Curcumin attenuates apoptosis in diabetic cardiomyopathy by up-regulating Sirt3 and inhibiting SOD2 acetylation

doi:10.21203/rs.3.rs-4700249/v1

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Curcumin attenuates apoptosis in diabetic cardiomyopathy by up-regulating Sirt3 and inhibiting SOD2 acetylation

https://doi.org/10.21203/rs.3.rs-4700249/v1

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Diabetic cardiomyopathy (DCM) is a major complication of type 2 diabetes mellitus (T2DM) that significantly contributes to morbidity and mortality. Streptozotocin (STZ) was used to induce the formation of a type 2 diabetes mellitus (DM) model in rats to research the effect of curcumin on diabetic cardiomyopathy. DM rats showed typical diabetic phenotypes such as increased blood glucose and impaired cardiac function. After curcumin treatment, the cardiac dysfunction and the serum levels of the DM rats were improved. At the same time, the apoptosis of cardiomyocytes decreased and the expression of Sirt3 increased. In vitro, H9c2 cells were cultured under high-glucose and high-fat (HG/HF) conditions, leading to cell apoptosis. Curcumin showed beneficial effects against the apoptosis of HG/HF H9c2 cells. However, after transfection of Sirt3-siRNA, the acetylation modification of superoxide dismutase 2 (SOD2) increased, and the anti-apoptotic effect induced by curcumin was eliminated. Our results showed that curcumin could attenuate diabetic cardiomyopathy by up-regulating Sirt3 and inhibiting SOD2 acetylation.

type 2 diabetes mellitus

diabetic cardiomyopathy

apoptosis

curcumin

Sirt3

Diabetes, about 90% of which is type 2 diabetes mellitus (T2DM), is one of the most serious health challenges in the world [1]. The number of 20–79 year olds people living with diabetes worldwide in 2021 is 536.6 million, which is more than 10.5% of the current world's adult population. Due to the rapid increase in the incidence of diabetes, it is expected to rise to 783.2 million by 2045 [2]. T2DM often causes a variety of complications, such as cardiovascular, renal, and neurologic [3]. These complications, especially cardiovascular diseases, are the leading causes of morbidity and mortality [4]. Diabetic cardiomyopathy refers to the damage to the structure and function of the heart that occurs without coronary artery disease or hypertension [5]. Although it is now clear that cardiomyocyte apoptosis is one of the important pathophysiological characteristics of advanced diabetic cardiomyopathy [6], the exact pathogenesis of cardiomyocyte apoptosis in diabetic cardiomyopathy has not been elucidated.

Curcumin is usually used as a food coloring agent, seasoning, and additives [7]. Numerous studies have revealed that curcumin has a therapeutic effect on a variety of chronic diseases [8, 9], such as tumors [10], diabetes [11], cardiovascular diseases [12], and neurological diseases [13], etc. Researchers have proved that supplementing curcumin can reduce the body weight [14], blood glucose, and lipid level [15] of STZ-induced diabetic rats, and improve the sensitivity of high-fat insulin [16]. Moreover, curcumin can protect diabetic cardiomyopathy by promoting autophagy and reducing apoptosis [17]. Curcumin reduces myocardial fibrosis, oxidative stress, inflammation, and apoptosis by activating the Akt/GSK-3β pathway [18]. Meanwhile, it relieves the oxidative stress of diabetic cardiomyopathy and inhibits cell apoptosis through Sirt1-Foxo1 and PI3K-Akt signaling pathways [19].

Sirt3 is a member of the Sirtuins family, mainly located in mitochondria [20]. It is closely related to a variety of physiological activities of cells, such as energy metabolism [21], oxidative stress [22], and apoptosis [23]. Studies have shown that Sirt3 is closely related to the treatment of diabetic cardiomyopathy. Many drugs can protect diabetic cardiomyopathy by activating Sirt3. For example, melatonin protects diabetic cardiomyopathy through MST1/Sirt3 signaling [24], and garlic activates Sirt3 to prevent diabetic damage [25]. In the hearts of obese mice fed a high-fat diet, the expression level of Sirt3 was low [26]. Sirt3 deacetylation can inhibit Bax-induced cardiomyocyte apoptosis under stress [27]. Superoxide dismutase 2 (SOD2) is a target of Sirt3 [28], which can be deacetylated by Sirt3 and then activated to play a role in antioxidant damage [29].

Our study aims to explore whether curcumin can improve the STZ-induced diabetic cardiomyopathy damage by activating Sirt3, and to provide new and strong evidence of curcumin treatment strategy for diabetic cardiomyopathy.

Animals and treatment

8-week-old adult male Sprague Dawley (SD) rats (n = 60) were purchased from the Shenzhen PKU-HKUST Medical Center and were randomly divided into the following 3 groups (n = 20, each group). (1) Control group (Con): the rats were fed on regular feed for 12 weeks. (2) diabetes mellitus group (DM): the rats were continuously fed with high-fat diet (HFD, Trophic Animal Feed High-Tech Co., Ltd., Nantong, China) for 8 weeks after the diabetes model was established. (3) diabetes mellitus + Curcumin group (Cur): the rats were continuously fed with HFD together with Curcumin (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China 100 mg/kg/day, p.o.) for 8 weeks after the diabetes model was established. Curcumin was initially dissolved in anhydrous ethanol and then diluted in sterile water. The rats were raised in a suitable rearing room (temperature: 20 ℃, humidity: 60%, 12 h light/dark cycle), freely fed and watered.

Establishment of type 2 diabetes mellitus model

After fed with HFD for 4 weeks, the DM and Cur group rats were injected streptozotocin (Sigma-Aldrich Ltd., St. Louis, MO, USA, 60 mg/kg/day) intraperitoneally for 3 days continuously. Streptozotocin (STZ) was dissolved in sodium citrate buffer (0.1 M, pH 4.2–4.5). Con group rats were injected sodium citrate buffer (0.1 M, pH 4.2–4.5, 0.25 ml/kg/day) intraperitoneally for 3 days continuously. One week after STZ injection, blood was collected from the tail vein, and fasting blood glucose was measured by blood glucose meter (Roche Ltd., Basel, Switzerland). A blood glucose level above 11.1 mmol/L was considered diabetes mellitus.

Echocardiography

The rats were anesthetized by inhaling 3% isoflurane (RWD Life Science Co. Ltd., Shenzhen, China), then lay on the operating console in the horizontal position and maintained the situation of anesthesia by 1% isoflurane. Echocardiography was manipulated with a 15 MHz linear transducer in M-mode, using a Vevo2100 High-Resolution Imaging System (Fujifilm Visual Sonics, Toronto, Canada). Echocardiography data included heart rate (HR), early ventricular filling peak velocity (E wave), late filling velocity during the systolic and diastolic phase (A wave), interventricular septum thickness (IVST), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS). All measurements were represented during 5 consecutive cardiac cycles. The ratio of E wave to A wave (E/A), LVEF and LVFS were calculated by computer algorithms.

Serum biochemical substances, and heart failure markers measurements

Glucose, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), lactate dehydrogenase (LDH), cardiac kinase (CK), and brain natriuretic peptide (BNP) were assessed using the rat ELISA Kits (Nanjing Jiancheng Biological Engineering Research Institute Co., Nanjing, China).

Hematoxylin-eosin (HE)

A standard Hematoxylin-eosin (HE, Sigma-Aldrich Ltd.) staining technique was processed. Briefly, heart tissues were fixed with 10% neutral formalin, dehydrated in ethanol, and then embedded. Subsequently, the tissues were sliced into 4-µm-thick sections and stained with hematoxylin-eosin (HE). 10 random fields of view were observed and photographed at 40 times magnification by an optical microscope, and then measured to evaluate the cardiomyocyte diameter by using Image-Pro Plus v.6.0. (Media Cybernetics, Silver Spring, MD, USA).

TUNEL assay

TUNEL staining kit (Roche Ltd.) was used to detect heart tissue sections or H9c2 cells. Cells with TUNEL-positive nuclei were counted under high magnification. The apoptotic index was shown as the ratio of apoptotic cells/the total number of cells×100%.

Immunofluorescence staining

Heart section specimens were fixed with 4% buffered paraformaldehyde, embedded in paraffin, dehydrated by an ascending series of ethanol, and cleaned with xylene. Cultured H9c2 cells were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 20 min, penetrated by blocking solution (1% BSA and 0.1%Triton-X in PBS), and incubated at room temperature for 2 h. The sections or H9c2 cells were then immunostained with a primary antibody against Sirt3 (1:100, Cell Signaling Technology, Boston, MA, USA) at 4 ℃ overnight. Next, the sections or H9c2 cells were incubated with goat anti-mouse IgG secondary antibody (1:50, Beyotime Biotechnology, Shanghai, China) for 1 h at room temperature. The nuclei were stained with DAPI (Beyotime Biotechnology) for 20 min. Finally, the results were obtained using a fluorescence microscope (Nikon Corporation, Otawara, Tochigi, Japan) and analyzed by Image-J software.

Cell culture and treatment

H9c2 cells (purchased from Shanghai Cellular Research Institute) were cultured in DMEM (Hyclone, Logan, UT, USA) with 10% FBS (Hyclone) and 1% penicillin/streptomycin (Hyclone) at 37℃ with 5% CO₂. In vitro, H9c2 cells were randomly allocated into the following groups: (1) control group (Con, 5.5 mM glucose); (2) high-glucose and high-fat group (HG/HF, 33 mM glucose,100 µM palmitate); (3–5) high-glucose and high-fat + different concentrations of curcumin groups (Cur 5 µM, Cur 10 µM, Cur 20 µM). In order to explore the role of Sirt3 in the high-glucose and high-fat environment by curcumin, H9c2 cells were randomly assigned to the following groups: (1) high-glucose and high-fat + NC-siRNA group (HG/HF + NC-siRNA); (2) high-glucose and high-fat + curcumin + NC-siRNA group (HG/HF + Cur + NC-siRNA); (3) high-glucose and high-fat + Sirt3-siRNA group (HG/HF + Sirt3-siRNA); (4) high-glucose and high-fat + curcumin + Sirt3-siRNA group(HG/HF + Cur + Sirt3-siRNA). Before treatment, cells of each group were cultured in low-glucose medium for 12h and then replaced with a new low-glucose medium. Cells were incubated with HG/HF, curcumin, NC-siRNA, or Sirt3-siRNA for 10 h.

Cell viability assay

To assess cell viability, Cell Counting Kit-8 ( Beyotime Biotechnology) was used according to the manufacturer’s instructions. Briefly, the cultured H9c2 cells were inoculated in a 96-well plastic plate for processing. After the treatment, replace the DMEM medium in each well, add 10 µl of CCK-8 reagent, and incubate for 2 h at 37 ℃ in the dark. The absorbance at 450 nm was measured by a microplate reader.

Small interfering RNA

After the cardiomyocytes reached 70%-80% confluency on the day of transfection. Specific double-stranded RNA oligonucleotides targeting Sirt3 (Sirt3-siRNA, sense, 5′-CCAUCUUUGAACUAGGCUUTT-3′, and anti-sense, 5′-AAGCCUAGUUCAAAGAUGGTT-3′; GenePharm, Shanghai, China) and nonspecific control RNA (NC-siRNA, sense, 5′-UUCUCCGAACGUGUCACGUTT-3′, and antisense, 5′-ACGUGACAC GUUCGGAGAATT-3′) were transfected into the cardiomyocytes with Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific Ltd., Waltham, MA, USA) in Opti-MEM medium (Thermo Fisher Scientific Ltd.). The culture medium was exchanged for HG/HF medium with or without curcumin 6 h after transfection.

Western blot analysis

The protein of heart tissue or H9c2 cells was extracted with RIPA lysate (KeyGEN BioTECH, Nanjing, China) containing phenylmethylsulfonyl fluoride (PMSF, KeyGEN BioTECH). Proteins were quantified using the BCA Protein Assay Kit. Equal amounts of protein samples (60 µg, 5–10 µl) were electrophoresed on SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with appropriate primary antibodies against β-actin (1:5000, 4970, Cell Signaling Technology), Bax (1:1000, 2772, Cell Signaling Technology), Bcl-2 (1:1000, 3498, Cell Signaling Technology), caspase-3 (1:1000, 14220, Cell Signaling Technology ), cleaved caspase-3 (1:1000, ab2302, abcam ), Ac-Sod2 (1:1000, ab137037 abcam ), Sod2 (1:500, sc-30080, Santa Cruz Biotechnology) and Sirt3 (1:1000, 5490, Cell Signaling Technology) overnight at 4 ℃. Next, the membranes were washed in TBST and reacted with rabbit anti-goat, goat anti-mouse, or goat anti-rabbit antibodies (Zhongshan Co., Beijing, China) for 2 h at 37 ℃. The blots were scanned and quantified by densitometry analysis using an image analyzer Quantity One System (Bio-Rad, Richmond, CA, USA).

Statistical analysis

Statistical analyses were performed using GraphPad Prism v8.0 (Graph Pad Software, San Diego, CA, USA). Results are presented as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test. As P < 0.05, differences were considered statistically.

Curcumin improves cardiac dysfunction and serum levels in STZ-induced type 2 diabetic rats

To assess the specific effects of curcumin on cardiac function of STZ-induced type 2 diabetic rats, the rats were arranged to undergo echocardiography at the 12th week. The statistical results of echocardiography were shown in Fig. 1A-C and Supplemental Table 1. There is no significant difference (p > 0.05) in heart rate (HR) between the three groups. Compared with the Con group, IVST, and LVESD were increased in the DM group (p < 0.05). Conversely, the LVEDD, LVEF, LVFS, and E/A were decreased in the DM group (p < 0.05). In the Cur group, IVST, and LVESD were decreased, while the LVEDD, LVEF, LVFS, and E/A were increased compared with the DM group (p < 0.05). After that, 12 rats were randomly selected from each group, body weight was measured, and venous blood was collected for serum biochemistry and heart failure biomarker detection. The glucose, TC, TC, LDL-C, LDH, CK, and BNP levels of the DM group at the 12th week were higher than those of the Con group. At the same time, these indicators of the Cur group were lower than those of the DM group (Fig. 1D-K, Sup Table 2). Highlighted in the figures were the baseline characteristics of echocardiography and serum studies. These results indicated that curcumin could be conducive to restoring the cardiac function and serum levels of STZ-induced type 2 diabetic rats.

Figure 1

Curcumin improves cardiac dysfunction and serum levels in type 2 diabetic rats. Body wight (A), LVEF (B), and LVFS (C) of rats in each groups. (D-K) Serum biochemical substances such as glucose, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and heart failure markers such as lactate dehydrogenase (LDH), cardiac kinase (CK), and brain natriuretic peptide (BNP) of rats in each groups. Data are shown as the mean ± SEM, n = 12. ^*P < 0.05, ^**P < 0.01 compared with Con; ^#P < 0.05, ^##P < 0.01 compared with DM.

Curcumin reduces myocardial apoptosis in STZ-induced type 2 diabetic rats

Hematoxylin-eosin (HE) staining (Fig. 2A) of representative heart tissue showed the effect of curcumin on myocardial histology in STZ-induced type 2 diabetic rats. Compared with the Con group, the number of cardiomyocytes in the DM group was reduced (p < 0.05). In the Cur group, the number of cardiomyocytes was higher than that in the DM group. As shown in Fig. 2B-C, the apoptosis of the DM group was higher than that of the Con group, and curcumin treatment could alleviate the apoptosis in diabetic cardiomyopathy (p < 0.05). The ratio of cleaved caspase-3/caspase-3 and the expression of Bax in the DM group increased significantly, and the expression of Bcl-2 decreased, indicating severe apoptosis (Fig. 2D-G, p < 0.05). Meanwhile, these markers of apoptosis in the Cur group were reversed (Fig. 2D-G, p < 0.05).

Figure 2

Curcumin alleviated myocardial apoptosis in type 2 diabetic rats. (A) HE to assess myocardial apoptosis, scale bars = 50 µm. (B) TUNEL staining of myocardial apoptosis. (C) myocardial apoptotic index (%). (D) Western blot of myocardial apoptosis markers. (E-G) Western blot analysis of the ratio of cleaved caspase-3/caspase-3, Bax, and Bcl-2. Data are shown as the mean ± SEM, n = 5. ^*P < 0.05, ^**P < 0.01 compared with Con; ^#P < 0.05, ^##P < 0.01 compared with DM.

Curcumin reduces myocardial apoptosis by up-regulating Sirt3 in STZ-induced type 2 diabetic rats

The representative immunofluorescence staining of heart tissues (Fig. 3A-B) indicated that the fluorescence intensity of Sirt3 in the DM group was reduced. In the Cur group, it has increased compared to the DM group. Moreover, the results of western blot showed the expression of Sirt3 was down-regulated and the ratio of Ac-Sod2/Sod2 increased in the DM group. While in the Cur group, the expression of Sirt3 was up-regulated and the ratio of Ac-Sod2/Sod2 decreased (Fig. 3C-E, p < 0.05). These results suggested that curcumin might prevent myocardial apoptosis by up-regulating Sirt3 and activating Sirt3/Sod2 pathway.

Figure 3

Curcumin alleviated myocardial apoptosis by up-regulating Sirt3 in type 2 diabetic rats. (A) Representative images of Sirt3 immunofluorescence intensity, scale bars = 50 µm. (B) The fluorescence intensity analysis of Sirt3. (C) Western blot of Sirt3, Ac-Sod2 and Sod2. (D-E) Western blot analysis of Sirt3 and the ratio of Ac-Sod2/Sod2. Data are shown as the mean ± SEM, n = 5. ^*P < 0.05, ^**P < 0.01 compared with Con; ^#P < 0.05, ^##P < 0.01 compared with DM.

Curcumin inhibits apoptosis of H9c2 cells by up-regulating Sirt3 in HG/HF situation

In order to further clarify the protective effect of curcumin in type 2 diabetic cardiomyopathy, an in vitro experiment with H9c2 cells was designed. As shown in Fig. 4A-B, not only did curcumin have no toxic effect on cell survival, but it also improved cell viability compared with the Con group or the HG/HF group. The results showed that curcumin at a concentration of 10 µM could exert the cytoprotective effect, and it was selected for follow-up research.

In the HG/HF group, the apoptosis index, the ratio of cleaved caspase-3/caspase-3, and the expression of Bax were higher than those in the control group, while the expression of Bcl2 was lower, indicating that cell apoptosis was aggravated. Curcumin at 10 µM could reduce cell apoptosis, the ratio of cleaved caspase-3/caspase-3, the expression of Bax, and up-regulate the expression of Bcl2 (Fig. 4C-D, F-I, p < 0.05).

In addition, the fluorescence intensity and expression of Sirt3 in the HG/HF group declined, while the ratio of Ac-Sod2/Sod2 was elevated. Once again, curcumin at 10 µM improved the fluorescence intensity and expression of Sirt3, and reduced the ratio of Ac-Sod2/Sod2 (Fig. 4C, E-F, J-K, p < 0.05).

Figure 4

Curcumin reduced apoptosis of H9c2 cells by up-regulating Sirt3. (A-B) The viability of H9c2 cells (%). (C) Representative images of TUNEL staining and Sirt3 immunofluorescence intensity, the apoptotic cells were detected by TUNEL (green), Sirt3 was presented by red, and the nuclei were marked by DAPI (blue), scale bars = 50 µm. (D) Cardiomyocyte apoptotic index (%). (E) The fluorescence intensity analysis of Sirt3. (F) Western blot of apoptosis markers, Sirt3, Ac-Sod2, and Sod2. (G-K) Western blot analysis of the ratio of cleaved caspase-3/caspase-3, Bax, Bcl-2, Sirt3, and the ratio of Ac-Sod/Sod2. Data are shown as the mean ± SEM, n = 5. ^*P < 0.05, ^**P < 0.01 compared with Con; ^#P < 0.05, ^##P < 0.01 compared with HG/HF; ^$P < 0.05, ^$$P < 0.01 compared with Cur (5 µM); ^&P < 0.05, ^&&P < 0.01 compared with Cur (10 µM).

Inhibition of Sirt3 eliminated the curcumin-induced anti-apoptosis of H9c2 cells in HG/HF situation

To further investigate that curcumin reduced cardiomyocyte apoptosis by up-regulating Sirt3, H9c2 were treated Sirt3-siRNA or NC-siRNA to inhibit Sirt3 and block the Sirt3/Sod2 pathway. Compare with Con or NC-siRNA group, Sirt3-siRNA did not affect cell survival, and did not affect the cell viability in HG/HF conditions with or without curcumin (Fig. 5A-B, p < 0.05).

Simultaneously, compared with the HG/HF + NC-siRNA group, the apoptosis index, the ratio of cleaved caspase-3/caspase-3, and the expression of Bax were declined in the HG/HF + Cur + NC-siRNA group, while the expression of Bcl2 was raised. However, these effects were suppressed by Sirt3-siRNA (Fig. 5C-D, F-I, p < 0.05). Meanwhile, the results showed that, compared with the HG/HF + NC-siRNA group, the expression of Sirt3 in the HG/HF + Cur + NC-siRNA group increased, and the ratio of Ac-Sod2/Sod2 decreased. Sirt3-siRNA attenuated curcumin-induced upregulation of Sirt3 expression, anti-apoptotic protection, and anti-heart failure function (Fig. 5C, E-F, J-K, p < 0.05).

Figure 5

Sirt3-siRNA inhibited the curcumin-induced anti-apoptosis in HG/HF treated H9c2 cells. (A-B) The viability of H9c2 cells (%). (C) Representative images of TUNEL staining and Sirt3 immunofluorescence intensity, the apoptotic cells were detected by TUNEL (green), Sirt3 was presented by red, and the nuclei were detected by DAPI (blue), scale bars = 50 µm. (D) Cardiomyocyte apoptotic index (%). (E) The immunofluorescence intensity analysis of Sirt3. (F) Western blot of apoptosis markers, Sirt3, Ac-Sod2, and Sod2. (G-K) Western blot analysis of the ratio of cleaved caspase-3/caspase-3, Bax, Bcl-2, Sirt3, and the ratio of Ac-Sod/Sod2. Data are shown as the mean ± SEM, n = 5. ^*P < 0.05, ^**P < 0.01 compared with NC-siRNA + HG/HF; ^#P < 0.05, ^##P < 0.01 compared with NC-siRNA + HG/HF + Cur; ^$P < 0.05, ^$$P < 0.01 compared with Sirt3-siRNA + HG/HF.

The high morbidity and mortality caused by diabetes and its complications have caused tremendous pressure and challenges to the global health system, and its development trend should not be ignored [30]. Diabetic cardiomyopathy (DCM) is one of the important complications of diabetes, and it is the main cause of morbidity and death in diabetic patients [31]. The diabetic heart is usually accompanied by metabolic disorders, abnormal subcellular components, and immunological changes, which can cause local inflammation, oxidative stress, mitochondrial dysfunction, and then induce cell apoptosis [6]. These changes can cause cardiomyocyte hypertrophy, myocardial interstitial fibrosis, left ventricular diastolic (systolic) dysfunction, decreased left ventricular ejection fraction (LVEF), and decreased cardiac output, leading to heart failure, and ultimately to the death of diabetic patients [32, 33]. Our results of Doppler echocardiography and serum test also indicated that. Meanwhile, after curcumin treatment, the cardiac function and serum biochemical indexes of the DM rats were improved. In DCM, cardiomyocyte apoptosis is often accompanied by cardiac inflammation and oxidative stress and is related to blood glucose levels [34]. Cardiomyocyte apoptosis usually plays an important causal role in the development of diabetic cardiomyopathy [35]. In our research, DM rats showed phenotypic changes with increased blood glucose and increased apoptosis. Curcumin showed the effects of alleviating blood glucose and apoptosis.

The peculiar chemical structure of curcumin allows it to interact with huge numbers of molecules and has a potential role in the prevention and treatment of several diseases [36]. Curcumin has multiple effects such as antibacterial, antidiabetic, antiviral, and anticancer [37, 38], and has been proven to improve inflammatory diseases such as cancer, cardiovascular disease, and osteoarthritis [36]. Curcumin has potent antioxidant, anti-inflammatory, and anti-proliferative properties in DCM [10]. It can inhibit oxidative stress and downregulates NF-kB expression [39]. Besides, it alleviates oxidative stress and inhibits apoptosis via Sirt1-Foxo1 and PI3K-Akt signaling pathways [19]. There is a logical correlation between the antioxidant, anti-inflammatory, anti-apoptotic and anti-fibrotic effects of curcumin. Our research focused on the apoptosis of cardiomyocytes and the anti-apoptotic effect of curcumin. Our results indicated that curcumin inhibited the myocardial apoptosis and the expression of apoptosis-related molecular Bax in DM rats.

Sirt3 is a member of the Sirtuins family, it locates in the mitochondria and widely expresses in the heart [40]. It is considered as a major mitochondrial deacetylase, and its depletion will damage the mitochondria and cardiac function in the heart [41]. Furthermore, Sirt3 plays an important role in the regulation of cardiomyocyte energy metabolism, oxidative stress, apoptosis, and autophagy [42, 43]. In addition, the Apelin-Sirt3 signaling pathway has been demonstrated to prevent and improve mitochondrial dysfunction in DCM [44]. Sirt3 deficiency can lead to defective trans-mitochondrial cristae alignment and impaired mitochondrial bioenergetics, which promotes cardiac dysfunction [45]. The PGC1α-Sirt3 signaling pathway has two functions. Firstly, this pathway enhances mitochondrial function, thereby improving energy metabolism. Secondly, it contributes to antioxidant and anti-inflammatory effects and suppressing cardiomyocyte apoptosis in DCM [46]. Moreover, Sirt3 mediates the deacetylation of Foxo3a, promotes the expression of antioxidant factors, and inhibits oxidative stress [47]. Meanwhile, Sirt3 reduces the expression of TIGAR by inhibiting p53 acetylation, reducing oxidative stress and apoptosis [48]. Our results further proved the anti-apoptotic ability of Sirt3 in DCM. And under the promotion of curcumin, Sirt3 deacetylated Sod2 to prevent oxidative damage and reduce cardiomyocyte apoptosis.

Competing interests

The authors declare that they have no competing interests.

Ethical approval and consent to participate

The animal experiments were approved by Shenzhen PKU-HKUST Medical Center (Guangdong, China), and strictly adhered to the procedures in the Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health Publication, No. 85 − 23, revised 1996).

Funding information

This work was supported by Shenzhen Key Medical Discipline Construction Fund (No. SZXK019) and Sanming Project of Medicine in Shenzhen (SZSM201911018).

Author Contribution

Conceived and designed the experiments: Y. W. Performed the experiments: W. Z. Analyzed the data: W. W. Contributed reagents/materials/analysis tools: HP. L. Wrote the paper: Y. W.

Data availability statement

All data generated or analyzed during this study are included in this published article.

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Curcumin attenuates apoptosis in diabetic cardiomyopathy by up-regulating Sirt3 and inhibiting SOD2 acetylation

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Abstract

Figures

Introduction

Material and methods

Animals and treatment

Establishment of type 2 diabetes mellitus model

Echocardiography

Serum biochemical substances, and heart failure markers measurements

Hematoxylin-eosin (HE)

TUNEL assay

Immunofluorescence staining

Cell culture and treatment

Cell viability assay

Small interfering RNA

Western blot analysis

Statistical analysis

Results

Curcumin improves cardiac dysfunction and serum levels in STZ-induced type 2 diabetic rats

Curcumin reduces myocardial apoptosis in STZ-induced type 2 diabetic rats

Curcumin reduces myocardial apoptosis by up-regulating Sirt3 in STZ-induced type 2 diabetic rats

Curcumin inhibits apoptosis of H9c2 cells by up-regulating Sirt3 in HG/HF situation

Inhibition of Sirt3 eliminated the curcumin-induced anti-apoptosis of H9c2 cells in HG/HF situation

Discussion

Declarations

Competing interests

Ethical approval and consent to participate

Funding information

Author Contribution

Data availability statement

References

Additional Declarations

Supplementary Files

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