Curcumin attenuates palmitic acid-induced cell apoptosis by inhibiting endoplasmic reticulum stress in H9C2 cardiomyocytes

Abstract

Diabetic cardiomyopathy is mediated by multiple molecular mechanisms including endoplasmic reticulum (ER) stress. Curcumin, a phenolic compound, has cytoprotective properties, but its potential protective action against diabetic cardiomyopathy and the related molecular mechanisms are not fully elucidated. In this study, we evaluated the effects of curcumin on cell viability and apoptosis in palmitic acid (PA)-treated H9C2 cardiomyocytes and investigated the signaling pathways involved. Treatment with PA reduced cell viability, induced apoptosis, enhanced apoptosis-related protein expression (Caspase 3 and BCL-2 associated X protein (BAX)), and activated ER stress marker protein expression (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)). Curcumin attenuated PA-induced reduction in cell viability and activation of apoptosis, Caspase 3 activity, BAX, CHOP, and GRP78 expression. 4-Phenylbutyric acid (4-PBA) attenuated the PA-induced effects on cell viability and apoptosis, similar to curcumin. Both curcumin and 4-PBA also attenuated PA-induced increase in ER stress protein (CHOP and GRP78) expression. Curcumin also protected against cytotoxicity, apoptosis, and ER stress induced by thapsigargin. These findings indicate that PA triggers apoptosis in H9C2 cells via ER stress pathways and curcumin protects against this phenomenon.

Introduction

Diabetic cardiomyopathy accounts for more than half of the diabetes-related morbidity and mortality cases. ¹ Elevated free fatty acid levels in the blood contribute to the development of various obesity-associated diseases, such as diabetic cardiomyopathy. Palmitic acid (PA), the main component of dietary saturated fat, causes metabolic diseases, such as diabetes and cardiovascular disease. ² Previous studies have reported that PA induces apoptosis in different cells, such as neurons, ³ granulosa cells, ⁴ hepatocytes, ⁵ endothelial cells, ⁶ and cardiomyocytes. ⁷ And the various effects mechanisms of PA-induced apoptosis involve oxidative stress, ⁸ damage to mitochondria, ⁹ and the inhibition of autophagy. ^10,11 However, the exact molecular mechanism of PA-induced apoptosis has not been fully understood. Recent findings suggest that endoplasmic reticulum (ER) stress also plays an important role in PA-triggered H9C2 cell apoptosis. ^{12 –14}

The ER stores calcium and is a site of protein synthesis and modification. ¹⁵ The excessive accumulation of unfolded or misfolded proteins in the ER can lead to unfolded protein response (UPR). The UPR is a fundamental homeostatic process that leads to the activation of transcription factor 6 (ATF6), inositol-requiring enzyme 1α (IRE1α), and pancreatic ER kinase (PERK). ¹⁶ Under normal physiological conditions, glucose-regulated protein 78-kDa (GRP78) interacts with PERK, IRE1α, and ATF6, which maintains the transmembrane proteins in an inactive state. If the UPR fails to repair the cellular damage, ER stress is prolonged and signaling is activated. ER stress often induces cell apoptosis via CCAAT/enhancer-binding protein homologous protein (CHOP). ¹⁷ 4-Phenylbutyric acid (4-PBA), an ER stress inhibitor, may decrease the expression of ER stress marker proteins.

Curcumin, a phenolic compound present in the turmeric roots, has therapeutic activities against diverse pathological conditions, such as cancer, atherosclerosis, and cardiovascular diseases. ^18,19 Previous studies have revealed that curcumin protects cells and tissues against ischemia-reperfusion injury by its antioxidant ²⁰ and antiinflammatory ²¹ properties. Further, several studies have suggested that curcumin protects the cell against damage by regulating autophagy. ^21,22 Some studies have also demonstrated that curcumin attenuates cell apoptosis through inhibiting ER stress. ^{23 –25} Nevertheless, it is still unclear whether curcumin protects H9C2 cells against PA-induced injury by inhibiting ER stress.

The aim of this study was to evaluate the effects of curcumin on PA-induced injury in H9C2 cells and to explore the possibility that curcumin ameliorated cell apoptosis by inhibiting ER stress.

Materials and methods

Materials

Curcumin (Figure 1), thapsigargin (TG), and 4-PBA were obtained from Sigma-Aldrich (St Louis, Missouri, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, New York, USA). Radioimmunoprecipitation assay (RIPA), phenylmethylsulfonyl fluoride (PMSF), and Tris-buffered saline-Tween-20 (TBST) were obtained from Solarbio (Beijing, China). The H9C2 cardiomyocyte cell line was purchased from the Chinese Academy of Sciences (Shanghai, China). Caspase 3 Activity Colorimetric Assay Kit, Total Protein Extraction Kit, and bicinchoninic acid (BCA) Protein Assay Kit were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Cell Counting Kit 8 (CCK 8) and the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Analysis Kit were purchased from Beijing Zoman Biotechnology Co., Ltd. (Beijing, China). The rabbit anti-rat primary antibodies against BCL-2 associated X protein (BAX; catalog # sc-4239) and β-actin (catalog # sc-517582) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). The rabbit anti-rat primary antibodies against CHOP (catalog # ab10444) and GRP78 (catalog # ab32618) were purchased from Abcam (Cambridge, UK). The goat anti-rabbit secondary antibodies were secured from Proteintech (Wuhan, China).

Figure 1.

The chemical structure of curcumin.

Cell culture and treatment

The H9C2 cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 μg/ml) in a humidified incubator at 37°C in the presence of 5% CO₂. Curcumin, 4-PBA, and TG were prepared in dimethyl sulfoxide (DMSO) and immediately diluted with the culture medium before the experiment. The final concentration of DMSO in the incubation mixture was not more than 0.1% (v/v). When the cells reached 70−80% confluence, they were exposed to various concentrations of curcumin (1.25−40 μM) and/or 400 μM PA. We selected 400 μM PA as the working concentration based on a preliminary study. An additional group of cells was exposed to TG (500 nM) and PA (400 μM) in the absence and presence of 4-PBA (500 nM) and curcumin (10 μM). After incubation for 24 h, the cells were collected to assess cell viability, apoptosis, Caspase 3 activity, and protein expression (by western blotting) was performed. The morphology of the treated H9C2 cells is shown in the Supplementary Figures S1 to S3.

Estimation of cell viability

The H9C2 cell viability was monitored by CCK 8 according to the manufacturer’s instructions. The cells were plated at a density of 2 × 10⁴ per well in 96-well plates. After incubation with different treatments for 24 h, 10 μl of CCK 8 was added to each well, and the cells were incubated for 2 h at 37°C. The resulting absorbance of the samples was measured at 405 nm using a microplate reader (Bio-Rad 680, Hercules, California, USA). The cell viability was calculated as follows: relative viability (%) = [A450(treated)− A450(blank)]/[A450 (control)− A450 (blank)]×100%.

Estimation of cell apoptosis

After treatment, the cells were harvested, washed with phosphate buffer solution (PBS), and digested with 0.25% trypsin, without ethylene diamine tetra acetic acid (EDTA). Thereafter, the cells were centrifuged at 500 × g for 5 min, washed twice with cold PBS, and density adjusted to 1 × 10⁵ cells/ml. The cells were then processed according to the manufacturer’s instructions. Briefly, the cells were initially resuspended in 50 µl of binding buffer, after which 5 µl of PI was added, and the mixture was allowed to stand for 15 min in the dark. Finally, 450 µl of binding buffer and 1 µl of Annexin V-FITC were added and incubated for 15 min in the dark. Apoptosis was detected by flow cytometry (FACS CaliburTM, BD Biosciences, San Jose, California, USA) within 1 h of the last incubation.

Western blotting

After treatment, the cells were collected and washed with ice-cold PBS and lysed with RIPA lysis buffer containing 1% PMSF, and the total protein concentration was measured by BCA assay according to the manufacturer’s instructions. For each sample, 30 µg of total protein was loaded into the wells of a 15% gel and the proteins then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in Tris-glycine running buffer (Solarbio, Beijing, China) at constant voltage (70 V; PowerPac Basic Power Supply, Bio-Rad) for 2 h. The proteins were subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, Massachusetts, USA) with a constant current of 400 mA (PowerPac™ Basic Power Supply) for 90 min under western blot transfer buffer (Solarbio, Beijing, China). After blocking in TBST with 10% skimmed milk for 2 h, the samples were incubated with primary antibody against BAX (1:500), β-actin (1:2000), CHOP (1:1000), or GRP78 (1:1000) overnight at 4°C. After washing, the membranes were incubated with a secondary antibody (1:4000) conjugated to horseradish peroxidase at 37°C for 30 min. The immunoreactive bands were visualized using a Super Signal West Pico kit (Proteintech, Wuhan, China) by Bio-Rad imaging system (Bio-Rad), according to the manufacturer’s instructions. The protein band densities were semi-quantified by densitometric analysis using ImageJ software 1.48 (National Institutes of Health, Bethesda, Maryland, USA).

Estimation of Caspase 3 activity

After the treatment, the cells were harvested by centrifugation and incubated in lysis buffer on ice for 15 min. The lysates were then centrifuged at 15000 r/min at 4°C for 15 min). The protein content was determined using the BCA assay, according to the manufacturer’s instructions. The aliquots were then incubated with the Caspase 3 substrate at 37°C in a microplate for 4 h and the absorbance was recorded at 405 nm using a microplate reader (Bio-Rad 680).

Statistical analysis

All numerical data were expressed as the mean ± standard error of mean from at least three separate experiments. Statistical comparisons were done using one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test, with p < 0.05 indicating significance. All data analyses were done with SPSS software version 13 (SPSS Inc., Chicago, Illinois, USA).

Results

Curcumin attenuated the cytotoxicity of PA in H9C2 cells

Curcumin alone had no effects on the viability of H9C2 cells at concentrations up to 10 μM, whereas at 20 and 40 μM, curcumin decreased the cell viability compared to that of the control group (Figure 2(a)). In addition, compared to the 400 μM PA group, the 2.5−10 μM curcumin enhanced the cell viability in a concentration-dependent manner, but 20 μM curcumin have a lower cell viability compared with 10 μM group and higher than PA-treated cells (Figure 2(b)).

Figure 2.

Curcumin attenuates PA-induced cytotoxicity in H9C2 cells. Viability of H9C2 cells incubated with various concentrations of curcumin alone for 24 h (a). Viability of H9C2 cells incubated with 400 μM PA alone and in the presence of different concentrations of curcumin for 24 h (b). The data are presented as the mean ± SEM of three independent experiments. The columns represent the corresponding experiments. The columns with different letters are significantly different (p < 0.05) from each other. CTL: control; CUR: different concentrations of curcumin; PA: 400 µM palmitic acid.

Curcumin attenuated PA-induced apoptosis in H9C2 cells

PA was added to the culture medium in the absence and presence of different concentrations of curcumin for 24 h, and then the cells were collected for analysis by flow cytometry analysis and apoptosis-related protein detection (Figure 3). The results showed that 400 μM PA triggered cell apoptosis by around 30% cell apoptosis (Figure 3(a)). Curcumin at a concentration of 2.5−20 μM inhibited apoptosis, and 10 μM curcumin exhibited the maximum protective effects (Figure 3(a)). In addition, curcumin inhibited the increase in Caspase 3 activity and BAX protein expression induced by PA (Figure 3(b) and (c)).

Figure 3.

Curcumin attenuates the PA-induced apoptosis (a) and the increase in Caspase 3 activity (b) and BAX expression (c) in H9C2 cells. All activities and protein expression were assessed after a 24 h incubation with 400 μM PA and varying concentrations of curcumin. BAX expression was normalized relative to that of β-actin. The data are represented as the mean ± SEM of three independent experiments. The columns represent the corresponding experiments. The columns with different letters are significantly different (p < 0.05) from each other. CTL: control; CUR: different concentrations of curcumin; PA: 400 µM palmitic acid; BAX: BCL-2 associated X protein.

Curcumin attenuated PA-induced ER stress in H9C2 cells

We detected the effects of curcumin on ER stress marker GRP78 and CHOP expression in the PA-treated H9C2 cells. PA markedly enhanced the expression of GRP78 and CHOP, and this expression was attenuated by curcumin in a concentration-dependent manner up to a concentration of 10 μM (Figure 4(a) to (c)).

Figure 4.

Curcumin attenuates PA-induced ER stress in H9C2 cells. The protein levels of ER stress markers including GRP78 (a, b) and CHOP (a, c) were measured by western blot. All protein expression was assessed after a 24 h incubation with 400 μM PA and varying concentrations of curcumin. GRP78 and CHOP expression was normalized relative to that of β-actin. The data are represented as the mean ± SEM of three independent experiments. The columns represent the corresponding experiments. The columns with different letters are significantly different (p < 0.05) from each other. CTL: control; CUR: different concentrations of Curcumin; PA: 400 µM palmitic acid; CHOP: CCAAT/enhancer-binding protein homologous protein; GRP78: glucose-regulated protein 78; ER: endoplasmic reticulum.

4-PBA attenuated the PA-induced cytotoxicity, apoptosis, and ER stress in H9C2 cells

To confirm the role of ER stress in PA-mediated H9C2 cell apoptosis, H9C2 cells were treated with PA and the ER stress inhibitor 4-PBA. Similar results were observed with the CCK 8 assay and flow cytometry (Figure 5(a) to (c)). 4-PBA significantly enhanced the cell viability and attenuated the apoptosis caused by PA at 24 h (Figure 5(a) to (c)). This compound also reduced the Caspase 3 activity and BAX expression in these cells (Figure 5(d) to (f)). In addition, western blot revealed that the expression of GRP78 and CHOP in PA-treated cells was markedly reduced after treatment with 4-PBA (Figure 5(d), (g), and (h)).

Figure 5.

4-PBA attenuates the PA-induced cytotoxicity, apoptosis, and ER stress in H9C2 cells. Cell viability was measured by CCK 8 assay (a). Apoptosis analysis was detected by flow cytometry (b, c). Western blot of BAX, GRP78, and CHOP expression are shown (d). The Caspase 3 activity of the H9C2 cells is shown (e). The relative BAX expression (f), GRP78 expression (g), and CHOP expression (h) are depicted. The proteins expression levels were normalized to β-actin. The data are represented as the mean ± SEM of three independent experiments. The columns represent the corresponding experiments. The columns with different letters are significantly different (p < 0.05) from each other. CTL: control, PA: 400 µM palmitic acid; 4P: 500 nM 4-PBA; 4-PBA: 4-phenylbutyric acid; CHOP: CCAAT/enhancer-binding protein homologous protein; GRP78: glucose-regulated protein 78; BAX: BCL-2 associated X protein; CCK 8: Cell Counting Kit 8; ER: endoplasmic reticulum.

Curcumin rescued TG-induced H9C2 cell viability decrease, apoptosis, and ER stress

To further examine the effects of curcumin on ER stress-induced cell apoptosis, we added TG to the culture medium to induce ER stress. In a manner similar to PA, TG was found to be cytotoxic to the H9C2 cells. PA induced apoptosis by increasing the Caspase 3 activity and BAX expression, and these were attenuated by curcumin (Figure 6(a) to (f)). Curcumin also attenuated the increase in GRP78 and CHOP expression in the TG-treated cells (Figure 6(d), (g), and (h)).

Figure 6.

Curcumin rescued TG-induced H9C2 cell viability reduction, apoptosis, and ER stress. Cell viability was measured by CCK 8 assay (a). Apoptosis analysis was detected by flow cytometry (b, c). Western blot assays of BAX, GRP78, and CHOP expression are shown (d). The Caspase 3 activity of H9C2 cells is shown (e). The relative BAX expression (f), GRP78 expression (g), and CHOP expression (h) are depicted. The proteins expression levels were normalized to β-actin. The data are represented as the mean ± SEM of three independent experiments. The columns represent the corresponding experiments. The columns with different letters are significantly different (p < 0.05) from each other. CTL: control; TG: 500 nM thapsigargin; CUR: 10 µM curcumin; CHOP: CCAAT/enhancer-binding protein homologous protein; GRP78: glucose-regulated protein 78; BAX: BCL-2 associated X protein; ER: endoplasmic reticulum.

Discussion

Previous studies have reported that PA induces myocardial lipotoxicity by causing oxidative stress, ⁸ damaging mitochondria, ⁹ inhibiting autophagy, ^10,11 and increasing ER stress. ^{12 –14} Further, ER stress plays a key role in the process of cell injury. Some studies have reported that PA-induced injury can be attenuated by inhibiting ER stress. ^13,14 Curcumin, a phenolic compound, can protect the cells against lipotoxicity. ^2,26,27 A recent study reported that curcumin inhibits ER stress caused by cerebral ischemia-reperfusion injury in rats. ²⁸ However, the mechanisms underlying the protective effects of curcumin on PA-induced myocardial lipotoxicity are not fully understood. Here, we used H9C2 cardiomyocytes to study the effects and molecular mechanisms of action of curcumin on PA-induced H9C2 cell injury.

We first detected the effects of curcumin on cell viability. The results showed that 1.25−10 μM curcumin had no effect on cell viability, but 20 and 40 μM curcumin inhibited H9C2 cell viability. Our results are consistent with previous studies that curcumin at low concentrations protects cells against apoptosis. ^22,29 On the other hand, high curcumin in concentrations cause death in many cell types, especially in cancer cells. ^30,31

Previous studies have reported that PA induces H9C2 cell injury and apoptosis. ^14,32 Recent studies have demonstrated that PA induces apoptosis in the H9C2 cells by increasing the levels of BAX and the activation of Caspase 3. ⁸ Caspase 3 plays a key role in the caspase-mediated apoptosis pathway, which is positively correlated with the rate of apoptosis. ³³ BAX, belonging to the BCL-2 family, also has a pro-apoptotic effect and has an important effect on cell apoptosis. ³⁴ Consistent with the previous findings, our study suggested that PA increases cell apoptosis and decreases cell viability. Furthermore, we found that PA-induced H9C2 cell apoptosis was related to greater Caspase 3 activity and upregulation of BAX.

Some studies have provided evidence that curcumin ameliorates H9C2 cell injury and apoptosis. ¹⁰ Consistent with these studies, in the current study, we found that curcumin improved cell viability and decreased cell apoptosis and the expression of apoptosis-related proteins, including BAX and Caspase 3. Curcumin also significantly reduced H9C2 cell apoptotic rate and improved cell viability at concentrations of 2.5−20 μM, with 10 μM curcumin providing the greatest protection. Based on these results, we concluded that curcumin suppresses apoptosis in the PA-treated cells.

The mechanism(s) involved in the protective action of curcumin in PA-induced cell injury and apoptosis are still not fully understood. Recent studies suggest that curcumin prevents diabetic cardiomyopathy by suppressing apoptosis through restoring autophagy. In vivo and in vitro studies have demonstrated that the protective actions of curcumin are associated with the activated c-Jun N-terminal kinase (JNK)-1 and AMP-activated protein kinase (AMPK) pathways. ¹⁰ Curcumin also plays a significant protective role against apoptosis through inhibiting ER stress. ^23,25,28 In our study, we found that PA enhanced the expression of ER stress marker genes, such as GRP78 and CHOP, suggesting that PA successfully induces ER stress in the H9C2 cells. This finding is consistent with a previous study showing that ER stress is involved in PA-induced H9C2 cell apoptosis. ^31,35,36 Interestingly, we also found that curcumin decreases the GRP78 and CHOP expression in a concentration-dependent manner. Therefore, our study demonstrated that curcumin inhibits PA-induced ER stress in myocardial injury.

We further studied the role of ER stress in PA-mediated H9C2 cell apoptosis. We found that the inhibition of ER stress by 4-PBA attenuated the PA-induced reduction in cell viability and increase in apoptosis. This was further supported by the decrease in the protein expression of CHOP, BAX, and Caspase 3 activity. This result is consistent with previous studies showing that PA induces ER stress and apoptosis in hepatoma cells, ^35,37 renal cells, ³⁶ and pancreatic islet β-cells. ³⁸

To further decipher the effects of curcumin on ER stress-mediated apoptosis, H9C2 cells were incubated with TG to induce ER stress. In these experiments, curcumin inhibited the expression of GRP78, CHOP, and BAX; decreased the Caspase 3 activity and apoptosis; and reversed the decrease in viability caused by TG. The results further supported the hypothesis that curcumin inhibits ER stress ^28,39,40 and cell apoptosis. ^22,33

In conclusion, our study demonstrated that PA inhibited cell viability and induced cell apoptosis in H9C2 cells by inducing ER stress. By ameliorating the ER stress, curcumin protected H9C2 cells against lipotoxicity. These results suggest a potential therapeutic application in which curcumin may be useful in attenuating ER stress and protecting against cardiomyopathy.

Supplemental material

Supplemental Material, Fig_S1 - Curcumin attenuates palmitic acid-induced cell apoptosis by inhibiting endoplasmic reticulum stress in H9C2 cardiomyocytes

Supplemental Material, Fig_S1 for Curcumin attenuates palmitic acid-induced cell apoptosis by inhibiting endoplasmic reticulum stress in H9C2 cardiomyocytes by G Guan, L Lei, Q Lv, Y Gong and L Yang in Human & Experimental Toxicology

Supplemental material

Supplemental Material, Fig_S2 - Curcumin attenuates palmitic acid-induced cell apoptosis by inhibiting endoplasmic reticulum stress in H9C2 cardiomyocytes

Supplemental Material, Fig_S2 for Curcumin attenuates palmitic acid-induced cell apoptosis by inhibiting endoplasmic reticulum stress in H9C2 cardiomyocytes by G Guan, L Lei, Q Lv, Y Gong and L Yang in Human & Experimental Toxicology

Supplemental material

Supplemental Material, Fig_S3 - Curcumin attenuates palmitic acid-induced cell apoptosis by inhibiting endoplasmic reticulum stress in H9C2 cardiomyocytes

Supplemental Material, Fig_S3 for Curcumin attenuates palmitic acid-induced cell apoptosis by inhibiting endoplasmic reticulum stress in H9C2 cardiomyocytes by G Guan, L Lei, Q Lv, Y Gong and L Yang in Human & Experimental Toxicology