
==== Front
Open Life Sci
Open Life Sci
biol
Open Life Sciences
2391-5412
De Gruyter

biol-2022-0957
10.1515/biol-2022-0957
Research Article
TAK-242 alleviates diabetic cardiomyopathy via inhibiting pyroptosis and TLR4/CaMKII/NLRP3 pathway
Zhao Xiaolong #
Zhang Jing #
Xu Feng
Shang Longqi
Liu Qingquan
Shen Chunjian shenchunjian1976@hotmail.com

School of Graduates, Dalian Medical University, Dalian, China
Medical Department, The Second Hospital of Dalian Medical University, Dalian City, China
Department of Nursing, The Second Affiliated Hospital of Shenyang Medical College, Shenyang City, China
Department of Cardiothoracic Surgery, The Fourth People’s Hospital of Shenyang, No. 20 Huanghe South Street, Shenyang, 110000, Liaoning, China
# The authors contributed equally to the paper.

10 9 2024
2024
19 1 2022095724 4 2024
19 7 2024
12 8 2024
© 2024 the author(s), published by De Gruyter
2024
the author(s), published by De Gruyter
https://creativecommons.org/licenses/by/4.0/ This work is licensed under the Creative Commons Attribution 4.0 International License.

Abstract

Diabetic cardiomyopathy (DCM) is identified as a progressive disease that may lead to irreparable heart failure. Toll-like receptor (TLR) signaling is believed to be implicated in the pathogenesis of DCM. This study intended to explore the potential impact of Toll-like receptor 4 (TLR4) on DCM in vitro and in vivo. Streptozotocin and HG medium were utilized to induce diabetes in animal and cell models, respectively. Selective TLR4 inhibitor TAK-242 and calcium/calmodulin-dependent protein kinase-II (CaMKII) inhibitor KN-93 were employed to explore the involvement of TLR4/CaMKII in DCM. TLR4 expression was increased in DCM hearts, while inhibition of TLR4 activation by TAK-242 improved cardiac function, attenuated heart hypertrophy, and fibrosis, as well as reduced oxidative stress and proinflammatory cytokine levels in rats, which were confirmed by Doppler echocardiography, hematoxylin and eosin staining, and Masson Trichome staining and specific enzyme-linked immunosorbent assay kits. Besides, the expression of hypertrophy-related molecules and oxidative stress damage were also inhibited by TAK-242. Furthermore, TAK-242 treatment reduced CaMKII phosphorylation accompanied by decreased expression of NOD-like pyrin domain-containing protein 3, gasdermin D (GSDMD), The N-terminal domain of Gasdermin D (GSDMD-N), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and Caspase-1 both in vivo and in vitro. Similar positive impacts on HG-induced pyroptosis were also observed with KN-93 treatment, and this was achieved without affecting TLR4 expression. Collectively, our work suggested that TAK-242 demonstrated substantial benefits against DCM both in vivo and in vitro, potentially attributed to the suppression of the TLR4-mediated CaMKII/NLRP3 pathway activity.

Keywords

CaMKII/NLRP3 pathway
diabetic cardiomyopathy
inflammasome
pyroptosis
TAK-242
TLR4
==== Body
pmc1 Introduction

Diabetes mellitus (DM) is a chronic condition that poses a considerable public health burden, and its global prevalence is on the rise [1]. Diabetic cardiomyopathy (DCM), first proposed by Rubler, refers to a heart muscle condition that occurs in individuals with diabetes [2]. DCM is identified as impaired cardiac function in the absence of concurrent cardiovascular conditions, including coronary heart disease, systemic hypertension, valvar disease, or other structural abnormalities in the heart [3]. Notably, the progression of DCM typically starts with impaired diastolic function of the left ventricle and cardiac hypertrophy, eventually leading to systolic dysfunction and heart failure, accompanied by cardiomyocyte loss and fibrosis accumulation in the heart [4,5]. Despite numerous studies conducted on DCM, our understanding of its pathophysiology remains incomplete.

Pyroptosis has been proven to play a significant role in the progression of DCM. Elevated glucose levels can induce cell pyroptosis [6], and inhibiting pyroptosis has shown effectiveness in alleviating DCM symptoms [7,8]. The assembly of an inflammasome complex, along with caspase-1 and IL-1β, serves as the initiation of pyroptosis [9,10]. It is well understood that inflammatory factor tumor necrosis factor-alpha (TNF-α) is involved in DCM pathogenesis on account of its role in initiating several intracellular death signaling, including pyroptosis [11]. It is reported that TNF-α forms a complex by binding to TNF receptor 1, and the complex is validated to be involved in pyroptosis initiation [12,13]. Studying pyroptosis enhances our comprehension of the mechanisms contributing to DCM progression, which reveals novel targets for advancing treatment approaches.

Toll-like receptors (TLRs) are membrane-bound receptors that play a crucial role in innate immunity and are involved in the regulation of various signal transduction pathways [14]. TLR4, in particular, has been implicated as a key mediator of inflammation in diabetes and may contribute to the development of diabetic complications [15,16]. Multiple studies have evidenced that hyperglycemia activates TLR4 and induces inflammation in DCM [17]. Suppressing TLR4-associated pathways has been proven effective in mitigating DCM [18,19]. Additionally, a previous study has shown that TLR4 mediates cell pyroptosis through various mechanisms [20]. Hence, targeting TLR4 presents a promising approach for treating DCM by effectively inhibiting pyroptosis, which could offer valuable targets for the development of innovative therapeutic interventions.

Ca2+/calmodulin-dependent protein kinase (CaMK) family members are essential in a variety of pathophysiological functions, such as survival, proliferation, cell differentiation, and inflammatory disorders [21]. Ca2+/calmodulin-dependent protein kinase-II (CaMKII), one prominent member, acts as a multifunctional serine/threonine kinase that controls expressions of the downstream genes via activating transcription factors [22]. CaMKII is involved in initiating NOD-like pyrin domain-containing protein 3 (NLRP3) inflammasome signaling in cardiomyocytes, which triggers inflammation in non-ischemic heart disease [23]. Notably, CaMKII/NLRP3 signaling is involved in the process of pyroptosis, and targeting NLRP3 through the CaMKII/cyclic AMP response element binding protein axis has demonstrated significant alleviation symptoms in Alzheimer’s mouse models by inhibiting pyroptosis [24]. However, further research is required to elucidate the mechanistic role of CaMKII/NLRP3 in DCM.

Recent studies have highlighted the intervention methods targeting pyroptosis as a potential future direction for preventing and treating DCM [6,25], but the mechanism related to pyroptosis needs to be further studied. The previous study has reported that TLR4 activates the CaMKII Mst1/2-Rac axis by inducing calcium influx [26]. Consequently, our current investigation primarily focused on the effects and underlying mechanisms of TLR4 on HG-stimulated cardiac damage. Our findings demonstrated that inhibiting TLR4 suppressed pyroptosis, primarily through its inhibitory impact on the CaMKII/NLRP3 pathway. This study offers a new mechanistic understanding of the protective effects of TAK-242 in DCM.

2 Materials and methods

2.1 Animal model

The experiments were carried out on male Sprague Dawley (SD) rats, aged 8 weeks, weighing between 150 and 170 g. All protocols involving animal subjects adhered to the laboratory animal guidelines established by the US National Institutes of Health and received approval from the Animal Care and Use Committee of Dalian Medical University (no: CSE202301007). The experimental rats were maintained under specific pathogen-free conditions, with a conventional room setup and a 12:12-h light/dark cycle. Throughout the study, the animals were provided free access to food and water.

Male SD rats (aged 8 weeks, weighing between 150 and 170 g) were assigned at random into one of four groups following acclimatization for 1 week: control group (n = 8), DM group (n = 16), control+TAK-242 group (n = 8), and DM+TAK-242 group (n = 8). The rats in the DM and DM+TAK-242 groups were intraperitoneally administered with streptozotocin (STZ; 50 mg/kg; Sigma-Aldrich, USA) dissolved in citrate acid buffer dilution (Sigma-Aldrich, USA) for three consecutive days. Rats in the control and TAK-242 groups received the citrate buffer injection. Fasting blood glucose (FBG) levels were measured 1 week after STZ injection, and the rats with FBG levels above 16.7 mM were selected for further investigation [27]. The rats in the control+TAK-242 and DM+TAK-242 groups received daily intraperitoneal injections of TAK-242 (3 mg/kg, dissolved in DMSO) for seven consecutive days starting from 3 weeks after STZ administration. Both the control group and the DM group of rats were administered an equivalent volume of DMSO via intraperitoneal injection. Following 8 weeks of STZ injection, the DM group was further divided into two subgroups: the DM group (n = 8) and the DM+KN-93 group (n = 8). The DM+KN-93 group received intraperitoneal injections of KN-93 at a dosage of 10 mg/kg/2 days for 8 weeks, while rats in the other group received vehicle injection. Blood glucose levels were regularly monitored throughout the experiment. Following the completion of the echocardiogram (ECG), blood samples were collected, and cardiac tissues were obtained for cardiac histology and molecular biology studies. All animals were sedated utilizing 2% isoflurane administered by inhalation and underwent ECG testing to evaluate ST segment alterations. Echocardiography was performed using small-animal ultrasonography (Vinno Co., China) to assess various cardiac functions, including ejection fraction (EF), fractional shortening (FS), peak E-to-peak A ratio (E/A), and left ventricular internal dimension at systole (LVIDs).

2.2 Histopathology

Midventricular heart samples were fixed in a solution of 4% formalin, dehydrated with alcohol, embedded in paraffin, and cut into 5 μm sections using a microtome for histological examination. Hematoxylin and eosin staining was conducted on several transverse slices of paraffin-embedded samples to visualize general tissue morphology. Additional paraffin slices were stained with Masson trichrome to assess collagen deposition [28]. The sections were analyzed under a light microscope (Olympus, Japan), and photomicrographs of the tissue sections were captured with an Olympus microscope camera.

2.3 Cell culture

H9c2 cardiomyocytes, derived from rats, were obtained from the American Type Culture Collection. The cell line was cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Cytiva, America) supplemented with 10% fetal bovine serum (Kangyuan Biotechnology, China) and 1% penicillin–streptomycin. The cells were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere. Cells between passages 3 and 8 were used for subsequent experiments. To simulate the hyperglycemic (HG) state of type-1 diabetes, a high glucose solution (30 mM glucose) was utilized. The cells were separated into two groups: normoglycemic (NG, 5.5 mM glucose) and HG. TAK-242 (1 mM) and KN-93 (5 μm) were added to determine the mechanism of action. According to the experimental design, the H9c2 cardiomyocytes were categorized into five groups based on the culture media and medications employed: NG (normal glucose), HG (high glucose), NG+TAK-242, HG+TAK-242, and HG+KN-93.

2.4 Cell Counting Kit-8 (CCK-8) assay

H9c2 cells were seeded onto a 96-well plate with a total amount of 1 × 104 cells per well. Then, the cells were exposed to HG treatment, with or without TAK-242 (1 mM) and KN-93 (5 μM) for 48 h. The proliferation of cells was evaluated utilizing a CCK-8 Kit (Beyotime Biotechnology, China) in strict accordance with the manufacturer’s instructions. Following TAK-242 or KN-93 treatment, 10 μL of CCK-8 reagent was added, and then the cells were further incubated for 2 h at 37°C. The measurement of absorbance was taken at a wavelength of 450 nm.

2.5 Measurement of reactive oxygen species (ROS)

Intracellular ROS levels were evaluated using the 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe kit (Beyotime, China). After treating the cells with HG and/or TAK-242 for 24 h, the cells were washed three times with serum-free medium. Subsequently, the cells were incubated with DCFH-DA in the absence of light for 30 min. The fluorescence intensity was measured using a flow cytometer (ACEA, China) for quantitative analysis.

2.6 Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining

The TUNEL staining agents (Beyotime, China) were used to stain the cell slides or tissue sections in accordance with the manufacturer’s instructions. After staining the nuclei with 4′,6-diamidino-2-phenylindole, the sections were sealed, and a fluorescent microscope (COIC, China) was used to capture the images.

2.7 Flow cytometry

Cell apoptosis was measured by employing flow cytometry. H9c2 cells were collected and stained with Annexin V-FITC and propidium iodide. The flow cytometer (ACEA, China) was utilized to evaluate the stained cells for the dead cells population following dual labeling for 15 min at room temperature and darkness [29].

2.8 Enzyme-linked immunosorbent assay (ELISA)

The levels of IL-1β and IL-18 in the cell culture supernatants or serum were determined using ELISA kits from Elabscience Biotechnology (China). The quantification was performed following the manufacturer’s instructions and guidelines provided with the kits [30].

2.9 Reverse transcriptase qPCR

The total RNA samples were extracted from cardiac tissues as well as H9c2 cells utilizing the AG RNAex Pro Reagent (Accurate Biology, China) as per the manufacturer’s instructions, followed by reverse transcription into complementary DNA using an RT reagent kit (ABclonal, USA). SYBR Green PCR Master Mix Kit (ABclonal, USA) was utilized to quantify TLR4, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-major histocompatibility complex (β-MHC), CollagenI, CollagenIII, connective tissue growth factor (CTGF), and β-actin mRNA levels. The reactions were performed on a real-time PCR equipment (Bioer Technology, China) with β-actin as the internal control. The primer sequences are depicted below.

TLR4 forward 5′-CCAGAGCCGTTGGTGTATCT-3′ and reverse 5′-AGAGCATTGTCCTCCCACTC-3′.

BNP forward 5′-AGTCCTAGCCAGTCTCCAGA-3′ and reverse 5′-ATCCGGTCTATCTTGTGCCC-3′.

ANP forward 5′-GCCGGTAGAAGATGAGGTCA-3′ and reverse 5′-AGCTGGATCTTCGTAGGCTC-3′.

β-MHC forward 5′-GCAGATCATCAAGGCCAAGG-3′ and reverse 5′-AGTTGCCTCTTGAGGTCCTC-3′.

Collagen forward 5′-ATGTGCCACTCTGACTGGAA-3′ and reverse 5′-TCCATCGGTCATGCTCTCTC-3′.

CollagenIII forward 5′-CCCCTGGTTCTTCTGGACAT-3′ and reverse 5′-TGGGCCTTTGATACCTGGAG-3′.

CTGF forward 5′-GTGAGTCCTTCCAAAGCAGC-3′, reverse 5′-TAGTTGGGTCTGGGCCAAAT-3′.

β-Actin forward 5′-GGATTCCTATGTGGGCGACGA-3′ and reverse 5′-GCGTACAGGGATAGCACAGC-3′.

2.10 Western blot

To extract total proteins, rat heart tissues, as well as H9c2 cells, were lysed utilizing an ice-cold radio-immunoprecipitation lysis buffer (Absin, China). A BCA detection kit (Absin, China) was employed to quantify the concentration of isolated proteins. The proteins were separated by performing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes using an electric transfer method. After the blocking step with a 5% nonfat dry milk solution for 1.5 h, the protein samples on the PVDF membrane were then incubated with the primary antibodies overnight at 4°C. This step allows the primary antibodies to specifically bind to their target proteins on the membrane for detection in subsequent steps. The following were the primary antibodies against various target proteins: TLR4 (A5258, 1:2,000, ABclonal), collagen I (14695-1-AP, 1:2,000, Proteintech), TGF-β (21898-1-AP, 1:2,500, Proteintech), α-S-adenosylmethionine (α-SAM) (14395-1-AP, 1:6,000, Proteintech), CaMKII (12666-2-AP, 1:2,000, Proteintech), p-CaMKII (AP0255, 1:4,000, ABclonal), NLRP3 (27458-1-AP, 1:1,000, Proteintech), gasdermin D (GSDMD) (20770-1-AP, 1:5,000, Proteintech), the N-terminal domain of gasdermin D (GSDMD-N) (ab215203, 1:1,000, Abcam), caspase-1 (22915-1-AP, 1:6,000, Proteintech), ASC (16087-1-AP, 1:3,000, Proteintech), and β-actin (81115-1-RR, 1:20,000, Proteintech). After conventional washing procedures, the PVDF membranes loaded with protein samples were incubated in HRP-conjugated secondary antibody (S0001, 1:3,000, Affinity) for 1.5 h at room temperature and visualized using an ECL buffer (Tanon, China). Image Lab software was applied to measure the strip absorbance values, which were normalized to the intensity of the β-actin band as the loading control [31].

2.11 Statistical analysis

Experimental data were analyzed and interpreted via utilizing GraphPad Prism 8. The results were presented in the format of mean ± standard deviation. The statistical analysis was carried out using either Student’s t-test or one-way analysis of variance by Tukey’s multiple comparison test. A p-value below 0.05 was deemed statistically significant.

3 Results

3.1 TLR4 was up-regulated in the hearts of DM rats and H9c2 cells exposed to HG

We first investigated the expression of TLR4 mRNA and protein levels in the hearts of DM rats and H9c2 cells exposed to HG. The results revealed a significant elevation of TLR4 expression in the heart tissues following STZ-induced DM, as illustrated in Figure 1a and b. Furthermore, a significant elevation in TLR4 levels was observed in H9c2 cells after HG stimulation (Figure 1c and d). TAK-242 is a specific inhibitor of TLR4. In both in vitro and in vivo experiments, the treatment of TAK-242 alone did not significantly alter the expression of TLR4, which was consistent with the previous report [32]. However, when TAK-242 was administered in conjunction with DM or HG induction, it significantly inhibited the expression of TLR4 at mRNA and protein levels (Figure 1a–d).

Figure 1 TLR4 was up-regulated in the hearts of DM rats and H9c2 cells exposed to HG. (a) TLR4 mRNA levels in the heart. (b) Protein band images and statistical analysis demonstrating TLR4 expression in the heart tissues of DM rats. (c) TLR4 mRNA levels in H9c2 cells. (d) Representative protein band pictures and statistical analysis demonstrating TLR4 expression in H9c2 cells. The in vivo experiment was repeated for eight times independently, and the in vitro for three times. *p < 0.05 versus (vs) control; # p < 0.05 vs DM.

3.2 STZ-induced heart injury was ameliorated for inhibition of TLR4

DCM is one of the most prevalent consequences of diabetes, frequently resulting in substantial morbidity and mortality [33]. STZ-induced heart damage is commonly associated with the development of cardiac hypertrophy and dysfunction [34]. As depicted in Figure 2a–c, the DM group demonstrated markedly elevated FBG levels and a heightened heart weight-to-body weight (HW/BW) ratio compared to the control group, suggesting the presence of diabetes-induced cardiac hypertrophy. Treatment with TAK-242 resulted in a lower HW/BW ratio when compared to the DM group. However, no statistically significant differences were observed in FBG or BW. These findings suggested that TAK-242 therapy improved heart hypertrophy in rats with diabetes.

Figure 2 STZ-induced heart injury was ameliorated for inhibition of TLR4. FBG (a), BW (b), HW/BW (c), and morphology (d) of the rat hearts. (e) Representative photographs of heart H&E staining from various groups, as well as measurement of cardiomyocyte area. (f) qRT-PCR to detect the mRNA levels of the hypertrophic makers, including ANP, BNP, and β-MHC. (g) Echocardiographic indicator results. (h) TAK-242 effects on STZ-induced EGC alterations in rats. The in vivo experiment was repeated for eight times independently. *p < 0.05 vs control; # p < 0.05 vs DM.

To further confirm the effect of TLR4 suppression on heart enlargement caused by STZ injection, heart size was measured (Figure 2d). Furthermore, myocyte area, as determined by H&E staining, revealed that TLR4 suppression played a critical role in reducing STZ-induced cardiac injury (Figure 2e). Additionally, mRNA levels of cardiac hypertrophy markers (ANP, BNP, and β-MHC) were significantly elevated in the DM group compared to the control group. TLR4 suppression with TAK-242 effectively prevented these elevations in comparison to the DM group (Figure 2f).

Moreover, cardiac function was measured utilizing the echocardiography. The results indicated that diabetes led to a notable decrease in EF, FS, and the E/A ratio, along with an increase in LVIDs compared to the control group. However, TLR4 inhibition with TAK-242 significantly improved EF, FS, and E/A ratio, while reducing LVIDs (Figure 2g). The ECG data in Figure 2h depicted that the DM group had a considerably higher ST-segment elevation compared to the control group. TAK-242 therapy clearly ameliorated the ECG abnormalities, demonstrating the protective effect of TLR4 inhibition.

3.3 TAK-242 attenuated fibrosis in DCM

In the context of STZ-induced DCM, DM rats exhibited higher levels of fibrosis when compared to the control group. Notably, TAK-242 treatment showed a reduction in the extent of fibrotic tissues (Figure 3a). Cardiac tissues from DM rats indicated a significant upregulation of Collagen I, Collagen III, and CTGF compared to the control. However, when comparing DM+TAK-242 to DM alone, there was a considerable downregulation of these genes (Figure 3b). Similarly, increased levels of Collagen I, α-SAM, and TGF-β proteins were observed following STZ induction, which subsequently decreased upon TAK-242 treatment (Figure 3c). These findings suggested that the attenuation of cardiac damage by TAK-242 was associated with the suppression of fibrosis.

Figure 3 TAK-242 attenuated fibrosis in DCM. (a) Representative images of Masson trichromatic staining, as well as a quantitative analysis of the fibrotic area ratio. (b) Relative mRNA expression levels of Collagen I, III, and CTGF. (c) Quantification of collagen I, α-SMA, and TGF-β protein levels was demonstrated using Western blot. The in vivo experiment was repeated for eight times independently. *p < 0.05 vs control and # p < 0.05 vs DM.

3.4 TAK-242 inhibited HG-induced ROS generation in vitro

To examine the potential antioxidative impact of TAK-242 therapy in vitro, DCFH-DA fluorescence was utilized to monitor ROS formation in H9c2 cells. Subsequently, confocal microscopy and flow cytometry were then employed to measure intracellular ROS levels. Our results showed that HG treatment significantly augmented ROS production in H9c2 cells. However, pretreatment with TAK-242 exhibited a remarkable reduction in HG-induced ROS production in H9c2 cells (Figure 4a and b).

Figure 4 TAK-242 inhibited HG-induced ROS generation in vitro. DCFH-DA staining (a), flow cytometry (b) were employed to access the intracellular ROS levels. The in vitro experiment was repeated for three times independently. *p < 0.05 vs NG; # p < 0.05 vs HG.

3.5 TAK-242 inhibited CaMKII phosphorylation and cardiomyocytes pyroptosis in DM rats

In the DM group, there was an elevated p-CaMKII/CaMKII ratio and increased expression of pyroptosis-related proteins, including NLRP3, GSDMD, GSDMD-N, caspase 1, and ASC compared to the control group. However, TAK-242 administration resulted in a reduction of the p-CaMKII/CaMKII ratio and decreased expression of NLRP3, GSDMD, GSDMD-N, caspase 1, and ASC when compared to the DM group (Figure 5a). The DM group displayed significantly elevated concentrations of IL-1β and IL-18 in the serum compared to the control group. However, TAK-242 administration considerably lowered the serum levels of these cytokines (Figure 5b). Furthermore, the experimental results revealed a minimal presence of TUNEL-positive cells in the control group. In contrast, the DM group exhibited a significant rise in the number of TUNEL-positive cells compared to the control group. Interestingly, the DM+TAK-242 group demonstrated a reduction in TUNEL-positive cells when compared to the DM group (Figure 5c).

Figure 5 TAK-242 inhibited CaMKII phosphorylation and cardiomyocytes pyroptosis in DM rats. (a) Western blot representative images and quantitative analysis of p-CaMKII/CaMKII, NLRP3, GSDMD, GSDMD-N, Caspase 1, and ASC levels. (b) TAK-242 effects on IL-1β and IL-18 levels in rat serum. (c) Illustration of TUNEL staining and examination of positive cells. The in vivo experiment was repeated for eight times independently. *p < 0.05 vs control and # p < 0.05 vs DM.

3.6 KN-93 inhibited the expression of pyroptosis-related proteins in DM rats

KN-93, a selective CaMKII inhibitor, was utilized to investigate the involvement of CaMKII in pyroptosis-related signaling. KN-93 treatment significantly decreased the p-CaMKII/CaMKII ratio as well as the protein levels of NLRP3, GSDMD, GSDMD-N, Caspase 1, and ASC when compared to the DM group. However, the intervention did not impact TLR4 expression (Figure 6a). In addition, the levels of pro-inflammatory variables such as IL-1β and IL-18 in blood were considerably lower in the DM+KN-93 group compared to the DM group (Figure 6b).

Figure 6 KN-93 inhibited the expression of pyroptosis-related proteins in DM rats. (a) Western blot representative images and quantitative analysis of TLR4, p-CaMKII/CaMKII, NLRP3, GSDMD, GSDMD-N, caspase 1, and ASC levels. (b) KN-93 effects on cardiac inflammatory cytokines in serum. The in vivo experiment was repeated for eight times independently. *p < 0.05 vs control and # p < 0.05 vs DM.

3.7 TAK-242 suppressed HG-induced pyroptosis in H9c2 cells

To further study the inhibitory effect of TAK-242 on HG-induced pyroptosis in H9c2 cells, the p-CaMKII/CaMKII ratio and the expression levels of NLRP3, GSDMD, GSDMD-N, Caspase 1, and ASC were measured. The results demonstrated a significant elevation of these proteins in response to HG stimulation. However, TAK-242 treatment effectively reduced the production of these key proteins, thereby inhibiting pyroptosis in H9c2 cells (Figure 7a). Moreover, HG stimulation led to a substantial increase in the production of IL-1β and IL-18 in the culture medium. However, treatment with TAK-242 effectively inhibited this rise in IL-1β and IL-18 levels (Figure 7b). In addition, incubation with HG substantially induced pyroptosis in H9c2 cells, as evidenced by an increase in TUNEL-positive cells. Conversely, TAK-242 treatment led to a decrease in TUNEL-positive cells (Figure 7c). Flow cytometry results further supported these findings, revealing an increased rate of pyroptosis in H9c2 cells following the HG challenge, which was ameliorated by TAK-242 treatment (Figure 7d). These findings suggest that TAK-242, by suppressing TLR4, attenuates HG-induced cardiomyocyte damage.

Figure 7 TAK-242 suppressed HG-induced pyroptosis in H9c2 cells. (a) The expression of pyroptosis pathway proteins was accessed using Western blot. (b) ELISA kits were used to detect the effect of TAK-242 on the levels of IL-1β and IL-18 in the supernatant of H9c2 cell culture media. (c) TUNEL staining assay representative images. (d) The proportion of dead cells was determined by flow cytometry. The in vitro experiment was repeated for three times independently. *p < 0.05 against NG and # p < 0.05 vs HG.

3.8 Inhibition of CaMKII relieved pyroptosis levels of H9c2 cells induced by HG

To confirm whether TLR4 exerted its effects via CaMKII-mediated pyroptosis, H9c2 cells were exposed to KN-93. Preliminary tests were conducted to assess the impact of TAK-242 and KN-93 on cell viability. The application of 1 mM TAK242 or 5 μM KN-93 did not result in a significant impact on cell viability. Therefore, we used 1 mM TAK242 or 5 μM KN-93 in the later experiments. In addition, we found that HG significantly reduced cell viability, while TAK-242 and KN-93 significantly alleviated HG-induced cell death (Figure S1).

The group given both HG and KN-93 exhibited a reduction in the enhanced levels of pyroptosis-related proteins induced by HG, while TLR4 expression remained unaffected (Figure 8a). Additionally, the ELISA analysis revealed that KN-93 significantly decreased the concentrations of IL-1β and IL-18 in the H9c2 cell culture supernatants (Figure 8b). These findings indicated that inhibiting CaMKII activation was beneficial for mitigating cell damage to some extent in vitro.

Figure 8 Inhibition of CaMKII relieved the pyroptosis levels of H9c2 cells induced by HG. (a) Western blot representative images and quantitative analysis of TLR4, p-CaMKII/CaMKII, NLRP3, GSDMD, GSDMD-N, caspase 1, and ASC levels. (b) ELISA was used to measure IL-1β and IL-18 levels in H9c2 cell culture supernatants. The in vitro experiment was repeated for three times independently. *p < 0.05 vs NG and # p < 0.05 vs HG.

4 Discussion

Our research illustrated that targeting TLR4 possessed therapeutic potential in alleviating heart injury associated with DCM. TAK-242 treatment was effective in reducing fibrosis in DCM and inhibiting ROS production induced by HG. Furthermore, TAK-242 was able to inhibit CaMKII phosphorylation, NLRP3 expression, and pyroptosis both in vivo and in vitro. These results indicated that focusing on TLR4 and the downstream CaMKII/NLRP3-mediated pyroptosis pathway could be a promising treatment approach for DCM.

Inhibition of TLR4 could be a potential treatment target for DCM [18,19]. The previous study has shown that GTS-21 exhibited cardioprotective effects by mitigating the TLR4/NF-κB pathway in rats with STZ-induced DCM [35]. Nicotinamide mononucleotide prevented CD36-mediated lipid accumulation and CD36-TLR4 interaction, thereby reducing inflammation, fibrosis, cardiac dysfunction, and whole-body insulin resistance [36]. Our study revealed that the expression of TLR4 was elevated in the left ventricular myocardium of DCM rats. Furthermore, to evaluate the role of TLR4 in DCM, we employed TAK-242, a well-known TLR4 inhibitor, to investigate whether inhibiting TLR4 could slow disease progression. Our findings provided additional evidence that hyperglycemia contributed to hypertrophy, fibrosis, oxidative stress, pyroptosis in cardiomyocytes, and eventually leading to cardiac failure. Treatment with TAK-242 reversed cardiac hypertrophy in rats, as indicated by reduced HW/BW ratio, decreased cross-sectional area of cardiomyocytes, and downregulation of hypertrophy-related genes, such as ANP, BNP, and β-MHC. Notably, no significant change was observed in blood glucose levels or body weight between the DM+TAK-242 group and the DM group, indicating that the preventive effects of TAK-242 on DCM were not dependent on metabolic regulation.

Diabetes-induced cardiomyopathy is characterized by myocardial fibrosis as its hallmark pathology [37,38]. Previous research demonstrated that suppression of TLR4 protected experimental rats from left ventricular dysfunction and cardiac fibrosis following myocardial infarction [39]. We observed significant impairments in diastolic function, as evidenced by reduced E/A ratio, and systolic dysfunction, indicated by reduced EF and FS, in the DM group compared to the control group in our current study. Besides, treatment with TAK-242 effectively restored these functional parameters. Moreover, the inhibition of TLR4 by TAK-242 led to a significant decrease in cardiac fibrosis by downregulating the expression of the TGF-β signaling pathway. This resulted in reduced levels of fibrotic markers, including α-SMA and collagen I, in the hearts of diabetic rats. Moreover, arrhythmia, defined by elevated ST segment, is a common consequence of DCM [40,41]. Remarkably, TAK-242 treatment effectively decreased diabetic-induced ST-segment elevation, indicating its protective impact on myocardial cells.

The study unveils a novel discovery suggesting that the inhibition of the CaMKII/NLRP3 axis shows promise in suppressing pyroptosis and mitigating myocardial damage in DCM. The significant role of CaMK family members, particularly CaMKII, in disease pathogenesis and their involvement in essential cellular processes make them compelling targets for therapeutic interventions [21]. In this study, we investigated the inhibitory effect of TLR4 on CaMKII protein and delved into the underlying mechanisms. Targeting the CaMKII/NLRP3 axis has been proven to mitigate sepsis-induced cardiac injury by reducing pyroptosis [42]. TLR4 signaling has been proven to be linked to CaMKII activation, with TLR4 acting as an upstream molecule in the CaMKII signaling pathway [43]. In our experimental model of DCM induced by STZ, inhibiting TLR4 improved cardiac dysfunction and reduced the expression of pyroptosis-related proteins in myocardial tissue. Additionally, TAK-242 treatment effectively reduced the degree of cardiomyocyte pyroptosis caused by HG in vitro. Importantly, TAK-242-mediated suppression of TLR4 led to a reduction in p-CaMKII expression, whereas KN-93 treatment had no effect on TLR4 expression. These results provide evidence that TLR4 functions as an upstream regulator of CaMKII activity in heart damage induced by HG. Hence, our findings revealed that TAK-242 inhibited CaMKII activity, thereby preventing CaMKII-mediated cardiomyocyte pyroptosis.

The prior investigation has presented evidence implicating the NLRP3 inflammasome, which comprises NLRP3, pro-caspase 1, and apoptosis-associated speck-like protein with a CARD domain (ASC), as an inflammatory mediator in the development of DCM [44]. Following NLRP3 inflammasome activation, active caspase-1 enhances its activity in maturing the progenitors of the inflammatory cytokines IL-1β and IL-18 [45,46]. Blocking TLR4 signaling has been confirmed to decrease NLRP3 expression via decreasing the expression of nuclear factor-kappa B (NF-κB) in HG-induced cardiac cells [47]. In our study, we discovered that HG dramatically stimulated the creation and activation of NLRP3 inflammasomes, which boosted the production of mature IL-1β and IL-18 in vitro and in vivo. TAK-242 effectively reversed the pyroptosis-related alterations in NLRP3, caspase 1, IL-1β, and IL-18 in diabetic rats and HG-exposed H9c2, thereby mitigating myocardial damage.

Furthermore, TAK242 was reported to reduce ROS accumulation in H9c2 cells [48], hence decreasing HG-induced cell damage. It is worth noting that there is a close relationship between ROS generation and the pyroptosis process. Intracellular oxidative stress and increased ROS generation promote pyroptosis via the NLRP3 inflammasome [49]. The interplay between ROS and pyroptosis aggravates the inflammation, which leads to significant cell damage. However, this investigation does not provide evidence regarding the specific mechanism by which ROS influences cardiac pyroptosis. Future work is needed to investigate the role of ROS in modulating cardiomyocyte pyroptosis.

This study has several limitations that should be acknowledged. First, ethical considerations prevented the assessment of TLR4 expression in the left ventricular tissue of diabetic patients. Thus, the direct relevance of our findings to human DCM remains to be determined. Second, our study primarily utilized TAK-242 to confirm its effect and did not employ genetically modified rats specifically targeting TLR4. Future studies should consider addressing these limitations to further investigate the mechanisms underlying DCM and identify potential therapeutic interventions.

5 Conclusion

In summary, this study demonstrated the significance of TLR4 in STZ-induced cardiac injury and explored the impact and underlying mechanism of TAK-242 on DCM in vitro and in vivo.

Notably, this research provides novel evidence demonstrating that TAK-242, a specific TLR4 inhibitor, effectively alleviates cardiomyocyte pyroptosis by inhibiting active CaMKII and subsequently restricting the activation of pyroptosis-related proteins. These findings imply that modulating TLR4 to target the CaMKII/NLRP3 signaling pathway could be a possible treatment option for DCM.

Supplementary Material

Supplementary Figure

Acknowledgements

The authors are grateful for the reviewer’s valuable comments that improved the manuscript.

Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

Funding information: This study was supported by the Health Commission of Shenyang.

Author contributions: Chunjian Shen designed the study. Xiaolong Zhao and Jing Zhang wrote the manuscript. Xiaolong Zhao, Jing Zhang, and Feng Xu performed the experiments. Xiaolong Zhao, Qingquan Liu, and Longqi Shang analyzed the data. Xiaolong Zhao prepared the figures. Chunjian Shen revised the manuscript. All authors reviewed the manuscript.

Conflict of interest: Authors state no conflict of interest.

Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
==== Refs
References

[1] Mannino GC, Andreozzi F, Sesti G. Pharmacogenetics of type 2 diabetes mellitus, the route toward tailored medicine. Diabetes Metab Res Rev. 2019;35(3):e3109.
Mannino GC Andreozzi F Sesti G Pharmacogenetics of type 2 diabetes mellitus, the route toward tailored medicine Diabetes Metab Res Rev 2019 35 3 e3109 30515958
[2] Jubaidi FF, Zainalabidin S, Mariappan V, Budin SB. Mitochondrial dysfunction in diabetic cardiomyopathy: The possible therapeutic roles of phenolic acids. Int J Mol Sci. 2020;21(17):6043.
Jubaidi FF Zainalabidin S Mariappan V Budin SB Mitochondrial dysfunction in diabetic cardiomyopathy: The possible therapeutic roles of phenolic acids Int J Mol Sci 2020 21 17 6043 32842567
[3] Sun L, Yu M, Zhou T, Zhang S, He G, Wang G, et al. Current advances in the study of diabetic cardiomyopathy: From clinicopathological features to molecular therapeutics (Review). Mol Med Rep. 2019;20(3):2051–62.
Sun L Yu M Zhou T Zhang S He G Wang G Current advances in the study of diabetic cardiomyopathy: From clinicopathological features to molecular therapeutics (Review) Mol Med Rep 2019 20 3 2051 62 31322242
[4] Ritchie RH, Abel ED. Basic mechanisms of diabetic heart disease. Circ Res. 2020;126(11):1501–25.
Ritchie RH Abel ED Basic mechanisms of diabetic heart disease Circ Res 2020 126 11 1501 25 32437308
[5] Paolillo S, Marsico F, Prastaro M, Renga F, Esposito L, De Martino F, et al. Diabetic cardiomyopathy: Definition, diagnosis, and therapeutic implications. Heart Fail Clin. 2019;15(3):341–7.
Paolillo S Marsico F Prastaro M Renga F Esposito L De Martino F Diabetic cardiomyopathy: Definition, diagnosis, and therapeutic implications Heart Fail Clin 2019 15 3 341 7 31079692
[6] Lu Y, Lu Y, Meng J, Wang Z. Pyroptosis and its regulation in diabetic cardiomyopathy. Front Physiol. 2021;12:791848.
Lu Y Lu Y Meng J Wang Z Pyroptosis and its regulation in diabetic cardiomyopathy Front Physiol 2021 12 791848 35145423
[7] Meng L, Lin H, Huang X, Weng J, Peng F, Wu S. METTL14 suppresses pyroptosis and diabetic cardiomyopathy by downregulating TINCR lncRNA. Cell Death Dis. 2022;13(1):38.
Meng L Lin H Huang X Weng J Peng F Wu S METTL14 suppresses pyroptosis and diabetic cardiomyopathy by downregulating TINCR lncRNA Cell Death Dis 2022 13 1 38 35013106
[8] Liu C, Yao Q, Hu T, Cai Z, Xie Q, Zhao J, et al. Cathepsin B deteriorates diabetic cardiomyopathy induced by streptozotocin via promoting NLRP3-mediated pyroptosis. Mol Ther Nucleic Acids. 2022;30:198–207.
Liu C Yao Q Hu T Cai Z Xie Q Zhao J Cathepsin B deteriorates diabetic cardiomyopathy induced by streptozotocin via promoting NLRP3-mediated pyroptosis Mol Ther Nucleic Acids 2022 30 198 207 36250207
[9] Aluganti Narasimhulu C, Singla DK. Amelioration of diabetes-induced inflammation mediated pyroptosis, sarcopenia, and adverse muscle remodelling by bone morphogenetic protein-7. J Cachexia Sarcopenia Muscle. 2021;12(2):403–20.
Aluganti Narasimhulu C Singla DK Amelioration of diabetes-induced inflammation mediated pyroptosis, sarcopenia, and adverse muscle remodelling by bone morphogenetic protein-7 J Cachexia Sarcopenia Muscle 2021 12 2 403 20 33463042
[10] Dessouki FBA, Kukreja RC, Singla DK. Stem cell-derived exosomes ameliorate doxorubicin-induced muscle toxicity through counteracting pyroptosis. Pharmaceuticals (Basel). 2020;13(12):450.
Dessouki FBA Kukreja RC Singla DK Stem cell-derived exosomes ameliorate doxorubicin-induced muscle toxicity through counteracting pyroptosis Pharmaceuticals (Basel) 2020 13 12 450 33316945
[11] Tanzer MC. A proteomic perspective on TNF-mediated signalling and cell death. Biochem Soc Trans. 2022;50(1):13–20.
Tanzer MC A proteomic perspective on TNF-mediated signalling and cell death Biochem Soc Trans 2022 50 1 13 20 35166321
[12] Huyghe J, Priem D, Bertrand MJM. Cell death checkpoints in the TNF pathway. Trends Immunol. 2023;44(8):628–43.
Huyghe J Priem D Bertrand MJM Cell death checkpoints in the TNF pathway Trends Immunol 2023 44 8 628 43 37357102
[13] Wu J, Lin S, Chen W, Lian G, Wu W, Chen A, et al. TNF-alpha contributes to sarcopenia through caspase-8/caspase-3/GSDME-mediated pyroptosis. Cell Death Discov. 2023;9(1):76.
Wu J Lin S Chen W Lian G Wu W Chen A TNF-alpha contributes to sarcopenia through caspase-8/caspase-3/GSDME-mediated pyroptosis Cell Death Discov 2023 9 1 76 36823174
[14] Sahoo BR. Structure of fish Toll-like receptors (TLR) and NOD-like receptors (NLR). Int J Biol Macromol. 2020;161:1602–17.
Sahoo BR Structure of fish Toll-like receptors (TLR) and NOD-like receptors (NLR) Int J Biol Macromol 2020 161 1602 17 32755705
[15] Shen X, Lan C, Lin Y, Zhang F, Zhang Y, Chen M, et al. Suppression of TLR4 prevents diabetic bone loss by regulating FTO-mediated m6A modification. Int Immunopharmacol. 2023;122:110510.
Shen X Lan C Lin Y Zhang F Zhang Y Chen M Suppression of TLR4 prevents diabetic bone loss by regulating FTO-mediated m6A modification Int Immunopharmacol 2023 122 110510 37413932
[16] Zhu L, Han J, Yuan R, Xue L, Pang W. Berberine ameliorates diabetic nephropathy by inhibiting TLR4/NF-κB pathway. Biol Res. 2018;51(1):9.
Zhu L Han J Yuan R Xue L Pang W Berberine ameliorates diabetic nephropathy by inhibiting TLR4/NF-κB pathway Biol Res 2018 51 1 9 29604956
[17] Wang Y, Luo W, Han J, Khan ZA, Fang Q, Jin Y, et al. MD2 activation by direct AGE interaction drives inflammatory diabetic cardiomyopathy. Nat Commun. 2020;11(1):2148.
Wang Y Luo W Han J Khan ZA Fang Q Jin Y MD2 activation by direct AGE interaction drives inflammatory diabetic cardiomyopathy Nat Commun 2020 11 1 2148 32358497
[18] Shi H, Zhou P, Ni Y-Q, Wang S-S, Song R, Shen A-L, et al. In vivo and in vitro studies of Danzhi Jiangtang capsules against diabetic cardiomyopathy via TLR4/MyD88/NF-κB signaling pathway. Saudi Pharm J. 2021;29(12):1432–40.
Shi H Zhou P Ni Y-Q Wang S-S Song R Shen A-L In vivo and in vitro studies of Danzhi Jiangtang capsules against diabetic cardiomyopathy via TLR4/MyD88/NF-κB signaling pathway Saudi Pharm J 2021 29 12 1432 40 35002381
[19] Yao J, Li Y, Jin Y, Chen Y, Tian L, He W. Synergistic cardioptotection by tilianin and syringin in diabetic cardiomyopathy involves interaction of TLR4/NF-κB/NLRP3 and PGC1a/SIRT3 pathways. Int Immunopharmacol. 2021;96:107728.
Yao J Li Y Jin Y Chen Y Tian L He W Synergistic cardioptotection by tilianin and syringin in diabetic cardiomyopathy involves interaction of TLR4/NF-κB/NLRP3 and PGC1a/SIRT3 pathways Int Immunopharmacol 2021 96 107728 33971494
[20] Wang J, Zhang F, Xu H, Yang H, Shao M, Xu S, et al. TLR4 aggravates microglial pyroptosis by promoting DDX3X-mediated NLRP3 inflammasome activation via JAK2/STAT1 pathway after spinal cord injury. Clin Transl Med. 2022;12(6):e894.
Wang J Zhang F Xu H Yang H Shao M Xu S TLR4 aggravates microglial pyroptosis by promoting DDX3X-mediated NLRP3 inflammasome activation via JAK2/STAT1 pathway after spinal cord injury Clin Transl Med 2022 12 6 e894 35692100
[21] Brzozowski JS, Skelding KA. The multi-functional calcium/calmodulin stimulated protein kinase (CaMK) family: Emerging targets for anti-cancer therapeutic intervention. Pharmaceuticals (Basel). 2019;12(1):8.
Brzozowski JS Skelding KA The multi-functional calcium/calmodulin stimulated protein kinase (CaMK) family: Emerging targets for anti-cancer therapeutic intervention Pharmaceuticals (Basel) 2019 12 1 8 30621060
[22] Reyes Gaido OE, Nkashama LJ, Schole KL, Wang Q, Umapathi P, Mesubi OO, et al. CaMKII as a therapeutic target in cardiovascular disease. Annu Rev Pharmacol Toxicol. 2023;63:249–72.
Reyes Gaido OE Nkashama LJ Schole KL Wang Q Umapathi P Mesubi OO CaMKII as a therapeutic target in cardiovascular disease Annu Rev Pharmacol Toxicol 2023 63 249 72 35973713
[23] Suetomi T, Miyamoto S, Brown JH. Inflammation in nonischemic heart disease: initiation by cardiomyocyte CaMKII and NLRP3 inflammasome signaling. Am J Physiol Heart Circ Physiol. 2019;317(5):H877–90.
Suetomi T Miyamoto S Brown JH Inflammation in nonischemic heart disease: initiation by cardiomyocyte CaMKII and NLRP3 inflammasome signaling Am J Physiol Heart Circ Physiol 2019 317 5 H877 90 31441689
[24] Song X, Cui Z, He J, Yang T, Sun X. κ‑opioid receptor agonist, U50488H, inhibits pyroptosis through NLRP3 via the Ca2+/CaMKII/CREB signaling pathway and improves synaptic plasticity in APP/PS1 mice. Mol Med Rep. 2021;24(1):529.
Song X Cui Z He J Yang T Sun X κ‑opioid receptor agonist, U50488H, inhibits pyroptosis through NLRP3 via the Ca2+/CaMKII/CREB signaling pathway and improves synaptic plasticity in APP/PS1 mice Mol Med Rep 2021 24 1 529 34036389
[25] Zhang L, Ai C, Bai M, Niu J, Zhang Z. NLRP3 inflammasome/pyroptosis: A key driving force in diabetic cardiomyopathy. Int J Mol Sci. 2022;23(18):10632.
Zhang L Ai C Bai M Niu J Zhang Z NLRP3 inflammasome/pyroptosis: A key driving force in diabetic cardiomyopathy Int J Mol Sci 2022 23 18 10632 36142531
[26] Geng J, Shi Y, Zhang J, Yang B, Wang P, Yuan W, et al. TLR4 signalling via Piezo1 engages and enhances the macrophage mediated host response during bacterial infection. Nat Commun. 2021;12(1):3519.
Geng J Shi Y Zhang J Yang B Wang P Yuan W TLR4 signalling via Piezo1 engages and enhances the macrophage mediated host response during bacterial infection Nat Commun 2021 12 1 3519 34112781
[27] Li M, Wang S, Liu X, Sheng Z, Li B, Li J, et al. Cadmium exposure decreases fasting blood glucose levels and exacerbates type-2 diabetes in a mouse model. Endocrine. 2022;76(1):53–61.
Li M Wang S Liu X Sheng Z Li B Li J Cadmium exposure decreases fasting blood glucose levels and exacerbates type-2 diabetes in a mouse model Endocrine. 2022 76 1 53 61 35041127
[28] Liu X, Liu Y, Tang L, Du C. Inhibition of farnesyl pyrophosphate synthase alleviates cardiomyopathy in diabetic rat. Cell Cycle. 2023;22(6):666–79.
Liu X Liu Y Tang L Du C Inhibition of farnesyl pyrophosphate synthase alleviates cardiomyopathy in diabetic rat Cell Cycle 2023 22 6 666 79 36310380
[29] Li F, Hu Z, Huang Y, Zhan H. Dexmedetomidine ameliorates diabetic cardiomyopathy by inhibiting ferroptosis through the Nrf2/GPX4 pathway. J Cardiothorac Surg. 2023;18(1):223.
Li F Hu Z Huang Y Zhan H Dexmedetomidine ameliorates diabetic cardiomyopathy by inhibiting ferroptosis through the Nrf2/GPX4 pathway J Cardiothorac Surg 2023 18 1 223 37430319
[30] Sun S, Dawuti A, Gong D, Wang R, Yuan T, Wang S, et al. Puerarin-V improve mitochondrial respiration and cardiac function in a rat model of diabetic cardiomyopathy via inhibiting pyroptosis pathway through P2X7 receptors. Int J Mol Sci. 2022;23(21):13015.
Sun S Dawuti A Gong D Wang R Yuan T Wang S Puerarin-V improve mitochondrial respiration and cardiac function in a rat model of diabetic cardiomyopathy via inhibiting pyroptosis pathway through P2X7 receptors Int J Mol Sci 2022 23 21 13015 36361807
[31] Li X, Pan F, He B, Fang C. Inhibition of ADAM10 ameliorates doxorubicin-induced cardiac remodeling by suppressing N-cadherin cleavage. Open Life Sci. 2021;16(1):856–66.
Li X Pan F He B Fang C Inhibition of ADAM10 ameliorates doxorubicin-induced cardiac remodeling by suppressing N-cadherin cleavage Open Life Sci 2021 16 1 856 66 34522779
[32] Zhong X, Xiao Q, Liu Z, Wang W, Lai C-H, Yang W, et al. TAK242 suppresses the TLR4 signaling pathway and ameliorates DCD liver IRI in rats. Mol Med Rep. 2019;20(3):2101–10.
Zhong X Xiao Q Liu Z Wang W Lai C-H Yang W TAK242 suppresses the TLR4 signaling pathway and ameliorates DCD liver IRI in rats Mol Med Rep 2019 20 3 2101 10 31257518
[33] Dillmann WH. Diabetic cardiomyopathy. Circ Res. 2019;124(8):1160–2.
Dillmann WH Diabetic cardiomyopathy Circ Res 2019 124 8 1160 2 30973809
[34] Zheng D, Chen L, Li G, Jin L, Wei Q, Liu Z, et al. Fucoxanthin ameliorated myocardial fibrosis in STZ-induced diabetic rats and cell hypertrophy in HG-induced H9c2 cells by alleviating oxidative stress and restoring mitophagy. Food Funct. 2022;13(18):9559–75.
Zheng D Chen L Li G Jin L Wei Q Liu Z Fucoxanthin ameliorated myocardial fibrosis in STZ-induced diabetic rats and cell hypertrophy in HG-induced H9c2 cells by alleviating oxidative stress and restoring mitophagy Food Funct 2022 13 18 9559 75 35997158
[35] Youssef ME, Abdelrazek HM, Moustafa YM. Cardioprotective role of GTS-21 by attenuating the TLR4/NF-kappaB pathway in streptozotocin-induced diabetic cardiomyopathy in rats. Naunyn Schmiedebergs Arch Pharmacol. 2021;394(1):11–31.
Youssef ME Abdelrazek HM Moustafa YM Cardioprotective role of GTS-21 by attenuating the TLR4/NF-kappaB pathway in streptozotocin-induced diabetic cardiomyopathy in rats Naunyn Schmiedebergs Arch Pharmacol 2021 394 1 11 31 32776158
[36] Wang S, Han Y, Liu R, Hou M, Neumann D, Zhang J, et al. Glycolysis-mediated activation of v-ATPase by nicotinamide mononucleotide ameliorates lipid-induced cardiomyopathy by repressing the CD36-TLR4 axis. Circ Res. 2024;134(5):505–25.
Wang S Han Y Liu R Hou M Neumann D Zhang J Glycolysis-mediated activation of v-ATPase by nicotinamide mononucleotide ameliorates lipid-induced cardiomyopathy by repressing the CD36-TLR4 axis Circ Res 2024 134 5 505 25 38422177
[37] Li W, Lou X, Zha Y, Qin Y, Zha J, Hong L, et al. Single-cell RNA-seq of heart reveals intercellular communication drivers of myocardial fibrosis in diabetic cardiomyopathy. Elife. 2023;12:e80479.
Li W Lou X Zha Y Qin Y Zha J Hong L Single-cell RNA-seq of heart reveals intercellular communication drivers of myocardial fibrosis in diabetic cardiomyopathy Elife 2023 12 e80479 37010266
[38] Cheng Y, Wang Y, Yin R, Xu Y, Zhang L, Zhang Y, et al. Central role of cardiac fibroblasts in myocardial fibrosis of diabetic cardiomyopathy. Front Endocrinol (Lausanne). 2023;14:1162754.
Cheng Y Wang Y Yin R Xu Y Zhang L Zhang Y Central role of cardiac fibroblasts in myocardial fibrosis of diabetic cardiomyopathy Front Endocrinol (Lausanne) 2023 14 1162754 37065745
[39] Guo X, Xue M, Li C-J, Yang W, Wang S-S, Ma Z-J, et al. Protective effects of triptolide on TLR4 mediated autoimmune and inflammatory response induced myocardial fibrosis in diabetic cardiomyopathy. J Ethnopharmacol. 2016;193:333–44.
Guo X Xue M Li C-J Yang W Wang S-S Ma Z-J Protective effects of triptolide on TLR4 mediated autoimmune and inflammatory response induced myocardial fibrosis in diabetic cardiomyopathy J Ethnopharmacol 2016 193 333 44 27558948
[40] Wen Y, Geng L, Zhou L, Pei X, Yang Z, Ding Z. Betulin alleviates on myocardial inflammation in diabetes mice via regulating Siti1/NLRP3/NF-κB pathway. Int Immunopharmacol. 2020;85:106653.
Wen Y Geng L Zhou L Pei X Yang Z Ding Z Betulin alleviates on myocardial inflammation in diabetes mice via regulating Siti1/NLRP3/NF-κB pathway Int Immunopharmacol 2020 85 106653 32531709
[41] Chen W, Zhang Y, Wang Z, Tan M, Lin J, Qian X, et al. Dapagliflozin alleviates myocardial ischemia/reperfusion injury by reducing ferroptosis via MAPK signaling inhibition. Front Pharmacol. 2023;14:1078205.
Chen W Zhang Y Wang Z Tan M Lin J Qian X Dapagliflozin alleviates myocardial ischemia/reperfusion injury by reducing ferroptosis via MAPK signaling inhibition Front Pharmacol 2023 14 1078205 36891270
[42] Cui S, Li Y, Zhang X, Wu B, Li M, Gao J, et al. FGF5 protects heart from sepsis injury by attenuating cardiomyocyte pyroptosis through inhibiting CaMKII/NFκB signaling. Biochem Biophys Res Commun. 2022;636(Pt 2):104–12.
Cui S Li Y Zhang X Wu B Li M Gao J FGF5 protects heart from sepsis injury by attenuating cardiomyocyte pyroptosis through inhibiting CaMKII/NFκB signaling Biochem Biophys Res Commun 2022 636 Pt 2 104 12 36368152
[43] Shuai W, Kong B, Fu H, Shen C, Huang H. Loss of MD1 increases vulnerability to ventricular arrhythmia in diet-induced obesity mice via enhanced activation of the TLR4/MyD88/CaMKII signaling pathway. Nutr Metab Cardiovasc Dis: NMCD. 2019;29(9):991–8.
Shuai W Kong B Fu H Shen C Huang H Loss of MD1 increases vulnerability to ventricular arrhythmia in diet-induced obesity mice via enhanced activation of the TLR4/MyD88/CaMKII signaling pathway Nutr Metab Cardiovasc Dis: NMCD 2019 29 9 991 8 31353205
[44] Sun Y, Ding S. NLRP3 inflammasome in diabetic cardiomyopathy and exercise intervention. Int J Mol Sci. 2021;22(24):13228.
Sun Y Ding S NLRP3 inflammasome in diabetic cardiomyopathy and exercise intervention Int J Mol Sci 2021 22 24 13228 34948026
[45] Man SM, Karki R, Kanneganti T-D. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol Rev. 2017;277(1):61–75.
Man SM Karki R Kanneganti T-D Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases Immunol Rev 2017 277 1 61 75 28462526
[46] Luo B, Huang F, Liu Y, Liang Y, Wei Z, Ke H, et al. NLRP3 inflammasome as a molecular marker in diabetic cardiomyopathy. Front Physiol. 2017;8:519.
Luo B Huang F Liu Y Liang Y Wei Z Ke H NLRP3 inflammasome as a molecular marker in diabetic cardiomyopathy Front Physiol 2017 8 519 28790925
[47] Huang Z, Zhuang X, Xie C, Hu X, Dong X, Guo Y, et al. Exogenous hydrogen sulfide attenuates high glucose-induced cardiotoxicity by inhibiting NLRP3 inflammasome activation by suppressing TLR4/NF-κB pathway in H9c2 cells. Cell Physiol Biochem. 2016;40(6):1578–90.
Huang Z Zhuang X Xie C Hu X Dong X Guo Y Exogenous hydrogen sulfide attenuates high glucose-induced cardiotoxicity by inhibiting NLRP3 inflammasome activation by suppressing TLR4/NF-κB pathway in H9c2 cells Cell Physiol Biochem 2016 40 6 1578 90 27997926
[48] He J, Huang L, Sun K, Li J, Han S, Gao X, et al. Oleuropein alleviates myocardial ischemia-reperfusion injury by suppressing oxidative stress and excessive autophagy via TLR4/MAPK signaling pathway. Chin Med. 2024;19(1):59.
He J Huang L Sun K Li J Han S Gao X Oleuropein alleviates myocardial ischemia-reperfusion injury by suppressing oxidative stress and excessive autophagy via TLR4/MAPK signaling pathway Chin Med 2024 19 1 59 38589925
[49] Sun L, Ma W, Gao W, Xing Y, Chen L, Xia Z, et al. Propofol directly induces caspase-1-dependent macrophage pyroptosis through the NLRP3-ASC inflammasome. Cell Death Dis. 2019;10(8):542.
Sun L Ma W Gao W Xing Y Chen L Xia Z Propofol directly induces caspase-1-dependent macrophage pyroptosis through the NLRP3-ASC inflammasome Cell Death Dis 2019 10 8 542 31316052
