as atherosclerosis, myocardial hypertrophy, and vascular remodeling. ¹³ , ¹⁴ Recently, Zhu et al. manifested that depletion of Mirt2 aggravated ischemia‐triggered myocardial infarction and cardiomyocyte apoptosis through reducing PDK1. ¹⁵ Gao et al. elucidated that HTR2A enhanced phosphorylations of PDK1 to activate AKT‐mTOR signaling and promoted cardiac hypertrophy. ¹⁶ Furthermore, it was revealed that deficiency of PDK1 in cardiac muscle results in HF and increased sensitivity to hypoxia. ¹⁷ This research explored the impact of PDK1 on Dox‐induced HF.

Dioscin is a kind of steroid sapogenin synthesized naturally by plants, belonging to spirosterol class. As an essential raw material for the synthesis of steroid hormones and steroidal contraceptives, Dioscin was commonly employed for the production of pregnenolone, progesterone, and cortisol drugs. ¹⁸ In the past few decades, pharmacological tests have implied that Dioscin has the effects of antitumor, modulating blood lipids, preventing platelets aggregation, and accelerating bile secretion; thus, it is mainly employed to treat cardiovascular disease, encephalitis, and cancers. ¹⁹ For instance, Dioscin suppressed coronary heart disease via decreasing oxidative stress by modulating p38 MAPK signaling. ²⁰ What is more, Dioscin was reported to treat myocardial infarction by promoting angiogenesis. ²¹ However, whether Dioscin protected the heart from damage remains unclear.

In this work, the effects of Dioscin on autophagy and PDK1/Akt/mTOR signaling were explored by in vivo and in vitro experiments. This research will offer a prospective therapeutic target and prognostic indicator for HF patients.

2. MATERIALS AND METHODS

2.1. Animals and HF model establishment

All of the animal experimental protocols were subject to approval by Jiangsu Province Hospital of Chinese Medicine (Approval No.: NU(2020) 3A‐152). Sixteen male C57/BL6 mice (8 weeks old) were gained from the Laboratory Animal (Nanjing).

HF model in mice was constructed using Dox. ²² Dox (cat. no. 25316‐40‐9; MilliporeSigma) was dissolved in saline to generate a stock solution of 100 μM and was diluted to a final concentration of 2 μM for all experiments unless otherwise specified. Briefly, following 7 days acclimation, mice were randomly divided into the Control (control, n = 8) and Dox group (HF group, n = 8). Mice in the HF group were intraperitoneally of 2.5 mg/kg injected with Dox six times in 2 weeks, and then were treated with 40 mg/kg of Dioscin for seven consecutive days by oral gavage. ²³ Dioscin (purity >98%) was purchased from Spring & Autumn Biological Engineering Co., Ltd. (Nanjing, China) All mice in control groups were treated with 0.5% CMC‐Na for seven consecutive days. Finally, the electrocardiograms of mice were detected before the animals were sacrificed. Mice were sacrificed under anesthesia to relieve pain. Heart tissues were gathered at −80°C for further analysis.

2.2. Echocardiographic assessment

Mice were anesthetized with 1% of the pentobarbital sodium with a dose of 50 mg/kg. The left ventricular systolic pressure (LVSP), left ventricular end‐diastolic pressure (LVEDP), and ±dp/dt max parameters of mice were assessed by the hemodynamic method.

2.3. Cell culture, transfection, and in vitro experimental design

Mouse myocardial cell line (HL‐1) was purchased from American Type Culture Collection (ATCC, Manassas, USA), cultured in the DMEM medium containing 10% FBS (Hyclone, USA), 100 μg/mL of streptomycin, and 100 U/mL of penicillin (Hyclone, USA), and incubated at 37°C with 5% CO₂. When the cells were grown to 70% confluence, the knockdown vector of PDK1 (shPDK1) and control vectors (shNC) were transfected using Lipofectamine 2000 (Invitrogen, USA). Cells were harvested after 48 h transfection for further experiments. To examine the role of Dioscin in myocardial injury cell model, HL‐1 cells were pretreated with 100 ng/mL Dioscin for 24 h before challenged with Dox (5 μM) for 24 h. ²³

2.4. Histopathology analysis

For HE staining, the heart tissues were fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) and then cut into 0.4 μm slices after staining with H&E. The pathological alterations were observed under a microscope. For Immunohistochemistry, the heart sections were gathered and incubated with the antibody according to previously reported recommendations. ²⁴

2.5. Biochemical analysis

The levels of the lipid peroxidation biomarker malondialdehyde (MDA) and the activity of antioxidant enzymes superoxide dismutase (SOD) were determined using an appropriate commercial kit (Bio‐Diagnostics Co, Egypt).

2.6. Western blotting

Total proteins were exacted from mouse heart tissues and myocardial cells with RIPA buffer (Beyotime). Subsequently, the proteins were separated by SDS‐PAGE, transferred onto PVDF membranes, and then blocked with 5% nonfat milk at 37°C for 1 h. Thereafter, the membranes were probed with primary antibodies p‐PDK1^Ser241 (1:500, ab131098, abcam), PDK1 (1:1000, ab202468, abcam), cleaved caspase 3 (1:1000, ab32042, Abcam), Bcl‐2 (1:1000, ab182858, Abcam), Beclin1 (1:1000, ab207612, Abcam), Atg5 (1:100, ab109490, Abcam), p‐Akt (1:500, ab38449, Abcam), p‐mTOR (1:1000, ab109268, Abcam), Akt (1:500, ab8805, Abcam), mTOR (1:500, ab32028, Abcam) and GAPDH (1:1000, 2803, CST) at 4°C overnight and secondary antibodies for nearly 2 h. Protein bands were visualized with ECL kit (Pierce) and quantified by Image J software.

2.7. Flow cytometry

HL‐1 cells were rinsed with cold PBS and then re‐suspended in Annexin V Binding Buffer. Next, HL‐1 cells were added with FITC‐Annexin V and PI (BD Pharmingen). Thereafter, data were assessed via flow cytometry with FACScalibur flow cytometer (Becton Dickinson).

2.8. Statistical analysis

The GraphPad Prism 7 software was employed for statistical analysis. The data were expressed as mean ± SD from at least three times of experiments independently. One‐way ANOVA followed by Tukey's post hoc test was used to compare statistical differences among various groups. p < 0.05 was deemed significant in statistics.

3. RESULTS

3.1. The effect of Dioscin on the cardiac function and structural changes in Dox‐induced HF mice

First, echocardiography revealed that HF model in mice was established successfully, evidenced by the obvious reduction in the level of LVSP and enhancement in the level of LVEDP in Dox‐induced HF mice compared with the control mice. Then, after Dioscin treatment, LVSP level was heightened, and LVEDP was reduced (Figure 1A,B). HE staining displayed that the left ventricular myocardial cells in the control group were arranged orderly, while those in the HF group were arranged loosely. The pathological changes were counteracted by Dioscin introduction (Figure 1C). These results expounded that Dioscin alleviated Dox‐induced cardiac damage in HF mice.

FIGURE 1.

Effect of Dioscin on the cardiac function and structural changes in DOX‐induced HF mice. (A,B) Echocardiographic analysis showed the effect of Dioscin on LVSP and LVEDP values. (C) HE exhibited that cardiomyocyte architecture in mice treated with Dox or Dox + Dioscin. *p < 0.05; **p < 0.01. N = 3 per group.

3.2. Dioscin suppresses Dox‐induced apoptosis and excessive autophagy in HF mice model

The levels of key molecules in the apoptotic pathway including Bcl‐2 and Cleaved‐caspase‐3 were examined via western blotting. As depicted in Figure 2A, the levels of pro‐apoptotic protein (cleaved caspase 3) were augmented, whereas the anti‐apoptotic protein (Bcl‐2) was lessened in Dox‐elicited HF mice, suggesting that the apoptotic signaling was activated. However, treatment with Dioscin reversed the levels of these proteins toward normal levels, suggesting that Dioscin had an anti‐apoptotic effect on HF mice model. Accumulating evidence has implied that autophagy dysfunction is the main cause of cardiac fibrosis. ²⁵ To further elucidated the role of autophagy in HF mice model, protein expression of classical autophagic markers including Beclin1 and Atg5 was measured by Western blotting. Results insinuated that protein levels of Beclin1 and Atg5 were elevated in Dox‐elicited cardiac tissue. Meanwhile, Dioscin treatment downregulated the levels of Beclin1 and Atg5, implying that Dioscin could attenuate autophagy (Figure 2B). These results expounded that autophagy in the Dioscin‐treated mice was suppressed compared with that in Dox administration mice, which thereby suggested that autophagy could be a key regulatory mechanism in the protective impact of Dioscin.

FIGURE 2.

Dioscin suppresses Dox‐induced apoptosis and excessive autophagy in HF mice model. (A) Western blotting detected the level of Bcl‐2, cleaved caspase 3 in mice treated with Dox or Dox + Dioscin. (B) Protein expression of Beclin1 and Atg5 were analyzed. *p < 0.05; **p < 0.01. N = 3 per group.

3.3. Dioscin protects HL‐1 cells from injuries caused by Dox

To further uncover the potential target of Dioscin on HF, the Dox‐stimulated HL‐1 cell model was established. Flow cytometry assay showed that the apoptosis augmented by Dox stimulation, which was substantially lessened by Dioscin (Figure 3A). Besides, Dioscin abrogated the enhancement of cleaved caspase 3 levels and reduction of Bcl‐2 induced by Dox (Figure 3B). Detection of MDA and SOD levels in HL‐1 cells demonstrated that Dioscin had an anti‐oxidant effect in vitro (Figure 3C,D). Taken together, Dioscin ameliorated Dox‐induced cell apoptosis and oxidative stress in HL‐1 cells.

FIGURE 3.

Dioscin protects HL‐1 cells from the injuries caused by Dox. HL‐1 cells were grouped into Control, Dox, and Dox + Dioscin groups. (A) Cell death was detected using flow cytometry assay. (B) Protein levels of cleaved caspase 3 and Bcl‐2. (C,D) Levels of MDA and SOD. *p < 0.05; **p < 0.01. N = 3 per group.

3.4. Dioscin activates Akt/mTOR signaling via PDK1

Subsequently, the mechanism underlying the effect of Dioscin was studied. It was displayed that the protein levels of PDK1, p‐Akt, and p‐mTOR were decreased following Dox treatment, which was neutralized by Dioscin. To confirm whether Dioscin regulated HF progression via the PDK1/Akt/mTOR pathway, PDK1 was silenced in HL‐1 cells. Western blotting showed that PDK1 deficiency reversed the promotion of Dioscin on p‐PDK1 (Ser241), p‐Akt, and p‐mTOR levels in Dox‐induced HL‐1 cells, suggesting that PDK1 activated the Akt/mTOR signaling (Figure 4A). Next, rapamycin (inhibitor of mTOR) was introduced. Similarly, the p‐Akt, and p‐mTOR protein level was decreased in Dox + Dioscin + rapamycin group compared with that in Dox + Dioscin group. In the meantime, the total Akt and mTOR protein levels were not affected. These results implicated that Dioscin could upregulate the activity of PDK1/Akt/mTOR pathway in Dox‐elicited HL‐1 cells.

FIGURE 4.

Dioscin activates Akt/mTOR signaling via PDK1. (A) The protein expression of p‐PDK1 (Ser241), p‐Akt, p‐mTOR, PDK1, Akt, and mTOR in HL‐1 cells in Control, Dox, Dox + Dioscin, and Dox + Dioscin + shPDK1 and Dox + Dioscin + rapamycin groups was detected. *p < 0.05. N = 3 per group.

3.5. Dioscin prevents cell apoptosis and autophagy in vitro through PDK1‐mediated Akt/mTOR signaling

It was elaborated that PDK1, as a target protein, played an essential function via the Akt/mTOR pathway in the processes of autophagy and apoptosis. ²⁶ The biological role of PDK1‐mediated Akt/mTOR signaling in HF progression was evaluated. As exhibited in Figure 5A, the suppressive effect of Dioscin on the apoptosis of HL‐1 cells under Dox stimulation was abated by PDK1 deficiency or rapamycin. Consistently, PDK1 knockdown attenuated the effects of Dioscin on the levels of cleaved caspase 3 and Bcl‐2, MDA and SOD. Similarly, inactivation of the Akt/mTOR signaling has same effects as PDK1 depletion (Figure 5B–D). Furthermore, western blotting showed that rapamycin or shPDK1 abrogated the effects of Dioscin on Beclin1 and Atg5 (Figure 5E). In sum, Dioscin prevented HL‐1 cells from Dox‐induced damages via activating the PDK1/Akt/mTOR pathway.

FIGURE 5.

Dioscin prevents cell apoptosis and provokes autophagy in vitro through PDK1‐mediated Akt/mTOR signaling. Cells were divided into Dox + Dioscin, Dox + Dioscin + shPDK1, and Dox + Dioscin + rapamycin groups. (A) The apoptosis of designated cells was detected. (B–D) The levels of cleaved caspase 3, Bcl‐2, MDA, and SOD levels. (E) Protein expression of Beclin1 and Atg5. *p < 0.05; **p < 0.01. N = 3 per group.

4. DISCUSSION

The present study provided evidence that Dioscin ameliorates Dox‐induced HF via inhibiting autophagy and apoptosis via in vivo and in vitro studies. It has been widely recognized that estradiol decreases the risk for cardiovascular disease, but estrogen replacement therapy is limited for its side effects. ²⁷ Dioscin, a natural steroid saponin isolated from the root bark of wild dioscorea nipponica, is currently widely employed for cardiovascular disease treatment. ²⁸ It was implied by Kong that Dioscin alleviated hypoxic‐caused cardiac dysfunction in myocardial infarction via elevating the level of lncRNA MANTIS. ²⁹ Dioscin improved myocardial infarction damage through adjusting BMP4/NOX1‐mediated inflammation. ³⁰ In addition, Dox was reported as an effective and commonly used to treat hematological and solid tumors. ³¹ However, Dox can lead to multiple‐organ toxicity in patients. As an anticancer agent, Dox can lead to long‐term dose‐dependent cardiotoxicity and HF. ³² This research elaborated that Dioscin protected heart function in Dox‐induced HF mice model and HL‐1 cell model, evidenced by improved LVSP and reduced LVEDP. The findings of this study indicated that Dioscin could suppress Dox‐induced HF and provided a new therapeutic method for HF.

Next, we further excavated the influences and mechanisms of Dioscin on autophagy. It was insinuated that autophagy is a dynamic process that participated in the concerted action of autophagy‐related genes, including Beclin‐1, Atg5, and LC3. ³³ Multiple studies proposed that disordered and reduced autophagy was involved with HF progression. Moxibustion impaired chronic HF via repressing autophagy by elevating mTOR expression. ³⁴ Rutin relieved Dox‐induced HF by reducing excessive autophagy and apoptosis. ³⁵ Previous studies disclosed that Dox‐triggered cardiotoxicity was implicated in the dysregulation of autophagy. Moreover, Dox administration promoted myocardial autophagy, which was closely related to damage to cardiac function. ³⁶ Excessive autophagy can induce cell death, which is called autophagic cell death. Several researches have elaborated that the suppression of Dox‐triggered excessive autophagy has vital beneficial impacts on cardiac damage in vivo. ³⁷ Herein, autophagy was activated by Dox in cardiac tissue, and Dioscin may provide protection by restraining excessive autophagy. Further, it was implied that Dioscin suppressed Dox‐induced apoptosis and excessive autophagy in HF mice model, and ameliorated Dox‐induced cell apoptosis and oxidative stress in HL‐1 cells. These findings unveiled that Dioscin protected against myocardial cell damage by repressing apoptosis and autophagy.

Subsequently, we further verified the mechanism of Dioscin in regulating autophagy and PDK1/Akt/mTOR signaling pathway in HF progression. It was illustrated that PDK1 was closely implicated in myocardial cell injury via the Akt/mTOR pathway in myocardial cell injury. To cite an instance, PDK1 has been well established to activate Akt/mTOR signaling, and deficiency of PDK1 triggered severe cardiomyopathy and aberrant electrophysiology. ³⁸ Moreover, LDHA might have effects on ischemia‐reperfusion injury and accelerate cardiomyocyte proliferation by affecting the expression of PDK1, Akt, and mTOR. ³⁹ Herein, it was demonstrated that Dox does not inhibited the p‐PDK1/PDK1 ratio, but directly inhibited the PDK1 protein. Moreover, Dioscin treatment partially reversed the PDK1 level decreased by Dox. Furthermore, rapamycin or shPDK1 reversed the activation of Dioscin on PDK1 and Akt/mTOR signaling in Dox‐elicited HL‐1 cells. Besides, the effects of Dioscin on Dox‐stimulated HL‐1 cell apoptosis, oxidative stress, and autophagy‐associated proteins Beclin1 and Atg5 were offset after co‐incubation with rapamycin or PDK1 silence, indicating that the mechanism of Dioscin in HF is mediated by PDK1/Akt/mTOR signaling.

In conclusion, the function and mechanism of Dioscin have been investigated, and our work demonstrated Dioscin exerted cardioprotective effect through inhibiting Dox‐induced apoptosis and excessive autophagy via regulating the PDK1/Akt/mTOR pathway. These discoveries provide a theoretical basis for studying the pharmacological mechanism of Dioscin and a novel therapeutic strategy for HF.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Yuan L, Ji H‐G, Yan X‐J, Liu M, Ding Y‐H, Chen X‐H. Dioscin ameliorates doxorubicin‐induced heart failure via inhibiting autophagy and apoptosis by controlling the PDK1‐mediated Akt/mTOR signaling pathway. Kaohsiung J Med Sci. 2023;39(10):1022–1029. 10.1002/kjm2.12740