Phytoformulation with hydroxycitric acid and capsaicin protects against high-fat-diet-induced obesity cardiomyopathy by reducing cardiac lipid deposition and ameliorating inflammation and apoptosis in the heart

Abstract

Background and aim

Phytoformulation therapy is a pioneering strategy for the treatment of metabolic disorders and related diseases. The aim of the present study was to investigate the protective effect of a phytoformulation consisting of hydroxycitric acid and capsaicin against obesity-related cardiomyopathy.

Experimental procedure

Sprague-Dawley rats were fed HFD for 21 weeks, and phytoformulation (100 mg/kg body weight) was administered orally for 45 days starting at week 16.

Results and conclusion

We found that HFD supplementation resulted in significant hyperglycemia and caused an increase in cardiac lipid deposition, inflammation and apoptosis in the heart. Phytoformulation therapy not only significantly decreased blood levels of glucose, cholesterol, triglycerides, free fatty acids, and inflammatory cytokines in obese rats, but also protected cardiac tissue, as shown by histological analysis. Conversely, phytoformulation therapy decreased mRNA levels for sterol regulatory element-binding factor 1, fatty acid synthase, acetyl-CoA carboxylase, and fatty acid binding protein 1 genes involved in fatty acid synthesis and absorption in obese rats. It increased the levels of lysosomal acid lipase, hormone-sensitive lipase, and lipoprotein lipase genes involved in fatty acid degradation in the heart. In addition, the phytoformulation improved cardiac inflammation and apoptosis by downregulating the genes nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB), tumour necrosis factor α, interleukin-6, toll-like receptor-4 (TLR-4), BCL2-associated X and caspase-3. In conclusion, our results show that the phytoformulation improved insulin sensitivity and attenuated myocardial lipid accumulation, inflammation, and apoptosis in the heart of HFD-induced obese rats by regulating fatty acid metabolism genes and downregulating NF-kB/TLR-4/caspase-3.

Graphical abstract

Highlights of the findings and novelties

• •Phytoformulation made with hydroxycitric acid and capsaicin alleviated HFD-induced dyslipidemia, hyperglycemia and IR.
• •Phytoformulation enhanced fatty acid catabolism and suppressed fatty acid synthesis and uptake in the heart.
• •Phytoformulation ameliorated cardiac inflammation and apoptosis by regulating the NF-kB/TLR4/BaX/caspase-3 pathway.
• •The proposed phytoformulation in this study could serve as protective agents against obesity-associated cardiomyopathy.

Abbreviations

ACC

acetyl-CoA carboxylase

BaX

BCL2 Associated X

CRP

C-reactive protein

CVD

cardiovascular diseases

FABP1

fatty acid binding protein 1

FAS

fatty acid synthase

FFA

free fatty acids

HDL

high-density lipoprotein

HFD

high-fat diet

HSL

hormone-sensitive lipase

IL-6

interleukin-6

IR

insulin resistance

LAL

lysosomal acid lipase

LDL

low-density lipoprotein

LPL

lipoprotein lipase

NF-kB

nuclear factor kappa-light-chain-enhancer of activated B cells

OCM

obesity cardiomyopathy

PPAR-α

peroxisome proliferator activated receptor-alpha

PL

phospholipids

SREBF1

sterol regulatory element-binding factor 1

TG

triglycerides

TLR-4

toll-like receptor-4

TNF-α

tumour necrosis factor α

1. Introduction

Obesity is a major independent risk factor for cardiovascular disease (CVD), such as "obesity cardiomyopathy (OCM)," which is characterized by structural and functional changes in the heart closely related to obesity.¹ Obesity and/or diabetes cause cardiac substrate metabolism to change such that fatty acid oxidation increases while lipids accumulate in the heart. These circumstances have led to the development of cardiac dysfunction, also known as lipotoxic cardiomyopathy, which is associated with abnormal lipid metabolism.² Further evidence that suggested that saturated fatty acid-induced inflammation in cardiomyocytes leads to OCM³ and, as reported by Ren et al.⁴ adipose tissue dysfunction, systemic inflammation, metabolic abnormalities (insulin resistance (IR), altered glucose transport, excess free fatty acids, and lipotoxicity), oxidative stress, autophagy/mitophagy malformation, myocardial fibrosis, apoptosis, and endothelial damage potentially contribute to OCM.

In addition to a distorted metabolic profile, a number of alterations in cardiac function and structure occur when lipids and adipose tissue accumulate in extreme amounts, and the ectopic presence of triglycerides (TG) and lipid metabolites has been associated with lipotoxicity and cardiomyocyte apoptosis.⁵ On the other hand, studies have shown that the effects of hyperlipidemia can lead to impaired cardiac structure, cardiomyocyte fibrosis,⁶ and even interstitial haemorrhage.⁷ A high-fat diet (HFD) may contribute to a number of pathological conditions by inducing cell apoptosis,⁸ ferroptosis, and autophagy.⁹ From a biochemical perspective, a HFD may provoke the body's inflammatory response,¹⁰ cause a mismatch between energy intake and consumption, release free fatty acids into the blood, and lead to a disorder of lipid metabolism.¹¹

In recent years, plant-derived compounds have received much attention as novel protective and even therapeutic approaches for the treatment of obesity and diabetes due to their bioactive constituents,12, 13, 14, 15 which have the potential to positively modulate molecular pathways and gene or protein expressions.^16,17 On the other hand, hydroxycitric acid (HCA) is a primary active constituent of Garcinia cambogia and comprises a citric acid with a hydroxyl group at the second carbon. HCA has two diastereomers because there are two chiral centres in the molecule. HCA is known to reduce weight gain and fat synthesis,¹⁸ due to its anti-obesity effects and various mechanisms of action in the treatment of obesity. Primarily, HCA is a competitive inhibitor of the enzyme ATP-citrate lyase, which converts mitochondria-derived citrate to acetyl-CoA, a precursor for the fatty acid and mevalonate synthesis pathways. Reduction of acetyl-CoA units results in lower fatty acid biosynthesis and lipogenesis.¹⁹ On the other hand, capsaicin is an important active ingredient found in chilli peppers (Capsicum annuum). Capsaicin, also known as trans-8-methyl-N-vanillyl-6-nonenamide, is a crystalline, off-white solid, lipophilic, colourless, and odourless²⁰ and has several therapeutic effects, including a reduction in body weight.²¹ Available scientific literature suggests that capsaicin has the potential to increase lipid oxidation, inhibit adipogenesis, induce thermogenesis, suppress appetite, and increase satiety, which is controlled by neural circuits in the hypothalamus.²¹

At the same time, combination drug therapy is an innovative and rational use of phytochemicals to develop safe and effective combination therapies that could be the future of metabolic disorder treatment. The concept of synergism is considered a form of combination treatment in which the presence of one element significantly enhances the function of other elements through different mechanisms.²² To the best of our knowledge, the combination of HCA and capsaicin in OCM has not been documented in the literature. Based on the therapeutic effects of HCA and capsaicin, we hypothesized that a phytoformulation containing these agents could alleviate OCM by affecting lipid metabolism, inflammation, and apoptosis in the heart. To support our hypothesis, we developed an obese high-fat diet (HFD) rat model and determined the effect of phytoformulation on lipotoxicity, inflammation, and apoptosis markers, as well as the corresponding mRNA expressions in the heart.

2. Materials and methods

2.1. Chemicals

HCA (CAS No.: 27750-10-3, ≥95 purity) and capsaicin (CAS No.: 404-86-4, 100% purity) were purchased commercially (Selleck Chemicals LLC, Houston, USA). All reagents used for this study were of analytical grade.

2.2. Preparation of the phytoformulation

The phytoformulation was prepared using HCA (45 mg) and capsaicin (45 mg) as active ingredients in water and various additives such as Tween-80 (0.5 mg), crospovidone (disintegrant; 2.5 mg), microcrystalline cellulose (diluent; 2.5 mg), colloidal silicon dioxide (lubricant; 1.5 mg), polyvinylpyrrolidone (binder; 2.5 mg) and sodium saccharin (sweetener; 0.5 mg) were used to improve the shelf life of the phytoformulation. At the same time, we tried different concentrations to prepare a phytoformulation, but an equal amount of each compound (in a 1:1 ratio) proved to be more effective; therefore, we established this form of phytoformulation for animal testing. The dosage of the phytoformulation (100 mg/kg body weight) was determined based on the toxicological evaluation performed in accordance with the fixed dosage technique described in the Organization for Economic Cooperation and Development Guideline 423.

2.3. Animals

Male Sprague-Dawley rats (weight 140–150 g; 6–8 weeks old) obtained from Nandha College of Pharmacy, Erode, Tamilnadu, India, were used for this study. According to a study by Maric et al.,²³ female rodents are less likely to gain weight and show less adiposity than males. Our previous studies^16,24 have also shown that male Sprague-Dawley rats showed remarkable characteristics of obesity when fed HFD; therefore, we selected male rats for this study. Before the start of the experiment, animals were acclimatized at a temperature of 22 ± 2 °C and humidity of 45–64% on a 12-h day/night cycle. The protocol of this study was approved by the Institutional Animal Ethical Committee (IAEC), Nandha College of Pharmacy (approval number: NCP/IAEC/2021-22/07).

2.4. Experimental settings

Group I (normal control) was fed a normal diet prepared according to the recommendations of AIN -93 containing all the required macro- and micronutrients, under sterile conditions and with unlimited access to water. The HFD was obtained commercially from the ICMR-National Institute of Nutrition, Hyderabad, India, and its composition was consistent with the dietary recommendations previously reported by Uddandrao et al. (2020).²⁴ The rats in the normal group were fed a standard diet, whereas the remaining rats were given a HFD for 16 weeks to become obese. Then, the obese rats were randomly divided into 3 groups of 6 rats each, and the corresponding drugs were administered from week 16 to week 21.^24,25

2.5. Experimental design

The study was divided into 4 groups, each consisting of 6 animals: Group I: normal control; Group II: HFD-induced OCM control; Group III: OCM + phytoformulation (100 mg/kg body weight) and Group IV: OCM + lorcaserin (10 mg/kg body weight). Normal saline (2 ml/kg body weight) for groups I and II and the corresponding drugs for III and IV were administered orally by gavage for a period of 45 days. Lorcaserin is a well-known anti-obesity drug, and Bohula et al.²⁶ demonstrated that lorcaserin resulted in sustained weight loss in a high-risk group of overweight or obese patients without increasing the rate of severe cardiovascular events and improved the metabolic profile. The primary objective of the study was to evaluate the effect of a phytoformulation against diet-induced changes in lipid metabolism and cardiovascular complications. Therefore, lorcaserin was included as a control, and the therapeutic efficacy of phytoformulation and lorcaserin was compared.

During the experimental period, the body weight and food intake of the rats were recorded every week. At the end of the experimental period, animals were kept fasting overnight, and blood was collected under mild anesthesia (pentobarbital sodium (40 mg/kg/body weight)) by retro-orbitol sinus puncture; then animals were sacrificed by cervical decapitation of the neck and organs were harvested, and heart weight and heart weight to body weight ratio were documented.

2.6. Determination of biochemical markers

Blood glucose levels were measured using commercially available kits, and serum insulin concentration was determined using an enzymatic immunoassay (Cat. No.: # ERINS, Thermo Fisher Scientific, India). The IR was evaluated using the homeostasis model assessment formula: HOMA = fasting serum insulin (mU/l) x fasting plasma glucose (mM)/22.5. In addition, serum levels of TG, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and free fatty acids (FFA) were measured using a fully automated biochemical analyzer (Selectra PRO M Lite, ELITech Group, France).

2.7. Determination of the cardiac lipid profile

Cardiac tissue was homogenized in a lipid extraction buffer consisting of 5 vol of isopropanol, 2 vol of water, and 2 vol of Triton X-100. The homogenates were shaken vigorously and then centrifuged at 24104×g for 5 min. The total cholesterol content was determined using ferric chloride-uranyl acetate reagent, FFA was estimated by copper nitrate-triethanolamine reagent, phospholipids (PL) were measured by digestion with perchloric acid, and TG was determined by a fully automated biochemical analyzer (Selectra PRO M Lite, ELITech Group, France).

2.8. Inflammatory biomarkers in plasma

Plasma biomarkers of inflammation, namely C-reactive protein (CRP) (cat. no.: E-EL-R0506), fibrinogen (cat. no.: E-EL-R1125), and homocysteine (cat. no.: E-BC-K143) were measured using commercially available kits (Elabscience, India).

2.9. Histopathological analysis of the heart

Heart tissue was removed from the sacrificed rats, washed with saline, and preserved in 10% formalin for histological examination. After embedding the tissue in paraffin, 5 μm thick slices of the paraffin-embedded specimens were prepared and stained with hematoxylin and eosin (H&E). Histoarchitectonic changes in the prepared sections were assessed using a light microscope.

2.10. RT-PCR analysis

Frozen heart tissue was thawed, homogenized, and total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and reverse transcribed with a DNA synthesis kit (Applied Biosystems, Foster City, USA) to produce cDNA. For semiquantitative PCR, 20 ng of cDNA was extracted. PCR amplification was performed for 38 cycles under the following conditions: denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, and extension at 72 °C for 1 min. The sequences of primers for fatty acid binding protein 1 (FABP1), sterol regulatory element-binding factor 1 (SREBF1), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), lysosomal acid lipase (LAL), hormone-sensitive lipase (HSL), lipoprotein lipase (LPL), peroxisome proliferator-activated receptor-alpha (PPAR-α), nuclear factor kappa-light chain enhancer of activated B cells (NF-kB), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), toll-like receptor-4 (TLR-4), BCL2 associated X (BaX) and caspase-3 were shown in Table 1. The housekeeping gene β-actin was used for normalization.

Table 1.

Sequences of the primers used for RT-PCR analysis.

Gene	Primer Sequence
FABP1	F 5′-CCATGACTGGGGAAAAAGTC-3′
	R 5′-GCCTTTGAAAGTTGTCACCAT-3′
FATP1	F 5′-TGCACAGCAGGTACTACCGCAT-3′
	R 5′-TGCGCAGTACCACCGTCAAC-3′
SREBF1	F 5′-AATCAGGACCATGCCG-3′
	R 5′-CTCAACCTATGAAAATAAAGTTTGC-3′
FAS	F 5′-GAGGACTTGGGTGCCGATTAC-3′
	R 5′-GCTGTGGATGATGTTGATGATAGAC-3′
ACC	F 5′-GCCTCCGTCAGCTCAGATAC-3′
	R 5′-ATGTGAAAGGCCAAACCATC-3′
LAL	F 5′-TGGAGGGACAAACCACTGA-3′
	R 5′-AAGGGAATCGGACCACTTG-3′
HSL	F 5′-CTTCTCCCTCTCGTCTGCTG-3′
	R 5′-AATGGTCCTCTGCCTCTGTC-3′
LPL	F 5′-GATCCGAGTGAAAGCCGGAG-3′
	R 5′-TTGTTTGTCCAGTGTCAGCCA-3′
PPAR-α	F 5′-TTAGAGGCGAGCCAAGACTG-3′
	R 5′- CAGAGCACCAATCTGTGATGA-3′
NF-kB	F 5′-ACCTTTGCTGGAAACACACC-3′
	R 5′-ATGGCCTCGGAAGTTTCTTT-3′
TNF-α	F5′-ATGTGGAACTGGCAGAGGAG-3′
	R5′-AGAAGAGGCTGAGGCACAGA-3′
IL-6	F 5′-AGCCAGAGTCATTCAGAGCA-3′
	R 5′-AGAGCATTGGAAGTTGGGGT-3′
TLR-4	F 5′-TGGCATCATCTTCATTGTCC-3′
	R 5′-CAGAGCATTGTCCTCCCACT-3′
BaX	F 5′-TCAGGATGCGTCCACCAAGAAG-3′
	R 5′-TGTGTCCACGGCGGCAATCATC-3′
Caspase-3	F 5′-AGCAAACCTCAGGGAAACATT-3′
	R 5′-CTCAGAAGCACACAAACAAAACT-3′
β-actin	F 5′-GGCACCACACTTTCTACAAT-3′
	R 5′-AGGTCTCAAACATGATCTGG-3′

2.11. Statistical analysis

Results of the study were presented as mean ± SD and n = 6. All group data were statistically analysed using SPSS 20.0 software. Statistical analysis was performed using one-way analysis of variance and the least-significant difference test. P values < 0.05 were considered significant.

3. Results

3.1. Phytoformulation protected against HFD-induced body weight gain, heart/body weight index and adipose tissue development

Fig. 1 shows the effects of phytoformulation on food intake, body weight, heart/body weight index, and adipose tissue development. The results showed that compared with rats receiving normal pellet diet, HFD supplementation significantly increased body weight and heart/body weight index and also showed a significant increase in food intake in the II group. However, in the obese rats given the phytoformulation, there was a suppression of body weight gain from week 18 (Fig. 1A), which decreased significantly (P < 0.05) at the end of the treatment period, and concomitant suppression of the heart/body weight ratio (Fig. 1B) and a decrease in food intake (Fig. 1C). In contrast, the obese control group showed a significant (P < 0.05) increase in adipose tissue formation and concomitant decreased adipose tissue development when the phytoformulation was administered to obese rats (Fig. 1D&E).

Fig. 1.

Effect of phytoformulation on (A) body weight, (B) heart-to-body weight ratio, (C) food intake and adipose tissue development (D) epididymal, (E) subcutaneous, (F) blood glucose level, (G) insulin and (H) IR in HFD-induced OCM rats. Values are expressed as mean ± SD, ∗P˂0.05.

3.2. Phytoformulation attenuates hyperglycemia

HFD-induced obese rats showed a significant (P < 0.05) increase in glucose (Fig. 1F), insulin (Fig. 1G) and IR (Fig. 1H) levels when compared with the normal control group. At the same time, supplementation of the phytoformulation showed a significant decrease in glucose, insulin, and IR levels in the obese rats compared with the untreated obese rats.

3.3. Phytoformulation improved dyslipidemia in both serum and heart

Compared with the normal control group, HFD supplementation in the obese control rats resulted in a significant increase in serum total cholesterol, TG, LDL, FFA, and a concomitant decrease in HDL levels (Fig. 2A–E); however, these effects were significantly (P < 0.05) attenuated in the obese rats by treatment with the phytoformulation (P < 0.05), suggesting that the phytoformulation may attenuate the HFD-induced serum lipid metabolism disturbance.

Fig. 2.

Effect of phytoformulation on serum hyperlipidemia markers (A) total cholesterol (B) triglycerides, (C) LDL, (D) HDL and (E) free fatty acids in obese rats. The phytoformulation suppressed the accumulation of lipids (F) total cholesterol, (G) triglycerides, (H) free fatty acids and increased the content of (I) phospholipids in the heart of obese rats. Values are expressed as mean ± SD, ∗P˂0.05.

The effects of phytoformulation on lipid accumulation in the heart of obese control and experimental rats are shown in Fig. 2. In the obese control group, there was a concomitant decrease in the levels of PL and a significant increase in the levels of total cholesterol, TG, and FFA (Fig. 2F–I). Interestingly, administration of the phytoformulation to obese rats resulted in a considerable (P < 0.05) restoration of these abnormalities in obese rats and underscores that the phytoformulation could prevent the deposition of lipids in cardiac tissue.

3.4. Phytoformulation suppressed the production of proinflammatory biomarkers

Fig. 3 illustrates the plasma levels of inflammatory biomarkers in control and experimental rats. The results of this study showed a significant (P < 0.05) increase in the levels of CRP (Fig. 3A), fibrinogen (Fig. 3B), and homocysteine (Fig. 3C) in the obese control rats compared with the normal control rats. On the other hand, administration of the phytoformulation to the obese rats resulted in a significant (P < 0.05) decrease in the levels of proinflammatory biomarkers compared with the untreated obese rats.

Fig. 3.

Phytoformulation suppressed the production of proinflammatory cytokines (A) C-reactive protein, (B) fibrinogen, and (C) homocysteine in obese rats. Values are expressed as mean ± SD, ∗P˂0.05.

3.5. Effect of phytoformulation on the histoarchitecture of the heart

Microscopic examination of the heart section in control rats showed normal histologic architecture of the heart with regularly arranged myocardial fibres and muscle bundles (Fig. 4A). In contrast, the HFD-induced obese rats had damaged cardiac sections characterized by disorganized myocardial fibres associated with interstitial edema with clogged blood vessels, inflammatory cellular collections, and myocardial fibrosis (Fig. 4B). However, in the obese rats treated with the phytoformulation, the histopathological abnormalities were not as severe; the heart showed soft inflammatory mononuclear cells and no areas of necrosis. The arrangement of cardiac fibres in rats treated with phytoformulation and lorcaserin was comparable to that of the control group (Fig. 4C&D).

Fig. 4.

The histopathological findings of the cardiac tissue of the control and experimental groups, (A) the normal control group showed normal structure of cardiomyocytes and muscle fibres, (B) the OCM control showed the remarkable pathological signs induced by HFD supplementation, (C) OCM + phytoformulation and (D) OCM + lorcaserin showed no significant histopathological abnormalities. H&E, scale bar = 40 μm, 40X. The graphs show the (E) the percentage of interstitial fibrosis and (F) the pathology score based on the severity of histopathological changes in the different groups, marked with <0.5 points (no changes), 1 (negligible), 2 (mild), 3 (moderate), >4 (severe). MC: Muscle fibres, S: the intracellular spaces, Yarrow: vesicular nuclei, Arrowhead: damage to cardiac muscle fibers, Curved yarrow: separation of cardiac muscle fibres, Dot Yarrow: inflammatory cells, Dash dot yarrow: fatty degeneration. Values are expressed as mean ± SD, ∗P˂0.05.

3.6. Phytoformulation regulates genes responsible for synthesis, uptake and catabolism of fatty acids

Analysis of RT-PCR revealed that in the obese control group, mRNA expressions of genes responsible for fatty acid synthesis (SREBF1, FAS, and ACC) and fatty acid uptake (FABP1) were significantly (P < 0.05) increased (Fig. 5A–D). At the same time, concomitant down-regulation of mRNA expressions of genes responsible for fatty acid catabolism (LAL, HSL, LPL and PPAR-α) when compared to normal control rats (Fig. 5E–H). On the other hand, phytoformulation treatment resulted in down-regulation of SREBF1, FAS, ACC, and FABP1 and concomitant up-regulation of LAL, HSL and LPL mRNA expressions (Fig. 5) in the heart of obese rats, suggesting that phytoformulation may inhibit the accumulation of lipids in the heart through regulation of fatty acid catabolism.

Fig. 5.

Phytoformulation downregulated genes (A) SREBF1, (B) FAS, (C) ACC, responsible for fatty acid synthesis, and gene (D) FABP1, responsible for fatty acid uptake in the heart of HFD-induced OCM rats. The phytoformulation suppressed the cardiac lipid accumulation by upregulating the genes (E) LAL, (F) HSL, and (G) LPL and (H) PPAR-α, which are responsible for fatty acid catabolism in HFD-induced OCM rats. Values are expressed as mean ± SD, ∗P˂0.05.

3.7. Phytoformulation improved inflammation and apoptosis in the heart of obese rats by downregulating the corresponding mRNA expressions

Fig. 6 shows the effect of phytoformulation on mRNA expressions of genes regulating inflammation and apoptosis in the heart of obese control and experimental rats. Supplementation of HFD rats resulted in upregulation of mRNA expressions of NF-kB (Fig. 6A), TNF-α (Fig. 6B), IL-6 (Fig. 6C), TLR-4 (Fig. 6D), BaX (Fig. 6E), and caspase-3 (Fig. 6F). At the same time, these mRNA expressions were successfully down regulated by phytoformulation in obese rats compared with untreated obese rats.

Fig. 6.

Phytoformulation inhibits inflammation and apoptosis in the heart of HFD-induced OCM rats by regulating mRNA expressions of (A) NF-kB, (B) TNF-α, (C) IL-6, (D) TLR-4, (E) BaX and (F) caspase-3. Values are expressed as mean ± SD, ∗P˂0.05.

4. Discussion

HFD-induced obesity contributes significantly to the development of cardiovascular disease, and cardiac tissue is the main target of obesity. HFD-induced OCM is characterized by abnormal cardiac structure and dysfunction due to lipid accumulation and inflammation in the heart.²⁷ To combat these clinical changes, the use of medicinal herbs and the plant based active compounds continues to be a complementary treatment option, including CVD, as demonstrated by various researchers.28, 29, 30 Therefore, the present study sought to evaluate the therapeutic efficacy of phytoformulation with HCA and capsaicin against HFD-induced OCM using a rat model.

Overeating contributes significantly to IR and manifests as the metabolic syndrome and type 2 diabetes, both of which are characterized by impaired insulin signal and a relative nonautoimmune insulin deficit leading to hyperglycemia. The cardiac metabolic syndrome, which includes IR, hyperglycemia, and hyperinsulinemia, is increasingly recognized as a key factor in the development of heart failure and is gaining importance with the epidemic increase in diabetes and obesity.^31,32 Reduced rates of myocardial glucose uptake and extraction are associated with type 2 diabetes and obesity, which have underlying impairments in insulin signaling. This suggests that the myocardium is affected by impaired insulin action in the same way as other tissues,^33,34 as in skeletal muscle and liver, insulin-regulated glucose entry into the myocyte is dependent on GLUT4 translocation to the cell membrane. In IR and heart failure, the expected metabolic switch from major fatty acid oxidation to glycolysis is abnormal and the myocardium continues to rely on FFA oxidation for energy.^31,35 Several studies have shown that herbal compounds have hypoglycemic effects, significantly attenuate hyperinsulinemia and IR, and combat obesity.^24,25,36 Consistent with these studies, we found in this study that supplementation with phytoformulation significantly suppressed weight gain and fat deposition in obese rats. In addition, phytoformulation was also effective in reducing IR, hyperinsulinemia, and hyperglycemia induced by HFD.

Hyperlipidemia is a well-known metabolic condition and one of the most important risk factors for CVD. Experimental studies have shown that hyperlipidemia increases the likelihood of nonischemic heart failure, whereas lowering serum lipids may ameliorate cardiac dysfunction.^32,37 In addition to directly affecting the systolic function and the electrophysiological response of the heart, hyperlipidemia also indirectly affects cardiac function by promoting the development of atherosclerosis. This effect may be related to the gradual accumulation of cardiac lipids and eventually leads to systemic oxidative stress, proinflammatory state, and mitochondrial dysfunction.³⁸ In addition, increasing evidence from recent studies suggests that serum lipids accumulate in the heart, contribute to oxidative stress and inflammatory cardiac fibrosis,³⁵ decrease autophagy and microvascular density, alter the cardiomyocyte mitochondrial function, and make the myocardium more susceptible to damage, cardiac dysfunction, and electrophysiological changes.³⁹ At the same time, lowering serum lipid levels could successfully reverse early ventricular dysfunction and protect the heart.³⁸ In addition, research on pharmacological effects of lipid-lowering agents on the heart may offer crucial insights into the processes by which hyperlipidemia directly affects the heart. A broad-based recent study has shown that early administration of lipid-lowering agents could reduce the incidence of CVD.⁴⁰ Similarly, the current study has also shown that phytoformulation significantly lowers blood cholesterol, TG, FFA, and LDL levels while increasing HDL levels. This highlights that the phytoformulation could protect against the accumulation of lipids in the heart by lowering the serum lipid levels.

The two significant alterations in cardiac energy metabolism that occur in obesity and diabetes include higher rates of fatty acid oxidation and lower rates of glucose consumption.⁴¹ This metabolic profile is the origin and result of a marked cardiac IR, which is associated with a decrease in cardiac efficiency and function and with the accumulation of potentially toxic lipid metabolites in the heart that can exacerbate IR and cardiac dysfunction.⁴² There are a number of factors that contribute to the increased rates of fatty acid oxidation in the heart observed in obesity and diabetes, including increased delivery and uptake of fatty acids in cardiomyocytes, increased transcription of enzymes that degrade fatty acids, decreased allosteric regulation of fatty acid uptake and oxidation in mitochondria, and increased regulation of various fatty acid oxidizing enzymes by posttranslational acetylation.⁴² Adoption of treatment strategies aimed at shifting choice of cardiac energy substrate from fatty acid oxidation to glucose may be able to prevent the cardiac dysfunction caused by obesity and diabetes, as demonstrated in a previous study.⁴³

In addition, we evaluated cardiac lipid profiles and cardiac mRNA expressions that are associated with fatty acid synthesis, uptake, and catabolism to validate the anti-hyperlipidemic effect of the phytoformulation observed in the serum lipid study. It has been said that the primary aetiology of OCM is thought to be a disturbance in cardiac lipid metabolism.²⁷ Moreover, supplementation with HFD resulted in significant accumulation of TG, FFA, and cholesterol in cardiac tissues, confirming lipotoxic cardiomyopathy, and these results are in agreement with those of the studies by Wang et al.⁴⁴ and Jansy et al.³² In the present study, abnormal cardiac lipid deposition was observed in obese rats but was reversed by administration of the phytoformulation. Lipid synthesis and degradation are highly controlled multistep processes that follow a series of specific enzyme reactions. Obesity leads to irregular transcription of the genes for lipid accumulation, synthesis, and degradation in the heart and to a disturbance of the dynamic balance of lipid synthesis and degradation in the myocardium.⁴⁵ Similarly, we discovered that administration of the phytoformulation to HFD-induced obese rats resulted in decreased mRNA levels of the genes for fatty acid uptake (FABP1) and fatty acid synthesis (SREBF1, FAS and ACC). In addition, phytoformulation increased mRNA expression of LAL, HSL, PPAR-α, and LPL genes involved in fatty acid catabolism. Therefore, we hypothesized that phytoformulation decreased fatty acid synthesis and uptake while promoting fatty acid degradation thereby attenuating HFD-induced myocardial lipid accumulation. Previous studies reported that PPAR-α activation is associated with enhanced FFA uptake, conversion to acyl-CoA derivatives, and further catabolism via the β-oxidation pathways. Moreover, the TG -lowering effect of PPAR-α is also due to enhanced lipolysis via activation of genes associated with fatty acid degradation.⁴⁶ The results of our study are consistent with these findings and support our hypothesis that phytoformulation promotes fatty acid degradation by regulating PPAR-α.

In addition, a systemic inflammatory state mediated by metabolic abnormalities impairs myocardial structure and function. Maladaptive myocardial remodeling and its clinical consequences are driven by the pro-inflammatory milieu triggered by circulating cytokines, excessive availability of metabolic substrates, and paracrine signaling from activated immune cells in the heart. Indeed, cardiac tissue homeostasis is disrupted when cytokines and nutrient metabolites activate inflammatory processes in different cardiac cell types via common pathways. As a result of the ensuing subcellular changes, a phenotype of metabolic cardiomyopathy gradually emerges, which manifests clinically as heart failure with preserved ejection fraction.⁴⁷ According to a common paradigm, caloric overload induces the release of proinflammatory mediators in extracardiac tissues, which in turn leads to systemic and cardiac inflammation.⁴⁸ In parallel, circulating inflammatory cytokines (TNF-α and IL-6) that activate evolutionarily conserved inflammatory regulators such as NF-kB reduce systemic and cardiac insulin sensitivity. This persistent low-grade inflammation is usually caused by metabolic disorders and obesity. Chronic inflammation leads to metabolic reprogramming of the heart and contributes to adverse remodeling and functional impairment, in contrast to acute inflammatory responses to cardiac tissue injury, which are essential regenerative processes.⁴⁷ Treatment of OCM might involve disentangling the inflammatory systems involved in adverse cardiac remodeling in the cardio-metabolic state and controlling them with systemic mediators.⁴⁹

As shown by the elevated levels of proinflammatory cytokines and related substances, including CRP, homocysteine, and fibrinogen, in the current study, HFD confirms the onset of low-grade inflammation in the obese rats. According to previous studies, homocysteine and fibrinogen play a crucial role in the development of atherosclerosis and are a major cause of cardiomyopathy. Moreover, when the inflammatory state was triggered by IL-6, the inflammatory indicator CRP was immediately released from the heart.^32,50 Interestingly, administration of phytoformulations to obese rats suppressed HFD-induced low-grade inflammation, as confirmed by the reduction of serum CRP, homocysteine, and fibrinogen levels. In addition, HFD supplementation also increased the mRNA levels of genes involved in inflammation such as NF-kB, TNF-α, and IL -6. However, phytoformulation therapy administered to obese rats’ downregulated mRNA levels of these inflammatory cytokines in the heart. Therefore, these results suggest that phytoformulation may be a potential candidate to attenuate HFD-induced inflammation via regulation of the NF-kB cascade.

On the other hand, cardiac lipid accumulation has been hypothesized to promote myocardial apoptosis.⁵¹ In addition, the development and progression of heart failure are also significantly mediated by cardiomyocyte inflammation, which is followed by apoptosis and fibrosis.⁵² In the present study, we examined the mRNA expression of apoptotic genes (TLR-4, BaX, and caspase-3) in the heart to investigate whether or not the apoptosis pathway was activated in an HFD-induced obese rat. From the results of this study, the obese control group had increased mRNA expression for TLR-4, BaX, and caspase-3 and these results are in consistent with a previous study by Katare et al.⁵³ that showed that activation of TLR-4 causes cardiac fibrosis, apoptosis, mitochondrial dysfunction, and inflammation in rats.⁵³ In addition, other studies showed that the expression of Bax, Bcl-2, and cleaved caspase-3 was increased in obese rats, suggesting an increase in myocardial apoptosis.^54,55 In agreement with these studies, our results also showed that the mRNA expressions of TLR-4, BaX, and caspase-3 were considerably down-regulated in obese rats treated with phytoformulation. Thus, results support the hypothesis that phytoformulation can prevent the activation of TLR-4, which causes cardiomyocyte apoptosis, and inhibit apoptosis in the heart by suppressing BaX and caspase-3. Therefore, this phytoformulation may be a promising candidate to prevent the progression of OCM and our working hypothesis is shown in Fig. 7.

Fig. 7.

A proposed mechanism for the effect of phytoformulation on HFD-induced OCM. Phytoformulation regulates mRNA expressions of LAL, HSL, PPAR-α and LPL, thereby enhancing fatty acid catabolism. At the same time, the phytoformulation down-regulates mRNA expression of SREBF1, FAS, ACC and FABP1, leading to suppression of fatty acid synthesis and uptake in the heart. Subsequently, the phytoformulation attenuated apoptosis and inflammation by decreasing mRNA expressions of NF-kB, TNF-α, IL-6, TLR-4, BaX and caspase-3 in cardiomyocytes. Therefore, it is hypothesized that the phytoformulation may be able to prevent cardiac lipid deposition by regulating fatty acid metabolism and subsequently controlling NF-kB/TLR4/BaX/caspase-3 signalling and attenuating inflammation and apoptosis in the heart.

5. Conclusion

In conclusion, this study shows that a phytoformulation containing HCA and capsaicin alleviated HFD-induced weight gain, dyslipidemia, hyperglycemia, hyperinsulinemia, IR, and systemic inflammation. Moreover, this phytoformulation attenuated cardiac lipid deposition by promoting genes responsible for fatty acid degradation and suppressing genes associated with fatty acid synthesis and uptake. In addition, it improved cardiac inflammation and apoptosis by regulating the NF-kB/TLR4/BaX/caspase-3 pathway. In view of these results, phytoformulations containing HCA and capsaicin could serve as protective agents against obesity and OCM, and they have significant potential to become nutraceuticals for the treatment of metabolic disorders. To the best of our knowledge, this is the first study to report the potential role of a phytoformulation combining HCA and capsaicin in the prevention of OCM. However, this phytoformulation may not yet be suitable as a mainstay therapy until further preclinical studies are conducted to investigate a broad range of mechanisms of action. In addition, a thorough investigation of acute and chronic toxicity in normal animals, as well as bioavailability and pharmacokinetics studies, is recommended.

Author contribution

V. V. Sathibabu Uddandrao: Conceptualization, Methodology, Writing - Original Draft, Investigation, Formal analysis, Project supervision and administration. P. Chandrasekaran: Investigation, Data curation. G. Saravanan: Investigation, Formal analysis & Critical review, P. Brahmanaidu: Investigation, Formal analysis. S. Sengottuvelu: Investigation. P. Ponmurugan: Formal analysis & Critical review. S. Vadivukkarasi: Investigation. Umesh Kumar: Formal analysis & Critical review. All authors are read and approved the final manuscript.

Funding

None

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.