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Luteolin ameliorates rat model of metabolic syndrome-induced cardiac injury by apoptosis suppression and autophagy promotion via NR4A2/p53 regulation

BMC Complementary Medicine and Therapies volume 25, Article number: 14 (2025) Cite this article

Abstract

Background

Reduced cardiac autophagy, inflammation, and apoptosis contribute to cardiovascular complications caused by metabolic syndrome (MetS). It is documented that the nuclear receptor 4A2 (NR4A2) could modulate autophagy and apoptosis in cardiac complications. The aim of this investigation was to assess the therapeutic potential of luteolin, with documented beneficial properties, against MetS-associated cardiac injury.

Methods

Forty male albino Wistar rats were divided into 5 groups randomly as controls, MetS, and MetS animals treated with luteolin (25, 50, 100 mg/kg ip). The animal’s weight, blood pressure, lipid profile, tolerance to glucose and insulin, and cardiac histopathology were evaluated. Moreover, troponin T, creatine kinase-myocardial band (CK-MB), inflammatory profile (IL-6, IL-1β, TNF-α), transforming growth factor-β1 (TGF-β1), oxidative stress, and matrix metalloproteinase-9 (MMP-9) were analyzed to determine the cardiac state. Cardiac NR4A2 and p53, as well as apoptotic (B-cell leukemia/lymphoma 2 [BCL-2], Caspase [CASP]-3, and CASP-9) and autophagic mediators (Sequestosome-1/p62, Microtubule-associated protein 1 A/1B-light chain 3 [LC3], and Beclin-1) were measured by RT-qPCR and ELISA.

Results

Luteolin remarkably restored MetS-induced biochemical derangements and related cardiac injury via the suppression of apoptosis, inflammation, and stress but promotion of autophagy (p-value < 0.001).

Conclusion

Current findings revealed the promising therapeutical properties of luteolin against MetS-associated cardiovascular risks.

Peer Review reports

Background

Metabolic syndrome (MetS) is a multifaceted condition manifested by a cluster of symptoms that tend to occur together, raising crucial perils for cardiac complications, stroke, and diabetes. The main characteristics of MetS encompass hypertension, elevated blood glucose levels, insulin resistance, abdominal obesity, and imbalances in cholesterol levels, in particular dysregulated levels of LDL and HDL [1]. These elements collectively contribute to the appearance and development of vascular issues, including cardiovascular, coronary, and cerebrovascular diseases [2]. It is widely suggested that pathological alterations in cardiac autophagy, inflammation, and apoptosis pivotally contribute to cardiac complications caused by MetS [3, 4].

Nuclear receptor 4A2 (NR4A2) is a member of the NR4A subfamily of nuclear receptors, which is encoded by immediate-early response genes and plays a role in regulating various cellular processes. The activity of these receptors can be influenced by factors such as their expression levels, posttranslational modifications, and interactions with other proteins [5]. Additionally, NR4A2 exhibits mobility within the cellular environment affecting protein stability and participating in vital intracellular processes (e.g. autophagy, apoptosis, and ER stress) by interacting with p53, ATG7, and ATG12 proteins [6, 7]. Importantly, NR4A2 in the cardiac tissue was initially found to be rapidly and strongly activated following beta-adrenergic stimulation, however, the implications of this increase in activity have not been extensively studied [8].

The therapeutic strategies of both T2D and CVD, which are often associated with MetS, involve a combination of lifestyle modifications and chemotherapy. Nonetheless, CVD-related mortality and morbidity rates have not significantly improved all over the world [2]. Thereby, continuous investigations have aimed to suggest novel therapeutic strategies for the management of the disease. Luteolin, a potent flavone, represents a variety of health-promoting properties such as antioxidative, anti-estrogenic, anti-mutagenic, anti-apoptotic, anti-allergic [9], anti-inflammatory [10], anti-tumorigenic [11], and anti-diabetic [12] functions. It is suggested that luteolin’s therapeutic properties result from its ability to alter key signaling pathways, such as AKT/GSK 3β, AKT/PKB, and NFκB-AF1 [13, 14]. Interestingly, previous studies have shown that luteolin is able to prevent cardiac damage caused by hyperlipidemia, metabolic syndrome, ischemia-reperfusion, and diabetes [15,16,17]. However, the modification of molecular mechanisms such as apoptosis, autophagy, and upstream regulators by luteolin has not been elucidated.

According to what was mentioned, MetS remains one of the global health concerns and efforts to introduce novel treatment strategies continue. Since recent studies have emphasized the molecular mechanisms underlying the disease initiation and progression, the current study has aimed to evaluate the therapeutic properties of luteolin against cardiac injury caused by MetS. In this regard, possible alterations in the levels of autophagy, apoptosis, inflammation, and oxidative stress were measured.

Materials and methods

Animals and experimental protocol

40 adult male albino Wistar rats, 8 weeks old, were provided by the animal laboratory of the university. The rats were maintained in standardized environmental conditions, including a consistent temperature range of 23 ± 2 °C, humidity between 50 and 60%, and a 12-hour light/dark cycle. The free access to water and standard pelleted food ad libitum was allowed for animals. Animals were acclimatized to the laboratory environment for two weeks and then they were partitioned into five groups each consisting of eight rats as follows. Control (CON) animals were given a conventional chow diet for 16 weeks, followed by intraperitoneal (i.p.) saline injections in the final three weeks preceding euthanasia. MetS animals were fed a high-fat diet supplemented with a high fructose regiment in their water containing 60 kcal% fat and 25% D-Fructose for 16 weeks and with i.p. saline injections in the last three weeks before sacrifice (Table 1). Finally, three treatment groups (MetS + Lut) received the same high-fat diet and high fructose for 16 weeks along with luteolin (25, 50, and 100 mg/kg, i.p.) in the final three weeks before sacrifice. The induction of MetS and treatment of animals was based on previously published studies [2, 18]. Finally, animals were sacrificed under xylazine (10 mg/Kg) and ketamine (100 mg/Kg) (Alfasan, Woerden, Holland, and Bremer Pharma GMBH, 34414 Warburg, Germany) anesthesia. Five ml blood samples were obtained by cardiac puncture, maintained at room temperature for 20 min, and centrifuged at 2000 RPM. The resulting serum samples were aliquoted and kept at -20 °C for further biochemical analyses. Moreover, cardiac tissues were removed, washed with sterile PBS, and kept at -80 °C for further analyses.

Table 1 Experimental design and treatment protocols
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Blood pressure and heart-to-body (H/B) weight ratio

The weekly assessment of systolic blood pressure (SBP) was performed by the CODA tail-cuff blood pressure system (BP-98 A; Softron, Tokyo, Japan) [19], taken at precisely the same time, between 9 AM and 11 AM, to avoid any interference from the circadian rhythm. The average of 6 to 8 blood pressure measurements was computed to determine the SBP value for each rat.

The total body weights were measured monthly. After sacrificing, cardiac tissues were removed and ventricles were isolated for precise weight measurement [2]. To assess the development of cardiac hypertrophy cardiac/body weight ratio was measured by the following formula [2]:

$$\:H-B\:ratio=\frac{ventricles\:weight}{body\:weight}\:$$

H: heart weight, B: body weight.

The analysis of metabolic indices

The ip glucose tolerance test (GTT) and insulin tolerance test [20] were performed at the end of the dietary challenge according to a previously published study [21]. Moreover, the biochemical components of plasma, including HDL, LDL, TG, TC, and random glucose were determined using MyBioSource’s (USA) kits, adhering strictly to the manufacturer’s guidelines.

Histopathological analysis

Heart tissues were isolated and fixed in formalin (10%), dehydrated by a graded series of alcohol, and then embedded in paraffin to obtain 5 mm thickness sections benefiting from microtome (Viabrembo, Milan, Italy). Next, sections were dewaxed with xylene, hydrated with ethanol, and rinsed with sterile water. Finally, staining with H&E (hematoxylin and eosin) was performed and assessed under a standard microscope (Nikon, Tokyo, Japan).

Real-time quantitative polymerase chain reaction

Cardiac and aortic tissues from rats were processed through homogenization. Total RNA extraction was achieved via the PureLink RNA Mini Kit (Catalog #: 12183018 A, Thermo Fisher, USA) according to the manufacturer’s guidelines. The purity and integrity of the RNA were determined using the Biotek Nanodrop system and 1.5% agarose gel electrophoresis, respectively. Following cDNA synthesis with a commercial cDNA Reverse Transcription Kit (Catalog #: 4368814, Thermo Fisher, USA), it was analyzed by the Real-Time PCR System (Applied Biosystems, USA) and SYBR Green (Catalog #: K0253, Thermo Fisher, USA). Expression data was normalized against GAPDH. Results were expressed as fold change using the 2−ΔΔCT method [20]. The primer sequences utilized in this investigation are detailed in Table 2. A two-step reaction procedure was implemented, beginning with a 95 °C denaturation phase lasting 5 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 15 s.

Table 2 Primer sequences
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Enzyme-linked immunosorbent assay

Rats’ heart and aorta tissues, equivalent to 500 mg, underwent homogenization utilizing a RIPA solution enriched with protease inhibitors. Cardiac markers Troponin T and Creatine Kinase MB (CK-MB), along with inflammatory markers (IL-6, IL-1β, and TNF-α) were evaluated using ELISA kits from Abcam (USA), based on the manufacturer’s recommendations. Moreover, the levels of apoptosis markers (CASP-3, CASP-9, and Bcl-2), extracellular matrix remodeling (matrix metalloproteinase-9 [MMP-9]), autophagic proteins (SQSTM1/p62, LC3, and Beclin-1 [BECN1]), as well as interstitial fibrosis indicator (TGF-β1) were assessed using ELISA kits from MyBioSource (USA), in line with the manufacturer’s protocols.

The measurement of markers related to oxidative stress

To assess the extent of oxidative stress in rat heart and aorta tissue samples that had undergone homogenization, quantitative analysis for the activity of catalase (CAT) and superoxide dismutase (SOD) enzymes and the levels of lipid peroxidation (malondialdehyde [MDA]) implemented. This was achieved by utilizing the ELISA assay kit from MyBioSource and according to the provided brochure.

Statistical analysis

Data are presented as the mean ± SD and statistical significance was determined using one-way and two-way ANOVA tests followed by Tukey’s post hoc test. SPSS version 24.0 (IBM, Chicago, IL, USA) was used for statistical analysis, and GraphPad Prism version 8 (San Diego, CA, USA) was used for the drawing of graphics. The p-value < 0.05 was considered significant.

Results

Luteolin reduced total body and H/B weight ratio

Fortunately, the treatments and experimental protocol of the present had ended with no mortality. The findings of the present study revealed that the body weight of rats in groups MetS and MetS + Lut25 significantly increased compared to the controls (p-value < 0.001). This is even though the doses of 50 mg/kg and 100 mg/kg caused a significant decrease compared to MetS rats in the 16th week of treatment (Fig. 1), although a significant difference was obtained when compared to the controls (p-value < 0.05). In addition, the H/B weight ratio of MetS and MetS + Lut25 groups had a significant increase of 42.15% and 39.31% compared to the controls (p-value < 0.001). However, animals treated with doses of 50 mg/Kg and 100 mg/Kg did not show significant differences with the control group (p-value > 0.05).

Fig. 1
figure 1

Luteolin reduced total body weight and heart-to-body weight ratio in rats with metabolic syndrome. The trend of weight changes during 16 weeks of treatment (A) and the ratio of heart tissue to total body weight (B) are shown. *: significant difference with Controls; **: significant difference with MetS; the p-value < 0.05 was considered significant.(N = 8 per each group)

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Luteolin improved the metabolic profile

The present study measured the levels of a variety of metabolic biomarkers including LDL, HDL, TG, TC, and Glucose, in all studied groups (Fig. 2). Moreover, the levels of GTT, ITT, and SBP were measured (Fig. 3). According to the results, MetS animals revealed a significant increase of 335.51%, 366.93%, 302.50%, and 59.35% in LDL, TG, TC, and glucose levels, respectively, compared to the control group (p-value < 0.0001). Conversely, a 28.57% decrease in HDL level was observed in MetS animals compared to the control group (p-value < 0.0001). Although the treatment with a dose of 25 mg/Kg of Lut was not able to significantly improve the levels of the mentioned biochemical markers (p-value > 0.05), both doses of 50 mg/Kg and 100 mg/Kg caused significant ameliorating effects compared to MetS group animals.

Fig. 2
figure 2

Luteolin ameliorated metabolic indices in rats with metabolic syndrome. The levels of LDL (A), HDL (B), TG (C), TC (D), and glucose (E) are depicted. *: significant difference with Controls; **: significant difference with MetS; the p-value < 0.05 was considered significant. .(N = 8 per each group)

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Fig. 3
figure 3

The measurement of glucose and insulin tolerance and systolic blood pressure. The levels of GTT (A), ITT (B), and SBP (C). *: significant difference with Controls; **: significant difference with MetS; the p-value < 0.05 was considered significant. .(N = 8 per each group for GTT and ITT measurements; For SBP measurement an average of 6–8 measurements was computed for each rat and each group contained 8 animals)

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In addition, the trend of SBP during the weeks of treatment in MetS and MetS + Lut25 has increased significantly compared to the control group (p-value < 0.001), although the doses of 50 mg/Kg and 100 mg/Kg of Lut were able to significantly reduce SBP compared to MetS (p-value < 0.001). Furthermore, the findings revealed that the MetS and MetS + Lut25 animals had a significant increase in GTT and ITT levels compared to the control group (p-value < 0.001), whereas the treatment of animals with doses of 50 mg/Kg and 100 mg/Kg of Lut showed a significant decrease compared to the MetS group (p-value < 0.0001, Fig. 3).

Luteolin improved the histopathology of cardiac tissue

Histopathological examinations revealed centrally located nuclei in striated cardiomyocytes and anastomosed/branched cell fibers arranged in linear arrays in control animals. However, lesser striation with intercalated discs and loss of anastomoses were observed in both MetS and MetS + Lut25 animals. These pathological alterations were approximately alleviated in the cardiac morphology of MetS + Lut50 animals. The histopathological findings of MetS + Lut100 animals revealed an appearance similar to controls (Fig. 4).

Fig. 4
figure 4

Histopathological analysis. The figure depicts the histomorphology of controls (A), MetS (B), MetS + Lut25 (C), MetS + Lut50 (D), and MetS + Lut100 (E). H&E staining was performed

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Luteolin suppressed apoptosis in the cardiac tissue of rats with MetS

The present study investigated apoptosis in the studied groups by measuring the gene expression and protein levels of BCL-2, CASP-3, and CASP-9 (Fig. 5). The findings revealed that in untreated MetS rats, gene expression and protein levels of BCL-2 were significantly reduced compared to controls (p-value < 0.05). Moreover, there was an increase of 29.46% and 35.19% in the expression of CASP-3 and CASP-9 genes in MetS animals compared to the controls (p-value < 0.001), as the level of CASP-3 and CASP-9 proteins in the MetS rats increased significantly compared to the controls. Interestingly, all studied doses of Lut were able to cause a significant difference with the MetS group in terms of the expression level of BCL-2, CASP-3, and CASP-9 genes, although they did not represent a significant difference with the control group (p-value > 0.05). However, doses of 50 mg/Kg and 100 mg/Kg of Lut were able to significantly improve the level of studied proteins involved in apoptosis (p-value < 0.05).

Fig. 5
figure 5

Lutolin suppressed apoptosis in rats with metabolic syndrome. BCL-2 gene expression (A) and protein (D) levels, CASP-3 and CASP-9 gene expression (B and C, respectively), and protein (E and F, respectively) levels. *: significant difference with Controls; **: significant difference with MetS; the p-value < 0.05 was considered significant.(N = 8 per each group, for gene expression measurements, the analysis was performed with three replicates)

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Autophagy was promoted in MetS animals upon the administration of Lut

ELISA and RT-qPCR approaches were applied to study the protein and gene expression of p62, LC3, and BECN1 (Fig. 6). The findings revealed that MetS caused a significant increase in p62 gene expression and a remarkable decrease in LC3 and BECN1 gene expression compared to controls. However, the administration of Lut, particularly doses of 50 mg/Kg and 100 mg/Kg, determined the recovery of the expression of genes involved in autophagy. Moreover, the level of p62 protein in the cardiac tissue of MetS rats increased by 3.29 times compared to controls (p-value < 0.0001). Although the doses of 50 mg/Kg and 100 mg/Kg of Lut were able to significantly reduce the level of this protein compared to the MetS animals, a significant difference was also found with the controls (p-value < 0.05). On the contrary, the level of LC3 and BECN1 proteins in the MetS group has decreased significantly compared to the controls (p-value < 0.001). Administration of doses of 50 mg/Kg and 100 mg/Kg of Lut caused a significant difference with both the MetS group and the control group (p-value < 0.05).

Fig. 6
figure 6

The promotion of autophagy by luteolin in MetS animals. p62 gene (A) and protein (D) levels, LC3 (B and E) and BECN1 (C and F) gene expression and protein levels, respectively. *: significant difference with Controls; **: significant difference with MetS; the p-value < 0.05 was considered significant. (N = 8 per each group, for gene expression measurements, the analysis was performed with three replicates)

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Lut modulated the expression of NR4A2 and p53 genes in the cardiac tissue of MetS animals

The findings of the present study showed a significant decrease of 28.54% in NR4A2 gene expression and 2.28 times increase in p53 gene expression in the cardiac tissue of MetS rats compared to controls (Fig. 7). This is even though the administration of none of the doses of Lut was able to cause a significant difference neither with the MetS group nor with the control group in terms of NR4A2 gene expression. However, the administration of Lut in all studied doses caused a significant decrease in p53 gene expression compared to MetS animals (p-value < 0.01), although only the dose of 100 mg/Kg caused no significant difference in comparison with the controls (p-value > 0.05).

Fig. 7
figure 7

The effect of luteolin on NR4A2 and p53 expression. NR4A2 (A) and p53 (B) gene expression levels. *: significant difference with Controls; **: significant difference with MetS; #: the significance of the difference neither with Controls nor MetS; the p-value < 0.05 was considered significant. (N = 24 per each group)

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Lut ameliorated inflammation in the cardiac tissue of MetS animals

The level of three inflammatory markers including IL-6, IL-1β, and TNF-α was measured by the ELISA method in cardiac tissue (Fig. 8). The findings showed that the levels of IL-6, IL-1β, and TNF-α in MetS animals significantly increased by 2.63 times, 1.72 times, and 2.05 times, respectively, when compared to the controls (p-value < 0.001). The administration of all three doses of Lut caused a significant decrease in the levels of IL-6 and IL-1β compared to the MetS rats, although only the doses of 50 mg/Kg and 100 mg/Kg caused a significant decrease in the levels of TNF-α compared to non-treated MetS animals (p-value < 0.05). In addition, the administration of all three doses of the substance revealed a significant difference from the controls in terms of inflammatory markers in the cardiac tissue (p-value < 0.05).

Fig. 8
figure 8

The suppression of inflammation by luteolin. The levels of IL-1β (A), IL-6 (B), and TNF-α (C) are depicted. *: significant difference with Controls; **: significant difference with MetS; the p-value < 0.05 was considered significant. (N = 8 per each group)

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Lut improved cardiac injury and stress in rats with MetS

Troponin T, CK-MB, MMP-9, and TGF-β were measured in the cardiac tissue to reveal MetS-induced cardiac injury (Table 3). In animals with MetS, there was a significant increase of 15.1-fold and 1.8-fold in the levels of Troponin T and CK-MB, respectively, when compared to controls (p-value < 0.0001). Moreover, a significant increase of 42.53% and 256.50% in the levels of MMP-9 and TGF-β in the cardiac tissue of MetS animals compared to the controls was obtained (p-value < 0.001). Interestingly, all the studied doses of Lut caused a significant difference with the non-treated MetS animals in terms of troponin T, CK-MB, MMP-9, and TGF-β levels, although a significant difference was also obtained in comparison with the controls (p-value < 0.001).

Table 3 Lut improved cardiac injury and oxidative stress caused by MetS
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In addition, the activity of SOD and CAT and the level of MDA were measured to clarify the state of oxidative stress in the cardiac tissue. The findings showed that MetS had caused a significant decrease in the activity of antioxidant enzymes, SOD and CAT (p-value < 0.05), while the level of the lipid peroxidation marker, MDA, had increased significantly (p-value < 0.001). Interestingly, the studied doses of Lut were able to cause a significant difference with the MetS rats in terms of stress markers, although a significant difference with the control group was also obtained in doses of 25 mg/Kg and 50 mg/Kg (p-value < 0.05).

Discussion

MetS is considered a chronic non-infective syndrome clinically characterized by a set of cardiovascular risk factors such as hypertension, insulin resistance, disrupted glucose metabolism, abdominal obesity, dyslipidemia, and cardiac injury [22, 23]. A pro-inflammatory state, hemodynamic dysfunction, ischemia, and oxidative stress are described as the main causative agents of mentioned risk factors [22, 23]. Although cardiovascular complications are considered a major cause of high mortality in patients with MetS, treatment of patients faces serious challenges worldwide. As a result, continuous attempts are being made to propose novel therapeutic strategies with appropriate efficacy and low adverse effects. Since oxidative stress and inflammation have been described as the main risk factors for cardiac injury in patients with MetS, the application of herbal compounds with anti-inflammatory and antioxidant properties has been the main focus of a variety of investigations [24, 25]. The present study administered three different doses of Lut in a rat model of MetS and evaluated alterations in metabolic markers, weight, apoptosis, autophagy, inflammation, stress, and cardiac injury to elucidate the therapeutic properties of Lut against MetS and its underlying mechanism.

This investigation revealed that Lut significantly reduced the weight of MetS rats and prevented cardiac hypertrophy. Excess weight and hypertension, which mostly coexist, are considered pivotal risk factors for cardiovascular diseases, which suppress cardiac remodeling and may be associated with left ventricular hypertrophy and dysfunction of cardiac tissue [26, 27]. The increase of cardiac enzymes and ischemic markers are among the characteristics of obese people. Concordantly, the findings of the present study showed that the animal models of MetS presented a higher level of troponin T and CK-MB activity compared to the controls. Troponin T and CK-MB are considered specific biomarkers of myocardium injury as troponin T levels elevate during myocardial damage [28], and the primary source of CK-MB is the myocardium [29]. Therefore, MetS was associated with damage to the myocardium and hypertension, pathological events that the doses of 50 mg/Kg and 100 mg/Kg of Lut showed preventive effects.

The current findings revealed that luteolin administration caused a significant improvement in MetS-induced histopathological alterations in cardiac tissue such as lesser cardiomyocytes striation with loss of anastomoses and intercalated discs, which are known as necrosis [30]. Moreover, tissue fibrosis is a process of the repair, scar tissue formation, and replacement of normal tissue and the overgrowth of cardiac tissue leads to massive deposition of extracellular matrix leading to a chronic inflammatory response and organ failure. Importantly, MMPs could degrade the extracellular matrix, hence MMP-9 is strongly connected to myocardial infarction mortality and left ventricle remodeling/dysfunction [31, 32]. Indeed, cardiac fibroblasts indispensably contribute to tissue homeostasis and in cardiac repair after injury [33], where TGF isoforms represent considerable participation in cardiac repair and remodeling, and justification of the phenotype and responsibility of cardiomyocytes, fibroblasts, and vascular cells [34]. Therefore, the ability of doses of 50 mg/Kg and 100 mg/Kg of Lut to significantly reduce the levels of MMP-9 and TGF-β1 can be assumed an indicator of the potential of this antioxidant in preventing myocardial fibrosis and maintaining cardiac function. Accordingly, several other herbals have been shown to prevent cardiac damage by reducing tissue fibrosis, improving blood pressure, and confronting obesity [35,36,37,38,39].

In addition, dyslipidemia, insulin resistance, diabetes, and glucose intolerance have been considered risk factors for MetS and major contributors to cardiac injury. Importantly, doses of 50 mg/Kg and 100 mg/Kg of Lut represented the potential to significantly improve metabolic indices of cardiac damage caused by MetS. Frequently, biochemical changes in MetS are accompanied by the induction of inflammation and oxidative stress, which in turn can aggravate cardiovascular damage. In fact, an increase in the serum levels of pro-inflammatory cytokines, particularly IL-1β, IL-6, and TNF-α, all of which originate from the chronically inflamed adipose tissue and are associated with cardiac stress have been assumed as crucial indicators of MetS-related cardiac injury [27]. Also, MetS is characterized by an elevation in cardiac stress leading to impaired inflammation, vascular function, and atherosclerosis [40]. Oxidative stress is defined as the imbalance of antioxidant defenses with oxidants, which may result in lipid peroxidation, DNA damage, and dysregulation of intracellular signaling pathways [41]. Importantly, Lut was able to reduce the level of inflammatory markers IL-1β, IL-6, and TNF-α, increase the activity of antioxidant enzymes SOD and CAT, and reduce lipid peroxidation. Anti-inflammatory and anti-oxidative stress activity are the main properties of herbal nutraceuticals, which suggests these compounds as a therapeutic strategy for a variety of chronic diseases [41,42,43].

A plethora of evidence has shown that MetS is associated with the death of cardiomyocytes and cardiac dysfunction [44,45,46]. Apoptosis and autophagy are assumed to be the two main forms of regulated cell death [47], although autophagy has a dual role and may induce cell survival [48]. In fact, autophagy is a recycling mechanism that degrades unnecessary organelles and cellular debris by autophagolysosomal structures and thus provides the energy required by the cell [49]. The findings of the present study showed that MetS caused a significant increase in the levels of CASP-3 and CASP-9 compared to the controls, although the levels of BCL-2 decreased significantly. BCL-2 is assumed to be an inhibitor of apoptosis, and its downregulation represents apoptotic cell death, while CASP-3 and CASP-9 are the main mediators of the apoptotic cascade [50]. Therefore, MetS induced apoptosis in the cardiac tissue, a pathological event that Lut had restored. Also, p62 is a negative regulator of autophagy, and LC3 and BECN1 are the main proteins promoting autophagic flux [51, 52]. The results of the present study showed that the disease had suppressed autophagy, although doses of 50 mg/Kg and 100 mg/Kg Lut induced autophagy in the cardiac tissue of rats with MetS. Recently, a study assumed NR4A2 and p53 as the upstream regulators of apoptosis and autophagy and potential targets for the treatment of cardiac injury in MetS (Fig. 9) [3].

Fig. 9
figure 9

Simplified schematic of altered intracellular pathways in MetS and Lut-treated MetS rats

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The present study used an animal model of MetS to evaluate the protective effects of Lut. Although animal models are a common approach to studying MetS, the final decision needs further studies, especially clinical trials. Moreover, not using methods such as western blot, electron microscopy, etc., along with not examining the upstream regulators of the studied genes, may be one of the limitations of the present study. In this regard, the findings of the present study showed that p53 levels were changed in MetS animals in accordance with apoptosis and opposite to autophagy, however, no significant difference was found in terms of NR4A2 expression between the studied groups. Such a contradiction may be caused by the lack of examination of NR4A2 protein level by the present study, hence further studies are necessary to clarify the role of this orphan receptor as an upstream regulator of apoptosis and autophagy and target therapy to confront cardiac damage caused by MetS.

Conclusion

These findings demonstrated that Lut could regulate biochemical and metabolic indicators, prevent cardiac hypertrophy and fibrosis, suppress inflammation and oxidative stress, halt apoptosis, and promote autophagy. Therefore, Lut may represent promising properties as a novel therapeutic strategy to prevent MetS-induced cardiac injury. Nevertheless, further studies are encouraged.

Data availability

Data are available upon reasonable request from the corresponding author.

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Acknowledgements

The authors would like to thank all who contributed to the preparation of this article.

Funding

Guangdong Medical Science and Technology Research Foundation (2023111145039974).

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Authors and Affiliations

  1. Department of Comprehensive, Maoming People’s Hospital, Maoming, 525000, China

    Xiyan Dai

  2. Department of MRI, Maoming People’s Hospital, Maoming, 525000, China

    Bo Liang

  3. Department of Cardiovascular Medicine, First Hospital of Northwest University, Xi’an, 710043, China

    Yaolin Sun

Authors
  1. Xiyan Dai
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  2. Bo Liang
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Contributions

Yaolin Sun contributed to the conception and design of the investigation, Xiyan Dai and Bo Liang performed the experiments and analyzed the data, and Xiyan Dai wrote the primary draft of the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Yaolin Sun.

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The current study was designed and performed according to the international guidelines related to animal care and handling and the National Institute of Health. The use and treatment of animals, as well as how to design the study and the procedures that were taken during this investigation, including animal use and care, were reviewed and approved by the Guangzhou Ruiye Animal Experiments Ethics Committee (No: A202309014). The study is reported in accordance with ARRIVE guidelines.

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Dai, X., Liang, B. & Sun, Y. Luteolin ameliorates rat model of metabolic syndrome-induced cardiac injury by apoptosis suppression and autophagy promotion via NR4A2/p53 regulation. BMC Complement Med Ther 25, 14 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12906-025-04749-6

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Keywords

BMC Complementary Medicine and Therapies

ISSN: 2662-7671

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