Boeravinone B alleviates gut dysbiosis during myocardial infarction‐induced cardiotoxicity in rats

Abstract Myocardial infarction (MI) is the most common heart disease, and also, it is one of the leading causes of death from cardiovascular disease. It is well known that MI causes additional injury during blood flow restoration in ischaemic myocardium. Boeravinone B (BB) is a well‐known antioxidant and anti‐inflammatory drug. We investigated the cardioprotective effect of BB drug against isoproterenol (ISO)‐induced MI in rats in this experimental study, along with we analysed its underlying mechanism. Adult Sprague Dawley (SD) rats were treated subcutaneously with ISO (45 mg/kg), then divided into groups and then given BB drug was administered orally. The cardioprotective effect of BB on ISO‐induced MI rats was analysed by estimating the heart injury markers, antioxidant pro‐inflammatory cytokines and inflammatory parameters. We also detected quantified expression of inflammation and apoptosis‐related marker protein family. We estimated the effect of BB drug on GUT microbiota in ISO‐induced MI rats and scrutinized the histopathological variations in heart tissues. BB treatment significantly (P < .001) diminished the level of heart markers such as lactate dehydrogenase (LDH), troponin (TnT), creatine kinase (CK) and creatine kinase isoenzymes MB (CK‐MB). BB treatment also altered the antioxidant parameters and reduced the pro‐inflammatory cytokines in the serum and tissues. Additionally, the histopathological aspects demonstrated that the pathological changes observed in the heart tissue of the ISO group rats were suppressed by the BB treatment to varying degrees. Furthermore, the expressions of caspase‐3, p53, caspase‐9, Bax, interleukin‐6 (IL‐6), cytochrome C, neutrophil gelatinase‐associated lipocalin (NGAL), tumour necrosis factor‐α (TNF‐α), nuclear factor kappa B (NF‐κB) and interleukin‐1β (IL‐1β) in the heart tissue were down‐regulated whereas the Bcl‐2 expression seemed to be enhanced. BB treatment not only alleviated ISO‐induced gut dysbiosis by its enhanced specified Firmicutesto‐Bacteroidetes (F/B) ratio but also maintained the relative abundance of major bacteria such as Clostridium IV, Butyricicoccus, Clostridium XIVs, Akkermansia and Roseburia. Collectively, our findings showed that the BB drug acted against myocardial infraction and prevented the damage by reducing the oxidative stress and controlling the inflammatory pathways, and gut microbiota.

The cardioprotective effect of BB on ISO-induced MI rats was analysed by estimating the heart injury markers, antioxidant pro-inflammatory cytokines and inflammatory parameters. We also detected quantified expression of inflammation and apoptosisrelated marker protein family. We estimated the effect of BB drug on GUT microbiota in ISO-induced MI rats and scrutinized the histopathological variations in heart tissues. BB treatment significantly (P < .001) diminished the level of heart markers such as lactate dehydrogenase (LDH), troponin (TnT), creatine kinase (CK) and creatine kinase isoenzymes MB (CK-MB). BB treatment also altered the antioxidant parameters and reduced the pro-inflammatory cytokines in the serum and tissues. Additionally, the histopathological aspects demonstrated that the pathological changes observed in the heart tissue of the ISO group rats were suppressed by the BB treatment to varying degrees. Furthermore, the expressions of caspase-3, p53, caspase-9, Bax, interleukin-6 (IL-6), cytochrome C, neutrophil gelatinase-associated lipocalin (NGAL), tumour necrosis factorα (TNFα), nuclear factor kappa B (NF-κB) and interleukin-1β (IL-1β) in the heart tissue were down-regulated whereas the Bcl-2 expression seemed to be enhanced. BB treatment not only alleviated ISO-induced gut dysbiosis by its enhanced specified Firmicutesto-Bacteroidetes (F/B) ratio but also maintained the relative abundance of major bacteria such as Clostridium IV, Butyricicoccus, Clostridium XIVs, Akkermansia and Roseburia. Collectively, our findings showed that the BB drug acted against myocardial infraction and prevented the damage by reducing the oxidative stress and controlling the inflammatory pathways, and gut microbiota.

| INTRODUC TI ON
Acute myocardial infarction is considered to be significant of ischaemic heart disease and it widely causes mortality and morbidity worldwide. 1,2 Myocardial infarction (MI) is a diseased condition which is generated as a result of the induction of necrosis that occurs as a result of imbalance between the myocardial demand and coronary blood supply. MI is an adverse health issue and it is one of the leading causes of higher mortality and morbidity rates in the Western world, including China. 2,3 MI-related morbidity and mortality have reached epidemic proportions, with 16.7 million deaths per year worldwide. 2 The clinical symptoms of myocardial dysfunction and necrosis include electrocardiographic changes, changes in blood pressure, left ventricular dysfunction and changes in heart beat rate.
These symptoms arise as a result of the boosting of cardiac specific proteins level in the circulation. 4,5 The level of cardiac troponins frequently correlated with myocardial inflammation and infarctionrelated proteins that increased the risk of heart attack. Moreover, MI also induces cardiac hypertrophy and myocardial fibrosis. The specific mechanism involved in MI has been associated with inflammation, oxidative stress and apoptosis. 6,7 Isoproterenol (ISO)-induced myocardial infarction is considered to be a significant technique for inducing stress and inflammation in the rat's heart and also it aids in the estimation of cardioprotective effect of the tested compound. ISO (1-[3,4-dihydroxyphenyl]-2 -isopropylamino ethanol hydrochloride) is a synthetic β-adrenergic catecholamine which is used in the infra maximal doses to regulate the heart function. 3,8 Supramaximal dose of ISO could induce stress in the myocardium by reducing the amount of stored energy molecules in cardiomyocytes, which ultimately induces the infarct such as necrosis and irreversible cellular damage. 9,10 Various synthetic drugs have been used for treating this condition of heart attack, but most of the treatments show more severe side effects which may be fatal for patients. Nowadays scientists are focussing mainly towards plant-based drugs to scrutinize the cardioprotective effect against myocardial infarction. Any natural compound with good antioxidant activity is thought to protect cardiac tissues from MI. Studies suggest that ISO-induced myocardial dysfunction in the heart of the rodent is similar to human myocardial ischaemia. 4,11 As a result, ISO is widely used to induce MI in rats to investigate the effects of different processed drugs on MI.
Oxidative stress is involved in damaging the myocardial structure during cardiac hypertrophy. Additionally, evidence suggests that oxidative stress is an indicative of reduced levels of endogenous antioxidants in myocardium. 5,8 During oxidative stress, the activity of endogenous antioxidants such as catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) was seen to be greatly decreased leading to the induction of lipid peroxidation.
Increased levels of lipid peroxides activated malondialdehyde (MDA) which boosted the production of free radicals thereby suppressed the endogenous antioxidant status. It is well recognized that ISO induces the generation of reactive oxygen species (ROS) or free radical, which further causes oxidative stress, as demonstrated by considerably boosted the tissue MDA (lipid peroxidation marker) and suppressed the level of endogenous antioxidant enzymes such CAT, GPx and SOD, which have significant role against myocardial infarction. 2,3,12 Previous studies suggested that the myocardial infarct size can be limited by enhancing the endogenous antioxidants level that reduces the generation of free radicals. During oxidative stress condition, the production of ROS and the secretion of pro-apoptotic factors are initiated by mitochondria into the cytosol which activates the apoptotic cell death and signs of endoplasmic reticulum stress. 1,13,14 The inflammatory response is essential in the progression of MI.
Previous investigations suggest that pro-inflammatory cytokines damage the myocardial tissue. Moreover, MI effects are elevated as a result of an increase in the level of pro-inflammatory cytokines which also lead the tissue infiltration through inflammatory cells. 14,15 During MI, neutrophils invade the infarcted region, where they induce and increase myocardial cell injury by secreting proteolytic enzymes, a variety of chemokines and inflammatory cytokines, along with the production of reactive oxygen species (ROS). 6,14 Chemokines, reactive oxygen species and cytokines all contribute to the loss of organ function. A rise in pro-inflammatory cytokines speeds up MI, which leads to serious congestive heart failure. IL-1β, TNFα and IL-6 are pro-inflammatory cytokines that increase the inflammatory response during MI disease. TNFα causes neutrophil migration into the ischaemic region of infarcted myocardial tissue. Other cytokines involved in the stress-induced inflammatory responses in myocardial tissue include IL-6 (pleiotropic cytokine). 7 MI-attributed adverse reaction is categorized by the activation of various cellular signalling molecules, such as nuclear factor kappa light chain boost-activated B cells (NF-κB). It is proved that apoptosis and inflammatory reactions are regulated by the NF-kB. In the MI diseased condition, NF-κB phosphorylation initiates the intracellular signalling reaction and ultimately causes the generation of pro-inflammatory cytokines and inflammation-related proteins that involve numerous pathophysiological alterations. 7,14 The gut microbiota is composed of five phyla such as apoptosis, Boeravinone B, gut microbiota, isoproterenol, myocardial infarction phyla, such as fungi and archaea, constitute less than 1% of the gut flora. 6,16 These microorganisms in the gut convert carbohydrates and proteins into the various short chain fatty acids (SCFAs). The generated metabolites such as propionates (formed via Bacteroidetes), butyrates (formed via Firmicutes) and acetates degrade indigestible polysaccharides, which are then consumed and digested in the gut's distal region. Therefore, gut flora use the host's dietary derived molecules and considerably subsidize to gastrointestinal tract (GIT) physiological homeostasis, but any alteration in the gut dysbiosis (micro-ecosystem) may pave a way for the pathophysiological function of host cardiovascular system. 17 The gut microbiota controls the host's toxicity response, according to evidence pertaining to the gut.
In cardiovascular patients, ISO causes gut dysbiosis, although the relationship between these microbiota changes and the magnitude of these side effects is still unknown. 16 Dysbiosis of the gastrointestinal microbiota elevates the levels of gut-derived toxins in the bloodstream, boosts the oxidative stress and inflammation and also affects intestinal absorption and utilization of micronutrient. 16,18 Boeravinone B is a rotenoid isolated from the Boerhaavia diffusa. 19 The herb has been long in use by our ancestors and proved to be an effective formula for gastric ailments such as dyspepsia, abdominal pain, gastritis and other gut-related disorders. Boeravinone B and C exhibited the P-gp inhibitory effect. 19 Another member of the rotenoid family such as Boeravinone G shows the antioxidant and genoprotective effect. 20 BB showed the various pharmacological effects for the treatment of acute myoskeletal dysfunctions, spondylosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, psoriasis and atherosclerosis in rodents. 20 BB showed the anti-inflammatory and antioxidant effect against various diseases, but its efficacy and activity are not yet analysed against the myocardial infarction. In this protocol, we tried to establish the correlation between the Boeravinone B on ISO-induced MI injury in rats associated with the alteration in the gut microbiota. In the current investigation, we scrutinized the potential effect of Boeravinone B against ISO-induced MI in rats and its underlying mechanism was also expounded based on the composition of gut microbiota, antiapoptotic, anti-inflammatory and antioxidant activities.

| Chemical and reagents
Boeravinone B was purchased from Sigma-Aldrich. All the chemicals and reagents used in the experimental study were analytical graded.

| Induction of myocardial infarction
Isoproterenol (ISO) was used in the induction of MI. To induce MI, ISO was dissolved in vehicle (saline) and 100 mg/kg dose subcutaneously injected into rats after every 24 hours for 2 days. The rats were sacrificed after 48 hours (after the 1st injection of ISO). 2

| Experimental protocol
The rats were divided into different groups with each group containing ten rats after the MI was successfully induced using ISO. Table 1 shows how the rats were divided into various classes.
All group rats were weighed, and blood samples were taken from the retro-orbital plexus after the last BB treatment. To separate the serum, the blood samples were centrifuged at 15 000 rpm for 10 minutes. For further use, the separated serum was immediately stored at -20°C. At the end of the protocol, all group rats were euthanized using mild anaesthesia and heart tissue immediately removed, washed (saline), socked and finally weighed. A small piece (5 µm) of heart tissue was stored in the formaldehyde (40%) for histopathology. Rest of the heart tissue was kept at −20°C for biochemical estimation.

| Nitric oxide and inducible nitric oxide
Nitric oxide (NO) and inducible nitric oxide synthase (iNOS) levels were estimated using the commercial kits following the manufacturing protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).  and high-density lipoprotein (HDL) were estimated using the commercial kits following the manufacturing protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The level of low-density protein (LDL) and very low-density lipoprotein (VLDL) was estimated using the given formula.

| Cardiac injury markers
The cardiac injury markers such as CK, TnT, CK-MB and LDH were determined using commercial kits following the manufacturing protocol Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

| Estimation of cardiac function
Pressure volume catheter (1.9F; Scisense Instruments) was used for estimating the cardiac function using the previous method.

| Gene expression analysis
Ribonucleic acid (RNA) isolation kit (Promega) was used for isolating RNA from the heart following the manufacturing protocol. M-MLV reverse transcriptase was used for performing the reverse transcription. SYBR Green qPCR SuperMix on a LightCycler ® 480 Real-Time PCR system was used to quantify real-time reverse transcription using 384-well plates following the manufacturing instruction. All primer sequences are presented in Table 2. GAPDH was used as the internal standard.  were added and vortexed for next 15 seconds and incubated for 10 minutes in a water bath to maintain the temperature of 70°C.

| DNA extraction and 16s rRNA estimation
Finally, 1.5-mL sterilized Eppendorf tubes were taken and the elution buffer (150 µL) was mixed with the columns and incubated at room temperature for the next 10 minutes and centrifuged for 1 minute at 14 000 rpm. Agarose gel electrophoresis and nanodrop methods were used for qualitative and quantitative analysis of the eluted DNA sample.

| Haemodynamic estimation
BL-420F Biological system (TME Technology Co., Ltd) was used for estimating haemodynamic variables using the previously reported method. Briefly, all group rats were anaesthetised and placed in the supine position. ECG devices were placed subcutaneously and connected to record the electrocardiograms. Pressure electricity transducer was used for estimating the ventricular contractile function with inserted Millar catheters into the left ventricular cavity through the right carotid artery. A catheter-mounted pressure electricity transducer was used to obtain femoral arterial blood pressure readings.

| Statistical analysis
GraphPad Prism 8 software (Graph Pad Software) was used for statistical analysis. For the statistical analysis, one-way analysis of variance (ANOVA) was used for comparing the difference. Tukey multiple comparison test was performed for comparison of the different groups. The results were significantly considered if *P < .05, **P < .01 and ***P < .001.

| Effect of BB drug on heart markers
The  Tukey multiple comparison test was performed for the comparison the different groups. The results were significant considered if *P < .05, **P < .01 and ***P < .001

| Effect of BB on nitric oxide and inducible nitric oxide
Isoproterenol-induced MI rats showed an increased level of NO and iNOS compared with normal control. BB suppressed the level of NO and iNOS at dose-dependent manner ( Figure 3).

| Effect of BB on lipid parameter
The

| Effect of BB on pro-inflammatory cytokines
Pro-inflammatory cytokines include IL-6, TNFα, INFγ and IL-1β in the serum and heart tissue. ISO-induced MI rats showed the boosted level of TNFα, INFγ, IL-6 and IL-1β in the serum ( Figure 6) and heart tissue ( Figure 7). BB treatment significantly (P < .001) suppressed the pro-inflammatory cytokines in the serum as well as heart tissue.

| Effect of BB on gut microbiota
The ability of BB to change the gut microbiome of faecal microbiota transplanted from the recipients was investigated at the end of the experimental study. In contrast to the standard control group rats, the ISO-induced MI rats had a lower relative abundance of Firmicutes and a higher relative abundance of Bacteroidetes, as   The removal of gut microbiota effectively eliminated ISO-induced injury in various organs, including the heart, which clearly states that gut microbiota plays a key role in the expansion and progression of MI in the heart caused by ISO. 16,27 Various dietary supplements already prove their protective effect against ISO-induced cardiac toxicity by targeting the gut microbiota. In this study, we estimated the cardioprotective effect of BB on the ISO-induced MI and also, we found that the BB treatment precludes the deleterious effect of gut dysbiosis including alteration of gut microbiota, gut leakiness and inflammation. Faecal microbiota transplantation involved in the gut microbiome and involved in improving cardiomyopathy and also involved in modulating the dysregulation induced by ISO in rats.

| Effect of BB on cardiac histopathology
As a result, altering homeostasis by targeting the gut microbiota may be a novel way to treat ISO-induced MI. In ISO-induced MI rats, the findings showed that the BB administration prevents gut microbiota dysbiosis and the deterioration of intestinal barrier integrity. 16,27,28 After cardiotoxicity, the structure of the gut microbiota decreases in richness and diversity and administration of dietary supplements modified the gut microbiota and helped in suppressing the cardiotoxicity. 28,29 In this experiment, we observed that the BB treatment significantly decreased and prevented changes in gut microbiota alpha richness and diversity. In ISO-induced MI rats, BB treatment increased microbiota richness and prevented alpha diversity reduction.  27,29 If bacterial absorption of mucin-derived nutrients exceeds new intake, the integrity of this vital barrier can be jeopardized, triggering an inflammatory response and increasing gut permeability. The abundance of various positively anti-inflammatory bacteria was significantly increased after BB therapy, indicating that it had a beneficial effect on MI in rats.
In this research, we discovered that in ISO-induced MI rats, reduced expression of Cdx2 and Muc2 and an increase in the number of goblet cells in the villi, which release glycoproteins, implying a weakness in mucus production capability as the secretory role of goblet cells, are exhausted. 2,22 This exhaustion may be as a result of continuous secretion of mucin-2 glycoprotein in large amounts from the goblet cells. BB treatment continuously maintained the goblet cell number and Akkermansia relative abundance, which further boosted the protection and repairment of mucus barrier.

| CON CLUS ION
The current study found that administering BB to rats reduced dysregulation and gut dysbiosis during ISO-induced myocardial infarction. BB treatment significantly reduced the heart parameters at dose-dependent manner. ISO treatment significantly reduced endogenous antioxidant enzymes and boosted the inflammatory reaction. BB treatment considerably altered the antioxidant enzymes and inflammatory mediators.
• Boeravinone B treatment reduced the level of MDA and increased the level of SOD, CAT and GPx.
• Boeravinone B treatment reduced the inflammatory reaction in the serum and heart tissue by suppressing of pro-inflammatory cytokines and inflammatory mediators in the serum and heart tissues.
• Boeravinone B treatment altered the cardiac function parameters.
• Boeravinone B treatment alleviated gut microbiota by maintaining the F/B ratio.
• Boeravinone B treatment considerably regulates gut microbiota and suggests the cardioprotective effect.

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.