Abstract

Oxidative stress, fibrosis, and inflammasome activation from advanced glycation end product (AGE)–receptor of advanced glycation end product (RAGE) interaction contribute to diabetic cardiomyopathy (DCM) formation and progression. Our study revealed the impact of β-caryophyllene (BCP) on activating cannabinoid type 2 receptors (CB2Rs) against diabetic complication, mainly cardiomyopathy and investigated the underlying cell signaling pathways in mice. The murine model of DCM was developed by feeding a high-fat diet with streptozotocin injections. After the development of diabetes, the animals received a 12-week oral BCP treatment at a dose of 50 mg/kg/body weight. BCP treatment showed significant improvement in glucose tolerance and insulin resistance and enhanced serum insulin levels in diabetic animals. BCP treatment effectively reversed the heart remodeling and restored the phosphorylated troponin I and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a expression. Ultrastructural examination showed reduced myocardial cell injury in DCM mice treated with BCP. The preserved myocytes were found to be associated with reduced expression of AGE/RAGE in DCM mice hearts. BCP treatment mitigated oxidative stress by inhibiting expression of NADPH oxidase 4 and activating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor erythroid 2–related factor 2 (Nrf2) signaling. Also, BCP suppressed cardiac fibrosis and endothelial-to-mesenchymal transition in DCM mice by inhibiting transforming growth factor β (TGF-β)/suppressor of mothers against decapentaplegic (Smad) signaling. Further, BCP treatment suppressed nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome activation in DCM mice and alleviated cellular injury to the pancreatic tissues evidenced by significant elevation of the number of insulin-positive cells. To demonstrate a CB2R-dependent mechanism of BCP, another group of DCM mice were pretreated with AM630, a CB2R antagonist. AM630 was observed to abrogate the beneficial effects of BCP in DCM mice. Taken together, BCP demonstrated the potential to protect the myocardium and pancreas of DCM mice mediating CB2R-dependent mechanisms.

SIGNIFICANCE STATEMENT BCP, a CB2R agonist, shows protection against DCM. BCP attenuates oxidative stress, inflammation, and fibrosis in DCM via activating CB2Rs. BCP mediating CB2R activation favorably modulates AGE/RAGE, PI3K/AKT/Nrf2β and TGF-β/Smad and (NLRP3) inflammasome in diabetic cardiomyopathy.

Introduction

Diabetic cardiomyopathy (DCM) is a condition that affects the heart muscle in individuals with diabetes, even without other risk factors such as high blood pressure or coronary artery disease. It is a significant contributor to heart failure in diabetic patients (Avogaro et al., 2004; Wang et al., 2016). Given the increased susceptibility of diabetic patients to heart failure compared with nondiabetic individuals, it is imperative to thoroughly understand the underlying physiological processes of DCM and identify new treatment options for its management (Preis et al., 2009; Parim et al., 2019). Various factors have been hypothesized to contribute to the development of DCM. These include the build-up of advanced glycation end product (AGE), increased oxidative stress, activation of inflammatory pathways, and changes in the composition of the extracellular matrix leading to cardiac fibrosis (Murtaza et al., 2019; El-Azab et al., 2022).

AGEs are crucial in understanding the pathophysiology of DCM. The interaction between AGEs and receptors of advanced glycation end product (RAGEs) has been found to have several effects. It leads to the generation of reactive oxygen species (ROS), activation of oxidative stress, and activation of nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome-mediated pyroptosis and inflammation. These effects have been linked to the onset of cardiovascular diseases in diabetic patients, as shown in studies (Goldin et al., 2006; Yan et al., 2010; Wan et al., 2022). Endothelial cells play a central role in the harmful effects of high blood sugar levels in diabetic cardiomyopathy. One important change that occurs is the transformation of endothelial cells into cardiac fibroblasts, which further contributes to the development of cardiac fibrosis (Okayama et al., 2012; Kovacic, 2018).

Oxidative stress plays a crucial role in the development of DCM in individuals with type 2 diabetes mellitus (Wilson et al., 2018). The kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2–related factor 2 (Nrf2) signaling pathway is widely recognized as a crucial mechanism for defending cells against oxidative stress (Cuadrado et al., 2018). Nrf2 activation has been shown to be helpful in various cardiovascular diseases (Chen et al., 2014; Cuadrado et al., 2018).

The existing literature suggests that hyperglycemia can stimulate NLRP3, leading to the autocatalytic activation of procaspase-1 and subsequent activation of caspase-1. The maturation of prointerleukin-1β and prointerleukin-18 is caused by activated caspase-1 (Luo et al., 2014). This process results in inflammation-driven programmed cell death, specifically pyroptosis, which plays a role in the development of DCM. There is growing evidence suggesting that inhibiting the NLRP3 inflammasome could potentially delay pyroptosis in diabetes and its associated complications (Song et al., 2019; Yang et al., 2019).

One of the most promising therapeutic targets explored in the past few years for many chronic diseases involving low onset immune-inflammatory and oxidative changes is the endocannabinoid system consisting of endocannabinoid metabolizing enzymes and cannabinoid receptors. Cannabinoid 1 receptor (CB1R) and cannabinoid 2 receptor (CB2R) are part of the G-protein coupled receptors (Zou and Kumar, 2018). In contrast to CB1Rs, which are primarily found in the central nervous system, CB2Rs have a more widespread presence in peripheral tissues and immune system cells (Pacher and Steffens, 2009; Han et al., 2017). There is a growing body of evidence that suggests CB2Rs play a protective role in various experimental models of cardiovascular diseases (Moris et al., 2015; Maslov et al., 2016; Maslov and Karpov, 2017).

Among the ligands that target cannabinoid receptors, β-caryophyllene (BCP), which is a sesquiterpene that occurs naturally, has garnered a significant attention due to its full functional CB2R agonism. It is widely present in various edible plants, such as cinnamon, basil, black pepper, oregano, and cloves (Gertsch et al., 2008). Pharmacologically, BCP has been identified as a CB2R selective full agonist with a pKi value of 155 nmol/L for human CB2Rs, with a lack of affinity to CB1R. This, in turn, causes the Gi/Go subunit of G-proteins to become activated (Gertsch et al., 2008).

It has been reported that activation of the CB2R increases insulin secretion, decreases inflammation and oxidative stress that are associated with hyperglycemia, and corrects hyperglycemia (Kumawat and Kaur, 2019). Previous research has demonstrated that BCP has the potential to reduce hyperglycemia and hyperlipidemia, boosting insulin production and enhancing insulin sensitivity, as well as exerting antioxidant and anti-inflammatory effects. Hence, BCP appears as an interesting natural compound with CB2R selectivity and affinity that attributes to its therapeutic promise for diabetes and its related complications (Suijun et al., 2014; Basha and Sankaranarayanan, 2016; Youssef et al., 2019; Hashiesh et al., 2020; Li et al., 2020; Kumawat and Kaur, 2022; Mamdouh Hashiesh et al., 2023). In addition, it has been demonstrated that BCP can reduce the severity of fibrosis in a variety of animal models (Calleja et al., 2013; Mahmoud et al., 2014; Gushiken et al., 2022). Consequently, in light of the fact that CB2Rs play a significant role in diabetes and based on our previous report, which revealed that BCP prevented DCM progression via inhibiting lipotoxicity-induced cardiac stress and inflammation, our objective was to further investigate the therapeutic potential of BCP in mice with DCM. Hitherto, the effect of BCP activating CB2Rs on the interaction between AGE and RAGE in DCM has not been fully understood. The present study was undertaken to test the hypothesis whether BCP can reduce oxidative stress, fibrosis, and inflammation and cell signaling regulatory pathways controlling these pathological events in a mice model of DCM. Given the CB2R selectivity and affinity of BCP, the present study also investigated CB2R-dependent pathways using a pharmacological challenge protocol wherein the animals were pretreated with a CB2R antagonist, AM630, to negate the protective effects of BCP.

Materials and MethodsAnimals and Experimental Protocol.

Male C57BL/6 mice aged 8 weeks were obtained from the United Arab Emirates University’s Laboratory Animal Research Facility. The mice used in the experiments were housed in a climate-controlled environment at a temperature of 25°C and a 12:12 light/dark cycle, with free access to food and water. Before the experiments were initiated, the animal experimental procedures were approved by the Animal Ethics Committee of United Arab Emirates University, UAE.

Briefly, the DCM was developed by feeding a high-fat diet (HFD) to the mice for 4 weeks, which efficiently induced insulin resistance (Andrikopoulos et al., 2008), followed by the intraperitoneal injection of streptozotocin (100 mg/kg) (dissolved in 0.1 M sodium citrate, pH 4.5, supplied from ChemCruz, USA), to achieve a murine model with insulin resistance, partial insulin deficiency, and hyperglycemia, as described previously (Gu et al., 2017; Wu et al., 2020). The normal groups were fed a normal matching chow diet (10 kcal% fat), and diabetic groups were fed a HFD (45% kcal fat, Research Diets D12451). After being administered streptozotocin, mice with DCM and fasting blood glucose levels ≥ 250 mg/dL were identified 1 week later. Then, the DCM and age-matched naïve mice were treated with BCP for 12 weeks while being maintained on either a HFD or a normal diet. These animals were randomly allocated into six groups with 15 animals in each group.

Group I (Naïve): Nondiabetic mice received light olive oil orally for 12 weeks while being maintained on a normal diet.

Groups II (BCP): Nondiabetic mice received BCP (50 mg/kg, supplied from Sigma, USA, dissolved in light olive oil) by oral gavage daily for 12 weeks while being maintained on a normal diet.

Group III (HFD): HFD mice received light olive oil orally for 12 weeks while being maintained on a HFD diet.

Group IV (DCM): Diabetic mice received light olive oil orally for 12 weeks while being maintained on a HFD diet.

Group V (DCM+BCP): Diabetic mice received BCP (50 mg/kg) by oral gavage daily for 12 weeks while being maintained on HFD diet.

Group VI (DCM+BCP+AM630): Diabetic mice were given oral gavage of AM630 (1.5 mg/kg), a specific CB2R antagonist, 30 minutes before receiving BCP (50 mg/kg) by oral gavage daily for 12 weeks while being maintained on a HFD diet. The BCP dose was chosen based on previous literature (Meeran et al., 2021; Franco-Arroyo et al., 2022).

Oral Glucose Tolerance Test.

After a 12-week intervention, an oral glucose tolerance test (OGTT) was performed following a 6-hour fasting period in the light phase. Following the administration of glucose loading (2 g/kg, b. wt.) to each group, blood glucose levels were observed at 0, 30, 60, 90, and 120 minutes. In addition, the area under the curve (AUC) values for OGTT were calculated using a specific formula.

AUC0–120 = [30 × (G0 + G30) + 30 × (G30 + G60) + 30 × (G60 + G90) + 30 × (G90 + G120)]/2.

Determination of Insulin Content and Homeostatic Index of Insulin Resistance.

A Mouse Insulin ELISA Detection Kit was used to quantify the serum insulin levels in accordance with the instructions provided with the kit (Cat. No. 10-1247-01, Mercodia, Uppsala, Sweden). By applying the given formula, we calculated the homeostasis model assessment of insulin resistance (HOMA-IR). This assessment serves as a valuable tool for quantifying insulin resistance, and it involves analyzing the fasting levels of insulin and glucose.

The formula for calculating the HOMA-IR is as follows: divide the product of fasting glucose (mg/dl) and fasting insulin (mU/L) by 405 (Karim et al., 2019).

Determination of Heart Weight Index.

After conducting the experiment, the weight of each mouse’s body and heart was carefully measured and recorded. The hearts were subsequently extracted, rinsed in chilled phosphate-buffered saline, drained using filter paper, and weighed. The heart weight index was used to assess the extent of cardiac hypertrophy. This index is calculated by dividing the heart weight (HW) by the body weight (BW).

Estimation of Antioxidant Enzymes Activity.

The activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) was measured using readily available kits. The measurements were carried out following the guidelines provided by the manufacturer (Cayman Chemical Company, Ann Arbor, MI, USA). Using a microplate reader, the absorbance of SOD was quantified at 450 nm, and the resulting activity was expressed in U/ml. The absorbance of CAT was measured at 540 nm using a microplate reader, and the activity was reported as nmol/min/ml.

Histopathological Analysis.

The heart and pancreas tissues collected from all experimental mice were promptly washed with ice-cold phosphate-buffered saline and subsequently preserved in 4% paraformaldehyde for 48 hours. After fixation, the tissues were processed by being embedded in paraffin, cut into sections that were 3 μm thick, and then placed on glass slides. Following a precise de-waxing process, the tissue sections underwent H&E staining. Subsequently, the stained sections were observed under a 60x light microscope and captured in photographs. The diameter of the cardiomyocytes at the level of the nucleus in longitudinal sections was then measured. To observe changes in myocardial interstitial and perivascular fibrosis, Masson’s trichrome staining (ab150686; Abcam, Cambridge, UK) and Picrosirius red staining (ab245887; Abcam, Cambridge, UK) were conducted. The collagen fibers were measured in sections stained with Masson trichrome (3 μm). The collagen volume fraction was determined by dividing the collagen area by the total ventricular area in the section and multiplying by 100 (Sun et al., 2004). The presence of fibrillar collagen was observed in the picrosirius-stained sections (5 μm) due to its distinct red coloration. The morphological analysis was conducted using a light microscope (BX41, Olympus), and the results were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Ultrastructural Observations.

The heart and pancreas were precisely dissected into small tissue pieces measuring 1mm3. These tissue pieces were then promptly submerged in a solution containing 2.5% glutaraldehyde, which was kept at a temperature of 4°C. Following the rinsing of the samples with 0.1 M phosphate-buffered saline, the tissues were then fixed in a 1% osmic acid solution. Ultrathin sections were prepared following a meticulous process of dehydration using gradient ethanol and carefully mounted on the grid. Next, the grids underwent counterstaining with a solution of 2% uranyl acetate and 0.2% lead citrate. The resulting samples were then captured using a Philips CM10 Transmission Electron Microscope (The Netherlands).

Immunofluorescence of the Pancreas.

Briefly, the pancreatic-embedded sections were subjected to a de-paraffinization process. They were then treated at 95°C in a water bath with citrate buffer at PH (6.1) for 30 minutes to retrieve antigens. A blocking agent was added to the section and left for 45 minutes to prevent any unspecific binding. Next, the sections were labeled with insulin and glucagon antibodies and stored at 4°C overnight. The following day, the sections were incubated with fluorescent secondary antibodies (Alexa Fluor488 for mouse and Alexa Fluor594 for rabbit) at a dilution of 1:500 for 1 hour at room temperature. After incubation, the sections were mounted using Vectashield mounting media. An EVOS FL fluorescent microscope from Thermo Fisher Scientific (Waltham, MA, USA) was used to capture the images. Then the pictures were quantified using Image J software from the National Institutes of Health. We calculated the proportions of β and α cells, which produce insulin and glucagon, respectively, to the total area of the islets.

Immunohistochemistry.

The heart sections underwent dewaxing and rehydration using a series of xylene and ethanol washes. Following antigen retrieval, the sections underwent incubation in a 0.3% hydrogen peroxide solution and were then stained using VECTASTAIN Elite ABC kits. After being treated with blocking reagent, the sections were exposed to a series of antibodies. The sections were treated with anti-sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) (1:50, Santa Cruz Biotechnology, Inc., catalog no. 376235), anticollagen I (1:50, Abcam, catalog no. 270993), anticollagen III (1:50, Abcam, catalog no. 7778), antifibronectin (1:50, Abcam, catalog no. 2413), antivimentin (1:50, Cell Signaling Technology, catalog no. 5741), anti-CD31 (1:50, Santa Cruz Biotechnology, Inc., catalog no. 376764), anti-vascular endothelial-cadherin (1:50, Santa Cruz Biotechnology, Inc., catalog no. 52751), anti-Nrf2 (1:50, Abcam, catalog no. 31163), anti-SOD2 (1:50, Cell Signaling Technology, catalog no. 13141), anti NADPH oxidase 4 (NOX4) (1:50, Thermo Fisher Scientific, Inc., catalog no. PA5-85479), anti-NLRP3 (1:50, Abcam, catalog no. 214185), anti-interleukin 18 (IL-18) (1:50, Abcam, catalog no. 191860) at 4°C overnight and then with secondary antibodies for 1 hour at room temperature. Immunoreactivity was detected with diaminobenzidine, and sections were counterstained with hematoxylin. A digital camera (BX43, Olympus Co. Ltd., Japan) was used to take the pictures. Using the ImageJ program (National Institutes of Health, USA), the positively brown area was quantified.

Protein Extraction and Western Blot Analysis.

The protein lysates from heart tissue were measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Tissue protein was separated by gel electrophoresis and subsequently transferred to a polyvinylidene difluoride membrane. Following this, the membrane was incubated with the designated primary antibodies overnight at 4°C (Table 1). Immunoreactive bands were identified using secondary antibodies conjugated to horseradish peroxidase, and their visualization was achieved through enhanced chemiluminescence. Quantification was performed using Image J software from the National Institutes of Health. For total protein expression, glyceraldehyde-3-phosphate dehydrogenase, β-tubulin, and vinculin were used as loading controls.

TABLE 1

The dilution, catalog, manufacturer, and source of primary antibodies used

Statistical Analysis.

The mean ± S.E.M. was used to express the results. To conduct statistical studies, GraphPad Prism 8 was used. Tukey–Kramer analysis was conducted after a one-way ANOVA for group comparisons. For comparing the two groups, a nonparametric ‘t’ test, the Mann–Whitney U test, was used. A statistically significant value was determined to be a value of P < 0.05.

ResultsCB2R Agonist BCP Improves Impaired Glucose Tolerance and Insulin Resistance.

The blood glucose level was noticeably higher in the HFD mice and much higher in the DCM mice compared with the naïve mice, as seen by Fig. 1A. Following treatment with BCP for a period of 12 weeks, the blood glucose level of the DCM mice was found to be significantly lower. Insulin levels were seen to be significantly lower in DCM mice compared with the naïve group, while they were found to be higher in HFD mice (Fig. 1B). The DCM mice that were given oral BCP treatment of a period of 12 weeks experienced a significant increase in their serum insulin levels. In contrast, the antihyperglycemic and insulinotropic effects of BCP were rendered ineffective in DCM mice by the administration of AM630, a CB2R antagonist.

Fig. 1.

Effect of BCP treatment on impaired glucose tolerance and insulin resistance in DCM mice. (A) Blood glucose level. (B) Serum insulin. (C) Blood glucose level during oral glucose tolerance test. (D) The AUC of oral glucose tolerance test. (E) HOMA-IR. Data are expressed as mean ± S.E.M., n = 6–10. aP < 0.05 compared with Naïve, bP < 0.05 compared with HFD, cP < 0.05 compared with DCM, dP < 0.05 compared with DCM+BCP.

To study the effect of BCP on glucose metabolism in peripheral tissues and insulin resistance, the OGTT (Fig. 1, C and D) and HOMA-IR (Fig. 1E) were measured, respectively. When compared with the naïve group, the DCM mice in the OGTT demonstrated considerably higher glucose excursions following the glucose challenge; however, the administration of BCP resulted in a significant decrease in both glucose excursions and the AUC. In a similar manner, when compared with the naïve mice, the HOMA-IR was clearly elevated in the HFD and DCM mice, whereas the BCP treatment completely improved the HOMA-IR. It is interesting to note that the protective effects of BCP on impaired glucose tolerance and insulin resistance were abrogated when AM630 was administered prior to BCP. Therefore, activation of the CB2R by BCP has the potential to repair glucose metabolism, improve insulin resistance, and increase insulin secretion in mice with DCM.

CB2R Agonist BCP Attenuates Myocardial Remodeling and Restores Cardiac Contractile Proteins Expression in DCM.

Compared with the naïve group, only HFD mice showed significantly higher BW. BCP treatment had no effect on BW in DCM mice (Supplemental Table 1). The significant increase in the HW/BW ratio (HW/BW) in the HFD and DCM mice is evidence of cardiac hypertrophy. This rise indicates that the cardiac function may be affected, which is consistent with the histological findings (Fig. 2B) that presented cardiac hypertrophy in the form of an increased cardiomyocyte diameter in the region of the cell nucleus in the HFD and DCM mice. An increase in the diameter of the cardiomyocytes was a pathological change that was mitigated by the administration of BCP treatment. On the other hand, the preadministration of AM630 negated the protective effects of BCP. Additionally, to ascertain the impact that BCP has on the expression of contractile protein, specifically phospho troponin I (TnI), was evaluated through western blot (as shown in Fig. 2, D and E), and the expression of SERCA2a was evaluated through immunohistochemistry (as shown in Fig. 2, F and G). It was found that the level of phosphorylated TnI and SERCA2a expression was significantly reduced in the heart of HFD mice, and it was further reduced in DCM mice. However, the level of expression was significantly restored by treatment with BCP. A prior administration of AM630 was able to reverse the protective effect that was observed with BCP. Taken together, these findings demonstrate that BCP has the potential to lessen cardiac hypertrophy and restore the phosphorylated TnI and SERCA2a expression in DCM mice via activation of CB2Rs.

Fig. 2.

Effect of BCP treatment on myocardial remodeling and cardiac contractile proteins expression in DCM mice. (A) HW/BW. (B) Representative H&E staining of the myocardium (scale bar: 20 μm, n = 3). (C) Quantitative analysis of cardiomyocyte diameter (D) Western blotting analysis of P-TnI. (E) Densitometric analysis of myocardial protein expression of P-TnI (n = 3). (F) Representative images of immunohistochemistry for SERCA2a in cardiac sections as quantified in (G) (scale bar: 50 μm, n = 5). Data are expressed as mean ± S.E.M. aP < 0.05 compared with Naive, bP < 0.05 compared with HFD, cP < 0.05 compared with DCM, dP < 0.05 compared with DCM+BCP. The black arrows indicate hypertrophic cardiomyocytes. P-TnI, phospho-troponin I.

CB2R Agonist BCP Preserves Myocardium Ultrastructure in DCM Mice.

The transmission electron microscopy technique was used to investigate the effects of BCP treatment on the ultrastructure of the myocardium (Fig. 3). The myocardium displayed consistent myofibrils and regularly distributed myofilaments, continuous sarcomeres, a clear Z band, and a continuous intercalated disc in both the naïve group and the BCP control group. There were also continuous intercalated discs. The mitochondria were arranged in a manner that was both close and uniform in density. Mitochondrial cristae were observed alongside normal capillary endothelial cells and a normal nucleus.

Fig. 3.

Representative images of transmission electron microscopic observation of myocardium ultrastructure show that BCP protects myocardial cell ultrastructure in DCM mice (scale bar: 1 μm, n = 3). BV, blood vessel; ID, intercalated disc; L, lipids; M, mitochondrial; MF, myofilament fibers; My; myelin figures; N, nucleus.

In the myocardium of mice that were fed a HFD, the arrangement of the myofilaments was partially irregular, myofibrils were partially disrupted, and separated intercalated discs were observed. A slight enlargement of the mitochondria was observed, and the ridge was both rich and regular. Additionally, a thickening of the capillary basement membrane, an accumulation of lipid droplets, and a nucleus that was partially condensed were observed simultaneously. Furthermore, in comparison with the naïve group, the DCM mice exhibited disorderly arrangements of myocardial cells, characterized by the disruption of myofilament fibers, the misalignment of the Z line, and the discontinuous intercalated disc. There were vacuoles, mitochondrial cristae that were disrupted, and numerous myelin figures, all of which suggested that membrane disruption and cellular damage had occurred in myocardial tissues. The mitochondria were swollen, and the arrangement of the ridges was not uniform. Both the capillary basement membrane and the appearance of lipid droplets were thickened in the myocardium of patients with DCM. An increase in heterochromatin accumulation and invaginations were present in the diabetic cardiomyocyte nucleus, which was one of the morphological abnormalities observed in this cell type.

BCP treatment, on the other hand, was able to reduce the severity of these pathologic changes in the ultrastructure of the myocardium in DCM mice. This was demonstrated by improved sarcomere integrity, aligned and clear Z lines, and a slight reduction in the amount of myofibril distortion. There was a complete integration of the mitochondria with a rich and regular ridge. A capillary basement membrane that was thinner, a nucleus that was slightly condensed, and a reduction in the number of lipid droplets were also observed. It was demonstrated by the irregular arrangement of myofibrils with sparse, distorted, and broken myofilament fibers; disrupted sarcomeres; obscured Z lines; and separated intercalated discs that this beneficial effect of BCP was significantly reversed by the prior treatment with AM630. It was observed that the mitochondrial structures exhibited a disorganized array of mitochondria, an increase in the number and fission of mitochondria, signs of swelling, and the shattering of cristae. It was also observed that the basement membrane of the blood vessel had thickened and lipid droplets had accumulated; condensed nucleus was also observed.

CB2R Agonist BCP Inhibits AGE/RAGE Expression in DCM.

AGEs play a significant part in the beginning stages of end-organ damage and the progression of this damage in patients with diabetes that affects the heart. As can be seen in Figs. 4, A and B, AGE and RAGE protein expressions were found to be elevated in the HFD mice, and these expressions were even higher in the DCM mice when compared with the naïve group. The administration of BCP resulted in a reduction in the expression of AGE and RAGE in the DCM mice, an effect that was prevented by the administration of AM630 in the beginning. The activation of the CB2R by BCP has the potential to suppress the expression of AGE and RAGE in DCM mice that has been induced by hyperglycemia.

Fig. 4.

Effect of BCP treatment on AGE/RAGE expression in DCM mice. (A) Western blotting analysis of AGE and RAGE. (B) Densitometric analysis of myocardial protein expressions of AGE and RAGE (n = 3). Data are expressed as mean ± S.E.M. aP < 0.05 compared with Naïve, bP < 0.05 compared with HFD, cP < 0.05 compared with DCM, dP < 0.05 compared with DCM+BCP.

CB2R Agonist BCP Augments Antioxidants Status in DCM.

Oxidative stress has a significant pathophysiological role in the onset and progression of DCM. To determine if the beneficial effects of BCP against DCM were linked to its antioxidative properties, we analyzed the antioxidant enzymes in cardiac tissue. We found that mice fed a HFD showed a significant reduction in SOD activity, and DCM mice displayed a further decrease in both SOD and CAT activities, which were significantly augmented by BCP treatment. The abrogation of this protective effect was achieved through the administration of AM630 before BCP treatment (Fig. 5, A and B). Consequently, these findings suggested that activation of the CB2R by BCP could reduce the amount of oxidative stress experienced by DCM mice by increasing the capacity of the endogenous antioxidant system.

Fig. 5.

Effect of BCP treatment on antioxidants enzymes in DCM mice. (A) SOD. (B) Catalase. Data are expressed as mean ± S.E.M., n = 6. aP < 0.05 compared with Naïve, bP < 0.05 compared with HFD, cP< 0.05 compared with DCM, dP < 0.05 compared with DCM+BCP.

CB2R Agonist BCP Ameliorates Myocardial Oxidative Damage in DCM Mice via Phosphoinositide 3-Kinase/Protein Kinase B/Nrf2 Signaling Pathway.

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/Nrf2 signaling pathway plays a significant part in the process of protecting cardiomyocytes from oxidative damage. When compared with the naïve group, the results of the Western blot showed that the expression levels of Nrf2, heme oxygenase-1 (HO-1), and SOD2 were shown to be downregulated, while the expression levels of Keap1 were found to be increased in the heart of the HFD mice and more in DCM mice. In addition to this, DCM mice exhibited a higher level of cardiac oxidative stress, which was demonstrated by an increased expression of NOX4 in comparison with naïve mice. Furthermore, when compared with naïve mice, the phosphorylation of PI3K and AKT was observed to be significantly downregulated in the HFD and DCM mice. While treatment with BCP led to a significant increase in the levels of myocardial protein expression of Nrf2, HO-1, SOD2, p-PI3K, and p-Akt, it also led to a significant decrease in the levels of myocardial protein expression produced by Keap1 and NOX4 in DCM mice (Fig. 6, A and B). Furthermore, the findings of the Western blot were supported by immunohistochemical investigations, which showed that the distribution of NOX4 protein was increased in the hearts of DCM mice, while the distribution of Nrf2 and SOD2 proteins was decreased in the hearts of HFD animals and further reduced in the myocardium of DCM animals. On the other hand, the administration of BCP led to a decrease in the distribution of NOX4 proteins while simultaneously leading to an increase in the distribution of Nrf2 and SOD2 proteins (Fig. 6, C and D). The administration of AM630 prior to the administration of BCP in DCM mice resulted in a significant reduction in the protective effect of BCP. When taken as a whole, BCP was able to activate PI3K/AKT/Nrf2 signaling in a manner that was dependent on the CB2R. This resulted in a reduction in the oxidative damage and changes that were observed in the antioxidant proteins of the hearts of DCM animals.

Fig. 6.

Effect of BCP treatment on myocardial oxidative damage and PI3K/AKT/Nrf2 signaling in DCM mice. (A) Western blotting analysis of NOX4, Nrf2, Keap1, HO-1, SOD2, p-PI3K, PI3K, and p-AKT. (B) Densitometric analysis of myocardial protein expressions of NOX4, Nrf2, Keap1, HO-1, SOD2, p-PI3K, PI3K, and p-AKT (n = 3). (C) Representative images of immunohistochemistry for Nrf2, SOD2, and NOX4 in cardiac sections as quantified in (D) (n = 5) (scale bar: 50 μm). Data are expressed as mean ± S.E.M. aP < 0.05 compared with Naïve, bP < 0.05 compared with HFD, cP < 0.05 compared with DCM, dP < 0.05 compared with DCM+BCP.

CB2R Agonist BCP Alleviates Cardiac Fibrosis in DCM.

The heart tissues of DCM mice were processed for Masson’s trichrome and Picrosirius red staining, Western blot, and immunohistochemistry of fibrotic markers. This was done to further evaluate the potential antifibrotic activity of BCP. Masson’s trichrome and Picrosirius red staining of the hearts of the HFD mice revealed an increased level of collagen deposition both in the interstitial and perivascular region. On the other hand, the hearts of the DCM mice showed a further increase in collagen deposition in both areas. However, the BCP treatment effectively reduced the amount of collagen deposition in the hearts of the DCM mice (Fig. 7, A–C). The administration of AM630 in DCM mice prior to the administration of BCP was able to counter the protective effect of BCP on collagen deposition.

Fig. 7.

Effect of BCP treatment on cardiac fibrosis in DCM mice. (A) Cardiac fibrosis in DCM mice (n = 3) is shown. Masson trichrome staining (collagen is blue; scale bar: 50 μm [A1]) and Picrosirius red staining (collagen fibers-stained bright red, bright-field [A2], dark-field [A3]; scale bar: 100 μm) show interstitial fibrosis. Masson trichrome staining (A4) (scale bar: 50 μm) and Picrosirius red staining (bright-field [A5], dark-field [A6]; scale bar: 100 μm) show perivascular fibrosis. (B) Quantitative analysis of Masson trichrome staining (collagen volume fraction). (C) Quantitative analysis of Picrosirius red staining (% fibrosis area). (D) Western blotting analysis of collagen I, collagen III, and fibronectin. (E) Densitometric analysis of myocardial protein expressions of collagen I, collagen III, and fibronectin (n = 3). (F) Representative images of immunohistochemistry for collagen I, collagen III, and fibronectin in cardiac sections as quantified in (G) (n = 5) (scale bar: 50 μm). (H) Western blotting analysis of MMP-2 and MMP-9. (I) Densitometric analysis of myocardial protein expressions of MMP-2 and MMP-9 (n = 3). Data are expressed as mean ± S.E.M. aP < 0.05 compared with Naïve, bP < 0.05 compared with HFD, cP < 0.05 compared with DCM, dP < 0.05 compared with DCM+BCP.

Additionally, the increased expressions of collagen I, collagen III, and fibronectin were observed through Western blot (Figs. 7, D and E) and immunohistochemical staining (Figs. 7, F and G) in the mice that were fed a HFD. This was even more pronounced in the DCM mice, which were saved by the administration of BCP when they were treated. It was determined that the protective effect of BCP on these fibrotic markers could be reversed by the prior administration of AM630.

Matrix metalloproteinases (MMPs) are the primary proteases that are accountable for the degradation of extracts from the extracellular matrix. In previous studies, it was hypothesized that excessive stimulation of specific MMPs, such as MMP-2 and MMP-9, could potentially facilitate the formation of fibroblasts and the accumulation of extracellular matrix, both of which are essential processes for the remodeling of the heart. In the present study, we found that the HFD mice had a slightly elevated expression of MMP-9 and that the DCM mice had further upregulated expression levels of both MMP-2 and MMP-9, which were inhibited by the administration of BCP. According to Fig. 7, H and I, the protective effects were nullified by the administration of AM630 in advance. By reducing collagen deposition and mitigating overactivated fibrotic markers in diabetes-associated cardiac fibrosis on a CB2-dependent mechanism, these findings collectively suggested that BCP had significant antifibrotic activity in DCM. This was accomplished by reducing the amount of collagen that was deposited.

CB2R Agonist BCP Inhibits Cardiac Endothelial-to-Mesenchymal Transition via Attenuation of Transforming Growth Factor β/Suppressor of Mothers against Decapentaplegic Signaling Pathway in DCM.

Furthermore, in comparison with the naïve mice, the HFD mice exhibited a decrease in the expression of endothelial markers (CD31, vascular endothelial-cadherin) and an increase in the expression of mesenchymal markers [α-smooth muscle actin (α-SMA), vimentin]. Furthermore, the DCM mice exhibited an even more pronounced decrease in the expression of endothelial markers and increase in the expression of mesenchymal markers. By administering BCP, the endothelial-to-mesenchymal transition (EndMT) phenotype transition was prevented from occurring. As shown in Fig. 8, the protective effect of BCP on EndMT transition was nullified by the treatment of AM630 in the past. When taken as a whole, the stimulation of the CB2R by BCP has the potential to repress the transition of myocardial EndMT in mice with DCM.