Betalain exerts cardioprotective and anti-inflammatory effects against the experimental model of heart failure

Abstract

Betalain is a natural plant pigment known to elicit various biological activities. However, studies on the protective effect of betalain against heart failure have not reported yet. The experimental model of heart failure was created in Wistar rats using isoproterenol (ISO). The animals were randomly assigned into four groups such as sham-control, ISO-induced heart failure, betalain pretreated before ISO induction (50 mg/kg/day), and betalain drug control group were maintained for 6 weeks. At the end of the experimental period, anti-oxidant enzymes, inflammatory markers, matrix proteins, cardiac-specific markers, and micro RNAs were elucidated using RT-PCR, and ELISA analysis. The results demonstrated that the rats induced with ISO displayed an abnormality in cardiac functions, increased oxidative stress markers (p < 0.01), inflammatory cytokines (p < 0.01) while abrogated the expression of miR-18a, and increased miR-199a. While betalain pre-treated rats prevented the cardiac failure significantly (p < 0.01) with improved anti-oxidant enzymes, abrogated the inflammatory signals with restored matrix proteins, cardiac biomarker genes, and attenuated miR-423 and miR-27 compared to heart failure rats. The results of the study suggest that the betalain treatment protected the hearts from failing via microRNA mediated activation the anti-inflammatory signaling and restoring the matrix protein modulation.

Introduction

Heart failure or acute heart arrest is characterized by reduced cardiac output, increased occlusion pressure in the pulmonary artery, blood vessel congestion preceded by abnormalities in the systolic and diastolic functions. ¹ It is considered to be a combination of structural, physiological, and functional alterations of the myocardium that weakens the heart muscles and fail to perform normally. ² Patients who suffer from heart failure do not have any previous heart-related dysfunction or complications ³ and are mostly associated with sudden and harsh discomfort in the heart. It is a prominent public health issue that burdens the global healthcare ⁴ and affects the economy of the individual. Advancements in cardiac care have dramatically improved the conditions after treatment but the extent of life has not improved.

The preliminary event in the onset of cardiac fibrosis and other forms of heart failure are the deposition of proteins concerned with extracellular matrix (ECM) in the myocardium. ⁵ The impairment in the systolic and diastolic functions of the heart is due to the disrupted architecture of the myocardium that affects the coordination in the excitation and contraction.^6,7 Hence, the key for the development of treatment strategies for heart failure is creating the knowledge base on the mechanisms underlying the progression and resolution of cardiac signaling mechanism. The ECM components of the cardiac interstitial area are disturbed and hence the network cannot hold the cardiac fibroblasts together and affects the proliferation and migration of these cells to cause heart failure.^8,9 Despite major drug discoveries that are in trail, it has been proclaiming on the use of plant-based molecules for various bodily ailments suggests a quest for drugs targeting cardiac failure and hence, in the present study, a plant pigment betalain was selected.

Betalain (BTA), a tyrosine-derived pigments majorly found in plants of the Caryophyllales Order and is being used as natural pigments in the food industry, and also as a nutrient supplement in diet-based therapies. ¹⁰ It is non-toxic and good biological absorption makes it a probable molecule in the treatment of diseases with pathological aspects of oxidative stress. Having positive effects on its function, BTA obtained from beetroot have antioxidant, anticancer, antilipidemic, and antimicrobial activities. ¹¹ The free radicals scavenging properties of betalain can be explained as having an intrinsic activity that is shared between the imino group and the tetrahydropyridinegroups ¹² that the nitrogen atoms have the electronic resonance system shared between them can take up the electrons and hence having the reducing properties.^13,14

Wherefore, in the present study, the protective effect of BTA was elucidated in induced heart failure in animals using isoproterenol (ISO), a synthetic non-selective β-adrenoceptor agonist, and is a credible method for experimental animal heart failure model ¹⁵ to create arrhythmic heart contraction and expansion, cardiomyocytes loss and cardiac fibrosis. ¹⁶ ISO induction generates free radicals that are toxic to cardiac fibroblasts and induce cardiotoxicity through ROS generation of superoxide, nitric oxide, hydrogen peroxide, and hence therapeutic strategies must include free radicals scavenging molecules that act as anti-oxidants in the reduction of cardio-associated oxidative stress. The present study was hypothesized that the administration of BTA could promisingly protect the heart from failure through restoring the antioxidant defense mechanism, improving the anti-inflammatory cytokines, and initiating the regulation of key microRNA signatures.

Materials and methods

Chemicals

Betalain, Isoproterenol hydrochloride, First Strand cDNA Synthesis Kit, Real-Time PCR Enzymes & Kits were from Sigma Aldrich, St. Louis, USA. Malondialdehyde (MDA) assay kit from Abcam, USA. Nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), Xanthine Oxidase, and glutathione peroxidase (GPx) kits were obtained from Cayman Chemicals, USA. ELISA kits for TNF-α, IFN-γ, IL-1β, CXCR4, IL-6, TGF-β, IL-21, IL-10 matrix metalloproteinase (MMP-2, -9) and tissue-inhibitor of MMPs (TIMP-1, -2) were obtained from Fine Biotech, Wuhan, China. Gene-specific primers were obtained from Eurofins, Germany. MicroRNAs, rno-miR-18a-5p, rno-miR-27b-3p, rno-miR-199a-5p, rno-miR-423-3pTaqMan™ MicroRNA assays were obtained from Thermofisher Scientific, USA. All other chemicals and buffers used were reagent grade.

Induction of heart failure

Wistar male rats weighing 130 g (±15 g) were used in the study. The animals were kept in polycarbonate cages in a temperature-controlled (25 ± 2°C) room with a 12-hour light-dark cycle with humidity maintained at 55 ± 5%. The animals received free access to regular standard rat chow and reverse osmosis water. The animal experimental protocol was followed strictly following the institutional guidelines (XJU2019NO076) and as per Animal Ethical Committee suggestions. For the experimentation, animals were divided randomly into four groups consisting of six animals as follows (Figure 1): Normal control rats; Rats received isoproterenol (ISO) as cardiac failure induced group (75 mg/Kg BW, intraperitoneally for 2 consecutive days); Rats receiving betalain (100 mg/kg, orally) a week before ISO administration as the betalain retreatment group; and as a drug control group, rats were administered with betalain alone for 4 weeks. All of the experimental rats were maintained for 6 weeks and the animals were assessed for the systolic blood pressure (SBP) and heart rate (HR) described earlier. ¹⁷ For the assessment of the protective effect of betalain, the rats were then killed, the heart tissue was dissected out, heart weight to body weight ratio was analyzed and a portion of heart tissue was used for histopathological analysis using H&E (Haemotoxylin and Eosin). ¹⁸

Figure 1.

The schematic picture of experimental protocol.

Oxidative markers and antioxidant enzyme analysis

The onset of oxidative response was estimated using the level of malondialdehyde, nitric oxide, Xanthine oxidase (XO), and the levels of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were assayed in the heart tissue homogenate using kits obtained from Cayman Chemicals, USA.

ELISA assays for cytokines and gelatinase

The levels of TNF-α, IFN-γ, IL-1β, CXCR4, IL-6, IL-21, TGF-β, and IL-10 and the matrix metalloproteinase (MMP-2, -9) and tissue inhibitor of MMPs (TIMP-1, -2) in the heart tissue homogenate were elucidated using commercial assay kits as per the manufacturer’s instruction.

Reverse transcription-PCR

To evaluate the microRNA level specific for cardiac failure, quantitative real-time RT-PCR was performed with miR-specific primers. Briefly, the total RNA was isolated from the heart tissues using Trizol reagent and quantified. ¹⁷ For quantitative detection of miR-18, miR-27a, miR-199a, and miR-423, TaqMan MicroRNA Reverse Transcription Kit, TaqMan miR specific assays and snoRNA assays were used according to the manufacturer’s instructions. SnoRNA was used as a housekeeping control. Besides, the mRNA expression of cardiac functioning markers was also elucidated from cDNA which was transcribed from a known amount of RNA, and the real-time PCR was done for specific genes using SYBR® Green/ROX master mix using the gene-specific primers (Online Supplementary Table 1). The mRNA expression in fold increase was calculated from the Ct values by the comparative ΔΔCT formula using GAPDH as the house-keeping gene.

Statistical analysis

Statistical significance was evaluated using SPSS software (version 17). One-way analysis of variance (ANOVA) and Tukey’s post hoc test was used for the comparison and the data were expressed as the Mean ± SD; the p-value of less than 0.05 was considered statistically significant.

Results

The present study aimed at evaluating the cardioprotective role of betalain by identifying the cellular response that has been involved in the experimental model of heart failure. Figure 2 shows the histopathological analysis of the heart tissues. Rats induced with cardiac failure using ISO displayed increased cell pathology with evident infarct and damaged tissue fibrils compared to control rats. Also, a significant increase in the HW/BW ratio (p < 0.01), SBP (p < 0.05), and HR (p < 0.05) was found in ISO induced rats compared to control. Whereas, betalain pre-treated rats demonstrated histology evidencing the lessen infarct of cardiomyocytes while normal SBP and HR were also apparent. Further no significant changes were observed in the rats in betalain alone treated group compared to control (Figure 2).

Figure 2.

(a) Histopathological analysis of heart tissues (inset picture A: control, B: ISO, C: BTA+ISO, D: BTA); (b) heart weight to body weight (HW/BW) ratio; (c) systolic blood pressure (SBP); (d) heart rate (HR) of saline control, ISO induced and betalain treated group of rats. Values are expressed as mean ± SE (n = 6). Statistical significance expressed as **p < 0.05, **p < 0.01 ISO compared to saline control, ^$p < 0.05 betalain + ISO compared to ISO-induced rats.

To explore the cardioprotection conferred by betalain through the association with oxidative stress, the levels of MDA, NO, and antioxidant enzymes were examined, and the results are presented in Figure 3. Rats suffered from heart failure exhibited a two-fold increase in the levels of MDA and XO; while, the cellular rescuing system of antioxidant enzymes, SOD, catalase and Gpx level were found decreased compared to control. While rats received betalain treatment substantially attenuated (p < 0.05) the levels of MDA and XO levels and improved the levels of antioxidant enzymes (Figure 3).

Figure 3.

(a) Malondialdehyde (MDA) level; (b) nitric oxide level; (c) superoxide dismutase (SOD); (d) catalase (CAT); (e) glutathione peroxidase (GPx); (f) xanthine oxidase in the saline control, ISO induced and betalain treated group of rats. Values are expressed as mean ± SE (n = 6). Statistical significance expressed as **p < 0.05, **p < 0.01 ISO compared to saline control, ^$p < 0.05 betalain + ISO compared to ISO-induced rats.

Furthermore, the activation of cytokines and the dysregulation of matrix-associated enzyme levels were estimated and the results were presented in Figures 4 and 5. The results demonstrated that the levels of cytokines, IL-6, IL-1β, IL-21, CXCR4, TNF-α were increased (p < 0.01) and the level of IL-10 was reduced in ISO-induced rats. On the other hand, the levels of IFN-γ, TGF-β, TIMP-1 (p < 0.001) were increased while the levels of MMP-2, MMP-9, and TIMP-3 were decreased in ISO rats compared to control. While these inflammatory cytokines and chemokines were regulated in the betalain pre-treatment with improved IL-10 levels (p < 0.01) point out that the protective mechanism has been activated by the drug through ameliorating inflammatory signaling and regulating matrix associated signals in cardiac tissues (Figures 4 and 5).

Figure 4.

(a to f) Cytokines levels in the saline control, ISO induced and betalain-treated group of rats. The detail of the experiment is given in the methodology section. Values are expressed as mean ± SE (n = 6). Statistical significance expressed as **p < 0.05, **p < 0.01 ISO compared to saline control, ^$p < 0.05 betalain + ISO compared to ISO-induced rats.

Figure 5.

(a to f) Cytokines, MMPs, and TIMPs levels in the saline control, ISO induced and betalain treated group of rats. Values are expressed as mean ± SE (n = 6). Statistical significance expressed as *p < 0.05, **p < 0.01, ***p < 0.001 ISO compared to saline control, ^$p < 0.05, ^$$p < 0.01 betalain + ISO compared to ISO-induced rats.

The onset of cardiac failure markers was elucidated using RT-PCR and the results are presented in Figure 6. Rats induced with ISO demonstrated a profound increase in heart-specific marker genes such as ANF (3.2-fold), β-MHC (2.8-fold), STAT3 (2.6-fold), AKT (2.4-fold), α-NCX1 (4.2-fold), and reduced SERCA2 mRNA compared to control. However, the dysregulated levels of these genes were found regularized in betalain pre-treatment suggest that the drug exerts a cardioprotective effect (Figure 6).

Figure 6.

(a to f) qRT-PCR analysis of cardiac marker genes in the saline control, ISO induced and betalain treated group of rats. The mRNA fold increase is compared with the housekeeping gene GAPDH. The detail of the experiment is given in the methodology section. Values are expressed as mean ± SE (n = 6). Statistical significance expressed as *p < 0.05, **p < 0.01, ***p < 0.001 ISO compared to saline control, ^$p < 0.05, ^$$p < 0.01 betalain + ISO compared to ISO-induced rats.

Figure 7 shows the expression level of microRNA and the results demonstrated the reduced level of miR-18 with increased miR-27a (2.6-fold), miR-199a (1.8-fold), and miR-423 (2.4-fold) in rats suffering from heart failure. On the contrary, the levels of these miRs were significantly not affected in pre-treatment of rats with betalain suggest that the interplay of microRNAs is one of the clues involved in the progression of heart failure (Figure 7).

Figure 7.

(a to d) Expression analysis of microRNAs, miR-18, miR-27a, miR-199, and miR-423, in the saline control, ISO induced and betalain treated group of rats. The detail of the experiment is given in the methodology section. Values are expressed as mean ± SE (n = 6). Statistical significance expressed as *p < 0.05, **p < 0.01 ISO compared to saline control, ^$p < 0.05 betalain + ISO compared to ISO-induced rats.

Discussion

Several studies have indicated the ISO-induced myocardial fibrosis and heart failure resulted in inflammatory response that ends up in myocardial infarction. In the present study, rats were induced with cardiac failure using ISO and used betalain to treat the animals to study the cardioprotective role of it by evaluating the myocardial infarct size, oxidative stress parameters, and the inflammatory pathway that leads to reduced cell functioning of the cardiac tissues.

ISO-treatment is the most reliable method for studying the effect of drugs on heart failure. ¹⁹ During the onset of cardiac failure using ISO, the normal functioning of the heart is disturbed with an increase in the heart weight to body weight ratio was observed compared to control group. The apparent increase in heart weight is due to the increase in heart size and the animals are suffering from extensive heart contraction failure. ²⁰ The extensive size is an indicator of the contractile dysfunction with uncontrolled differences in the SBP and HR due to cardiac failure. On the contrary, the normalization of the blood pressure was attained in rat myocardium due to the pretreatment of BTA.^21–23

It has been reported that in chronic heart disease, accumulated reactive oxygen species due to reduced activity of anti-oxidant enzymes trigger the oxidative stress and results in cardiac necrosis.^24,25 ROS would cause peroxidation of lipids and proteins into advanced glycation end products (AGEs) and its deposition on the cardiac tissues on circulation would cause changes in the heart structure and results in damage to the cardiac tissues fibrils and weakening of the heart. This would alter the cardiac functioning and an increase in the heart rate and the systolic blood pressure. While the betalain treatment alleviates the ROS and protected the heart evidences its protective mechanism probably through the NF-κB (p65) mediated signaling.

MDA is a well identified biomarker of cardiac damage and it was extensively utilized as an indictor of cardiac damage. MDA regulated cardiac damage was reported in the cardiac damage. The intracellular antioxidants are the first line defense for the oxidative stress and it is vital to fight against cardiac damages. SOD, CAT, GSH and GPx, are the prime antioxidants in the cells that protect against oxidative stress.^26,27 The levels of NO and lipid peroxidation product MDA and XO have increased in the animals that were induced with ISO and treatment with BTA has decreased the ROS with increased activity of the antioxidant enzymes of SOD, catalase, GPx, and probably through the conversion of ROS into hydrogen peroxide and oxygen. ²⁸ Thus, the cardioprotective effects of BTA have been exerted through the induction of its anti-oxidant property ¹³ and have effectively reduced the oxidative stress in chronic heart failure on the cardiomyocytes.^29,30 Also, histopathological findings on BTA treatment have shown that the pretreated myocardium has reduced damage to the myocardial fibrils with the cell morphology retaining their normal shape and size or that the toxic effects of ISO had fewer effects on the cardiac architecture.

The main pathological factors that are responsible for the development of cardiovascular diseases are the inflammation associated with hypertension, lipid deposition, and diabetes. ³¹ ISO induction has increased the inflammatory cytokines of IL-1β, IL-6, and TNF-α^32,33 to execute the cardiotoxicity in the animals. As observed in in vitro studies, ³⁴ betalain could inhibit the IL-1 beta-mediated induction of IL-6 and pro-inflammatory actions of TNF-alpha on cardiomyocytes ¹⁸ utilizing the anti-inflammatory role for cardioprotection.

The increased level of CXCR4 in cardiac failure animals was observed with the decreased contractility of the cardiomyocytes, which are the responsive element for the activation of the beta-adrenergic receptor. ³⁵ The role of chemokine, CXCL12, and its receptor (CXCR4) on the cardiac dysfunction has been reported with a dual function depends on the stages of the heart stating whether it is failing or recovering.^36,37 Hence, the corrective action of betalain in the present study speculates that cardiac dysfunction is reversed by activating the beta-receptor. ³⁸ Controlling the levels of superoxide and NO by betalain has indirectly reduced the expression of IL-21 ³⁹ that is playing the prominent role in the activation of these chemokines discussed and also the matrix metalloproteases ⁴⁰ that would increase the tissue fibrosis by increasing the expression of collagen. ³⁹ The resultant anti-inflammation event by betalain ⁴⁰ is marked by an increase in the anti-inflammatory cytokines, IL-10^41,42 thus might have stopped the ECM deposition in failing heart.

The onset of myocardial infarction reported with the simultaneous activation of inflammatory cytokines that are followed by an increased level of MMPs. ⁴³ Especially, TNF-α increases the production of MMP-2 and MMP-9 in animal models. ⁴⁴ They are known to be involved in the progression of heart failure by the dissolution of the tissue matrix collagen and enhance myocardial rupture and decrease heart function. ⁴⁵ Betalain treatment has increased the expression of natural inhibitors of MMPs, the TIMPs in the pathway to recover the myocardial infarct remodeling by reducing the MMPs actions. ⁴⁶ ECM is maintained by the cardiac fibroblasts and their activation leads to the formation of tissue fibrosis in the heart. IFN-γ increased in the affected animals transiently activates the Stat-3 to phosphorylate it, with TGF-beta activates the process further, ⁴⁷ to translocate into the nucleus ⁴⁸ to express collagens that are necessary for the cardiac remodeling. In the myogenic differentiation, PI3K-AKT ⁴⁹ and JAK2-STAT3 ⁵⁰ pathways have a role by the mediation of Myocyte Enhancer Factor 2 transcriptional regulatory protein (MEF2). MEF2 is important for the expression of various genes of cardiac muscle^51,52 and skeletal gene expression ⁵³ partially mediate the JAK2-STAT2 pathway.

An increased number of genes like ANF, β-myosin heavy chain ⁵⁴ have participated in the cardiac remodeling in cardiac hypertrophy which are speculated to influence of epigenetic mechanism of HDAC on these gene expressions. Sodium calcium exchanger (Ncx1) gene, under the influence of HAT/transcriptional activator, p300, was up-regulated in cardiac failure animals and the increased ratio of NCX1/SERCA indicates that the contractile function of the myocytes has decreased. ⁵⁴ To explain the interplay between the ISO-induced cardiac phenotypes to the expression of miRNAs that influence the pathological events, we have analyzed the expression of miRNAs. On that, we observed a decrease in the levels of expression of miR-18 a marker known to decrease in cardiac hypertrophy ⁵⁵ and a simultaneous increase in miR-423 indicating heart failure.^56–58

Cardiac contractility is based on the expression of the β-myosin heavy chain (β-MHC) and is influenced by the pathological conditions and hence expressed in the model of heart failure and subsequent cardiac hypertrophy. The factors that regulate the β-MHC gene expression were regulated by miRNAs. The expression analysis showed over-expression of miR-27a and is correlating with the conditions of β-MHC up-regulation together with ISO-induced stress conditions. ⁵⁹ Similarly, the extent of cardiac hypertrophy was evidenced with the over-expression of miR-199a that is specific to the cardiomyocytes ⁵⁸ and is a known marker for hypertrophy in cardiac muscles. ⁶⁰ Conversely, rats with BTA administration have displayed regularized microRNA signatures that prove the direct effect of the drug in imparting the cardioprotection. Thus in the present study, the ISO-induced cardiotoxicity has been better managed with the treatment of betalain that could act as a cardioprotective agent in suppressing oxidative stress, inflammation, and cardiac remodeling.

Hence, the present study results demonstrate the use of BTA could effectively give protection to the cardiomyocytes from cell damage due to oxidation and inflammation and prevent the subsequent tissue remodeling and cardiac fibrosis. The results projected here are from in-depth analysis of the experiments done at the molecular level to understand the interplay between the miRNA and the protein in elucidating the probable pathway for cardioprotection. Overall, the present study proposes a candidate molecule for future studies in elucidating the clear molecular mechanism of heart failure.

Supplemental material

Supplemental Material, sj-pdf-1-het-10.1177_09603271211027933 - Betalain exerts cardioprotective and anti-inflammatory effects against the experimental model of heart failure

Supplemental Material, sj-pdf-1-het-10.1177_09603271211027933 for Betalain exerts cardioprotective and anti-inflammatory effects against the experimental model of heart failure by Y Gao, X Liang, Z Tian, Y Ma and C Sun in Human & Experimental Toxicology