Background
Doxorubicin (DOX) is one of the most commonly used chemotherapeutic drugs for a wide range of cancers, such as leukemia, soft tissue sarcomas and breast cancer [
1]. However, the rate of DOX-induced cardiotoxicity (DIC) is reported to be as high as 57%, and the mortality rate from these heart diseases is reported to be 8.2 times higher than that in healthy people [
2]. Currently, dexrazoxane is the only FDA approved drug for the protection against cardiotoxicity. However, the Committee for Medicinal Products for Human Use (CHMP) in the UK has published the outcome of a referral that recommends several restrictions on dexrazoxane use in both children and adults with cancer. Therefore, there is an urgent need to identify the underlying mechanism of DIC and novel therapeutic agents that can prevent and/or reverse DOX-induced cardiovascular adverse effects. Recently, inflammation induced by doxorubicin has been identified as a high risk factor for developing heart failure and drugs with anti-inflammatory properties are attractive therapeutics for alleviating DIC [
3,
4].
Nuclear factor-κB (NF-κB)-mediated inflammatory pathway is a classic signaling cascade that controls the expressions of pro-inflammatory genes in multiple types of cells. In the resting state, inactive NF-κB is sequestered in the cytoplasm by IκB (inhibitor of NF-κB). Under stimulation of pro-inflammatory signals, IKKs (IκB kinases) are activated which phosphorylates IκB on serine residues. Phosphorylated IκB will be degraded and active NF-κB dimers will be released and translocated into nucleus, inducing expressions of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin 8 (IL-8). Several studies have confirmed that levels of inflammatory cytokines are increased in myocardial cells under doxorubicin-stimulated condition [
5,
6]. Furthermore, hyper-activation of NF-κB is implicated in inflammatory responses in DIC [
5,
7]. Nevertheless, the regulatory mechanisms of NF-κB transcriptional activity in DIC are still unclear.
The mammalian target of rapamycin (mTOR) is an atypical serine/threonine kinase, which regulates physiological homeostasis at the cellular and holistic level. Transcription factor EB (TFEB) plays a master role in governing basic cellular processes [
8]. The mTOR-mediated phosphorylation negatively regulates TFEB nuclear translocation and activity [
9]. It is of note that rapamycin analogues are therapeutically used as immuno-suppressants, and TFEB was also validated to have potential anti-inflammatory effect [
10]. In this study, we further investigated whether mTOR-TFEB pathway plays a role in DIC-related inflammation. A profound understanding of this mechanism will provide a basis for discovery of novel targets as well as the therapeutic agents in alleviating DIC.
Salvia miltiorrhiza Bunge has been widely applied in traditional Chinese medicine (TCM) for a long history in the treatment of various inflammatory diseases [
11]. As a dietary supplement, it is the first TCM to be documented in USP 37-NF32. Dihydrotanshinone I (DHT) is the one of major active diterpenes in the liposoluble extract [
12]. Studies have shown that DHT possesses a variety of anti-tumor and anti-inflammatory properties [
13‐
16]. However, whether DHT could alleviate DIC and whether the protective effect is medicated by the anti-inflammatory pathway remain elusive. In this study, we established an in vitro DOX-stimulated H9C2 model and an in vivo DIC mice model. The anti-inflammatory mechanism of DHT was investigated. This study will provide insight into the anti-inflammatory strategies and drug combination therapy in the management of DIC.
Methods
Reagents and chemicals
Saline was purchased from SiYao Co., Ltd. (Shijiazhuang, China). Doxorubicin was bought from ApexBio Technology LLC (Houston, TX, United States). DHT was purchased from Shanghai Shidande SDHTdard Technical Service Co., Ltd. (Shanghai, China). Rapamycin was purchased from Sangong Pharmaceutical Co., Ltd. (Shanghai, China). 4% paraformaldehyde was from Beijing Applygen Technology Inc. (Beijing, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich LLC (Shanghai, China). PDTC was purchased from Abmole China Branch. MHY1485 was purchased from Selleck (Houston, TX, United States). Dulbecco’s Modification of Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin, streptomycin, 0.1 mol/L sodium cacodylate buffer, sodium carboxymethylcellulose and DAPI were purchased from Beijing BioDee Biotechnology Co., Ltd. (Beijing, China). GFP-TFEB lentiviral vector were bought from Hanbio Technology Co., Ltd. (Shanghai, China). All other chemicals were purchased from commercial sources.
Establishment of DIC model in Zebrafish, in mice and pharmacological treatments
The DIC model in zebrafish was established according to the methods described in a previous study [
17]. Briefly, after the heart had formed and circulation had begun 1 day postfertilization (dpf), zebrafish were treated with 100 μM DOX or/and 10 nM DHT, and phenotypic changes, including fraction shortening (FS), the blood speed of the tail vein, heart rate and survival rate were respectively assessed at three dpf.
C57BL/6 male mice (18 g ± 2 g) were purchased from Beijing SPF Biotechnology Co., Ltd., China (Beijing, China). All mice were housed at temperature 22 ± 2 °C, with proper humidity, lighting (12 h light/12 h dark cycle), and free access to food and water. All mice were randomized into four groups as follows: 16 mice in the DOX group, DHT-treated group or positive drug (Rapamycin, Rapa)-treated group, respectively; 10 mice in Saline group. DIC model was induced via tail vein injection with DOX (5 mg·kg
− 1) once weekly for 4 weeks and sham group was given with saline (0.9% NaCl) at the same time. This mice model has been described in previous study [
18]. Besides, as the positive control drug, Rapamycin, an inhibitor of mTOR, was widely reported to be able to alleviate cardiac inflammation [
19,
20]. Each drug was orally and daily administered for 4 weeks starting on 1 week after the last DOX injection. The doses of DHT (20 mg·kg
− 1) and Rapamycin (2.81 mg·kg
− 1) were determined based on previous studies [
21,
22]. Given that DHT and Rapamycin are both poorly soluble in water, we applied 0.5% aqueous solution of a sodium carboxymethylcellulose (CMC) as the suitable vehicle as previous literature described. And the Saline group and the DOX group received the same volume of vehicle. All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publications No.85–23) and with Beijing University of Chinese Medicine Animal Care Committee (approve code BUCM-4-2,018,101,504-4068). After study, all mice were anaesthetized by isoflurane inhalation 2% and then euthanized by cervical dislocation.
Echocardiographic assessment of cardiac functions
After 4 weeks’ administration, cardiac function was examined by Transthoracic echocardiography (Vevo TM 2100; Visual Sonics, Canada). Left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD) were assessed for at least three uninterrupted cardiac cycles. Then mice were sacrificed, heart and blood were collected.
Histological examination
For histological analysis, hearts were immersed in 4% paraformaldehyde for at least 24 h, then were embedded in paraffin and cut into 4 μm serial slices. Paraffin sections were stained with Hematoxylin-Eosin (HE) and were monitored under an optical microscope at 400 × magnification.
Malondialdehyde (MDA) assay and superoxide dismutase (SOD) assay
MDA and SOD content commonly reflect the level of oxidative stress. Evidences showed that oxidative stress and inflammation are regarded as essential partners presenting simultaneously and interact with each other in diverse pathological conditions. MDA content and SOD activity in plasma were detected by following the instructions of commercially available kits (Jiancheng, Nanjing, China). MDA level was expressed as nmol/L in plasma. SOD activity was expressed as U/mL in plasma.
Immunochemistry
The slices were dewaxed with xylene and gradient alcohol hydration, then were added with drops of 3% H2O2 for resting 15 min to block endogenous peroxidase. After washing with phosphate buffered saline (PBS) for three times, the cartilage slices were added with heated sodium citrate buffer for microwaving 6 min to antigen repair, which was repeated twice. After cooling and washing with PBS, the cartilage slices were treated with 5% goat serum for 10 min, to obstruct the binding of nonspecific antibody, then added with drops of the first antibodies of TNF-α. Later the slices were incubated at 37 °C for about 30 min and washed with PBS. After reacting with the second antibody, the slices were incubated at 37 °C for another 30 min and washed with PBS, later followed by staining for 10 min with diaminobenzidine (DAB), washing with running pure water, re-staining with hematoxylin and mounting with neutral balsam. Finally, we observed the images under a microscope and picture-taking.
Flow cytometry
Flow cytometry was performed referring to literature concerned [
23]. Hearts from mice (three mice per group) were minced into small pieces around 1 mm
3 followed by digestion with 40% collagenase II (17101–015; Invitrogen, CA, United States) and 0.25% Trypsin (17104–019; Invitrogen, CA, United States) for 10 min. To disperse solid tissue into single cells, the enzymes (mainly collagenase and protease) were used to digest collagen fibers and elastic fibers, and to hydrolyze proteins and mucopolysaccharide substances that tight junction structure of tissue cells [
24,
25]. Then cells were put into centrifuge at a force of 300 g for 5 min. After washing two times with PBS, cells were resuspended with 200 μL PBS, then stained with 0.75 μg anti-CD11b, 1 μg anti-F4/80, 1 μg anti-CD86 and 1 μg anti-CD206 antibodies. Followed by examining on a FACS Canto II flow cytometer (BD Biosciences). Data were analyzed by using the FlowJo software (FlowJo, LLC, Ashland, OR, USA). The antibodies were listed in Table
1.
Table 1
Antibodies used in Flow cytometry
PE-Cy7 Rat Anti-CD 11b Clone M1/70 | 561,098; BD PMG, United States |
F4/80 (BM8.1) Rat mAb (APC Conjugate) | 86,007; Cell Signaling Technology, Germany |
PE anti-mouse CD86 | 105,105; BioLegend, United States |
FITC anti-mouse CD206 (MMR) Antibody | 141,703; BioLegend, United States |
Establishment of LPS-induced RAW264.7 cell model and pharmacological treatments
RAW 264.7 macrophages in our study were obtained from China Infrastructure of Cell Line Resources. RAW 264.7 macrophages were cultured in DMEM supplemented with 10% FBS at 37 °C in a humidified atmosphere (5% CO2 and 9% O2). To evaluate the effects of DHT on LPS-induced RAW264.7, cells were stimulated with LPS (1 μ g/mL) for 24 h with or without DHT (added to media 2 h before treating with LPS). The concentrations of DHT in culture media in different cell groups were 10, 50 and 100 nM, respectively.
Detection for TNF-α in the supernatant
Cell supernatants were collected from LPS-induced RAW 264.7 (with or without DHT pretreatment). The concentrations of TNF-α and IL-1β in the supernatants were determined by using ELISA kits bought from Boster Biological Technology co.ltd and the manufacturer’s instructions were followed. TNF-α and IL-1β levels were expressed as pg/mL.
Establishment of DOX-stimulated H9C2 cell model and pharmacological treatments
H9C2 cells in our study were obtained from China Infrastructure of Cell Line Resources. The establishment of DOX-stimulated H9C2 cell model has been mentioned in our previous study [
26]. For pharmacological treatments, we set up various groups: Control group, DOX group (with DOX 1 μM), DOX + DHT group (with DHT 10 nM), DOX + DHT + MHY1485 group (with DHT 10 nM and MHY1485 5 μM) and DOX + PDTC group (with PDTC 100 nM). For western blot analysis, H9C2 cells were cultured in 10 cm petri dishes. MHY1485, an agonist of mTOR, was applied to verified whether DHT can inhibit mTOR, sequentially lead to downregulation of NF-κB-mediated inflammatory response. PDTC, a NF-κB inhibitor, was set as the positive drug in vitro experiment.
GFP-TFEB lentiviral vector transfection
The specific methods have been described in our previous study [
26].
Assessment of apoptosis by a Hoechst 33258 staining kit
H9C2 cells were fixed with ice-cold 4% paraformaldehyde for 20 min after washed with 0.1 mol/L sodium cacodylate buffer. Next, the cells were washed three times with 0.1 mol/L sodium cacodylate buffer before stained with Hoechst 33258 (Beijing Solarbio Science & Technology Co., Ltd., China) for 15 min in the dark. Finally, an inverted fluorescence microscope (Olympus; BX50-FLA; Japan) was applied to visualize the apoptotic cells.
Immunofluorescence
Prior to incubation with primary antibody Cleaved caspase-3 (9664; Cell Signaling Technology, Germany) in a diluted concentration of 1:150 at 4 °C overnight, the paraffin-embedded sections need to be deparaffinized, inactivated with 0.3% hydrogen peroxide for 15 min and blocked with normal goat serum for 10 mins at room temperature, and then incubated with secondary antibody goat anti-rabbit IgG polyclonal (ab15007; Abcam, United States) for one hour at room temperature and dark place, followed by DAPI staining at room temperature for 5 min in the dark. Finally, sections were washed and fixed with antifade mounting medium. The optical microscope was used for photographing at 400 × magnification (Leica Microsystems GmbH).
H9C2 cells were grown on a laser confocal dish for the specified time, and then fixed with 4% paraformaldehyde for 15 min, followed by permeabilization (0.5% Triton X–100 in 0.1 mol/L sodium cacodylate buffer) for 20 min and blocked with normal goat serum for one hour. Later, cells were incubated with primary antibody overnight at 4 °C, followed by incubation with secondary antibody in the dark at room temperature for one hour. After being washed 3 times with PBS, cells were counterstained with DAPI (5 μg/mL) for 30 min. Images were then taken with a confocal microscopy.
Western blot analysis
Protein samples were prepared and detected according to our previously described methods [
26]. The antibodies we used were listed in Table
2.
Table 2
Primary antibodies used in western blot
p-NF-κB | ab97726; Abcam, United States |
NF-κB | CST8242; Cell Signaling Technology, Germany |
TNF-α | ab205587; Abcam, United States |
IL-8 | ABM40268; Abbkine; United States |
COX2 | ab15191; Abcam; United States |
p-IKKα/β | CST2697T; Cell Signaling Technology, Germany |
IKKα | CST2682; Cell Signaling Technology, Germany |
IKKβ | CST8943; Cell Signaling Technology, Germany |
mTOR | CST2983; Cell Signaling Technology, Germany |
p-mTOR | CST2971; Cell Signaling Technology; Germany |
S6K1 | ab9366; Abcam, United States |
p-S6K1 | ab2571; Abcam, United States |
GAPDH | ab8245; Abcam, United States |
rabbit IgG H&L | ab16284; HRP; Abcam, United States |
mouse IgG H&L | ab97250; HRP; Abcam, United States |
TFEB | 13,372–1-AP; Proteintech; United States |
Data and statistical analysis
Statistical analyses were performed on GraphPad Prism software 6.0 (San Diego, CA, USA). All results were expressed as the mean ± SD. Comparisons between two groups were performed with the unpaired two-tailed t-test. Multiple comparisons were analyzed using ANOVA followed by Bonferroni-corrected post hoc test. The difference was considered statistically significant when
P < 0.05. The statistical analysis abided the recommendations of the experimental design and analysis in pharmacology [
27].
Discussion
In the present study, in vivo and in vitro experiments were conducted to explore the protective effects of DHT against DIC and its underlying anti-inflammation mechanisms. The main findings are as follows: (1) DHT could improve cardiac functions evidenced by inhibiting the activation of M1 macrophages and excessive release of pro-inflammatory cytokines both in vivo (mouse and zebrafish) and in vitro (H9C2 and RAW264.7); (2) DHT could inhibit NF-κB-mediated inflammatory response via mTOR-TFEB pathway; (3) The TFEB-IKK-NF-κB axis plays a vital role in regulating DIC-related inflammation.
A growing body of evidence supports that DOX, as an effective chemotherapy drug, is a double-edged sword [
32]. Indeed, irreversible injury to nontarget tissues often complicates cancer care by limiting therapeutic dosages of DOX and lowering the quality of patients’ life during and after DOX treatment [
33]. Particularly, the heart is a preferential target of DOX and cardiotoxicity is one of the most severe side effects induced by DOX. Alterations in the administration of the drug, applications of liposomal DOX, and assistants with cardio-protective agents are the strategies that have been applied for the prevention or alleviation of cardiotoxicity. However, drugs that are commonly adopted to reduce DIC often have adverse effects. It is of great importance to explore novel therapeutic strategies with fewer side effects. A correlation between inflammation and adverse cardiovascular outcomes in DOX induced toxicity has been documented [
34,
35]. Elevation of pro-inflammatory cytokines has been linked with worse DIC outcome in multiple studies [
5]. Application of Chinese herbs that are able to mitigate inflammation without compromising the anti-tumor effect is of particular interest for the treatment of DIC.
Salvia miltiorrhiza Bunge is a well-known traditional herb with a long history of clinical application for the treatment of cardiovascular diseases [
36,
37]. Its active ingredients have been widely investigated since last century [
38]. For a long time, investigations into the bioactivities of DHT were mainly focused on cytotoxicity to various tumor cells [
16,
39]. Emerged evidence pointed out that DHT has potential efficacy for curing cardiovascular diseases [
40‐
42], however its anti-DIC effect, either in vivo or in vitro, has not been elucidated yet. The results of our study demonstrated that DHT treatment improved heart function in a DIC zebrafish model and in a DIC mouse model. It’s reported that DHT could play a therapeutic role in various inflammatory diseases, including atherosclerosis, allergic inflammation and colitis [
43,
44]. Besides, DHT could modulate immune cell function, such as suppression of the release of cytokines [
45]. To date, there have been no researches on the anti-inflammatory of DHT during application of DOX. Recruitment and activation of M1 macrophages have been reported to play a major role in DIC-related inflammation [
34,
35]. Our in vivo
and in vitro results showed that DHT could suppress accumulation of macrophages and activation of M1 macrophages under DOX-stimulation. Although it is still unclear as to the origin of heart macrophages, recent studies have suggested that these macrophages are derived from either the proliferation of resident macrophages or the differentiation of blood monocytes [
46]. The in vitro results showed that expression of NF-κB and secretion of pro-inflammatory cytokines by macrophages were also inhibited by DHT. These data demonstrated that DHT could suppress inflammation by inhibiting activation of macrophages. Although pro-inflammatory cytokines are generally produced by activated macrophages, myocardial cells can also produce inflammatory agents through NF-κB-dependent pathway under pathological conditions. It’s noteworthy that NF-κB-mediated inflammatory response has been demonstrated as a pivotal pathway in DIC model [
47,
48]. The involvement of pro-inflammatory cytokines driven by the activation of NF-κB can lead to the severe myocardial injury manifested by the dramatic reduction of the heart function [
6,
49,
50]. Herein, the NF-κB pathway is believed to be one of the most attractive targets for DIC patients [
48]. In current study, both in vivo and in vitro data showed that DHT suppressed cardiac levels of activated NF-κB as well as downstream inflammatory genes, including TNF-α, IL-8 and COX2 under DOX stimulation. The effect of DHT on the upstream regulative pathway was further investigated.
The mTOR protein is a serine/threonine kinase that regulates a variety of cellular functions. Update studies suggest that it is also an important regulator of inflammation responses. A number of studies have indicated that pharmacological inhibition of mTOR can provide anti-inflammatory protection [
20,
30,
51]. Rapamycin is a specific inhibitor of mTOR and was applied as positive control drug in this study. Intriguingly, rapamycin dramatically improved cardiac functions and inhibited inflammatory response in DIC models. DHT had similar inhibitory effect on mTOR as rapamycin, providing evidence that mTOR is a potential pharmacological target of inflammation response in DIC. Previous study reported that mTOR inhibitors augmented the anti-inflammatory activities of regulatory T cells and reduced the production of pro-inflammatory cytokines by macrophages [
52]. In this study, we focused primarily on the inflammatory regulatory effects and mechanisms of mTOR signaling pathway in cardiomyocytes. The mTOR agonist, MHY1485, was applied to DOX-stimulated H9C2 cells. After co-incubation with MHY1485, the effects of DHT on NF-κB, TNF-α, COX2 and nuclear TFEB were abrogated, suggesting that the protective mechanism of DHT on inflammatory response is mainly mediated by mTOR-NF-κB signaling pathway, moreover, TFEB plays pivotal roles in this signaling pathway.
TFEB has been recently identified as serving critical and diverse roles in immune systems [
8]. Then, to verify how the TFEB participates in mTOR-NF-κB pathway, loss/gain of the function of TFEB were performed. We found that DOX treatment reduced the expression of nuclear TFEB, and up-regulated phosphorylation of IKKα/β and NF-κB, suggesting that there might be a link between TFEB and NF-κB activation. When H9C2 cells were transfected with lentiviral vector carrying GFP-TFEB, TFEB overexpression downregulated the expressions of activated IKKα/β and NF-κB, further indicating that the IKK-NF-κB signaling axis is directly inhibited by TFEB. Targeting TFEB using pharmacological agents may, therefore, hold great promise against cardiac inflammatory complications. Intriguingly, DHT treatment promoted nuclear localization of TFEB and downregulated the expressions of p-IKKα/β and p-NF-κB, while inhibiting TFEB through application of mTOR agonist could abolish the effects of DHT on p-NF-κB. These data demonstrated that DHT inhibited NF-κB transcriptional activity via TFEB-IKK signaling pathway. Taken together, our data offered the evidence that DHT inhibited NF-κB-mediated inflammatory response through mTOR-TFEB-IKK signaling pathway.
In the present study, we also investigated the anti-apoptotic effects of DHT. Though apoptotic cells could trigger or potentiate inflammation, the underlying mechanism remains to be clarified [
31]. In consistent with previous reports [
53‐
55], we found that DOX could induce apoptosis and DHT has anti-apoptotic effects. We further explored the role of mTOR in apoptosis [
56]. Intriguingly, co-incubation of cells with mTOR agonist abolished the inhibitory effects of DHT on apoptosis, suggesting that DHT exerted anti-apoptosis effects via mTOR signaling pathway. The mTOR signaling may serve as the co-regulator of both inflammatory and apoptotic pathway. The underlying molecular mechanism warrants further investigation.
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