Hesperidin alleviates hypothyroidism-related cardiac dysfunction by targeting cardiac miRNAs, Nrf2/NF-κB signaling, oxidative stress and inflammation

Abstract

Background: Hypothyroidism is a frequent endocrine health issue that is linked to adverse cardiovascular events. Accumulating evidence suggests that thyroid hormone replacement does not fully reverse the cardiovascular complications associated with the disease despite normalization of serum thyroid hormone levels, indicating a need for adjunctive, complementary, or alternative therapies. Hesperidin (HSD) has diverse pharmacological activities, however, its therapeutic potential on the crosstalk between hypothyroidism and cardiac dysfunction has not been previously reported.

Methods: This study aimed to investigate the cardioprotective efficacy of HSD on carbimazole (CMZ)-induced hypothyroidism in rats in comparison to the traditional thyroid hormone replacement therapy; levothyroxine (LT4). Male Wistar albino rats were divided into four groups: normal control (NC), CMZ (30 mg/kg), CMZ + HSD (30 mg/kg CMZ + 200 mg/kg HSD), and CMZ + LT4 (30 mg/kg CMZ + 0.045 mg/kg). All doses were given orally and daily for 9 weeks.

Results: CMZ intake resulted in a significant decrease in thyroid hormones (THs) levels with a subsequent increase in serum thyroid stimulating hormone and cardiac enzymes activities, dyslipidemia, and body weight gain. Cardiac tissues revealed marked oxidative stress, inflammation, and structural degenerative lesions. As well, cardiac expression of miRNAs-92a and -499 was elevated while that of miRNA-21 was depleted, reflecting an interdependence between hypothyroidism and the development of cardiac dysfunction. Despite HSD and LT4 effectively alleviating the THs profile, only HSD offered substantial protection from hypothyroidism-associated cardiac inflammation and injury through its potent impact on the transcriptional miRNAs level and Nrf2/NF-κB protein expression (key regulators of the redox biomarkers and the inflammatory mediators).

Conclusion: HSD provides dual thyroprotective and cardioprotective effects that enhance THs bioavailability and functionality in the cardiovascular system.

Keywords: cardiac dysfunction; hesperidin; hypothyroidism; inflammation; miRNAs; oxidative stress.

FIGURE 1

Effect of HSD and LT4 on the serum lipid profile, cardiovascular risk indices, and cardiac redox status of CMZ-induced hypothyroidism in rats. Data were normalized to remove variations in absolute values (Supplementary Table S1) and presented in a heat map to show the pattern across the different groups (n = 6). Abbreviations: NC, normal control rats; CMZ, carbimazole-induced hypothyroid rats; CMZ + HSD, hypothyroid rats treated with hesperidin; CMZ + LT4, hypothyroid rats treated with levothyroxine; T.G., triglycerides; Total Ch., total cholesterol; HDL-Ch., high density lipoprotein cholesterol; LDL-Ch., low density lipoprotein cholesterol; vLDL-Ch., very low density lipoprotein cholesterol; CVR1, cardiovascular risk index 1; CVR2, cardiovascular risk index 2; AAI, anti-atherogenic index; MDA, malondialdehyde; NO, nitric oxide; GSH, reduced glutathione; SOD, superoxide dismutase; CAT, catalase.

FIGURE 2

Effect of HSD and LT4 on the cardiac gene expression level of miRNAs-92a, -499, and -21, NOS (endothelial and inducible), and caspase-3/BAX apoptotic markers of CMZ-induced hypothyroidism in rats. Data were normalized corresponding to the mean values of the controls (n = 6). Absolute values in Supplementary Table S2. Abbreviations: NC, normal control rats; CMZ, carbimazole-induced hypothyroid rats; CMZ + HSD, hypothyroid rats treated with hesperidin; CMZ + LT4, hypothyroid rats treated with levothyroxine; miRNA, microRNA; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; BAX, BCL2-associated X protein.

FIGURE 3

(A–C) Effect of HSD and LT4 on the cardiac protein expression level of Nrf2 and NF-κB of CMZ-induced hypothyroidism in rats. Data are mean ± SEM (n = 6). Abbreviations: NC, normal control rats; CMZ, carbimazole-induced hypothyroid rats; CMZ + HSD, hypothyroid rats treated with hesperidin; CMZ + LT4, hypothyroid rats treated with levothyroxine; Nrf2, nuclear factor erythroid 2-related factor 2; NF-κB, nuclear factor kappa B.

FIGURE 4

Cardiac histopathological alterations caused by CMZ-induced hypothyroidism and the effect of HSD and LT4 treatments (H&E staining). (A) Cardiomyocytes of the control group showing the normal intactness of the cardiac muscle fibers with proper striations (thin arrows) and regular vesicular nuclei (arrowhead). (B–D) Heart tissues of the CMZ-hypothyroid rats. (B) Showing myocytes losing striations (arrowhead), with congested blood vessels (arrows) and focal necrotic areas (F), (C) showing loss of continuity with adjacent myocytes (thin arrows) and myocytes losing their nuclei (arrowhead), fragmented cardiomyocytes with pyknotic nuclei (thick arrow), and vacuolation (V), and (D) showing irregular muscle fibers (arrow) and extensive inflammatory cellular infiltrations (star) associated with vacuolations (V). (E) Revealing potent improvement of the myocardial fibers after HSD administration. (F) Showing congested blood vessels (thick arrow) and inflammatory cellular infiltration (arrowhead) that are still apparent in the cardiomyocytes of LT4-treated rats. The histopathological scores are indicated in Table 4.

FIGURE 5

Cardiac ultrastructural abnormalities caused by CMZ-induced hypothyroidism and the effect of HSD and LT4 treatments. (A,B) Cardiomyocytes of the control group showing regular striations of the myofibrils (arrow), a normal shape of mitochondria (M), and a proper distribution of chromatin in the nucleus (N). (C,D) Cardiomyocytes of the CMZ-hypothyroid rats revealing fragmented (thick arrow) and degenerated (star) myofibrils, irregular aggregations of electron-dense mitochondria (M) with ill-defined cristae, shrunken nucleus (N) with dense clumps of heterochromatin (thin arrow), swelling of smooth endoplasmic reticulum (arrowhead), and appearance of vacuoles (V). (E) Cardiomyocytes of the HSD-treated group showing marked amelioration of the myofibrillar striations (arrow), regular shape of mitochondria (M), and euchromatic nucleus (N) with few cytoplasmic vacuolations (V). (F) Cardiomyocytes of the LT4-treated group showing improvement in the myofibrillar striations (thick arrow) and mitochondria (M), while the nucleus has dense clumps of heterochromatin (thin arrow) with few vacuoles in the sarcoplasm (V). Ultrastructural abnormality scores are in Table 4.

FIGURE 6

Effect of CMZ-induced hypothyroidism and its treatment with HSD or LT4 on the cardiac immunoexpression of (A–E) Gal-3, (F–J) MMP-9, and their quantification using H-score. (A) Cardiac tissue of the normal control group showing negative immunostaining toward Gal-3, (B) strong brown immunopositive reactivity of Gal-3 in the cardiomyocytes of CMZ-hypothyroid rats, (C) absence of reactivity for Gal-3 in cardiac myofibers of the HSD-treated group, while (D) showing mild cytoplasmic immune expression of Gal-3 in the cardiac tissues of the LT4-treated group (arrowheads). (F) Negative immunoreactivity of MMP-9 in the cardiomyocytes of the control group, (G) significant immune expression of MMP-9 that is localized in the damaged areas of the cardiomyocytes of the CMZ-hypothyroid group (arrowheads), (H) myofibers from the HSD-treated rats with very weak MMP-9 expression (arrowhead), and (I) revealing faint MMP-9 staining in the cardiac tissue fibers of the LT4-treated group (arrowheads).

FIGURE 7

A proposed mechanistic model summarises the multimodal effects of HSD in alleviating hypothyroidism-related cardiac dysfunction. Abbreviations: BAX, BCL2-associated X protein; CAT, catalase; CMZ, carbimazole; DIO1, deiodinase type-1; DIO3, deiodinase type-3; DIT, Diiodothyronine; eNOS, endothelial nitric oxide synthase; ETC, electron transport chain; Gal-3, galectin-3; GSH, glutathione; IL, interleukin; iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; MiRNA, microRNA; MIT, monoiodothyronine; MMI, methimazole; MMP-9, matrix metalloproteinase-9; MyHC7, myosin heavy chain-7; NF-κB, nuclear factor kappa-B; Nrf2, nuclear factor erythroid 2-related factor 2; PPAR-α, peroxisome proliferator activated receptor alpha; ROS, reactive oxygen species; RXR, retinoid-X-receptor; SOD, superoxide dismutase; SREBP2, sterol regulatory element-binding protein-2; TGF-β, Transforming growth factor beta; TPO, thyroid peroxidase; TR, thyroid hormone receptor; TRE, thyroid hormone response element; (*), factors lead to cardiac dysfunction in hypothyroidism; Brown arrows, effects of hesperidin; Blue arrows: effects of levothyroxine.