Curcumin alleviates oxidative stress and inhibits apoptosis in diabetic cardiomyopathy via Sirt1‐Foxo1 and PI3K‐Akt signalling pathways

Abstract Diabetes is a disorder of glucose metabolism, and over 90% are type 2 diabetes. Diabetic cardiomyopathy (DCM) is one of the type 2 diabetes complications, usually accompanied by changes in myocardial structure and function, together with cardiomyocyte apoptosis. Our study investigated the effect of curcumin on regulating oxidative stress (OS) and apoptosis in DCM. In vivo, diabetes was induced in an experimental rat model by streptozoticin (STZ) together with high‐glucose and high‐fat (HG/HF) diet feeding. In vitro, H9c2 cardiomyocytes were cultured with high‐glucose and saturated free fatty acid palmitate. Curcumin was orally or directly administered to rats or cells, respectively. Streptozoticin ‐induced diabetic rats showed metabolism abnormalities and elevated markers of OS (superoxide dismutase [SOD], malondialdehyde [MDA], gp91phox, Cyt‐Cyto C), enhanced cell apoptosis (Bax/Bcl‐2, Cleaved caspase‐3, TUNEL‐positive cells), together with reduced Akt phosphorylation and increased Foxo1 acetylation. Curcumin attenuated the myocardial dysfunction, OS and apoptosis in the heart of diabetic rats. Curcumin treatment also enhanced phosphorylation of Akt and inhibited acetylation of Foxo1. These results strongly suggest that apoptosis was increased in the heart of diabetic rats, and curcumin played a role in diabetic cardiomyopathy treatment by modulating the Sirt1‐Foxo1 and PI3K‐Akt pathways.

Diabetic patients usually accompanied by metabolic disorders, which directly lead to diabetic cardiomyopathy (DCM). 3,4 Diabetic cardiomyopathy is characterized by cardiac hypertrophy, changes in ventricular structure, as well as diastolic and systolic dysfunction. 5 It increases the risk of heart failure developing in diabetic patients, 6 mostly resulting from cardiomyocyte apoptosis. 7 One of the most important causes of cardiomyocyte apoptosis is oxidative stress (OS), 8,9 accompanied by the reduction of antioxidants (such as superoxide dismutase, SOD) and the large production of high-energy oxidative intermediates (such as reactive oxygen species, ROS), thus increase the apoptosis of cardiomyocyte. 10,11 Curcumin is a natural polyphenol isolated from the root of Curcuma longa, and it has antioxidant, anti-inflammatory, anti-apoptosis and anti-carcinogenic properties. 12,13 Recent research revealed a powerful role for curcumin including alleviating myocardial apoptosis and improving cardiac function in experimental diabetic rats. 14 Curcumin participates in regulation by involvement in a complex molecular regulatory network, including protein kinase C, c-Jun N-terminal kinase (JNK) and advanced glycation end-product pathways. 14,15 In vitro, curcumin can prevent OS induced by NADPH oxidase (NOX) and inhibit cardiomyocyte apoptosis induced by high glucose. 16 ROS produced by NOX plays an important role in controlling metabolism and regulating insulin secretion under physiological conditions. Increased ROS levels in abnormal conditions such as HG/HF intake may induce apoptosis via JNK, PI3K-Akt and other signalling pathways.
Sirtuin 1 (Sirt1) is a highly conserved nicotinamide adenosine dinucleotide (NAD) -dependent deacetylase, which plays regulatory role in metabolism and ageing. Sirt1 can deacetylate histones and several transcription regulators in the nucleus, as well as specific proteins in the cytoplasm and mitochondria, including Sirt1 inhibiting transcription factors (NF-κB, MMP-9, FOXO3a and p53), eNOS, PGC-1α and AMPK. 17 Sirt1 regulates the metabolism of fat and glucose by deacetylating the target proteins, thus acting as crucial regulator of cellular anti-stress, energy metabolism and tumorigenesis, delaying the onset of age-related disease and extending a healthy lifespan. 18 In vivo, emerging evidence from mouse models indicates Sirt1 is a potent protector from ageing-associated pathologies, such as cancer, liver steatosis, neurodegeneration, cardiovascular disease and diabetes. 19 However, there are still a few details that remain unclear, such as the mechanism of curcumin in inhibiting apoptosis and the relationship among multiple signalling pathways mentioned above. Our study investigated the effect of curcumin on regulating apoptosis in DCM.

| Animals
The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 8523, revised 1996) 2-4.5). Rats in control group (n = 10) were injected with citrate buffer alone after being fed a 4-week basic diet. One week after STZ injection, oral glucose tolerance test (OGTT) was performed to confirm STZ injectioninduced hyperglycaemia. Rats were not allowed to eat after 10 PM to make a fasting state. The next morning, rats were orally administrated by 75 g of liquid glucose, and blood glucose (in mmol/L) from tail vein blood samples were measured with a blood glucose meter at 30, 60, 90 and 120 minutes later. Rats with high blood glucose levels (≥13.5 mmol/L) and poor glucose tolerance were considered as diabetic rats. Body weight and blood glucose of rats were recorded every week during the whole experiment. Diabetic model (DM) rats (5 died) were randomly divided into two groups (15 in each group). Curcumin (purchased from Biochem Partner) was orally administered by drinking water (100 mg/kg/d) to treated DM rats (DM + Cur). Untreated DM rats and control rats (Con) received a 1% carboxyl methyl cellulose-Na solution. All rats were sacrificed at age 12 weeks.

| Histopathology
After measurement of heart weight, half rats of each group were used for histochemistry experiments. The entire rat hearts were immersed in 4% paraformaldehyde and placed in treatment boxes.
After a series of gradient alcohol dehydration, they were embedded in paraffin blocks. Paraffin tissues were cut into 4-μm-thick sections, dewaxed in xylene, rehydrated by reducing ethanol concentration, washed in phosphate-buffered saline, then stained with Masson trichrome stain. After staining, the slides were dehydrated by gradient concentration of ethanol and xylene.

| Assessment of cell viability
Cell viability was assessed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. H9c2 cells were cultured in DMEM containing 10% foetal bovine serum for 24 hours in 96-well culture plates at 1 × 10 4 cell per well. Cells were divided into groups of Con, Mannitol, HG/HF, Con with curcumin, HG/HF with curcumin, Con with two inhibitors, as well as combinations of HG/HF, curcumin, inhibitors. MTT was added at a final concentration of 0.5 mg/mL for 4 hours Thereafter, 100 μL DMSO was added to each well and absorbance was measured by using a microplate reader (Thermo Fisher Scientific, USA) with wavelength 490 nm. Cell viability is described as the ratio of the optical density of samples to that of the control.

| TUNEL assay
Apoptosis of tissue and cell was assessed by TUNEL assay. Apoptotic detection kit (Roche) was used to detect DNA fragments in the nucleus in situ. TUNEL-positive cells and total cells were counted, while the ratio was calculated.

| Quantification of SOD and MDA
Hearts or cells were collected and homogenized in phosphatebuffered saline. The amount of SOD and MDA was measured by Superoxide Dismutase (SOD) assay kit and Malondialdehyde (MDA) assay kit (Nanjing jiancheng Biotech, Nanjing, China).

| Western blot analysis
Total protein was separated from hearts or cells, separated by SDS-PAGE, and transferred to PVDF membranes. After being blocked with 5% non-fat dry milk at room temperature (RT) for 2 hours, the protein was incubated with antibodies against

| Statistical analysis
Statistical analyses were performed using GraphPad Prism v5.0 (Graph Pad Software, San Diego, CA, USA). All data are presented as mean ± SEM. One-way ANOVA was used to assess the differences between the groups, and then post hoc testing was conducted through Bonferroni correction as appropriate. P < 0.05 was considered statistically significant.

| Curcumin improves cardiac function in STZinduced type 2 diabetes rats
STZ-induced DM rats showed typical characteristics of type 2 diabetes, including increased blood glucose and decreased body weight ( Figure 1A,B, P < 0.01 vs. Con), while curcumin treatment improved these features. There is no significant difference in survival time of different groups ( Figure 1C). Moreover, the glucose tolerance status of DM and DM + Cur rats on OGTT is worse than that of control

| Curcumin alleviates oxidative stress and inhibits apoptosis of cardiomyocytes in type 2 diabetes rats
TUNEL-positive cardiomyocytes in DM were more numerous (

| Curcumin inhibits apoptosis via Sirt1-Foxo1 and PI3K-Akt signalling in type 2 diabetes rats
For further study of the molecular mechanism, we found that the protein expression levels of Sirt1, PI3K and the phosphorylation of

| Curcumin inhibits apoptosis of H9c2 cardiomyocytes via Sirt1-Foxo1 and PI3K-Akt signalling pathways in HG/HF situation
To corroborate the effect of curcumin on apoptosis in vitro, H9c2 cardiomyocytes were exposed to HG/HF and treated with gradient concentrations of curcumin. Mannitol was used as a control to exclude the effect of HG/HF-induced hyperosmolality on cells.
Curcumin alone or hyperosmolality had no effect on cell viability ( Figure 4A). Once again, the cell viability of HG/HF cells was greatly decreased, and all doses of curcumin could effectively improve viability ( Figure 4B, P < 0.01).
In addition, the proportion of TUNEL-positive cells in HG/HF was much higher than that in Con, while the proportion in HG/HF + Cur was significantly lower than that of HG/HF ( Figure 4C

| Inhibitors of Sirt1-Foxo1 and PI3K-Akt signalling pathways blocked the rescue of curcumin
To further confirm that curcumin could inhibit cardiomyocyte apoptosis via Sirt1-Foxo1 and PI3K-Akt pathways, we used specific inhibitors EX527 (Sirt1 inhibitor) and LY294002 (PI3K inhibitor) to block these two pathways.
The hyperosmolality effect of inhibitors was eliminated by com- Inhibitors did restrain curcumin effect but could not completely prevent it ( Figure 5K-N).
In addition, the expression of Sirt1 and PI3K was significantly reduced by EX527 and LY294002, respectively, and the two pathways Therefore, curcumin could activate Sirt1-Foxo1 and PI3K-Akt pathways, thereby reducing OS and inhibiting apoptosis.

| D ISCUSS I ON
Diabetes is one of the most common non-communicable disease that threaten human health and life. 20 In our study, we used STZ-induced HG/HF fed rats as a type 2 diabetes model. The model rats showed In previous research, the combination of STZ induction with HG/ HF intake was considered a general strategy for obtaining an animal model of the type 2 diabetes because it causes increased blood glucose, weight loss and a positive OGTT test, thus simulating the real course of human type 2 diabetes mellitus. 21,22 Our test results indicated that the modelling was successful, and the method of STZinduced type 2 diabetic rats was feasible. Type 2 diabetes has been shown to cause decreased cardiac function and disordered arrangement of cardiomyocytes, heart failure and even life-threatening. 23,24 Essentially, heart failure is due to type 2 diabetes leading to OS damage and cardiomyocyte apoptosis. 25,26 Our results confirmed these conclusions.
Curcumin has been previously shown to exert antioxidant, anti-inflammatory properties and anti-carcinogenic activity, which has attracted the attention of researchers. 27,28 We found that curcumin could significantly improve diabetic cardiomyopathy by reducing OS and apoptosis in cardiomyocytes. Firstly, curcumin eliminated elevated blood glucose and delayed the onset of cardiovascular complications by controlling metabolic abnormalities. 29 Our results confirmed that curcumin was involved in the control of glycemic metabolism. Furthermore, curcumin activated Sirt1-Foxo1 and PI3K-Akt signalling pathways in the heart of diabetic rats. Several studies have pointed out that Sirt1 has a cardioprotective effect, and its expression in the heart is down-regulated by many stress stimuli that collectively drive the pathogenesis of myocardial infarction. 36 In our data, the expression of Sirt1 reduced in the heart of DM rats and HG/HF H9c2 cells, and recovered after curcumin treatment. Akt has a well-defined role in regulating cardiovascular function (such as heart growth, myocardial contractile function and coronary angiogenesis) and can promote cell survival by inhibiting multiple targets in the apoptosis signalling cascade. Studies have shown that PI3K/Akt signalling pathway is involved in the growth, metabolism and apoptosis of myocardial cells, protecting the heart through PIK3CA. 43 Additionally, the PI3K-Akt-FoxO3a pathway can prevent STZ-induced cardiac function deterioration and structural cardiomyopathy in diabetic rats and reduce the apoptosis of diabetic cardiomyocytes. 44 The decrease of AKT-Foxo1 phosphorylation level is closely related to the occurrence of insulin resistance and apoptosis in DCM, and it plays an important role in inhibiting apoptosis and improving the cardiac function of DCM. 45 We reported for the first time that curcumin can play a protective role by activating the Contribution to reagents/materials/analysis tools. Bin-cheng Ren and Yu-fei Zhang: Writing the paper.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
All experimental procedures for animals were approved by the

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.