Quercetin improves diabetic cardiomyopathy by inhibiting pyroptosis via the Nrf2 pathway

doi:10.21203/rs.3.rs-1876714/v1

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Quercetin improves diabetic cardiomyopathy by inhibiting pyroptosis via the Nrf2 pathway

https://doi.org/10.21203/rs.3.rs-1876714/v1

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Introduction: The aim of the study was to investigate whether quercetin can promote the translocation of Nuclear factor erythroid-2 related factor 2 (Nrf2) into the nucleus to inhibit pyroptosis progression and improve diabetic cardiomyopathy.

Research design and methods:The protective effect of quercetin against diabetic cardiomyopathy was evaluated using a diabetic rat model by analyzing the expression of pyroptosis pathway proteins, myocardial cell apoptosis rate, degree of myocardial fibrosis, and serum inflammatory indexes in the hearts of rats in each group. The expression of Nrf2 in the nucleus of myocardial tissue and H9C2 cells were analyzed to clarify the role of quercetin in promoting the translocation of Nrf2 into the nucleus. In addition, the Nrf2 inhibitor ml385 was co-incubated with the myocardial tissue to confirm that quercetin inhibits diabetes-induced cardiomyocyte pyroptosis via the Nrf2 pathway.

Results: Quercetin promoted the translocation of Nrf2 into the nucleus in the cardiac cells of diabetic rats, increased the expression of antioxidant proteins HO-1, GCLC, and SOD, reduced the accumulation of ROS and the level of cell apoptosis in diabetic cardiomyocytes, and alleviated the cardiac fibrosis caused by diabetes. The therapeutic effects of quercetin were further validated in H9C2 cardiomyocytes. Additionally, ml385 prevented the beneficial effects of quercetin on diabetic cardiomyopathy, further indicating that the inhibition of pyroptosis by quercetin requires the participation of the Nrf2 pathway.

Conclusions: Quercetin promotes the translocation of Nrf2 into the nucleus, increases the expression of antioxidant factors in cells, and inhibits the progression of cell pyroptosis thereby alleviating diabetic cardiomyopathy.

Quercetin

Pyroptosis

Diabetic cardiomyopathy

Nrf2

Diabetic cardiomyopathy is a common complication of diabetes mellitus, with a potentially insidious early stage and late-stage complications such as endothelial damage to the coronary vessels and decreased myocardial contractility^[1]. Cardiac myocytes remain in a high-glucose state for an extended period, resulting in insulin resistance, increased glucose autoxidation, protein glycosylation, lipid peroxidation, intracellular peroxide accumulation, cardiomyocyte death, and increased cardiac fibrosis^[2,3].

Pyroptosis is a distinct caspase-dependent inflammatory cell necrosis characterized by cell lysis and the release of cytosolic contents into the surrounding environment, resulting in a strong inflammatory response^[4,5]. Various stimuli can induce pyroptosis in cells. Pi et al. discovered that molybdenum can induce pyroptosis in renal tubular epithelial cells and that drugs that prevent peroxide accumulation can significantly reduce the expression of NLR family pyrin domain containing 3 (NLRP3) and other substances involved in the classic pyroptosis pathway and reduce epithelial cell damage^[6]. In cardiovascular disease, substances such as high fat and urea can cause cardiomyocytes to undergo pyroptosis, which causes a cascade reaction and the release of a large number of inflammatory mediators, resulting in the development of a local inflammatory microenvironment in the myocardium^[7,8]. Therefore, preventing the development of pyroptosis can slow the progression of diabetic cardiomyopathy.

Quercetin is a natural flavonoid that is found in a wide range of plants. Previous research has indicated that quercetin has antioxidant and anti-inflammatory properties^[9]. Sul et al. demonstrated that quercetin prevents LPS-induced oxidative stress and inflammation by modulating NOX2/ROS/NF-kB in lung epithelial cells^[10]. Nuclear factor erythroid-2 related factor 2 (Nrf2) is the primary regulator of cellular resistance to oxidative stress. When the body is subjected to oxidative stress, Nrf2 in the cell translocates to the nucleus, where it promotes the expression of a wide range of antioxidant factors, thereby maintaining the body's redox balance ^[11,12]. As far as we know, there are no reports on whether quercetin can promote the translocation of Nrf2 into the nucleus and inhibit the progression of diabetes-induced pyroptosis. Therefore, this study aims to investigate this issue.

1.1 Construction of the diabetic rat model

2-month-old Sprague Dawley male rats were purchased from the Bengbu Medical College Experimental Animal Center . The diabetic rat model was established by high-fat feeding for four weeks and intraperitoneal injection of streptozotocin (50 mg/kg). A diabetic rat model was considered successful when the fasting blood glucose level was 11 mmol/L on two measurements, or 14 mmol/L in a single session. The rats were divided into the normal (CON-Group), diabetic (DM-Group), interval dosing + diabetic rat (Qod-Group), and daily dosing + diabetic (Qd-Group) groups with six rats each. Rats in each group were incubated for 6 months under the same room conditions. Among the administered rats, quercetin solution was used instead of daily drinking water (160 mg/mg. kg^-1.d^-1). All animals were raised in the Bengbu Medical College Animal Experiment Center, with different groups raised in different cages. The experimental environment was managed by the Animal Center: a room temperature of 26 °C and 12 h light-dark cycles were maintained, and the breeding environment was cleaned regularly by professionals. The experiment was approved by the Medical Ethics Committee of the First Affiliated Hospital of Bengbu Medical College (BYYFY-2021KY03).

1.2 Myocardial tissue sections

After the experimental rats were anesthetized with ether, they were humanely killed by cervical dislocation. Myocardial tissue of approximately 1 cm² was extracted from the apical part of the heart, fixed with 4% paraformaldehyde, embedded in paraffin. The myocardial sections were stained with masson and HE according to the instructions of staining reagents. All remaining animal carcasses were sent to the animal room for unified disposal.

1.3 ELISA to detect serum indicators

Venous blood was drawn from the tail veins of the rats, and the supernatant was collected after centrifugation and stored in a refrigerator at −80 °C for cryopreservation. Serum inflammatory markers and indicators of myocardial damage, including NF-kB, IL-1, TNF-α , and brain natriuretic peptide (BNP), were estimated according to the ELISA kit procedure; each serum sample was analyzed six times.

1.4 High sugar cardiomyocyte treatment

Rat H9C2 cardiomyocytes were purchased from the Shanghai iCell Biological Company, China, and were stored and cultured in the Cardiovascular Laboratory of Bengbu Medical College. The cells were divided into a normal medium group (Con-G, with medium glucose concentration of 5.5 mmol/L) and a high-glucose medium group (H-G, with medium glucose concentration of 30 mmol/L,).

1.5 Cardiomyocyte nucleoprotein extraction

After digestion and centrifugation of cells, cell plasma protein extraction reagent was added according to the manufacturer’s instructions and mixed well in an ice bath for 10 min; the resulting mixture was centrifuged at 12,000 g for 15 min, and the supernatant was removed. Subsequently, the cellular nucleoprotein extraction reagent was added, and mixed thoroughly in an ice bath for 10 minutes; the mixture was centrifuged at 12000 g for 15 minutes, and the supernatant (cellular nucleoprotein) collected.

1.6 Mitochondrial membrane potential measurement

A membrane potential staining solution was formed by adding 2 µl of JC-1 solution to 900 µl of JC-1 solvent and 100 µl of JC-1 staining buffer and mixing thoroughly. The treated cardiomyocytes were removed from the incubator, their media removed, and the staining solution and culture medium were added at a 1:1 ratio. Cells were incubated for 20 min, the staining solution was washed away, and the mitochondrial membrane potential was observed under a fluorescence microscope.

1.7 Tunel assay

Cell slides or tissue frozen sections were fixed with paraformaldehyde and rinsed three times with PBS. After 15 minutes of 0.5% Triton permeabilization membrane, rinsed three times with PBS, dripped the prepared stain on the surface of cells or slices, and incubated in an incubator for 1 hour in the dark. After three washes with PBS, the cells were counterstained with DAPI and observed under a microscope.

1.8 Main reagents

The following reagents were purchased: Nuclear Protein Extraction Kit (R0050), ml385 (IM1020) ,Quercetin (SQ8030) (Solarbio Life Science, Beijing, China); GCLC Rabbit Polyclonal Antibody (AF6969), Nrf2 Rabbit Polyclonal Antibody (AF7623), HO-1 Rabbit Polyclonal Antibody, One Step TUNEL Apoptosis Assay Kit (C1086), and Reactive Oxygen Species Assay Kit (S0033s), Enhanced mitochondrial membrane potential assay kit with JC-1(C2003S), Cell Counting Kit-8(C0037) (Beyotime Biotechnology, Shanghai, China); NLRP3 Rabbit Polyclonal Antibody (GB13457), Caspase-1 Rabbit Polyclonal Antibody (GB11383) (Servicebio, Wuhan, China); DMEM high glucose medium (SH30003; Hyclone, Utah, USA) . mRNA primers (Tsingke Biotechnology, Nanjing, China). HE Staining Kit(G1001), Masson's Trichrome Stain Kit(GP1032) (Servicebio,Wuhan,China). BNP(SEKF-0205),TNF-α (SEKR-0009),IL-1(SEKR-0002),NF-kB(

K009537P) (Solarbio Life Science, Beijing, China).

1.9 Statistical methods

SPSS software (version 25.0) was used to process all statistical data. All experiments were conducted at least three times, and data are expressed as mean ± SD. Student’s t-test was used to determine the statistical difference between the two groups of data. Images were analyzed using ImageJ software. Statistical significance was set at P < 0.05.

2.1 Quercetin improves myocardial fibrosis in diabetic rats

ELISA was used to identify inflammatory markers and indicators of myocardial damage, including NF-kB, IL-1, TNF-α, and BNP, in the serum of diabetic rats. The results showed that feeding quercetin significantly reduced the levels of inflammatory factors in the serum of diabetic rats, and decreased the secretion of BNP in the heart (Fig. 1A). Diabetes significantly increased the expression of pyroptosis proteins and decreased the expression of SOD proteins in the myocardial tissue (Fig. 1B and 1C). However, quercetin significantly inhibited the progression of pyroptosis in the diabetic myocardium, and the therapeutic effect became more pronounced as the frequency of administration increased (Fig. 1B and 1C). The results from analysis of the tissue sections further indicated that quercetin significantly reduced diabetes-induced cardiomyocyte apoptosis and inhibited the expression of collagen II and the progression of myocardial fibrosis (Fig. 1C and 1D). Additionally, we extracted nuclear proteins from the heart tissue and found that quercetin significantly increased the expression of Nrf2 protein in cardiomyocyte nuclei and activated the expression of downstream antioxidant factors HO-1 and GCLC (Fig. 1F). Therefore, we speculated that quercetin can ameliorate diabetic myocardial injury via the Nrf2 pathway.

2.2 Quercetin reduces high-glucose-induced cardiomyocyte injury

Further analysis was performed to better understand the mechanism of action of quercetin in cardiomyocytes. According to the CCK-8 data, the ideal dose of quercetin to increase cardiomyocyte activity is 12 µM (Fig. 2A). WB results revealed that quercetin dramatically reduced high-glucose-induced pyroptosis protein expression in cells, enhanced SOD protein expression, and successfully suppressed pyroptosis (Fig. 2B and 2C). Quercetin could also reduce the accumulation of ROS in cardiomyocytes and mitochondrial damage caused by high glucose, as well as ameliorate cell damage caused by high glucose, according to the estimates of mitochondrial membrane potential and intracellular reactive oxygen species in cardiomyocytes（Fig. 2D).

2.3 Quercetin promotes the translocation of Nrf2 into the nucleus

Nuclear proteins were extracted from H9C2 cells and the expression of Nrf2 protein in the nucleus was determined. This demonstrated that quercetin considerably boosted Nrf2 expression in the nucleus and accelerated the translocation of Nrf2 into the nucleus of cardiomyocytes（Fig. 3A). The expression of downstream antioxidant factors HO-1 and GCLC was also markedly elevated by the translocation of Nrf2 into the nucleus (Fig. 3B and 3C). The degree of cell viability in high-glucose media was dramatically increased by the overexpression of these antioxidant proteins (Fig. 3D). These results suggest that the inhibitory effect of quercetin on pyroptosis is related to the activation of the Nrf2 pathway.

2.4 ml385 inhibits the translocation of Nrf2 into the nucleus

As ml385 effectively blocks the Nrf2 pathway, 20 µM of ml385 was added to the medium to determine whether quercetin inhibited the pyroptosis pathway by inhibiting Nrf2. ml385 significantly inhibited mRNA expression of the antioxidant factors HO-1 and GCLC in the Nrf2 pathway (Fig. 4A). Further experiments confirmed that the addition of ml385 significantly inhibited the effect of quercetin against pyroptosis, increased the expression levels of pyroptosis proteins and decreased the expression levels of antioxidant proteins HO-1 and GCLC (Fig. 4B and 4C). Addition of ml385 abolished quercetin’s ability to inhibit apoptosis under high-glucose conditions (Fig. 4D). The results of these experiments further demonstrated that the Nrf2 pathway is necessary for quercetin to prevent pyroptosis.

Diabetes creates a high-glucose environment in the myocardium, causing myocytes to increase fatty acid uptake and oxidation in order to generate ATP. β-oxidation does not fully metabolize long-chain fatty acids, causing lipid accumulation and lipotoxicity in cells^[13-15]. High sugar levels promote the accumulation of cholesterol, fatty acids, and other substances in the blood, activate inflammatory factors such as TNF, IL-6, and IL-1, and worsen myocardial damage^[16,17]. Furthermore, these factors can activate the pyroptotic pathway, resulting in cell membrane rupture, release of numerous inflammatory factors, increased cardiac fibrosis, and a local inflammatory microenvironment^[18,19]. Therefore, inhibition of cardiomyocyte pyroptosis is an effective way to treat diabetic cardiomyopathy. This experiment confirmed that diabetes can lead to pyroptosis in rat cardiomyocytes and promote an increase in inflammatory factors NF-kB, IL-1, and TNF-α . Long-term inflammatory stimulation promotes cardiomyocyte apoptosis and increases collagen expression, thereby aggravating myocardial fibrosis. Accumulation of various factors reduces cardiac function and increases BNP secretion. Following quercetin administration, the expression of pyroptosis proteins in the myocardium was markedly decreased, the level of antioxidant proteins such as SOD was elevated, the incidence of myocardial cell apoptosis in diabetes was inhibited, and the progression of myocardial fibrosis was slowed. Moreover, the degree of inhibition of pyroptosis and fibrosis became more pronounced as the frequency of quercetin administration increased.

Further analysis of Nrf2 protein expression in the myocardial nucleus revealed that quercetin might enhance the translocation of Nrf2 into the nucleus, boost the expression of downstream antioxidant proteins HO-1 and GCLC, and inhibit ROS build-up and myocardial apoptosis. Studies have shown that Nrf2 is an important antioxidant and exerts a protective effect on the myocardium^[20,21]. Cen et al. discovered that mitoQ can promote Nrf2 nuclear translocation, scavenge intracellular peroxides, and upregulate downstream antioxidant response elements like HO-1 and quinone oxidoreductase to reduce cell damage^[22]. Quercetin is cytoprotective because it reduces the accumulation of intracellular ROS and increases the content of antioxidant enzymes^[23]. El-Fa et al. discovered that quercetin could reduce oxidative stress, downregulate the expression of inflammatory factors NF-κB and IL-6, reduce cell pyroptosis, and reduce galactose-induced kidney damage^[24].

However, to the best of our knowledge, no study to date has clarifies the relationship between quercetin and the Nrf2 pathway and whether the drug can inhibit pyroptosis in the diabetic myocardium via this pathway. Quercetin can promote the translocation of Nrf2 into the nucleus, enhance the expression of HO-1 and GCLC, and lessen the buildup of ROS in the myocardium brought on by diabetes, according to this study’s results at the rat myocardial tissue and cell levels. By promoting the translocation of Nrf2 into the nucleus, quercetin inhibits diabetes-induced pyroptosis, alleviates mitochondrial damage, and increases cardiomyocyte activity. However, the efficacy of quercetin in preventing pyroptosis was considerably diminished after the addition of ml385. This further demonstrated that the Nrf2 pathway is necessary for quercetin to prevent pyroptosis.

Diabetic cardiomyopathy is a common complication in patients with diabetes. The mechanism of diabetic cardiomyopathy remains unknown, and no specific therapeutic drugs are available.

This study found that quercetin can promote the translocation of Nrf2 into cells, increase the expression of downstream antioxidant factors HO-1 and GCLC, clear the accumulation of intracellular ROS, and inhibit the process of pyroptosis in diabetic cardiomyopathy. Quercetin can reduce the stimulation of inflammatory factors in myocardial tissue, delay the development of myocardial fibrosis, and safeguard heart function. This study elucidates the pathogenesis of diabetic cardiomyopathy and provides a new direction for the treatment of diabetes.

AREs: antioxidant response elements

NQO-1: quinone oxidoreductase

GCLC: glutamate cysteine ligase

HO-1: heme oxygenase

Nrf2: nuclear factor erythroid-2 related factor 2

Competing interests

All authors declared no competing financial interests.

Ethics approval

Ethical approval was given by the medical ethics committee of Bengbu Medical College with the following reference number :( BYYFY-2021KY03).

Funding

The present study was supported by research grants from Science and Technology Project of Anhui Province(1804h08020280).

Availability of data and materials

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Authors' contributions

Zhang Wei and Zhou Jing are responsible for the main experimental process; Kang Pinfang and Shi Chao are responsible for the arrangement of the experimental data; Qian Shaohuan is responsible for the experimental design and integration. All authors reviewed the manuscript.

Lee MMY, McMurray JJV, Lorenzo-Almorós A, et al. Diabetic cardiomyopathy. Heart. 2019 ;105(4):337-345.
Nakamura M, Sadoshima J. Cardiomyopathy in obesity, insulin resistance and diabetes. J Physiol. 2020;598(14):2977-2993.
Tang SG, Liu XY, Wang SP, et al. Trimetazidine prevents diabetic cardiomyopathy by inhibiting Nox2/TRPC3-induced oxidative stress. J Pharmacol Sci. 2019 Apr;139(4):311-318.
Yu P, Zhang X, Liu N, et al Pyroptosis: mechanisms and diseases. Signal Transduct Target Ther. 2021;6(1):128.
Tan Y, Chen Q, Li X, et al. Pyroptosis: a new paradigm of cell death for fighting against cancer. J Exp Clin Cancer Res. 2021;40(1):153.
Pi S, Nie G, Wei Z, et al. Inhibition of ROS/NLRP3/Caspase-1 mediated pyroptosis alleviates excess molybdenum-induced apoptosis in duck renal tubular epithelial cells. Ecotoxicol Environ Saf. 2021 ;208:111528.
Jiang M, Sun X, Liu S, et al. Caspase-11-Gasdermin D-Mediated Pyroptosis Is Involved in the Pathogenesis of Atherosclerosis. Front Pharmacol.2021;12:657486.
Sun Z, Chai Q, Zhang Z, et al. Inhibition of SGLT1 protects against glycemic variability-induced cardiac damage and pyroptosis of cardiomyocytes in diabetic mice. Life Sci. 2021;271:119116.
Chen KTJ, Anantha M, Leung AWY,et al. Characterization of a liposomal copper(II)-quercetin formulation suitable for parenteral use. Drug Deliv Transl Res. 2020;10(1):202-215.
Sul OJ, Ra SW. Quercetin Prevents LPS-Induced Oxidative Stress and Inflammation by Modulating NOX2/ROS/NF-kB in Lung Epithelial Cells. Molecules. 2021;26(22):6949.
Sánchez-de-Diego C, Pedraza L, Pimenta-Lopes C, et al. NRF2 function in osteocytes is required for bone homeostasis and drives osteocytic gene expression. Redox Biol. 2021;40:101845.
Li L, Fu J, Liu D, Sun J, et al. Hepatocyte-specific Nrf2 deficiency mitigates high-fat diet-induced hepatic steatosis: Involvement of reduced PPARγ expression. Redox Biol. 2020;30:101412.
Sun Y, Zhou S, Guo H, et al. Protective effects of sulforaphane on type 2 diabetes-induced cardiomyopathy via AMPK-mediated activation of lipid metabolic pathways and NRF2 function. Metabolism. 2020;102:154002.
ALTamimi JZ, BinMowyna MN, AlFaris NA, et al. Fisetin protects against streptozotocin-induced diabetic cardiomyopathy in rats by suppressing fatty acid oxidation and inhibiting protein kinase R. Saudi Pharm J. 2021;29(1):27-42.
Wang Z, Zhu Y, Zhang Y, et al. Protective effects of AS-IV on diabetic cardiomyopathy by improving myocardial lipid metabolism in rat models of T2DM. Biomed Pharmacother. 2020 ;127:110081.
Wang L, Cai Y, Jian L, et al. Impact of peroxisome proliferator-activated receptor-α on diabetic cardiomyopathy. Cardiovasc Diabetol. 2021 ;20(1):2.
Karwi QG, Sun Q, Lopaschuk GD. The Contribution of Cardiac Fatty Acid Oxidation to Diabetic Cardiomyopathy Severity. Cells. 2021;10(11):3259.
Tan Y, Zhang Z, Zheng C, et al. Mechanisms of diabetic cardiomyopathy and potential therapeutic strategies: preclinical and clinical evidence. Nat Rev Cardiol. 2020;17(9):585-607.
Zhang B, Li X, Liu G, et al. Peroxiredomin-4 ameliorates lipotoxicity-induced oxidative stress and apoptosis in diabetic cardiomyopathy. Biomed Pharmacother. 2021;141:111780.
Fox DB, Garcia NMG, McKinney BJ, et al. NRF2 activation promotes the recurrence of dormant tumour cells through regulation of redox and nucleotide metabolism. Nat Metab. 2020;2(4):318-334.
Jiang N, Zhao H, Han Y, Li L,et al. HIF-1α ameliorates tubular injury in diabetic nephropathy via HO-1-mediated control of mitochondrial dynamics. Cell Prolif. 2020 ;53(11):e12909.
Cen M, Ouyang W, Zhang W, et al. MitoQ protects against hyperpermeability of endothelium barrier in acute lung injury via an Nrf2-dependent mechanism. Redox Biol. 2021;41:101936.
Zeng H, Guo X, Zhou F, et al. Quercetin alleviates ethanol-induced liver steatosis associated with improvement of lipophagy. Food Chem Toxicol. 2019;125:21-28.
El-Far AH, Lebda MA, Noreldin AE, et al. Quercetin Attenuates Pancreatic and Renal D-Galactose-Induced Aging-Related Oxidative Alterations in Rats. Int J Mol Sci. 2020;21(12):4348.

No competing interests reported.

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Quercetin improves diabetic cardiomyopathy by inhibiting pyroptosis via the Nrf2 pathway

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