BSA stabilized AuNCs were synthesized according to previous reports with slight modifications. Briefly, HAuCl₄ (1 ml, 50 mM, 37°C) and 0.25 g of BSA were mixed in a 10 ml water solution. After the mixture was dissolved as a clear solution, 1 M of NaOH solution was added and the mixture was transferred to a water bath and incubated at 37°C for 12 h. The solution was filtered by a 0.22 µm filter and dialyzed against (10,000 molecular weight cut-off dialysis membrane) with three times distilled water. The solution was filtered. Then, 1 mg of Cur was dissolved in the as obtained BSA-AuNCs (1 ml). For comparison, another 1 mg of Cur was dissolved in a water solution.

H9c2 cardiomyocytes derived from rat myocardium were cultured in Dulbecco’s modified Eagle’s medium (DMEM), all of them are containing 10% fetal bovine serum and 1% penicillin-streptomycin. The culture incubator was placed in 5% CO₂ at 37°C. We used 0.25% trypsin-EDTA (Gibco, Invitrogen, United States) to pass the cells after the cell density reached 80%. The frequency of medium change was 3 days.

The MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was used to estimate the proliferation of H9c2 cells. Briefly, the cells were plated in 96-well plates (5 repeat wells) at a density of 1 × 104 cells/well for 24 h, the cells were serum-starved overnight in a medium containing 0.5% fetal bovine serum. Then, the different concentrations of Cur and AuCur were added for 1 day, respectively. Afterward, 20 μl of MTT solution [5 mg/ml in phosphate buffer solution (PBS)] was added into each well for 4 h. Subsequently, the supernatant was discarded, and 150 μl Dimethyl sulfoxide (DMSO) was added to each well. Shake on a shaker for 15 min. Finally, quantitative detection was performed on a microplate reader at the wavelength of 490 nm.

In situ cells were stained with oil red O staining solution (G1262, Solarbio Biotechnology, Beijing, China) to observe the accumulation of lipid droplets in cells. Briefly, we removed the cell culture, then washed it with PBS 2 times, and added Oro Fixative 20–30 min fixation solution. The fixed solution was removed and washed with distilled water two times. The cells were soaked in 60% isopropanol for 5 min. Then, isopropyl alcohol was removed and a newly prepared Oro Stain was added to soak for 10–20 min. The cells were washed with water 2–5 times until there was no excess dye. Mayer hematoxylin staining solution was added and the nuclei were restained for 1–2 min. After washing the cells again 2–5 times, ORO Buffer was added for 1 min. Finally, the cells were covered in distilled water and examined under a microscope.

The intracellular ROS was analyzed with ROS assay kit (S0033, Beyotime, Shanghai, China) respectively according to the manufacturer’s instructions. Briefly, We washed the cells with PBS. Then, ROS capturing reagent DCFH-DA (2,7-Dichlorodi -hydrofluorescein diacetate) was added and incubated at 37°C for 30 min without light. Finally, fluorescence confocal microscopy (Leica TSC SP5 confocal unit) was used for observation.

In each group, H9c2 cells were washed 3 times by PBS after incubating for a certain time. Then, these cells were fixed by 4% Paraformaldehyde (PFA) for 30 min. Subsequently, the cells were washed by PBS 3 times and then blocked by 5% goat serum for 2 h. After that, these cells were incubated in a culture medium with primary PPARα (1:200, AF5301, Affinity, Biosciences) antibodies, Drp1 (1:50,8570S, Cell Signaling Technology) antibodies, and anti-β-Tubulin antibodies for staying overnight at 4°C. Then, the cells were rinsed with PBS 3 times. Subsequently, these cells were incubated with secondary antibodies (Alexa Fluor 546-labeled anti-rabbit IgG) and the anti-mouse IgG (Alexa Fluor 488-labeled anti-mouse IgG) for 2 h and washed 3times by PBS. Finally, the cells were stained by DAPI for 15 min. Then, the cells imaging experiments were observed by fluorescence microscope.

Apoptotic cell death in the heart was detected in situ by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) staining of fragmented DNA using an In situ Cell Apoptosis Detection Kit (Beyotime, Shanghai, China). The procedure was performed according to the manufacturer’s instructions. Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by 0.3% Triton X-100 and then labeled by incubation (1 h, 37°C) with terminal deoxynucleotidyltransferase and nucleotide mixture containing fluorescein isothiocyanate-conjugated dUTP. The number of TUNEL positive cells was observed under the fluorescence microscope.

The treated cells were collected and lysed with Radioimmunoprecipitation assay buffer (Beyotime, China). Then, the protein concentration was detected by the BCA protein assay kit (Pierce, IL, United States). Equal aliquots of protein (5 μg/ul, 10 μl) were heated at 100°C for 10 min and then fractionated by 10% SDS-PAGE gels. Then, they were put on the PVDF films and treated by TBS-T (with 1% BSA) for 2 h. The proteins were transferred electrophoretically from the gels to PVDF membranes, which were then treated with anti-rabbit CD36, PPARα, dynamin-related protein 1 (Drp1), Bax, Bcl-2 antibodies, and with anti-mouse GAPDH, antibodies overnight at 4°C. Subsequently, they were treated with a corresponding secondary antibody for 2 h. Immunoreactive proteins were revealed using an enhanced chemiluminescence kit (Pierce Chemical, Rockford, IL, United States). Expression of GAPDH was used as the control. The autoradiograms were carried out on an Alpha Innotech Photodocumentation System (Alpha Innotech, Hayward, CA, United States). The relative absorbance of the bands, which representing the amount of protein expression, was analyzed using Quantity One software (Bio-Rad Laboratories).

All experiments have performed a minimum of three times, and the data were analyzed using GraphPad Prism 8 software (GraphPad Software, Inc.). Data are presented as the means ± SD and were analyzed using a t-test. p < 0.05 was considered to indicate a statistically significant difference.

For comparison of Cur and AuCur, the synthesized product was characterized by Scanning Electron Microscope (SEM) and Atomic Force Microscope (AFM). SEM images showed that Cur (Figure 1A) exhibited as large aggregates, indicating Cur themselves were not well-dispersed. On the other hand, with the assistance of AuNCs (Figure 1B), AuCur is homogenously dispersed as small particles (Figure 1C), which will facilitate the adsorption efficiency of AuCur in the body. The high-magnification morphology of AuCur and AuNCs was further characterized by AFM. It presented that Cur was not uniformly distributed (Figure 1D), whereas uniformly dispersed small particles (Figure 1E) could be observed in the AuCur samples. We further found that Cur precipitated as large aggregates within 1 hour (Figure 1F), whereas AuCur remained stable after a month (Figure 1G). This reveals the poorly dispersed and unstable properties of Cur in contrast to AuCur, which is in agreement with SEM and AFM studies. Since the unstable drugs may not survive the bio culture, the more stable AuCur are more promising for long-term use.

0–100 μM of the agents including the Cur group and the AuCur group was used for cell viability assay to check the drug toxicity. The cells used for the assay were myocardial cell line H9C2. After 24 h of drug (Cur) intervention, the cell viability decreased significantly at concentrations greater than 10 μM of Cur (Figure 2A). So, Cur with a concentration of 10 μM was selected for further experiments. In the case of AuCur, tested at same conditions than Cur, concentrations greater than 10 μM did not have a significant effect on cell viability. Indeed, AuCur remained safe up to 100 μM (Figure 2B).

To study the reduction of lipid accumulation In H9c2 cells, H9c2 cells were pretreated with 0.3 μM palmitic acid (PA) for 24 h. Then they were divided into five groups: control group, PA group, Au group, Cur group, and AuCur group (The last three groups were all added to PA). The reducing of lipid accumulation rate of the Cur group and AuCur group was significantly higher than that of the palmitic acid group. In addition, AuCur had a better effect than Cur in reducing lipid accumulation, and the difference was statistically significant (Figures 3A,B). The result was shown in the WB experiment. After palmitic acid treatment, the gray value of the bands increased but decreased after drug treatment. And the result shows that the gray value of AuCur is lower than using Cur (Figures 3C,E,F). As can be seen from the immunofluorescence images, the fluorescence intensity of PA group increased, while that of the Cur group and AuCur group decreased, and the fluorescence intensity of AuCur was weaker than that of Cur group. The difference was statistically significant (Figures 3D,G). The expression of the Lipid transport factor PPARα has increased in PA cultured H9c2 cells. Both Cur and AuCur can ameliorate these phenomena. This indicates that Cur and AuCur block the overexpression of lipid transporters and have a therapeutic effect on lipid accumulation caused by high lipids Therefore, AuCur is more effective at preventing PPARα from overexpressing than Cur.

To investigate the changes in H9C2 cells after palmitic acid treatment, we detected the changes in ROS levels. After adding 0.3 mM PA to cardiomyocytes for 24 h, ROS was detected with DCFH-DA. The results showed that the fluorescence intensity after palmitic acid treatment was significantly increased compared with that of the untreated group, indicating that the ROS level was significantly increased. The other three groups were treated with PA and then treated with three different drugs. The results showed that the fluorescence intensity of the Au group was no significant change. Both the Cur group and the AuCur group had therapeutic effects, and the treatment effect of the AuCur group was better than that of the Cur group, with statistical significance (Figures 4A,E). According to the above results, Cur slightly reduced the ROS, in contrast to AuCur which induce a significant reduction of the ROS. Then we explored changes in the level of mitochondrial fission-related factor Drp1. In the WB experiment, Drp1 increased after PA treatment, Cur and AuCur prevented this phenomenon, and AuCur showed a higher therapeutic effect (Figures 4B,D). Similarly, immunofluorescence results showed that Drp1 was significantly increased after PA treatment. After treatment with Au, Cur, and AuCur, the results showed that AuCur significantly reduced the fluorescence intensity, while the effect of Cur was not as effective as that of AuCur (Figures 4C,F). These results showed that Cur prevented the abnormal increase in the expression of the mitochondrial division factor Drp1 and was more effective when clumped with gold nanoparticles.

In the fluorescence assay that could mark apoptotic cells, the number of apoptotic cells labeled green after treatment with palmitic acid increased, and the green fluorescence was decreased after treatment with Cur and AuCur, and AuCur showed better anti-apoptotic effect than Cur alone (Figures 5A,E). Then we used WB assay to detect three key proteins related to apoptosis and analyze their expression levels. Caspase-3 and Bax are closely related to apoptosis, and their expression is increased when apoptosis occurs, while Bcl-2 is on the contrary. We showed that the ratios of Caspase-3 and Bax to Bcl-2 increased after palmitic acid treatment compared with the untreated group, while both Cur and AuCur prevented this phenomenon, and AuCur showed better effect than Cur (Figures 5B–D). Therefore, AuCur was more effective than Cur in preventing the PA-induced apoptosis of cardiomyocytes.

We used PPARα agonist WY14643 to study the effect of AuCur on PPARα in the treatment of lipotoxic cardiomyopathy. We found that the number of intracellular lipid droplets decreased significantly after AuCur treatment, but increased with the addition of PPARα agonist WY14643 (WY) (Figures 6A,B). The expression of PPARα was detected by WB, and the experiment showed that the expression of PPARα was decreased after AuCur treatment, and the addition of WY14643 could counteract the therapeutic effect of AuCur (Figures 6C,F). The expression of PPARα was detected by immunofluorescence assay. As expected, the fluorescence intensity of PPARα decreased in the AuCur group, and the addition of WY14643 again neutralized the therapeutic effect (Figures 6D,E). PPARα, as a key factor in lipid metabolism, plays a key role in the treatment of DCM. The results showed that AuCur affected lipid accumulation by altering PPARα.

To explore the role of PPARα in the treatment of high lipid-induced ROS elevation by AuCur, ROS fluorescence was filmed. The results showed that PA increased the green fluorescence of ROS, decreased the green fluorescence after AuCur treatment, and increased the green fluorescence after WY and AuCur combined treatment (Figures 7A,B). Similarly, in the immunofluorescence experiment of Drp1, PA increased Drp1 (red fluorescence), while the red fluorescence decreased after AuCur treatment, while the addition of WY increased the red fluorescence (Figures 7D,E). In the WB, Drp1 expression was enhanced under the action of PA, and AuCur inhibited this enhancement, but the inhibitory effect of AuCur was canceled under the action of WY (Figures 7C,F). In terms of the inhibitory effect of AuCur on apoptosis, we also found that AuCur combined with PPARα agonist had no therapeutic effect on the increase of Bax, a decrease of Bcl-2, and an increase of Caspase-3 induced by PA therapy (Figures 7G–I). After high-fat induction, ROS increased, while AuCur inhibited ROS, Drp1, and apoptosis. WY and AuCur simultaneously increased ROS, Drp1, and apoptosis again. These results suggested that the inhibitory effect of AuCur on palmitic acid-induced myocardial damage was neutralized by the PPARα agonist.