1,25(OH)2D3 ameliorates doxorubicin‑induced cardiomyopathy by inhibiting the NLRP3 inflammasome and oxidative stress
- Authors:
- Published online on: July 11, 2023 https://doi.org/10.3892/etm.2023.12112
- Article Number: 413
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Copyright: © Gu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
As a classical antitumor drug, doxorubicin (DOX) serves a key role in chemotherapy against a number of tumor types such as leukemia, lymphoma and breast cancer (1,2). However, DOX treatment may also induce cardiomyopathy, which adversely affects patient outcome (3,4). DOX-induced cardiomyopathy causes severe cardiac dysfunction, which mainly manifests as a decrease in cardiac ejection fraction (5). DOX-induced cardiomyopathy is an acute complication of long-term chemotherapy and can lead to an increased mortality rate in patients (6). The pathogenesis of DOX-induced cardiomyopathy is complex and remains unclear. Due to the lack of specific therapeutic drugs, patients with DOX-induced cardiomyopathy can only been given symptomatic relief and supportive treatment (7). Therefore, it is necessary to explore the pathophysiological processes of DOX-induced cardiomyopathy and investigate potential efficacious approaches for treatment.
1,25(OH)2D3 is a metabolic derivative of vitamin D3 that is also a steroid hormone (8). 25-hydroxyvitamin D3 [25(OH)D3] is the major circulating form of vitamin D. 25(OH)D3 metabolizes to 1,25(OH)2D3 by catalysis of the renal 25(OH)D1α hydroxylase (CYP27B1) (9). 1,25(OH)2D3 serves an important role in regulating calcium homeostasis, bone metabolism and the immune system (10,11). Previous studies have reported that CYP27B1 is mainly expressed in the kidney, but an increasing number of studies have reported that CYP27B1 is widely expressed in multiple organs and various cell types, such as the lungs, intestines, skin, keratinocytes, macrophages and osteoblasts, indicating that the targets of 1,25(OH)2D3 are extensive and complex (12,13). Vitamin D has also been reported to exert protective effects against cardiovascular disease (14) and diabetic cardiomyopathy (15). Patients with dilated cardiomyopathy tend to have lower levels of 25-hydroxyvitamin D3 in the serum compared with those subjects without dilated cardiomyopathy (16). The mechanism by which vitamin D protects against cardiomyopathy is mainly attributed to its antioxidant, anti-inflammatory and inhibitory effects on the renin-angiotensin system (17,18). However, whether 1,25(OH)2D3 can protect against the pathophysiological processes of DOX-induced cardiomyopathy and its mechanisms remain unknown.
The nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a protein complex that recognizes a diverse array of extracellular and intracellular signals, including damage-associated molecular patterns and pathogen-associated molecular patterns (19). Activation of the NLRP3 inflammasome pathway induces production of proinflammatory cytokines, such as IL-1β and IL-18, to generate a proinflammatory microenvironment and inflamm-ageing (20). However, the relationship between 1,25(OH)2D3 and the NLRP3 inflammasome pathway in DOX-induced cardiomyopathy remains unclear.
Oxidative stress has recently been widely recognized to be an important factor for DOX-induced cardiomyopathy (21). An imbalance between reactive oxygen species (ROS) and the antioxidant pathway may lead to ROS accumulation in cardiomyocytes. This can then induce damage to mitochondrial membrane proteins and DNA, thereby inhibiting ATP production in mitochondria by disrupting mitochondrial oxidative phosphorylation (22). Increased levels of ROS can cause lipid peroxidation in mitochondria, apoptosis and an excessive inflammatory response in cardiomyocytes, ultimately resulting in cardiac dysfunction (23). A previous study has demonstrated the antioxidant effect of 1,25(OH)2D3 in a number of disease settings, such as osteoporosis (24) and chronic kidney disease (25), in addition to skin senescence (18). Vitamin D3 1α(OH)ase knockout mice demonstrated a significant increase in ROS and H2O2 levels, in addition to aggravation of colon inflammation due to the extensive secretion of senescence-associated inflammatory cytokines (26). A previous study has reported that 1,25(OH)2D3 can activate nuclear erythroid 2-related factor 2 (Nrf2) pathways to activate enhancer of zeste homolog 2-mediated H3K27me3 histone modification, mediated by EZH2, to regulate downstream target genes (such as p16, p21, p53 and p19) transcriptional expression (27). However, little is known about the mechanism by which 1,25(OH)2D3 regulates DOX-induced cardiomyopathy.
To investigate this issue and its underlying molecular mechanisms, 10-week-old wild-type mice were administered DOX to construct a DOX-induced cardiomyopathy model in the present study. These mice were also administered with 1,25(OH)2D3. Echocardiography, histological and molecular analysis were used to investigate the effect of 1,25(OH)2D3 on DOX-induced cardiomyopathy and its potential mechanism.
Materials and methods
Drugs
DOX (cat. no. A603456) was purchased from Sangon Biotech Co., Ltd. DOX was diluted with normal saline to a concentration of 2 mg/ml for the in vivo study and to 43 µM for in vitro study. 1,25(OH)2D3 (cat. no. D1530) was purchased from Sigma-Aldrich; Merck KGaA. 1,25(OH)2D3 was diluted with normal saline to a concentration of 0.25 µg/ml for the in vivo study and 24 µM for the in vitro study.
Animals and experimental procedure
All animal studies were approved by Animal Ethical and Welfare Committee of Nanjing Medical University (approval no. IACUC-1707002) and were performed in accordance with the institutional and national regulations.
C57BL/6J mice (male; n=30; 10 weeks old; 23.81±0.34 g) were purchased from the Model Animal Research Center of Nanjing University and housed at 22-24˚C and in a relative humidity of 50±10% under a 12-h light/dark cycle. All the mice had free access to standard chow and water.
The schematic diagram of experimental procedure in the present study is presented in Fig. 1A. After a 1-week acclimation period, mice were randomly divided into three groups (n=10/group). Mice in the DOX-treated group (DOX) were injected intraperitoneally with DOX at a dose of 8 mg/kg per week for 4 weeks and with saline every other day for 4 weeks (28). Mice in the DOX + 1,25(OH)2D3-treated group [DOX + 1,25(OH)2D3] were injected intraperitoneally with DOX at the aforementioned dose and with 1,25(OH)2D3 at a dose of 1 µg/kg every other day for 4 weeks (29). Mice in the Sham group were injected intraperitoneally with saline every other day for 4 weeks. The body weight of each mouse in the Sham, DOX or DOX + 1,25(OH)2D3 groups was measured every week. All mice were sacrificed by CO2 (50% volume replacement rate/min) 4 weeks after the first injection of DOX when echocardiography and serum collection was complete.
The humane endpoints used to determine when animals should be immediately euthanized prior to the scheduled experimental endpoint were as follows: Weight loss of 20%; appetite loss for 24 h or poor appetite (<50% of normal appetite) for 3 days; weakness (inability to eat, drink or stand or extreme difficulty standing); impending death (depressive state with hypothermia); severe diseases of the cutaneous system (uncurable confrontation wound or repetitive self-injury); and severe diseases of the circulatory system (severe hemorrhage or rapid heart rate with marked dyspnea). No mice reached these humane endpoints or died prior to the scheduled experimental endpoint during this 4-week period.
Echocardiography
Echocardiography was performed in the Animal Core Facility of Nanjing Medical University (Nanjing, China) using the high-frequency color ultrasound system (40 µm resolution, 500 frames per second) Vevo 2100 (VisualSonics, Inc.). Cardiac function was evaluated in week 4 after the first injection of DOX was administered. Mice were anesthetized by inhaling isoflurane at a 1:1 concentration with oxygen (4% induction concentration, 1.5% maintenance concentration). Anesthetized mice were fixed in the supine position on a constant-temperature workbench at 25-26˚C. Hair on the chest area was removed using a leather knife and a simulated electrocardiogram was linked. Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured in the long axis and short axis, respectively. The following formulas were used to calculate left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), respectively.
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Enzyme-linked immunosorbent assay (ELISA)
Serum samples of mice were collected in the Animal Core Facility of Nanjing Medical University in week 4 after the first injection of DOX. Briefly, mice were anesthetized by the intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg), the hair around the eyeball was clipped and blood was then collected by removing the eyeball. A 0.75-ml volume of blood was collected from each mouse. Subsequently, mice were euthanized using CO2. Serum was obtained by centrifugation (1,500 x g for 10 min at 4˚C) after the blood was left for 1 h at 4˚C for component separation. Brain natriuretic peptide (BNP; cat. no. YFXEM00760) and cardiac troponin T (cTnT; cat. no. YFXEM00789) levels in serum were quantified using ELISA kits (Nanjing Yifeixue Biotechnology Co., Ltd.) according to the manufacturer's protocols.
Measurement of antioxidant enzymes and lipid peroxidation
Myocardial tissues from the left ventricle were prepared as a 10% homogenate with normal saline, followed by centrifugation at 4˚C at 2,000 x g for 15 min. Malondialdehyde (MDA; cat. no. A003-1-1), glutathione peroxidase (GSH-Px; cat. no. A005-1-1) and total superoxide dismutase (T-SOD; cat. no. A001-1-1) in the tissue supernatants were measured using assay kits according to the manufacturer's protocols (Nanjing Jiancheng Bioengineering Institute).
Masson's trichrome staining
Hearts were harvested and fixed in 4% paraformaldehyde at room temperature for 24 h. After standard paraffin embedding (75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol for 2 h, 95% alcohol for 1 h, ethyl alcohol I for 30 min, ethyl alcohol II for 30 min, ethyl alcohol and xylene for 10 min, xylene I for 10 min, xylene II for 10 min, 65˚C melted paraffin I for 1 h, 65˚C melted paraffin II for 1 h and 65˚C melted paraffin III for 1 h), heart tissues were cut into 4-µm sequential sections. Masson's trichrome staining (iron hematoxylin for 3 min, lichun red acid fuchsin for 8 min and aniline blue for 5 min, at room temperature) was used to evaluate the degree of myocardial fibrosis. The representative histopathological samples were imaged using a light microscope (Nikon Corporation) and analyzed using Image Pro-Plus software (version 6.0; Media Cybernetics, Inc.). A total of three images from randomly selected non-overlapping fields were captured per section at x400 magnification and the collagen tissue area was expressed as the percentage of the full ventricle area.
Immunohistochemistry
The 4-µm heart paraffin sections were deparaffinized, rehydrated in alcohol (ethyl alcohol for 1 min, 95% alcohol I for 1 min, 95% alcohol II for 1 min and 70% alcohol for 1 min) and washed in tap water. The tissue sections were then boiled for antigen retrieval in a citrate-EDTA antigen retrieval solution (cat. no. P0083; Beyotime Institute of Biotechnology) in a steamer for 30 min at 95-100˚C. Endogenous peroxidase activity was then blocked with methanol and hydrogen peroxide (1:10) for 10 min at room temperature. After washing with TBS (pH 7.6), the sections were blocked with 10% normal goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) for 1 h at room temperature, then incubated with the corresponding primary antibodies overnight at 4˚C. The dilutions of primary antibodies were as follows: IL-1β (cat. no. ab9722; 1 µg/ml; Abcam), IL-6 (cat. no. A0286; 1:200; ABclonal Biotech Co., Ltd.) and TNF-α (cat. no. GTX110520; 1:200; GeneTex, Inc.). After washing with PBS, the sections were incubated with a secondary antibody (MaxVision™ HRP-polymer anti-rabbit IHC kit; ready to use; cat. no. KIT-5004; Fuzhou Maixin Biotech Co., Ltd.) for 30 min at room temperature. Subsequently, 3,3'-diaminobenzidine was used as a chromogen. Finally, tissue sections were counterstained with hematoxylin at room temperature for 3 min and sealed with neutral balsam. The representative histopathological samples (magnification, x400, three random slides/sample) were imaged using a light microscope (Nikon Corporation) and analyzed using Fiji (version 2.3.0), an image processing package based on ImageJ (National Institutes of Health) (30).
Western blotting
Total protein was obtained from left ventricular myocardial tissues by sonication, centrifugation and heat denaturation. Briefly, heart tissues were placed into 4˚C RIPA lysis buffer (cat. No. P0013B; Beyotime Institute of Biotechnology) containing proteinase and phosphatase inhibitors (cat. No. 4906845001; Roche Diagnostics) and phenylmethanesulfonyl fluoride (cat. No. ST506; Beyotime Institute of Biotechnology) for protein extraction. Subsequently, the tissues were homogenized using a FastPrep-24 5G homogenizer (MP Biomedicals, LLC) at 4˚C for 2 min with 4.0-10 m/sec oscillation speed, and was centrifuged at 12,000 x g at 4˚C for 10 min. After quantification using a Pierce BCA Protein Assay Kit (cat. no. PI23225; Thermo Fisher Scientific, Inc.), the supernatant was mixed with loading buffer and heated to 95-100˚C for 5 min. An equal amount (30 µg/lane) of protein was used for western blotting. Proteins were resolved by SDS-PAGE using 8-15 % gradient gels and then transferred onto PVDF membranes. Membranes were blocked with 5 % non-fat milk for 60 min at room temperature and then incubated with the corresponding primary antibodies overnight at 4˚C. The dilutions of primary antibodies used were as follows: Collagen I (cat. No. GTX26308; 1:1,000; GeneTex, Inc.), α-SMA (α-smooth muscle actin; cat. No. ab5694; 1:1,000; Abcam), IL-1β (cat. no. ab9722; 0.25 µg/ml; Abcam), IL-6 (cat. No. A0286; 1:1,000; Abclonal Biotech Co., Ltd.), TNF-α (cat. No. GTX110520; 1:500; GeneTex, Inc.), SOD1 (cat. No. ab16831; 1:2,000; Abcam), SOD2 (cat. No. NB100-1992; 1:2,000; Novus Biologicals, LLC), Nrf2 (cat. No. GTX135165; 1:1,000; GeneTex, Inc.), KEAP1 (Kelch-like ECH-associated protein 1; cat. No. ab119403; 1:1,000; Abcam), Caspase-1 (cat. No. 83383; 1:500; Cell Signaling Technology, Inc.), NLRP3 (cat. No. ab214185; 1:1,000; Abcam), ASC (caspase recruitment domain; cat. No. GTX55818; 1:1,000; GeneTex, Inc.) and GAPDH (cat. No. 2118; 1:1,000; Cell Signaling Technology, Inc.). After washing with TBST (containing 0.25% Tween20), the membrane was incubated with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (cat. No. SA00001-1 or cat. No. SA00001-2; 1:2,000; ProteinTech Group Inc.) for 60 min at room temperature. The membrane was washed three times in TBS with 0.1 % Tween20 (cat. No. ST671; Beyotime Institute of Biotechnology), before being visualized with an enhanced chemiluminescent solution (cat. No. WBKLS0050; MilliporeSigma) and imaged using an Uvitec Alliance mini-chemiluminescence device (Uvitec). GAPDH was used as a loading control. The intensity of the protein bands was quantified using ImageJ software (version 1.44; National Institutes of Health).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RNA was extracted from left ventricular myocardial tissues using TRIzol™ Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcription was performed using the 5X All-In-One RT MasterMix (Applied Biological Materials Inc.) according to the manufacturer's protocols. qPCR was performed using the Hieff™ qPCR SYBR® Green Master Mix (Shanghai Yeasen Biotechnology Co., Ltd.) on StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol (a two-step amplification: Initial denaturation at 95˚C for 5 min, followed by 40 cycles of 95˚C for 10 sec and 60˚C for 30 sec). Quantification of mRNA was carried out using the 2-ΔΔCq method and normalized to GAPDH (31). Primer sequences used for the RT-qPCR reactions are presented in Table I.
Cell culture and treatment
Human cardiomyocyte-like AC16 cells were purchased from MilliporeSigma (cat. no. SCC109) and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin and incubated in 5% CO2 at 37˚C. To model cardiotoxicity in vitro, AC16 cells were treated with 1 µM DOX (32) in the absence or presence of 1,25(OH)2D3 for 24 h at 37˚C after serum starvation [DOX treatment group or DOX + 1,25(OH)2D3 treatment group, respectively]. Untreated AC16 cells served as the Control group.
Cell viability assays
Cell viability was assayed with the Enhanced Cell Counting Kit (CCK)-8 (Beyotime Institute of Biotechnology) according to the manufacturer's protocols. AC16 cells were seeded into a 96-well plate (2x104 cells/well) and incubated until adherence was achieved. Cell culture medium was then replaced using DMEM. To evaluate the potential toxicity of 1,25(OH)2D3 towards AC16 cells, AC16 cells were treated with 1,25(OH)2D3 in triplicate at various concentrations (0, 25, 50, 100, 200 and 400 nM) for 24 h at 37˚C. To evaluate the protective effects of 1,25(OH)2D3 against DOX-induced cytotoxicity, AC16 cells were treated with 1 µM DOX and various concentrations of 1,25(OH)2D3 (0, 25, 50, 100 and 200 nM) in triplicate for 24 h at 37˚C. Subsequently, 10 µl enhanced CCK-8 solution was added into each well, cells were incubated for 2 h at 37˚C and the absorbance at 450 nm was measured.
ROS analysis
ROS production in AC16 cells was assessed using 2',7'-dichlorofluorescin diacetate (DCFH-DA; cat. no. 35845; MilliporeSigma). Following starvation treatment and induction of cardiotoxicity, AC16 cells were incubated with 10 µM DCFH-DA in the dark at 37˚C for 30 min and then washed twice with PBS. Oxidized 2',7'-dichlorofluorescin fluorescence (representing ROS level) was detected immediately by fluorescent microscopy (Guangzhou Micro-shot Technology Co., Ltd.). A total of three images from randomly selected non-overlapping fields were captured and the intensity of fluorescence was quantified using ImageJ software (version 1.44; National Institutes of Health).
Chromatin immunoprecipitation (ChIP) assay
AC16 cells were collected for ChIP assay using the High-Sensitivity ChIP kit (cat. no. 9003; Cell Signaling Technology, Inc.) following the manufacturer's protocol. H2AK119Ub (cat. no. 8240), H3K4me3 (cat. no. 9751) and H3K27me3 (cat. no. 9733) were purchased from Cell Signaling Technology Inc., which were used for the ChIP assay. Briefly, two 10-cm dishes containing 1x107 cells in 10 ml culture media were used for each ChIP experiment. Cells were crosslinked with 1% formaldehyde for 10 min at room temperature on an orbital shaker (60 rpm). The formaldehyde was then quenched by adding glycine (75 mM), the cells were incubated at room temperature for 10 min and rinsed twice with PBS. Cells were scraped and transferred into 15-ml Falcon tubes, before being centrifuged at 350 x g for 5 min at 4˚C. Cells were resuspended in 15 ml Buffer A [20 mM HEPES (pH=7.4), 10 mM EDTA, 0.5 mM EGTA and 0.25% Triton X-100] and incubated at 4˚C for 5 min. The cells were then centrifuged at 350 x g for 5 min at 4˚C and resuspended in 15 ml Buffer B [20 mM HEPES (pH=7.4), 150 mM NaCl, 10 mM EDTA and 0.5 mM EGTA] and incubated at 4˚C for 5 min on a rotating wheel. Cells were centrifuged again at 350 x g for 5 min at 4˚C and resuspended in 1 ml Buffer C [20 mM HEPES (pH=7.4), 10 mM EDTA, 0.5 mM EGTA, 0.1% SDS and 1X Protease Inhibitor Cocktail (cat. no. #5871; Cell Signaling Technology). After incubating for 10 min on ice, the cells were transferred into a 15-ml sonication tube. Sonication was performed using a Bioruptor (Bioruptor PLUS; Diagenode S.A.) using the following protocol: 10 Cycles of 30-sec sonication (150 Hz, at 4˚C), followed by 20 sec off, at high power for a total of 20 cycles with a 10-min pause and incubation on ice every 10 cycles. The sonicated chromatin was centrifuged at 16,000 x g for 10 min at 4˚C. For each immunoprecipitation reaction, chromatin (10 µg DNA), measured via Nanodrop2000 (Thermo Fisher Scientific, Inc.), was diluted to a total of 500 µl with 1X ChIP Buffer + PIC. Next, 5 µg antibodies were added into chromatin (10 µg DNA) in 500 µl 1X ChIP Buffer + PIC. Immunoprecipitated samples were incubated on an oscillating platform at 4˚C overnight. The following day, 30 µl Protein G Magnetic Beads were added immediately into each immunoprecipitation reaction and incubated on an oscillating platform at 4˚C for 2 h. Each sample tube was placed on a magnetic separator for 2 min to precipitate the Protein G Magnetic Beads until the solution was cleared and the supernatant was removed. The protein G magnetic beads were then washed using low- and high-salt wash. They were then re-suspended in 150 µl 1X ChIP eluting buffer before the chromatin was eluted from the samples using a vortex (200 x g at 65˚C for 30 min). Samples were centrifuged at 10,000 x g at 4˚C for 10 sec to remove trace amounts of evaporated sample from the centrifuge tube cap. The eluted chromatin supernatant was then transferred to a new tube. Un-crosslinking was performed by adding 6 µl 5M NaCl and 2 µl Proteinase K to each sample and incubating at 65˚C for 2 h. DNA Binding Buffer (750 µl) was added to each DNA sample and swirled to mix, before 450 µl sample was transferred to a DNA centrifuge column in the collection tube and centrifuged at 16,000 x g at 4˚C for 30 sec. DNA Wash Buffer (750 µl) was added to the centrifuge column in the collection tube and the samples were centrifuged again at 16,000 x g at 4˚C for 30 sec. DNA Elution Buffer (50 µl) was added to each centrifuge column, placed in a clean 1.5-ml microcentrifuge tube and centrifuged at 16,000 x g at 4˚C for 30 sec to elute the DNA. The eluate was purified for subsequent qPCR assay. DNA purification was performed as described in the Chip kit instructions (cat. no. 9003; Cell Signaling Technology, Inc.). qPCR was performed using the Hieff™ qPCR SYBR® Green Master Mix (Shanghai Yeasen Biotechnology Co., Ltd.) on StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol (a two-step amplification: Initial denaturation at 95˚C for 5 min, followed by 40 cycles of 95˚C for 10 sec and 60˚C for 30 sec). Quantification of mRNA was carried out using the 2-ΔΔCq method and normalized to input. In this experiment, the qPCR method was used to quantify the Chip experiment. The enrichment level was calculated by the formula 2% x 2 [C(T) 2% input-C(T) IP sample] with reference to operating instructions of the Chip kit (cat. no. 9003; Cell Signaling Technology, Inc.). For DNA gel assay, 2 X Taq Master Mix (Dye Plus) (Vazyme, P112-01) was used for PCR assay. The following DNA amplification procedure was applied: i) Initial denaturation 95˚C for 5 min; ii) denaturation 95˚C for 30 sec; ii) refolding at 62˚C for 30 sec; iv) extension at 72˚C for 30 sec; v) repeat of step ii)-iv) for 34 cycles; and vi) terminal extension at 72˚C for 5 min. DNA products were detected using 2% agarose gel electrophoresis. GelDoc XR System (Bio-Rad) was used for gel imaging. Primer sequences used for ChIP-qPCR reactions are presented in Table II.
Statistical analysis
Data from ≥ three independent experiments were expressed as the mean ± standard deviation. All statistical analyses were performed using the GraphPad Prism software (version 7.0; GraphPad Software, Inc.; Dotmatics). Statistical differences among treatment groups were analyzed using a one-way ANOVA followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.
Results
1,25(OH)2D3 alleviates DOX-induced cardiac dysfunction and injury
Mice were administered with DOX to construct a DOX-induced cardiomyopathy model whilst 1,25(OH)2D3 was administered to mice as previously described (Fig. 1A). The body weight of each mouse in the Sham, DOX and DOX + 1,25(OH)2D3 groups were measured each week, which yielded no significant differences among the three treatment groups (Fig. 1B). Echocardiography images of mice on week 4 indicated that ventricular wall motion amplitude markedly decreased after DOX administration, which was alleviated by 1,25(OH)2D3 (Fig. 1C). LVEF and LVFS were observed to be significantly decreased after DOX administration compared with those in the sham group (Fig. 1D and E). Compared with those in the DOX-treated group, 1,25(OH)2D3 treatment significantly increased LVEF and LVFS (Fig. 1D and E). Treatment with 1,25(OH)2D3 also significantly reduced the serum levels of BNP in mice with DOX-induced cardiomyopathy (Fig. 1F). Analysis of cTnT levels in serum was performed to detect signs of myocardial injury, which demonstrated that cTnT levels were significantly increased after DOX administration compared with those in the Sham group, which was in turn significantly reversed by 1,25(OH)2D3 treatment (Fig. 1G). These results suggest that 1,25(OH)2D3 treatment can alleviate DOX-induced cardiac dysfunction and injury.
1,25(OH)2D3 inhibits the activation of a pro-fibrotic and inflammatory microenvironment in DOX-induced cardiomyopathy
As 1,25(OH)2D3 was found to alleviate DOX-induced cardiac dysfunction and injury, it was therefore hypothesized that 1,25(OH)2D3 may reverse the fibrotic and inflammatory microenvironment in a model of DOX-induced cardiomyopathy. Masson's trichrome staining was used to evaluate the extent of myocardial fibrosis. 1,25(OH)2D3 was demonstrated to significantly inhibit collagen deposition in the heart tissues of mice with DOX-induced cardiac dysfunction (Fig. 2A and B). The mRNA expression levels of α-SMA, periostin (POSTN), Fibronectin and Vimentin in mice with DOX-induced cardiomyopathy were also found to be significantly reduced after 1,25(OH)2D3 treatment (Fig. 2C). In addition, it was demonstrated through western blotting that 1,25(OH)2D3 significantly downregulated α-SMA and collagen I protein expression levels compared with those in the DOX group (Fig. 2D and E). These results suggest the anti-fibrotic effect of 1,25(OH)2D3 against DOX-induced cardiomyopathy.
Immunohistochemistry staining was subsequently used to examine whether 1,25(OH)2D3 can reduce the production of inflammatory mediators in DOX-induced cardiomyopathy. The results demonstrated that the expression levels of IL-1β, IL-6 and TNF-α were significantly decreased in the DOX + 1,25(OH)2D3-treated group compared with those in the DOX-treated group (Fig. 2F and G). These findings were confirmed by western blotting, which demonstrated significantly reduced IL-1β, IL-6 and TNF-α protein expression levels in mice with DOX-induced cardiomyopathy treated with 1,25(OH)2D3 (Fig. 2H and I). These results suggest that 1,25(OH)2D3 inhibited the activation of a pro-fibrotic and inflammatory microenvironment in a mouse model of DOX-induced cardiomyopathy.
Increases in oxidative stress and the NLRP3 inflammasome pathways are ameliorated by 1,25(OH)2D3 treatment in DOX-induced cardiomyopathy
Although 1,25(OH)2D3 inhibited the activation of a pro-fibrotic and inflammatory microenvironment, the detailed mechanism underlying the regulation of this process remain poorly understood. Previous studies have reported that mitochondrial dysfunction and excessive activation of oxidative stress can aggravate DOX-induced cardiomyopathy (33,34). Furthermore, another previous study demonstrated that 1,25(OH)2D3 can significantly inhibit the production of ROS in skin and liver senescent cells (18). It was therefore hypothesized that 1,25(OH)2D3 administration could inhibit oxidative stress. Consequently, the associated pathway in DOX-induced cardiomyopathy was next assessed. To clarify this hypothesis, cell viability was analyzed using the CCK-8 kit to determine the minimal treatment concentration of 1,25(OH)2D3 that can cause toxicity in AC16 cells. CCK-8 results demonstrated that cell viability was not affected upon treatment with different concentrations of 1,25(OH)2D3 (0, 25, 50, 100, 200 and 400 nM), suggesting that no cytotoxicity to 1,25(OH)2D3 was demonstrated in AC16 cells up to a dose of 400 nM (Fig. 3A). Cell viability was found to be significantly decreased when the AC16 cells were treated with DOX (Fig. 3B). Doses of 50, 100 and 200 nM 1,25(OH)2D3 exhibited a significant protective effect against DOX-induced cytotoxicity in AC16 cells (Fig. 3B). In particular, the 100 and 200 nM doses of 1,25(OH)2D3 exhibited significantly increased protective effects on cell viability compared with that after treatment with the 50 nM dose of 1,25(OH)2D3 (Fig. 3B). Doubling the 1,25(OH)2D3 concentration to 200 nM had no significant effects on cell viability compared with that after 100 nM 1,25(OH)2D3 treatment (Fig. 3B). Therefore, 100 nM 1,25(OH)2D3 was selected as the safe and effective treatment dose for subsequent experiments.
Intracellular ROS levels in AC16 cells were analyzed using DCFH-DA staining, which demonstrated that DOX treatment significantly increased the intracellular ROS levels compared with those in control cells (Fig. 3C and D). Treatment of AC16 cells with 100 nM 1,25(OH)2D3 significantly decreased intracellular ROS levels compared with cells treated with DOX only (Fig. 3C and D). Oxidative stress was also measured in cardiac tissue homogenates of mice on the 4th week after the first injection of DOX was administered. Compared with those in the Sham group, cardiac tissues from DOX-treatment mice demonstrated a significantly increased MDA level but decreased T-SOD and GSH-Px levels (Fig. 3E-G). Mice in the DOX + 1,25(OH)2D3-treated group demonstrated significantly lower MDA levels, but markedly higher T-SOD and significantly higher GSH-Px levels, compared with those in the DOX-treated group (Fig. 3E-G). To further determine the potential mechanism of action underlying this process, protein expression levels of KEAP1, Nrf2, SOD1 and SOD2 were analyzed using western blotting. Compared with those in the DOX-treated group, 1,25(OH)2D3 treatment significantly increased SOD1, SOD2 and Nrf2 protein expression, whilst significantly decreasing KEAP1 expression (Fig. 3H and I). Taken together, these results suggest that 1,25(OH)2D3 reduced oxidative stress in DOX-induced cardiomyopathy by inhibiting the KEAP1/Nrf2 pathway.
The NLRP3 inflammasome has received considerable attention in the field of inflammation research (35,36). Levels of NLRP3 inflammasome-associated proteins in DOX-induced cardiomyopathy mice were therefore next analyzed, which demonstrated that the protein expression levels of NLRP3, ASC and Caspase-1 were significantly decreased with 1,25(OH)2D3 treatment compared with those in the DOX-only treatment group (Fig. 3J and K).
Taken together, these results suggested that 1,25(OH)2D3 ameliorated oxidative stress and inhibited the NLRP3 inflammasome pathway to alleviate the pathophysiological processes associated with DOX-induced cardiomyopathy.
1,25(OH)2D3 treatment affects histone modification levels in NLRP3 and Nrf2 promoters in DOX-induced cardiomyopathy
Previous studies have reported that 1,25(OH)2D3 can affect histone modifications to regulate downstream target gene expression and impact transcriptional regulation (37). To clarify the effect of 1,25(OH)2D3 on the level of histone modification in the NLRP3 promoter, ChIP assay was used to assess the levels of H3K4 (H3K4me3) and H3K27 (H3K27me3) trimethylation, in addition to H2AK119 monoubiquitination (H2AK119Ub), in the NLRP3 promoter region. The results demonstrated that 1,25(OH)2D3 treatment significantly decreased the level of H3K4me3 whilst significantly increasing the levels of H3K27me3 and H2AK119Ub in the NLRP3 promoter region, compared with that in the DOX-treated group (Figs. 4A-C and S1A). H3K4me3 modification may loosen chromatin and activate transcriptional expression (38). By contrast, H3K27me3 and H2AK119Ub modification may cause chromatin compression and inhibit the transcription of downstream genes (39). These results suggested that the inhibition of NLRP3 transcription by 1,25(OH)2D3 may have occurred due to a decrease in the levels of histone modification that is conducive to transcription activation and an increase in the levels of histone modifications that favor transcriptional inhibition in the NLRP3 promoter region.
The levels of different histone modifications in the Nrf2 promoter region were also evaluated. Treatment with 1,25(OH)2D3 significantly increased the level of H3K4me3 whilst significantly decreasing the levels of H3K27me3 and H2AK119Ub in the Nrf2 promoter region in a model of DOX-induced cardiomyopathy, suggesting that 1,25(OH)2D3 stimulation increased the transcriptional level of Nrf2 (Figs. 4D-F and S1B). These results contrast with those observed in the NLRP3 promoter. Therefore, these data suggest that 1,25(OH)2D3 treatment could significantly affect chromatin accessibility in cardiomyocytes to participate in the progression of cardiomyopathy, but the regulation of this process is likely to be complex.
Discussion
DOX, an anthracycline antibiotic derived from Streptomyces, is widely used for the treatment of multiple types of cancer, such as leukemia, lung and breast cancer (40). However, the toxic effects of DOX on cardiomyocytes limits its clinical application (3). The pathogenesis of DOX-induced cardiomyopathy remains poorly understood and there is a lack of effective therapeutic drugs (7). Therefore, it is particularly important to explore its mechanism and search for potential and effective treatment.
1,25(OH)2D3 is a metabolic derivative of vitamin D3 and its production needs catalysis by the CYP27B1 enzyme (9). 1,25(OH)2D3 binds to the vitamin D receptor (VDR) to induce downstream effects, such as anti-oxidative stress and anti-cell senescence (17,18). VDR and CYP27B1 are expressed in multiple organ and cell types, including the heart, suggesting that the targets of 1,25(OH)2D3 are wide-ranging (41). Previous studies have reported that 1 µg/kg 1,25(OH)2D3 exhibits antioxidant and anti-inflammatory effects on certain organs, such as the skin, liver and kidneys, in addition to impacting a number of diseases, such as arthrosis (by promoting apoptosis of fibroblast-like synoviocytes) and breast cancer (by inhibiting oxidative stress and inducing tumor cellular senescence) in mice (18,29,42). Oxidative stress and inflammation can also serve vital roles in DOX-induced cardiomyopathy (34). However, a potential risk of vitamin D overproduction is that it can cause high calcium and phosphorus levels, which are harmful to health, resulting in hyperparathyroidism, vascular calcification and arrhythmia (43). Previous studies have reported that 1 µg/kg 1,25(OH)2D3 has no effect on serum levels of calcium and phosphorus in mice, suggesting that 1 µg/kg can be used as a safe dose in mouse studies (18,29). Considering the impact of 1,25(OH)2D3 on multiple organ systems and the safety data reported in previous studies (18,29,42), the aforementioned dose (1 µg/kg) was used to study the potential effects of 1,25(OH)2D3 in a mouse model of DOX-induced cardiomyopathy.
The present study demonstrated that 1,25(OH)2D3 treatment could increase the levels of H3K27me3 and H2AK119Ub and decrease the level of H3K4me3 in the NLRP3 promoter region in DOX-induced cardiomyopathy. By contrast, treatment with 1,25(OH)2D3 decreased the levels of H3K27me3 and H2AK119Ub and increased the level of H3K4me3 in the Nrf2 promoter region. Therefore, 1,25(OH)2D3 may inhibit the activation of the NLRP3 inflammasome and KEAP1/Nrf2-induced oxidative stress, in turn alleviating the pathophysiological processes of DOX-induced cardiomyopathy.
The NLRP3 inflammasome is an important part of the inflammasome family and serves a critical role in inflammatory responses to multiple exogenous and endogenous factors (35). The NLRP3 inflammasome contains an NLRP3 protein that interacts with its adapter apoptosis-associated speck-like protein containing an ASC to recruit and activate caspase-1, which processes pro-IL-1β to mature IL-1β to trigger an inflammatory response (44). Cao et al (45) previously reported that 1,25(OH)2D3 can alleviate colitis disease progression by inhibiting the NLRP3 pathway, which is consistent with findings of the present study. The present study also demonstrated the inhibitory effect of 1,25(OH)2D3 on the NLRP3 inflammasome pathway in DOX-induced cardiomyopathy. However, Tulk et al (46) reported that 1,25(OH)2D3 can enhance the secretion of IL-1β in THP-1 cells by activating the NLRP3 inflammasome pathway after phorbol 12-myristate 13-acetate stimulation. Conflicting results obtained using different cell lines suggest that 1,25(OH)2D3 may serve different roles depending on the cell type or under different pathological conditions, further indicating the complex role of 1,25(OH)2D3 in different cell types.
Epigenetic modification serves as another regulatory process of gene transcription (47,48). Epigenetic regulation mechanisms, including CpG island DNA methylation, histone modification and non-coding RNA regulation, are considered key to regulating the transcription of the NLRP3 inflammasome (49). Lecoeur et al (50) reported that NLRP3 promoters exhibit a high level of histone H3K9/14 deacetylation and a high level of histone H3K4 demethylation during Leishmania amazonensis infection. As an important type of histone modification, histone ubiquitination also serves an important role in transcriptional regulation (39). Histone ubiquitination has been reported to serve an important role in DNA damage repair (51), stem cell regulation (52) and DNA replication (53). Previous studies have reported that 1,25(OH)2D3 can increase the level of H3K9me2 in the bone morphogenic protein 2 (BMP2) promoter region and regulates the transcriptional expression of the BMP2 gene through binding to VDR (54,39). However, to the best of our knowledge, no studies have assessed whether 1,25(OH)2D3 can affect histone modifications in the NLRP3 promoter region. In the present study, it was demonstrated that 1,25(OH)2D3 decreased H3K4me3 levels whilst increasing H3K27me3 and H2AK119Ub levels in the NLRP3 promoter region. A decrease in the levels of histone modifications favoring transcriptional activation and an increase in the levels of histone modifications favoring transcriptional inhibition can lead to significant chromatin compression in the NLRP3 promoter region to inhibit transcription.
In conclusion, the present study demonstrated that 1,25(OH)2D3 can regulate histone modification in NLRP3 and Nrf2 promoters, inhibit activation of the NLRP3 inflammasome pathway and oxidative stress in cardiomyocytes, in addition to alleviating the pathophysiological processes of DOX-induced cardiomyopathy. Therefore, 1,25(OH)2D3 may serve to be a future potential therapeutic drug for the treatment of DOX-induced cardiomyopathy.
Supplementary Material
1,25(OH)2D3 treatment affects histone modification levels in NLRP3 and Nrf2 promoters in DOX-induced cardiomyopathy. The DNA gels of different histone modifications in the (A) NLRP3 and (B) Nrf2 promoter regions in AC16 cells after treating with DOX or DOX + 1,25(OH)2D3. NLRP3, nod-like receptor family pyrin domain containing 3; Nrf2, nuclear erythroid 2-related factor 2; DOX, doxorubicin hydrochloride.
Acknowledgements
The authors would like to thank Professor Zhijian Yang (Nanjing Medical University, Nanjing, China) for his assistance in ethics writing.
Funding
Funding: The present study was supported by the Natural Science Foundation of Jiangsu Province (grant no. BK20210101), the Scientific Research Project of Gusu Health Talent Plan (grant no. GSWS2022067), the General Project of Suzhou Science and Technology Administration (grant no. SKYD2022131), the Research Project of Gusu School of Nanjing Medical University (grant no. GSKY20210214), the Key Project of Jiangsu Provincial Health Commission (grant no. ZDA2020023), the General Project of Wuxi Traditional Chinese Medicine Administration (grant no. ZYKJ202013) and the General Project of Wuxi Science and Technology Administration (grant no. N20202019).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
XG, XW, YD and JZ contributed to study conception and design. XG, LZ, JY, LC, CS and BL performed experiments. XG, LZ, JY and XW generated figures and performed data analysis. XG and YD confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
All animal studies were approved by Animal Ethical and Welfare Committee of Nanjing Medical University (approval no. IACUC-1707002).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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