Male C57BL/6J mice (8 weeks-old) were purchased from Aisaikesi (Shenyang). The mice were randomly divided into four groups. Sham group, Naringin group, DOX group and Naringin + DOX group. The mice were administered Naringin (50 mg/kg) solution dissolved in 0.5% CMC-Na or 0.5% CMC-Na via oral gavage for 22 days and at seventh day the mice were intraperitoneal injected with Doxorubicin hydrochloride (1 mg/kg) or saline for consecutive 15 days (Figure 1B). At the end of treatment, all mice were intraperitoneally anesthetized to collect the hearts and blood samples for determining heart injury and serum biomarker levels. All procedures were approved by the Animal Care and Use Committee of Dalian Medical University.

Hematoxylin & eosin (H&E) staining were carried out according to experiment protocol. Briefly, after the mice hearts were fixed with 4% formaldehyde overnight, they were embedded in paraffin and made into 5 μm sections. The cardiac tissues transversely sectioned from middle segment were stained with H&E for the assessment of myofilament morphology and injury.

For the assessment of the cardiomyocyte cross-sectional area, sections were stained with 50 μg/ml WGA for 60 min, and then it was calculated by measuring 15–20 cells per slid.

NRVMs were isolated from 1-2-day-old Sprague-Dawley (SD) rats. The heart tissue was cut into pieces and washed with precooled phosphate buffer saline (PBS), and then digested continuously with 0.25% trypsin. The cells were pre-incubated with Dulbecco’s Minimum Essential Medium/F12 (DMEM/F12) containing 10% (vol/vol) fetal bovine serum (FBS) (Life-Lab, AC03L055) and 1% penicillin-streptomycin (PS). Then, the harvested cells were incubated on 100 mm² culture dishes at 37°C with 5% CO₂. H9C2 cells were obtained from American type culture collection (ATCC). H9C2 cells were cultured in the DMEM containing d-Glucose-(4.5 g/L) with 10% (vol/vol) FBS and 1% PS at 37°C with 5% CO_2.

NRVMs and H9C2 cells were treated with Naringin at a dose of 50 μM in culture medium after cell generation, Doxorubicin hydrochloride was administrated at a dose of 5 μM after Naringin treatment.

TUNEL Assay kit was used to evaluate cardiac apoptosis according to the manufacturer’s protocol (US EVERBRIGHT, T6014). The apoptotic cells were visualized by fluorescence microscopy and apoptosis was indicated as the ratio of TUNEL-positive nuclei/total stained cell nuclei in five images per heart tissue.

Catalase (CAT), malondialdehyde (MDA), activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and Total Glutathione peroxidase (GSH-Px) were measured respectively using assay kits (CAT, MDA, LDH, and SOD kits: Nanjing Jiancheng Company, China; GSH-Px kit: Solarbio, China). The absorbance was detected using EnSpire microplatereader (Tecan, Switzerland).

Total RNA was isolated from NRVMs or heart tissues using TRIzol (Invitrogen) according to the manufacturer’s protocol. cDNA was used for PCR amplification with gene-specific primers for cybb and nox4. First-strand cDNA was produced from 1 µg of total RNA from each sample using PrimeScript RT reagent kit according to the manufacturer’s protocol (Yeasen, 11141ES60). The mRNA levels of genes were analyzed using SYBR Green Premix on an Applied Biosystems 7,500 Fast thermocycler. actn2 was used as the endogenous control.

Table Primer sequences

Gene Forward primer (5′–3′) Reverse primer (5′–3′)

Cybb GCT​ACG​CCT​TCA​ACA​CCA​AG AGT​TCG​TCC​CCT​TCT​CCT​GT

Nox4 CGG​GAT​TTG​CTA​CTG​CCT​CCA​T TGA​CTC​CTC​AAA​TGG​GCT​TCC

Actn2 ATG​CGG​TTC​CAC​AAG​ATT​GC AGC​CCT​TCT​TTG​GCA​GAT​GTT

Immunofluorescence staining was performed to detect the expression levels of Bcl-2 (Arigo) and γ-H2AX (abcam) of the cells. After washing with PBS three times, cells were fixed with 4% paraformaldehyde for 30 min, permeated with 0.1% Triton X-100 for 15 min, and then blocked with 3% fetal bovine serum albumin. Cells were incubated with the primary antibodies (anti-Bcl-2, anti-γ-H2AX) overnight at 4°C in a humidified chamber, and then incubated with a green fluorescent secondary antibody (diluted 1:200; Beyotime) for 2 h at room temperature. At last, the slide was staining with DAPI for 10 min and added anti-fluorescence quenching agent. The images were analyzed using a Nikon microscope (Tokyo, Japan).

MitoSOX Red Assay kit was used to detect the mitochondrial superoxide. H9C2 cells were grown till about 70% confluence and the fresh media were added before experiments. MitoSOX was added to a concentration of 5 μM according to manufacturer’s protocol. Before analysis with confocal microscope, cells were kept 30 min in CO₂ incubator at 37°C. The digital images were taken by an inverted confocal laser scanning microscope.

H9C2 cells were seeded in a 96-well plate and incubated with DOX for 24 h. Cell viability and Cytotoxicity assay was evaluated by Cell Counting Kit-8 (CCK8).

H9C2 cells were lysed with radioimmunoprecipitation assay (RIPA) buffer supplemented with PMSF (PMSF: RIPA = 1:100), and the supernatants were collected by centrifugation at 13,300 g for 15 min at 4°C. The proteins were separated by 12.5% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes by the Bio-Rad Western blotting system. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with the primary antibodies (anti-Bcl-2, anti-β-actin) overnight at 4°C and then followed by incubation with secondary antibodies (1:2,500) for 1 h at room temperature. Protein bands were visualized using an ECL kit and Image-Pro Plus 6.0 software was applied to quantify the band intensity values.

In the experiments with si-ECHS1 inhibition, H9C2 cells were divided into five groups: Control, NR (50 μM), DOX (5 μM), NR + DOX (50 μM), NR + DOX (50 μM)+si-ECHS1. si-ECHS1 (5′-3': GGG ACC AUA UCA CCC GGA UTT; 5′-3': AUU CCG GGU GAU AUG GUC CCT T) was transfected with LipFiter 3.0 (Hanbio) for 24 h, followed by subsequent operations.

The levels of inflammation factors in heart tissue or H9C2 cells was measured using IL-1β (Elabscience, China), IL-6 (Elabscience, China) and TNF-α (Elabscience, China) ELISA kits according to the manufacturer’s recommendations, The OD is measured spectrophotometrically at a wavelength of 450 nm, the contents of IL-1β, IL-6 and TNF-α were calculated according to the standard curve.

Results were expressed as mean ± standard deviation (SD). When the data were normally distributed, the difference between the two groups was analyzed by independent t-test, and the comparison between multiple groups was analyzed by one-way ANOVA. Differences were considered to be significant at p < 0.05.

To demonstrate the role of Naringin in myocardial injury induced by doxorubicin. Mice were treated Naringin by gavage and then intraperitoneal injection of doxorubicin at a cumulative dose of 15 mg/kg for 2 weeks. The results showed that the heart weight and size of mice treated with doxorubicin significantly decreased, while Naringin increased the heart weight and size of mice treated with doxorubicin (Figures 2A,B). However, the heart weight/body weight ratio of mice did not different among the four groups (Figure 2C), possibly because the body weight of mice also decreased after treatment with doxorubicin (Figure 2D). The tibia length of the mice did not change, so the heart weight/tibia length (HW/TL) results were the same as the heart weight of the mice (Figure 2E). We also did WGA staining to observe the size of myocardial cells in each group. The results showed that myocardial cells in DOX group were significantly smaller, which was consistent with the results in the literature. However, after Naringin treatment, the size of mouse cardiomyocytes increased (Figure 2F). Thus, Naringin could protect the size of mouse cardiomyocytes induced by doxorubicin.

There are vacuoles of different sizes in the cytoplasm and nucleus of the denatured cells, and the cells are honeycomb or reticular. Severe denaturation, vesicle mutual fusion into a large vesicle, the nucleus hung in the center, or was squeezed in the side, cytoplasm blank, shape such as balloon. Vacuolation occurs in heart tissue from chemotherapy patients and doxorubicin-induced mice. Here, a large number of vacuolated cardiomyocytes were found in doxorubicin-induced mouse heart tissues by H&E staining, while the number of vacuolated cardiomyocytes was significantly reduced after treatment with Naringin (Figure 3A). Subsequently, we measured the degree of LDH, a marker of myocardial injury. The results showed that doxorubicin promoted the increase of LDH concentration, which was decreased after Naringin treatment (Figure 3B). Doxorubicin could induce apoptosis of cardiomyocytes. Oxidative stress is also a pathway by which cardiomyocytes are damaged by doxorubicin. We detected the concentration level of CAT, GSH-PX, MDA and SOD. They play a crucial role in the balance of oxidation and antioxidant in the organism and is closely related to the occurrence and development of many diseases. In this experiment, the concentration of CAT, GSH-PX and SOD was inhibited by doxorubicin, while the concentration of CAT, GSH-PX and SOD increased after Naringin treatment (Figures 3C–E). The concentration of MDA was increased by doxorubicin, while the concentration of MDA decreased after Naringin treatment (Figure 3F). The mRNA expression levels of oxidative stress related factors NOX2 (cybb) and nox4 were also detected. The results showed that DOX could significantly promote the expression of cybb and nox4, while Naringin inhibited this phenomenon (Figures 3G,H). Next, TUNEL assay was performed to verify whether Naringin has protective effect on doxorubicin-induced apoptosis of cardiomyocytes. The results showed that doxorubicin could indeed induce apoptosis of mouse cardiomyocytes. Naringin, on the other hand, protects cardiomyocytes from apoptosis (Figure 3I). And we examined the marker of apoptosis bcl-2 and Bax by Western Blot. The results showed that Naringin could inhibited the expression of Bax and increase the expression of Bcl-2 induced by DOX (Figure 3J). Local tissue non-specific inflammation caused by DOX chemotherapy. We examined the level of IL-1β, IL-6 and THF-α. The results showed that Naringin could inhibited the level of IL-1β, IL-6 and THF-α induced by DOX (Figures 3K–M). These results suggested that Naringin could reduce cardiac injury by inhibiting apoptosis, inflammation and oxidative stress induced by doxorubicin in vivo.

We have shown that Naringin protects against doxorubicin-induced myocardial damage in vivo. Next, we verified this conclusion in vitro experiments. First, we used CCK8 assay to test whether Naringin inhibited the cardiotoxicity of doxorubicin. The results showed that doxorubicin could inhibit the proliferation of H9C2 cells and promote their death. Naringin, on the other hand, could protect H9C2 to some extent (Figure 4A). Next, Western blot experiments were used to observe the apoptosis and ROS. The results showed DOX induced the expression of Bax and NOX2, while Naringin inhibited the expression of Bax and NOX2 induced by DOX in H9C2 cells (Figure 4B). And we used IF to detect the expression of bcl-2. The results showed DOX decreased the expression of Bcl-2, while Naringin increased the expression of Bcl-2 induced by DOX in H9C2 cells (Figure 4C). MitoSox™ Red reagent is a novel fluorescent dye that specifically targets mitochondria in living cells. MitoSox™ red reagents are oxidized by superoxide and produce red fluorescence. Next, we used MitoSox™ to observe the effects of DOX and Naringin on oxidative stress. The results showed that the mitochondria of cells induced by doxorubicin showed a large amount of red color, while the red color decreased after treatment with Naringin (Figure 4D). γ-H2AX could detect genomic damage as well as environmental and physical damage caused by cytotoxic chemicals. In this study, doxorubicin could promote the expression of γ-H2AX in the nucleus, while Naringin could inhibit this phenomenon (Figure 4E). We examined the level of IL-1β, IL-6 and THF-α. The results showed that Naringin could inhibited the level of IL-1β, IL-6 and THF-α induced by DOX (Figures 4F,G).

We have proved Naringin inhibits the effect of DOX on apoptosis, inflammation and Oxidative stress in vitro. Next we looked at what proteins were involved. Oxygen free radicals produced by DOX cause cell damage and could peroxidize unsaturated fatty acids. ECHS1 mutations lead to decreased fatty acid mitochondrial β-oxidation activity, thereby reducing the formation of important energy substrates (such as acetyl CoA), reducing the production of ATP, leading to energy deficiency in the catabolic state (such as fever, after viral infection), and increasing organs. The susceptibility to dysfunction is especially obvious in tissues and organs that rely on fatty acids and ketone bodies as energy sources (such as heart tissue) (Haack et al., 2015). So we guess whether ECHS1 plays a role in the above results. First of all, we verified that DOX could inhibit the expression of ECHS1 in tissues and cells, and after treatment with Naringin, the expression of ECHS1 increased significantly (Figures 5A–C). Next, we constructed the siRNA of ECHS1, and verified by WB that ECHS1 did interfere with the sequence effectively (Figure 5D). Finally, we transfected si-ECHS1 in the presence of both DOX and Naringin, and found that after transfection of si-ECHS1, the activity of caspase3 increased (Figure 5E), the expression of apoptosis-related protein bcl-2 decreased, the expression of BAX increased, and the expression of oxidative stress-related protein NOX2 increased (Figure 5F). The concentration of SOD was inhibited by DOX, while the concentration of SOD increased after Naringin treatment. However, the level of SOD was decreased again after ECHS1 knockdown (Figure 5G). Then we measured the degree of LDH. The results showed that DOX promoted the increase of LDH concentration, which was decreased after Naringin treatment. However, the level of LDH was increased again after ECHS1 knockdown (Figure 5H). We examined the level of IL-1β, IL-6 and THF-α. The results showed that Naringin could inhibited the level of IL-1β, IL-6 and THF-α induced by DOX. However, the level of IL-1β, IL-6 and THF-α was increased again after echs1 knockdown (Figures 5I–K).