Home Involvement of SIRT3/FOXO1 and TLR4/NF-κB/NLRP3 pathways in protective effects of Δ(9)-tetrahydrocannabinol on diabetic cardiomyopathy in rats
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Involvement of SIRT3/FOXO1 and TLR4/NF-κB/NLRP3 pathways in protective effects of Δ(9)-tetrahydrocannabinol on diabetic cardiomyopathy in rats

  • Haiping Zhang ORCID logo , Xiaoli Hui ORCID logo and Hua Xu ORCID logo EMAIL logo
Published/Copyright: March 6, 2025
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 50 Issue 2

Abstract

Objectives

Diabetic cardiomyopathy (DCM) is a chronic complication of type 2 diabetes mellitus, leading to heart failure. Addressing DCM requires a comprehensive therapeutic approach. This study examines the protective role of Δ(9)-tetrahydrocannabinol (THC) in DCM by assessing its impact on cardiac function, inflammation, oxidative stress, and mitochondrial health and exploring the involvement of the SIRT3/FOXO1 and TLR4/NF-κB/NLRP3 signaling pathways.

Methods

Over a four-week period, THC (1.5 mg/kg, administered intraperitoneally) was given to type-2 diabetic Sprague-Dawley rats. Cardiac function was evaluated using a carotid catheter in vivo while mitochondrial integrity was assessed through fluorometric techniques. Moreover, cardiac biochemical biomarkers and the protein expression of key signaling proteins were measured via ELISA and immunoblotting.

Results

Compared to untreated diabetic rats, THC treatment showed pronounced protective effects, improving cardiac function and reducing markers of DCM. It significantly lowered the expression of NF-κB, NLRP3, IL-1β, and TNF-α while reducing diabetes-induced oxidative stress in the heart, as evidenced by decreased malondialdehyde levels and increased glutathione and catalase levels. THC also prevented mitochondrial membrane depolarization and reactive oxygen species production and substantially suppressed the upregulation of TLR4 and MyD88 while increasing FOXO1 expression in diabetic rats. Inhibition of the SIRT3/FOXO1 pathway using 3-TYP reversed the cardioprotective effects of THC, negating its impact on mitochondrial function and the expression of NF-κB/NLRP3/IL-1β without influencing TLR4/MyD88.

Conclusions

These results underscore that THC offers significant protection against diabetes-induced cardiac damage by mitigating functional and biochemical changes associated with DCM, primarily through modulation of the TLR4/NF-κB/NLRP3 and SIRT3/FOXO1/mitochondrial pathways.

Keywords: diabetes; cannabinoid; mitochondria; cardiomyopathy; inflammation; mechanism

Introduction

Diabetic cardiomyopathy (DCM) represents a major cardiovascular complication associated with type 2 diabetes mellitus, characterized by cardiac structural and functional alterations independent of higher blood pressure or coronary artery disease [1]. The pathophysiology of DCM is multifactorial, encompassing metabolic derangements, oxidative stress, inflammation, and mitochondrial dysfunction [2]. These factors collectively contribute to the development of myocardial fibrosis, cardiomyocyte apoptosis, and, ultimately, heart failure [2], 3]. Given the increasing prevalence of diabetes worldwide, there is a dominant need for effective therapeutic strategies to mitigate the progression of DCM and improve the quality of life for diabetic patients.

Mitochondrial dysfunction plays a central role in the progression of myocardial injuries in DCM [2]. In diabetic conditions, mitochondria often undergo structural abnormalities, leading to impaired energy metabolism and increased production of reactive oxygen species (ROS) [4]. Sirtuin 3 (SIRT3), a key mitochondrial deacetylase, regulates mitochondrial homeostasis and biogenesis by modulating the acetylation status of proteins involved in oxidative phosphorylation, such as forkhead box O1 (FOXO1), as well as proteins related to antioxidant defense and fatty acid metabolism [5]. Conversely, activation of Toll-like receptor 4 (TLR4) in DCM triggers downstream signaling through myeloid differentiation primary response 88 (MyD88), leading to the activation of nuclear factor kappa B (NF-κB), which upregulates NOD-like receptor protein 3 (NLRP3) inflammasome expression, exacerbating inflammatory damage in cardiomyocytes [6], [7], [8]. In the pathogenesis of DCM, the balance or interplay between the TLR4/NF-κB/NLRP3 and SIRT3/FOXO1 pathways is critical in modulating the inflammatory and metabolic responses that contribute to cardiac dysfunction. Importantly, fine-tuning FOXO1 activity by SIRT3 may prevent mitochondrial dysfunction and inflammasome activation in DCM, making it a potential therapeutic target to reduce myocardial damage in diabetes [9].

Cannabinoids, particularly Δ(9)-tetrahydrocannabinol (THC), have garnered significant attention for their potential therapeutic properties in various pathological conditions, including their anti-inflammatory, antioxidant, antidiabetic, and cardioprotective effects [10]. THC acts primarily through the activation of cannabinoid receptors, which are expressed in multiple organs, including the heart [11]. By mitigating detrimental pathways and preserving cellular functional integrity, THC addresses critical mechanisms underlying DCM. Its diverse cytoprotective properties reduce ROS production and regulate pro-inflammatory cytokines activation, thereby protecting mitochondrial function in diabetic conditions [12]. Previous studies have highlighted the benefits of THC in reducing myocardial ischemia-reperfusion injury, attenuating oxidative stress, and modulating inflammatory responses [13]. However, its specific role in DCM, particularly its impact on mitochondrial function and related signaling pathways, remains inadequately understood. This study addresses this gap by investigating the protective effects of THC on DCM in a rat model of type-2 diabetes. Specifically, we evaluated the impact of THC on cardiac function, oxidative stress, inflammasome-inflammatory response, and mitochondrial health. Moreover, we explored the molecular mechanisms involved, focusing on the SIRT3/FOXO1 and TLR4/NF-κB/NLRP3 pathways, which are pivotal in the pathogenesis of DCM.

Materials and methods

Animals and experimental design

Male Sprague-Dawley rats (180–220 g) were used in this study and housed in standard laboratory conditions (12-h light/dark cycle, temperature 22 ± 2 °C, and humidity 55 ± 5 %) with free access to standard chow and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee under ethical approval number XJTU1AF-CRF-2022-036 and conducted following the National Institutes of Health guidelines for the care and use of laboratory animals. Type-2 diabetes was induced by feeding the rats a high-fat diet (HFD; 60 % fat) (Research Diets Ltd, USA) for 8 weeks. Following the HFD regimen, a single intraperitoneal injection of streptozotocin (STZ, 35 mg/kg; Sigma-Aldrich, USA) dissolved in citrate buffer (pH=4.5) was administered to confirm diabetes. Rats with fasting blood glucose levels>250 mg/dL, measured using a glucometer (Accu-Chek, Roche Diagnostics), were considered diabetic.

The experimental rats were divided into five groups including Control: age-matched non-diabetic rats, DCM: untreated diabetic rats receiving high-fat diet and STZ treatment, DCM+THC: diabetic rats treated with THC (1.5 mg/kg/day, i.p.) [14] for 4 weeks, DCM+3-TYP: diabetic rats receiving 3-TYP (a SIRT3 inhibitor; 50 mg/kg/day, i.p.) [15] for 4 weeks, and DCM+THC+3-TYP: diabetic rats treated with THC and 3-TYP (Cayman Chemical, USA). Notably, 3-TYP was used in this study to block the activation of the SIRT3/FOXO1 pathway. The age-matched non-diabetic control group and the untreated DCM group received intraperitoneal injections of equivalent volumes of the vehicle. The dose of THC used in this study was based on its effective dose reported in previous research [14] and was lower than the toxic dose documented in earlier toxicological studies of THC [16].

Assessment of cardiac function

At the end of the treatment period, cardiac function was assessed prior to sacrificing the animals using a carotid catheterization technique. Rats were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). A saline-filled polyethylene catheter (Millar Co., USA) was inserted into the carotid artery and advanced into the left ventricle to measure left ventricular end-systolic pressure (LVEsP) and left ventricular end-diastolic pressure (LVEdP), using a pressure transducer (Harvard Apparatus, USA). Left ventricular developed pressure (LVDP) was calculated as the difference between LVEsP and LVEdP.

Analysis of serum CK-MB

The serum level of cardiac injury biomarker, creatine kinase-MB (CK-MB), was measured using a commercial ELISA kit (no. ab285275, Abcam, USA), following the manufacturer’s instructions. Approximately 2 mL of blood samples were collected from the tails of rats and allowed to clot at room temperature for 40 min. The samples were then centrifuged at 2,500 rpm for 15 min at 4 °C, and prepared for ELISA assay. The absorbance values of the solution were read at 450 nm spectrophotometrically (NanoDrop 8000, Thermo Scientific, USA).

Tissue sample preparation

Rat heart was immediately isolated under deep anesthesia and the left ventricle was quickly excised and rinsed in ice-cold phosphate-buffered saline (PBS, pH=7.4) to remove blood and debris. The tissue was minced into small pieces in buffer containing protease and phosphatase inhibitors (Sigma-Aldrich, USA) and homogenized on ice at a concentration of 10 % w/v. The resulting homogenate was centrifuged at 10,000 rpm for 20 min at 4 °C to separate the supernatant.

Measurement of oxidative stress and antioxidant markers

Malondialdehyde (MDA) levels in cardiac supernatant were quantified using the thiobarbituric acid reactive substances (TBARS) assay, which measures the reaction of MDA with thiobarbituric acid to form a colored complex, using a commercially available kit (no. 10009055, Cayman Chemical, USA), following the manufacturer’s instructions. The absorbance of the supernatant was measured spectrophotometrically at 532 nm. The reduced glutathione (GSH) levels were determined using a colorimetric assay (no. 703002, Cayman Chemical, USA) that employs 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) to measure the formation of a colored product, thionitrobenzoic acid (TNB). The absorbance of TNB was measured at 405 nm to provide an accurate estimation of GSH in the samples. Additionally, catalase activity was measured spectrophotometrically according to the instructions of a catalase assay kit (no. 707002, Cayman Chemical, USA). Results were normalized to protein content, determined via the Bradford assay (Bio-Rad, Canada).

Measurement of inflammatory and inflammasome markers

Inflammatory and inflammasome markers, including NF-κB (no. MBS453975), tumor necrosis factor-α (TNF-α) (no. MBS175904), NLRP3 (no. MBS2706815), and interleukin-1 beta (IL-1β) (no. MBS825017), were quantified using commercially available sandwich ELISA kits (Mybiosource Inc., USA), following the manufacturer’s instructions. Each assay involved the addition of substrate solutions that resulted in a measurable color change, with absorbance values read at a wavelength of 450 nm. Results were expressed in terms of concentration (pg/mg protein) and normalized to total protein content, determined using the Bradford assay.

Mitochondrial function assessment

For mitochondrial extraction, the PBS minced left ventricle tissue (pH=7.4) was homogenized on ice in an isolation buffer containing 10 mM Tris-HCl (pH=7.4), 250 mM sucrose, 1 mM EDTA, and 1 % bovine serum albumin (BSA), supplemented with protease inhibitor. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C. The mitochondrial pellet was carefully collected, resuspended in an isolation buffer without BSA, and centrifuged again at 10,000 rpm for 10 min at 4 °C to purify the mitochondria. The final mitochondrial pellet was resuspended in a small volume of buffer and kept on ice for use. Protein concentration in the mitochondrial fraction was quantified using a bicinchoninic acid (BCA) protein assay. Mitochondrial function was assessed by evaluating mitochondrial ROS levels, membrane potential, and ATP production. Mitochondrial ROS levels were measured using the DCFDA assay (no. 21884, Sigma-Aldrich, USA), where isolated mitochondria were incubated with DCFDA reagent, allowing for the detection of ROS through fluorescence, indicating oxidative stress within the mitochondria. Mitochondrial membrane potential was assessed using the JC-10 assay (no. MAK160, Sigma-Aldrich, USA), which differentiates between healthy and depolarized mitochondria based on the fluorescence emission shift of the red/green dye, reflecting changes in membrane integrity. Finally, ATP levels were quantified using a luciferase bioluminescence assay (no. MAK473, Sigma-Aldrich, USA), where the emitted light correlates with ATP concentration, following the kit instructions.

Western blotting

Protein expression levels of TLR4, MyD88, SIRT3, FOXO1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were analyzed by Western blotting. Cardiac tissue samples were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. 20 micrograms of proteins were separated by SDS-PAGE and subsequently transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5 % non-fat milk and incubated with primary antibodies (1:1,000 dilution; Abcam, USA) overnight at 4 °C. After thorough washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and protein expression was visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, USA). The intensity of the protein bands was normalized to that of GAPDH and semi-quantified to assess relative expression levels.

Statistical analysis

Data were presented as mean ± standard deviation (SD). Statistical comparisons between groups were performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. A p-value <0.05 was considered statistically significant. All analyses were conducted using GraphPad Prism v9.5.1 software (GraphPad Software, USA).

Results

Effects of THC on cardiac function and cardiac CK-MB levels

Cardiac function was significantly attenuated and serum level of CK-MB was significantly elevated in untreated diabetic rats (DCM group) compared to age-matched non-diabetic control rats (p<0.001), indicating cardiac injury associated with diabetes (Table 1). In contrast, treatment with THC resulted in a notable increase in LVEsP and LVDP, and a significant decrease in LVEdP and CK-MB levels, indicating improved cardiac systolic function, contractility, and diastolic filling and reduced ventricular stiffness and cardiac damage (p<0.001). However, the inhibition of the SIRT3 pathway through the administration of 3-TYP significantly reversed the positive effects of THC on LVEdP (p=0.025), LVEsP (p=0.004), LVDP (p=0.002), and CK-MB levels (p<0.001) indicating the critical role of the SIRT3 pathway in mediating THC’s protective effects in diabetic cardiomyopathy (Table 1).

Table 1:

Effects of THC and SIRT3 inhibition on cardiac function and injury.

Variables Groups
Control DCM DCM+THC DCM+3-TYP DCM+THC+3-TYP
LVEdP, mmHg 11.8 ± 2.6 25.9 ± 3.4a 15.9 ± 3.3b 28.7 ± 4.2 22.5 ± 3.7c
LVEsP, mmHg 115.0 ± 7.9 73.2 ± 9.5a 104.3 ± 6.1b 80.1 ± 6.8 88.9 ± 6.3d
LVDP, mmHg 92.5 ± 7.0 40.8 ± 7.0a 78.9 ± 6.6b 44.2 ± 5.9 58.2 ± 6.9d
CK-MB, U/L 75.7 ± 22.5 452.0 ± 64.8a 244.2 ± 55.0b 568.7 ± 79.0 441.7 ± 88.2e

  1. Data are presented as mean ± SD, and n=6 per group. ap<0.001 vs. control group; bp<0.001 vs. DCM group; cp<0.05, dp<0.01, and cp<0.001 vs. DCM+THC group. LVEdP, left ventricular end-diastolic pressure; LVEsP, left ventricular end-systolic pressure; LVDP, left ventricular developed pressure; CK-MB, creatine kinase-MB; THC, Δ(9)-tetrahydrocannabinol; DCM, diabetic cardiomyopathy; 3-TYP, a SIRT3 inhibitor.

Effects of THC on cardiac oxidative stress markers

Cardiac MDA levels, a marker of lipid peroxidation, were significantly elevated in untreated diabetic rats compared to age-matched non-diabetic control rats (p<0.001), indicating increased oxidative stress (Figure 1A). However, treatment with THC resulted in a significant reduction in MDA levels in rats with DCM compared to the DCM group (p<0.001), suggesting a protective effect against this oxidative damage. In contrast, GSH (p<0.001) and catalase (p=0.003) levels were significantly decreased in the DCM group (Figure 1B and C). THC treatment led to a marked increase in GSH and catalase levels in the DCM+THC group comparable to the control group (p=0.002), indicating enhanced antioxidant capacity in the context of diabetic cardiomyopathy. Conversely, the administration of 3-TYP in the DCM+THC+3-TYP group resulted in increased MDA levels (p<0.001) and decreased GSH and catalase levels (p<0.05), underscoring the reversal of THC’s antioxidative effects through SIRT3/FOXO1 pathway inhibition.

Figure 1: Effects of THC and SIRT3 inhibition on cardiac oxidative stress markers. (A) Malondialdehyde, MDA, (B) reduced glutathione, GSH, and (C) catalase. Data are presented as mean ± SD and n=6 per group. ** p<0.01 and *** p<0.001 vs. control group; ++ p<0.01 and +++ p<0.001 vs. DCM group; # p<0.05 and ## p<0.01 vs. DCM + THC group. THC, Δ(9)-tetrahydrocannabinol; DCM, diabetic cardiomyopathy; 3-TYP, a SIRT3 inhibitor.
Figure 1:

Effects of THC and SIRT3 inhibition on cardiac oxidative stress markers. (A) Malondialdehyde, MDA, (B) reduced glutathione, GSH, and (C) catalase. Data are presented as mean ± SD and n=6 per group. ** p<0.01 and *** p<0.001 vs. control group; ++ p<0.01 and +++ p<0.001 vs. DCM group; # p<0.05 and ## p<0.01 vs. DCM + THC group. THC, Δ(9)-tetrahydrocannabinol; DCM, diabetic cardiomyopathy; 3-TYP, a SIRT3 inhibitor.

Effects of THC on cardiac inflammatory and inflammasome markers

Levels of NF-κB, TNF-α, NLRP3, and IL-1β were significantly elevated in the untreated diabetic rats compared to the age-matched non-diabetic control group (p<0.001), indicating a heightened cardiac inflammatory state associated with diabetes (Figure 2). Treatment of DCM rats with THC resulted in a significant reduction in the levels of inflammatory markers and NLRP3 inflammasome, demonstrating its anti-inflammatory effects. Specifically, NF-κB and TNF-α levels were markedly decreased (p<0.001), suggesting the downregulation of pro-inflammatory signaling pathways. Additionally, THC treatment led to a notable decrease in NLRP3 and IL-1β levels (p<0.001), indicating reduced inflammasome activation and subsequent cytokine release. However, administration of 3-TYP in the DCM+THC+3-TYP group significantly increased the levels of NF-κB, NLRP3 (p<0.001), TNF-α, and IL-1β (p=0.002), highlighting the reversal of THC’s anti-inflammatory effects due to SIRT3 pathway inhibition.

Figure 2: Effects of THC and SIRT3 inhibition on cardiac inflammatory and inflammasome markers. (A) Nuclear factor kappa-B, NF-κB, (B) tumor necrosis factor-alpha, TNF-α, (C) NOD-like receptor protein 3, NLRP3, and (D) interleukin-1 beta, IL-1β. Data are presented as mean ± SD and n=6 per group. *** p<0.001 vs. control group; +++ p<0.001 vs. DCM group; ## p<0.01, ### p<0.001 vs. DCM + THC group. THC, Δ(9)-tetrahydrocannabinol; DCM, diabetic cardiomyopathy; 3-TYP, a SIRT3 inhibitor.
Figure 2:

Effects of THC and SIRT3 inhibition on cardiac inflammatory and inflammasome markers. (A) Nuclear factor kappa-B, NF-κB, (B) tumor necrosis factor-alpha, TNF-α, (C) NOD-like receptor protein 3, NLRP3, and (D) interleukin-1 beta, IL-1β. Data are presented as mean ± SD and n=6 per group. *** p<0.001 vs. control group; +++ p<0.001 vs. DCM group; ## p<0.01, ### p<0.001 vs. DCM + THC group. THC, Δ(9)-tetrahydrocannabinol; DCM, diabetic cardiomyopathy; 3-TYP, a SIRT3 inhibitor.

Effects of THC on cardiac mitochondrial function

In untreated diabetic rats, mitochondrial ROS levels were significantly elevated (p<0.001), indicating heightened oxidative stress within the mitochondria (Figure 3A). Treatment of diabetic rats with THC resulted in a marked reduction in mitochondrial ROS levels (p<0.001). Additionally, the mitochondrial membrane potential was significantly improved and ATP production was also increased in THC-treated diabetic rats (p<0.001), reflecting enhanced mitochondrial function and energy metabolism compared to the DCM group (Figure 3B and C). Conversely, the administration of 3-TYP to the THC-treated diabetic rats resulted in elevated mitochondrial ROS levels (p=0.023) and decreased membrane potential (p=0.05) and ATP production (p=0.002), indicating that inhibition of the SIRT3 pathway reversed the beneficial effects of THC on mitochondrial function.

Figure 3: Effects of THC and SIRT3 inhibition on cardiac mitochondrial function. (A) Mitochondrial ROS levels, (B) mitochondrial membrane potential changes, and (C) ATP production levels. Data are presented as mean ± SD and n=6 per group. **p<0.01 and ***p<0.001 vs. control group;+p<0.05, ++p<0.01, and +++p<0.001 vs. DCM group; #p<0.05, ##p<0.01 vs. DCM+THC group. THC, Δ(9)-tetrahydrocannabinol; DCM, diabetic cardiomyopathy; 3-TYP, a SIRT3 inhibitor; ROS, reactive oxygen species.
Figure 3:

Effects of THC and SIRT3 inhibition on cardiac mitochondrial function. (A) Mitochondrial ROS levels, (B) mitochondrial membrane potential changes, and (C) ATP production levels. Data are presented as mean ± SD and n=6 per group. **p<0.01 and ***p<0.001 vs. control group;+p<0.05, ++p<0.01, and +++p<0.001 vs. DCM group; #p<0.05, ##p<0.01 vs. DCM+THC group. THC, Δ(9)-tetrahydrocannabinol; DCM, diabetic cardiomyopathy; 3-TYP, a SIRT3 inhibitor; ROS, reactive oxygen species.

Effects of THC on cardiac protein expression

In untreated diabetic rats, as depicted in Figure 4, there was a significant upregulation of TLR4 and MyD88 protein expression (p<0.001), indicating enhanced TLR4-mediated inflammatory signaling (Figure 4A and B and 4C). Conversely, treatment with THC in the DCM+THC group resulted in a notable downregulation of TLR4 and MyD88 (p<0.001), reflecting a reduction in this inflammatory response. Additionally, SIRT3 and FOXO1 proteins levels were significantly increased in the DCM+THC group in comparison to the DCM group (p<0.001), highlighting THC’s potential role in promoting mitochondrial function and cellular stress resilience (Figure 4D and E). However, the administration of 3-TYP in the DCM+THC+3-TYP group resulted in reduced SIRT3 (p<0.001) and FOXO1 (p=0.01) expression levels, without affecting on the expression of TLR4 (p=0.12) and MyD88 (p=0.16), indicating that inhibition of the SIRT3 pathway did not counter the protective effects of THC through TLR4 pathway.

Figure 4: Effects of THC and SIRT3 inhibition on cardiac signaling protein expression. (A) Immunoblotting, (B) Toll-like receptor 4, TLR4, (C) myeloid differentiation primary response 88, MyD88, (D) sirtuin-3 (SIRT3), and (E) forkhead box O1, FOXO1. Data are presented as mean ± SD and n=6 per group. ***p<0.001 vs. control group; +++p<0.001 vs. DCM group; #p<0.05, ###p<0.001 vs. DCM+THC group. THC, Δ(9)-tetrahydrocannabinol; DCM, diabetic cardiomyopathy; 3-TYP, a SIRT3 inhibitor.
Figure 4:

Effects of THC and SIRT3 inhibition on cardiac signaling protein expression. (A) Immunoblotting, (B) Toll-like receptor 4, TLR4, (C) myeloid differentiation primary response 88, MyD88, (D) sirtuin-3 (SIRT3), and (E) forkhead box O1, FOXO1. Data are presented as mean ± SD and n=6 per group. ***p<0.001 vs. control group; +++p<0.001 vs. DCM group; #p<0.05, ###p<0.001 vs. DCM+THC group. THC, Δ(9)-tetrahydrocannabinol; DCM, diabetic cardiomyopathy; 3-TYP, a SIRT3 inhibitor.

Discussion

The findings of this study demonstrated that THC significantly improves cardiac function while reducing oxidative stress, inflammation, and inflammasome activation and preserving mitochondrial integrity in a rat model of type 2 diabetes. The protective effects are closely associated with the activation of SIRT3 and FOXO1 alongside the inhibition of TLR4-mediated inflammatory signaling. These findings suggest that THC holds significant therapeutic potential for DCM by modulating the SIRT3/FOXO1 and TLR4/NF-κB/NLRP3 pathways. Our study advances the current understanding of cannabinoid-mediated cardioprotection in DCM by offering a comprehensive evaluation of the interplay between mitochondrial and inflammatory pathways.

In healthy, non-diabetic subjects, THC’s effects on mitochondrial health are less well characterized than in disease models but could still offer benefits in reducing mitochondrial deterioration due to aging, transient stress, or environmental toxins [12], 17]. By modulating oxidative stress, THC may prevent oxidative damage to mitochondrial proteins, lipids, and DNA, thereby preserving mitochondrial function even under physiological stress [12]. Studies on the nervous system suggest that activation of cannabinoid receptors CB1 and CB2 can improve mitochondrial function by reducing excitotoxicity and cytokine-induced stress while enhancing antioxidant defenses [18]. These effects could also benefit non-diabetic individuals, supporting overall mitochondrial health. Conversely, earlier investigations have established THC’s potential for cardiac protection across various disease models, including myocardial ischemia and diabetic condition. For instance, studies have shown that THC improves cardiac function, reduces infarct size, and decreases creatine kinase levels in ischemic models, highlighting its efficacy in mitigating ischemic injury [10], 13], 19], 20]. The endocannabinoid system has also been implicated in reducing oxidative stress and inflammation in diabetic models, with activation of CB1 and CB2 receptors shown to attenuate cytokine production and oxidative damage [18], 21], 22]. However, while prior studies have identified the involvement of the endocannabinoid system in reducing oxidative stress and inflammation, they have largely overlooked the distinct roles of mitochondrial and TLR4 signaling in these processes. Our findings corroborate these results while advancing the understanding by exploring mitochondrial regulation – specifically through SIRT3 and FOXO1 activation – as a central mechanism of THC’s protective effects in DCM, a topic that has not been extensively explored in this context.

Mitochondrial dysfunction is a hallmark of DCM, driving oxidative stress and inflammation that exacerbate cardiac injury [23]. Damaged mitochondria not only fail to meet the heart’s energy demands but also amplify inflammation by releasing damage-associated molecular patterns (DAMPs) that activate inflammasomes such as NLRP3 [24], 25]. Our study demonstrates that THC mitigates these effects by preserving mitochondrial membrane potential, reducing ROS production, and enhancing cardioprotective mechanisms, primarily through SIRT3/FOXO1 pathway activation. Although other sirtuins like SIRT1 and SIRT4 play essential roles in mitochondrial homeostasis, SIRT3’s role in mitochondrial health is well-established, as its deacetylase activity regulates critical mitochondrial proteins and maintains adaptive redox balance [26], 27]. Thus, SIRT3 is most directly linked to the functional optimization of mitochondria. FOXO1, a downstream target of SIRT3, further contributes to mitochondrial quality by promoting the clearance of dysfunctional mitochondria through the activation of mitophagy and by upregulating the expression of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase. Additionally, FOXO1 can activate peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), promoting the formation of new, healthy mitochondria, which is beneficial for maintaining mitochondrial function in diabetic cardiomyopathy [9], 28]. Our findings align with prior reports highlighting the protective roles of SIRT3 and FOXO1 in cardiovascular diseases, but they extend these observations by demonstrating their involvement in THC-mediated cardioprotection. Interestingly, the inhibition of SIRT3 with 3-TYP abrogated THC’s beneficial effects on mitochondrial function, redox balance, and pro-inflammatory mediators, confirming the pivotal role of the SIRT3/FOXO1 pathway in mediating these effects.

Our findings further indicate that DCM is associated with the activation of both general inflammatory pathways (such as NF-κB and TNF-α) and inflammasome-related pathways (such as NLRP3 and IL-1β), as reported in previous literature [6], 25]. In addition to its mitochondrial actions, THC exerts potent anti-inflammatory effects. These findings align with earlier research showing that cannabinoids attenuate NF-κB signaling and inflammasome activation in diabetic and ischemic models [29]. However, our study uniquely demonstrates that THC’s anti-inflammatory effects are partially dependent on SIRT3 activation. Specifically, while SIRT3 inhibition reversed THC’s effects on NF-κB and NLRP3, it did not affect the downregulation of TLR4 and MyD88 by THC. This observation suggests that THC modulates the TLR4/MyD88 pathway independently of SIRT3, indicating the involvement of additional, yet-to-be-identified molecular targets. The independent regulation of the TLR4/MyD88 and SIRT3/FOXO1 pathways by THC underscores its versatility as a therapeutic agent. While the SIRT3/FOXO1 axis primarily governs mitochondrial protection and oxidative stress reduction, the TLR4/MyD88 pathway appears to mediate upstream inflammatory signaling. These findings highlight the diversity of THC’s cardioprotective actions and underscore the need for a multifaceted approach to targeting DCM, as single-pathway interventions may not fully address the complex interplay of oxidative and inflammatory processes in the disease.

In conclusion, this study demonstrates that THC has significant therapeutic potential in DCM by improving cardiac function, reducing oxidative stress and inflammation, and preserving mitochondrial integrity. The cardioprotective effects of THC are largely mediated through the modulation of the TLR4/NF-κB/NLRP3 and SIRT3/FOXO1 pathways. Notably, while the anti-inflammatory effects of THC are largely reliant on SIRT3 activation, its effects on the TLR4/MyD88 axis appear to be independent of SIRT3, suggesting the involvement of additional mechanisms in its cardioprotective actions. Further research into the molecular interactions between THC and these signaling pathways will be critical in refining its potential clinical applications.

Corresponding author: Dr. Hua Xu, PhD, Department of Tumor Radiotherapy, Xi’an International Medical Center, Xi’an, 710100, China, E-mail:

  1. Research ethics: All experimental procedures were approved by the Institutional Animal Care and Use Committee under ethical approval number XJTU1AF-CRF-2022-036 and conducted following the National Institutes of Health guidelines for the care and use of laboratory animals.

  2. Informed consent: Not applicable.

  3. Author contributions: HZ and HX performed conceptualization. HZ, XH, and HX involved in validation, research, resources, data reviewing, and writing. HX reviewed and edited the final draft. All authors read and approved the final manuscript.

  4. Use of Large Language Models, AI and Machine Learning Tools: None declared.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: None declared.

  7. Data availability: The data can be obtained on request from the corresponding author.

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Received: 2024-10-08
Accepted: 2025-01-02
Published Online: 2025-03-06

© 2025 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/tjb-2024-0258
Keywords for this article
diabetes; cannabinoid; mitochondria; cardiomyopathy; inflammation; mechanism
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Review
  3. Interaction of fetuin-A with obesity related insulin resistance and diabetes mellitus
  4. Research Articles
  5. Preanalytical errors in pediatric blood sampling: a systematic review of common challenges and risks
  6. The promotive role of reticulocalbin 3 (RCN3) in the pathogenesis of keloid via TGFβ1/Smad2/Smad7 signaling pathway in vitro
  7. Investigation into drug resistance to cisplatin in cancer stem cell-enriched population in non-small cell lung cancer
  8. The cytotoxic and antiproliferative effect of Polygala saponin XLIV on the human colorectal carcinoma cell line
  9. Development of HEK293T cell reference materials for β-thalassemia genetic testing using prime editing
  10. Investigation of propofol, fentanyl, and midazolam-related toxicity and the protective effect of midazolam on THLE-2 cell lines
  11. Leucine-rich α-2-glycoprotein 1 can be a novel angiogenic mediator in autosomal dominant polycystic kidney disease
  12. Asiaticoside reverses the inhibition effect of miR-184 on proliferation, migration and AKT phosphorylation of HTR-8/Svneo cells
  13. Perioperative D-dimer levels and head and neck cancer surgery: a prospective observational study
  14. Comprehensive analysis of KRT15 in pan-cancer and verification in lung adenocarcinoma
  15. Age and sex-dependent effects of nitrosative stress on the osmotic fragility of human red blood cells
  16. Evaluation of oxidative stress parameters in older patients with urinary incontinence
  17. Determination of reference change values for thyroid-related biomarkers: TSH, fT3, fT4, Tg, Anti-Tg, and Anti-TPO
  18. An evaluation of serum boron level in pregnancies with severe pre-eclampsia
  19. Involvement of SIRT3/FOXO1 and TLR4/NF-κB/NLRP3 pathways in protective effects of Δ(9)-tetrahydrocannabinol on diabetic cardiomyopathy in rats
  20. Evaluating the potential therapeutic effect of Rosa damascena Mill. essential oil on acetic acid-induced ulcerative colitis in rats
  21. Assessment of blood collection competency in phlebotomists: a survey study
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