Ameliorating effect of rutin against diclofenac-induced cardiac injury in rats with underlying function of FABP3, MYL3, and ANP

Abstract Diclofenac is a widely prescribed anti-inflammatory drug having cardiovascular complications as one of the main liabilities that restrict its therapeutic use. We aimed to investigate for any role of rutin against diclofenac-induced cardiac injury with underlying mechanisms as there is no such precedent to date. The effect of rutin (10 and 20 mg/kg) was evaluated upon concomitant oral administration for fifteen days with diclofenac (10 mg/kg). Rutin significantly attenuated diclofenac-induced alterations in the serum cardiac markers (LDH, CK-MB, and SGOT), serum cytokine levels (TNF-α and IL-6), and oxidative stress markers (MDA and GSH) in the cardiac tissue. Histopathological examination and Scanning Electron Microscopy (SEM) findings displayed a marked effect of rutin to prevent diclofenac-mediated cardiac injury. Altered protein expression of myocardial injury markers (cTnT, FABP3, and ANP) and apoptotic markers (Bcl-2 and Caspase-3) in the cardiac tissue upon diclofenac treatment was considerably shielded by rutin treatment. MYL3 was unaffected due to diclofenac or rutin treatment. Rutin also significantly improved diclofenac-induced gastrointestinal and hepatic alterations based on the observed ameliorative effects in key mediators, oxidative stress markers, histopathology examination, and SEM findings. Overall results suggest that rutin can protect the diclofenac-induced cardiac injury by lowering oxidative stress, inhibiting inflammation, and reducing apoptosis. Further research work directs toward the development of phytotherapeutics for cardioprotection.


Introduction
Cardiovascular diseases are the leading cause of death worldwide, with more than seventeen million deaths every year (World Health Organization 2021). Drug-induced cardiotoxicity is considered as one of the crucial factors for this high mortality rate (Kelleni and Abdelbasset 2018). Nowadays, it has become mandatory to evaluate cardiovascular safety during the preclinical and clinical stages of the drug development process (Avila et al. 2020, Lam andWu 2021). A high attrition rate of new chemical candidates has been observed in the drug development process. Many drugs are withdrawn from the market due to cardiotoxicity. However, several essential drugs are even available on the market through modification of dose regimens (Ferdinandy et al. 2019). Diclofenac is a widely prescribed drug among nonsteroidal anti-inflammatory drugs (NSAIDs). Cardiovascular complications take the shine away from its therapeutic use (Spitz 2013, Ghosh et al. 2015. Long-term consumption causes cardiac injuries followed by the aggravation of cardiovascular diseases, mainly increasing the risk of myocardial infarction and/or worsening congestive heart failure and stroke (Hudson et al. 2007, Waksman et al. 2007). Based on the various cardiovascular risks of NSAIDs, diclofenac belongs to the higher risk group, and naproxen categorizes as the lower-risk group (Mart ın Arias et al. 2019). In recent times, diclofenac has been recommended to use at the lowest dose level and/ or for a shorter duration (Spitz 2013, Ghosh et al. 2015.
Under these circumstances, plant-based natural products can complement the pharmacological activity of a drug as well as can combat its dose-dependent side effects. In this direction, rutin (3, 3 0 , 4 0 , 5, 7-pentahydroxyflavone-3-rhamnoglucoside) is reported to have several biological activities that can be beneficial to aid the anti-inflammatory activity of diclofenac as well as to counteract its liabilities (Prince and Priya 2010, Punithavathi et al. 2010, Arjumand et al. 2011, Khan et al. 2012, Alonso-Castro et al. 2013, Yoo et al. 2014. We reported earlier that rutin could augment the efficacy of diclofenac using a rat model (Dogra et al. 2020). However, limited reports of diclofenac-induced cardiotoxicity in the preclinical model are available to date, considering oral administration and repeated dose treatment (Abdel-Daim et al. 2018, Arafa et al. 2020. It is reported in the literature that diclofenac could induce cardiotoxicity in rodents by increasing the reactive oxygen species (ROS), thereby reducing the anti-oxidant defense in the cardiac tissue (Abdulmajeed et al. 2015, Abdel-Daim et al. 2018. Furthermore, excessive ROS production triggers cardiomyocyte apoptosis by increasing caspase-3 activity and inhibiting B-cell lymphoma 2 (Bcl-2) expression resulting cardiomyopathy (Takahashi et al. 2004, Jin et al. 2013. In this pursuit, we aimed in the present study to explore for any cardioprotective effect of rutin with the possible underlying mechanisms using diclofenac-induced cardiac injury in the rat model.

Experimental animals and ethical prerequisites
Healthy adult male Wistar rats (8-10 weeks of age; 130 to 160 g of body weight) were housed and maintained in the animal house under normal laboratory conditions. Rats were given a standard pellet diet (M/s Ashirwad Industries, Chandigarh, India) with water ad libitum. Our Institutional Animal Ethics Committee endorsed the present study protocol (Approval no. 1981/77/8/2020).

Experimental dose and dose formulation
The experimental dose of diclofenac in the present study was 10 mg/kg based on its human dose of 100 mg/day (https:// www.rxlist.com/consumer_diclofenac_cataflam/drugs-condition.htm). The same dose level of diclofenac is also reported to induce toxicities by several researchers (Owumi and Dim 2019, Alabi and Akomolafe 2020, Elsherif et al., Owumi et al. 2020. The experimental dose of rutin here was 10 and 20 mg/kg/day based on our previous experimentations for rutin's effect on the pharmacokinetics/efficacy of diclofenac in the rat model (Dogra et al. 2020). The dose formulation of diclofenac and rutin was prepared individually. An aqueous suspension containing sodium carboxymethylcellulose (0.25%, w/v) was used as a vehicle. The dose-volume was 10 mL/kg. Freshly prepared dose formulations were given through oral gavage.

Study design
A total of 30 animals were randomly divided into five groups (n ¼ 6). Diclofenac was treated at 10 mg/kg orally for 15 consecutive days to induce cardiac injury. Rutin was given at 10 or 20 mg/kg for 15 consecutive days through the oral route. Treatment protocols for each group were as follows: Group-1: Control (vehicle), Group-2: Diclofenac (10 mg/kg), Group-3: Diclofenac (10 mg/kg) and rutin (10 mg/kg), Group-4: Diclofenac (10 mg/kg) and rutin (20 mg/kg), and Group-5: Rutin (20 mg/kg). After 15 days of treatment, animals were fasted overnight (10 h) and on the next day (16 th day of experiment), collected the blood sample from the retroorbital plexus to obtain serum. Next, sacrificed the animals by carbon dioxide euthanasia (70% of chamber volume per min) followed by cervical dislocation and then dissected the animals to obtain the heart, stomach, and liver. The organ to body weight ratio was estimated ( Figure S1, Supplementary Material). Parts of tissue were kept for histopathology and SEM examination, whereas the remaining tissue was snap-frozen and stored (À80 C) for further studies.

Oxidative stress markers
MDA content was measured in the cardiac tissue homogenate (100 mg/mL), prepared using 1.15% potassium chloride in water (w/v). GSH content was determined in the cardiac tissue homogenate, which was prepared using 50 mg of each tissue in 100 mM of sodium phosphate buffer containing 5 mM of ethylenediaminetetraacetic acid (EDTA) (750 mL) and 25 %(v/v) of orthophosphoric acid in water (200 mL). Both the above-mentioned studies were performed using our earlier reported protocol (Dogra et al. 2021). Detailed methodologies are given in Supplementary Material.

Inflammatory cytokine levels
Serum level of tumor necrosis factor a (TNF-a) and interleukin 6 (IL-6) was quantified using respective ELISA kits (rat TNF-a ELISA kit, sensitivity: 2.5 U/L, Lot no. 1818268B, Invitrogen and rat IL-6 ELISA kit, sensitivity: 12.0 pg/mL, Lot no. 166226040, Invitrogen) as per the manufacturer's protocol.

Histopathology
The histopathological examination was performed using the hematoxylin-eosin (H & E) staining method. Briefly, the cardiac tissue was fixed in 10% neutral buffered formalin solution, embedded in paraffin, sectioned by microtome, dehydrated using the mixture of ethanol & water, and stained with H & E dye. The slides were examined under the light microscope using 40Â magnification (Make: Magnus Opto Systems; Model: INVI). Semi-quantitative analysis was also done by scoring for particular parameters, including inflammatory cells infiltration, red blood cell (RBC) congestion, myocardial necrosis, and myocardial fibrosis (Awad et al. 2021). Grading was done from normal (zero) to severe (three and above) (Khafaga and El-Sayed 2018). The data were expressed as mean ± S.E.M (n ¼ 6). Data were compared between the following groups: control vs. rutin alone or diclofenac alone; diclofenac alone vs. combination of diclofenac and rutin. NS represents statistically insignificant. Statistical significance level: Ã p < 0.05, ÃÃ p < 0.01, and ÃÃÃ p < 0.001. Figure 2. Effects of rutin on oxidative stress markers and inflammatory cytokines: (A) MDA content in cardiac tissue, (B) GSH level in cardiac tissue, (C) TNF-a level in serum, and (D) IL-6 level in serum. Each value is expressed as mean ± S.E.M (n ¼ 6). Data were compared between the following groups: control vs. rutin alone or diclofenac alone; diclofenac alone vs. combination of diclofenac and rutin. NS represents statistically insignificant. Statistical significance level: Ã p < 0.05, ÃÃ p < 0.01, and ÃÃÃ p < 0.001.

SEM
SEM examination was performed for cardiac tissue. Briefly, tissue was processed using the following sequences: (a) perfused with 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 24 h at 4 C, (b) washed once with the same buffer, (c) fixed in 1% osmium tetroxide in 100 mM of sodium phosphate buffer (pH 7.4) for 1 h at 4 C, (d) dehydrated with ethanol ranging from 10 to 100%, (e) immersed in 1-2 mL of hexamethyldisilazane (HMDS) for 10 min, and (f) air-dried at room temperature to remove the HMDS.
Finally, gold-sputtered samples were mounted on a sample stub to examine in the SEM instrument (Make: Jeol; Model: JSM-IT 300).

Western Blotting
Protein expression of cTnT, ANP, FABP3, MYL3, Bcl-2, and procaspase-3 were evaluated in the cardiac tissue homogenate by Western Blot analysis as per our earlier reported protocol (Dogra et al. 2021). The cardiac tissue homogenate was prepared using radioimmunoprecipitation buffer with protease inhibitor cocktail, phenylmethylsulphonyl fluoride (2 mM), sodium orthovanadate (0.5 mM), and sodium fluoride (50 mM), followed by centrifugation at 3000 rpm for 15 min and then, protein level was estimated by Bradford method. The protein was separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to the polyvinylidene difluoride membrane (100 volts, 120 min, 4 C). The membrane was blocked with 3% BSA (room temperature, 2 h) to avoid nonspecific antibody binding. The membrane was now incubated with respective primary antibodies overnight at 4 C. Post-primary incubation, the membrane was washed thrice with Tris-buffered saline and then re-incubated with chemiluminescent horseradish peroxidaseconjugated secondary antibody (room temperature, 1 h). After that, the membrane was washed again with Tris-buffered saline thrice, and imaging was done using the Chemidoc imaging system (Make: Syngene, Maryland, USA; Model: G: BOX, XT-4). Densitometry analysis was done by Image J software.

Effect of rutin against diclofenac-mediated gastric alterations
The effect of rutin on the stomach was also examined by the following investigations: (a) estimation of prostaglandin E 2 (PGE 2 ) and thromboxane B 2 (TXB 2 ) was done (Rat PGE 2 ELISA Kit, Lot no. 201703 and Rat TXB 2 ELISA Kit, Lot no. 201906, SunRed Biotechnology) as per the manufacturer's protocol where gastric tissue homogenate (250 mg/mL) was prepared using Dulbecco's phosphate-buffered saline (10 mM, pH 7.4); (b) histopathological examination and scoring (Koc et al. 2020); (c) SEM analysis.

Effect of rutin against diclofenac-mediated hepatic changes
In addition to the above-mentioned studies, the effect of rutin on the liver was also examined through the following investigations: (a) MDA and GSH content in the liver tissue homogenate as mentioned above for cardiac tissue; (b) histopathological examination and scoring (Khafaga and El-Sayed 2018); and (c) SEM analysis.

Statistical analysis
The statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey's test (GraphPad Prism 5 software). Experimental data are represented as mean ± standard error mean (S.E.M) for each group of animals. Data were compared between the following groups: control vs. rutin alone or diclofenac alone; diclofenac alone vs. combination of diclofenac and rutin. The p values less than 0.05, 0.01, and 0.001 were considered statistically significant.

Rutin attenuated MDA and GSH content in cardiac tissue
MDA (p < 0.001) and GSH (p < 0.01) content in cardiac tissue was notably enhanced and reduced, respectively upon diclofenac treatment (Figure 2). Simultaneously treatment of rutin (20 mg/kg) with diclofenac significantly declined and enhanced the MDA content (p < 0.05) (Figure 2(A)) and GSH level (p < 0.05) content (Figure 2(B)), respectively, compared  to diclofenac alone. However, these improved effect of rutin at 10 mg/kg lacks statistical significance.

Rutin prevented histopathological alterations in cardiac tissue
Histopathological examination of cardiac tissue was performed using the following standard features: inflammatory cells infiltration, RBC congestion, myocardial necrosis, and myocardial fibrosis (Figure 3). The scoring data were evaluated following the same statistical procedure using earlier reported literature (Khafaga and El-Sayed 2018, Nishi et al. 2018, Bhatt et al. 2021, Korkut Celikates et al. 2021). Diclofenac's treatment caused significant up-regulation of all the above-mentioned scoring parameters as compared to control (p < 0.001) ( Figure  4). In comparison to only diclofenac treatment, rutin at 10 mg/ kg showed significant improvement of only myocardial necrosis (p < 0.05), but rutin at 20 mg/kg significantly down-regulated all the histopathological parameters like inflammatory cell infiltration (p < 0.05), RBC congestion (p < 0.001), myocardial fibrosis (p < 0.001), and myocardial necrosis (p < 0.001) as compared to the diclofenac alone treatment.
Rutin preserved modification of surface architecture in cardiac tissue SEM analysis of cardiac tissue ( Figure S2, Supplementary Material) was carried out to understand the histopathological findings better. Presences of ridge and pit patterns were the foremost criteria to assess tissue surface architecture. Diclofenac's treatment caused the mark disappearance of Figure 6. The representative H & E-stained images of gastric tissue with a scale bar : (A) represents control group, (B) represents rutin alone at 20 mg/kg, (C) represents diclofenac alone at 10 mg/kg, (D) represents diclofenac in combination with rutin at 10 mg/kg, and (E) represents diclofenac in combination with rutin at 20 mg/kg. ridge and pit patterns. However, the ridge and pit orientation remained intact upon treatment of rutin along with diclofenac. Rutin at 20 mg/kg exhibited a more pronounced effect to preserve the diclofenac-mediated alterations in tissue surface architecture.

Rutin regulated protein expression of myocardial injury markers and apoptotic markers in cardiac tissue
Western Blot analysis of cardiac tissue lysate was performed to assess the regulation of the following protein expression: cTnT, ANP, FABP3, MYL3, Bcl-2, and procaspase-3 ( Figure 5). Diclofenac treatment caused considerable up-regulation of cTnT compared to control (p < 0.05) (Figure 5(A)). Concomitant treatment of rutin at 10 and 20 mg/kg with diclofenac significantly down-regulated the cTnT expression level compared to the diclofenac alone (p < 0.05).
ANP was substantially up-regulated due to only diclofenac treatment (p < 0.001) (Figure 5(B)). Simultaneous administration of rutin at 10 mg/kg (p < 0.001) and 20 mg/kg (p < 0.01) with diclofenac showed remarkable down-regulation of diclofenac treatment-mediated elevation of ANP expression.
MYL3 expression was assessed in all the experimental groups, and the observed effect indicated a lack of contribution by this protein during diclofenac-induced cardiac injury ( Figure 5(D)).
The protein expression of Bcl-2 was significantly downregulated in the diclofenac alone group compared to the control group (p < 0.001) ( Figure 5(E)). Although rutin treatment at 10 mg/kg did not considerably affect the up-regulation of Bcl-2 protein expression, it showed substantial effect at 20 mg/kg (p < 0.01) in combination with diclofenac.
Diclofenac treatment resulted in a significant decline in the protein expression of procaspase-3 compared to control (p < 0.05) (Figure 5(F)). Among the experimental dose levels of rutin, concomitant treatment of rutin with diclofenac at only 20 mg/kg (p < 0.05) up-regulated the procaspase-3 protein expression in a significant manner.
Rutin improved the pathophysiology of gastric tissue PGE 2 and TXB 2 levels in stomach tissue were notably declined upon treatment with diclofenac alone compared to the control p < 0.01 and p < 0.05, respectively ( Figure S3 and Figure S4, Supplementary Material). Rutin at only 20 mg/kg significantly improved the diclofenac-mediated alteration in the PGE 2 and TXB 2 levels (p < 0.05). Histopathological examination of gastric tissue illustrated that rutin in combination with diclofenac significantly prevented diclofenac-mediated gastric damage ( Figure 6). These assessments are based on the following standard features: inflammatory cell infiltration, epithelial cell erosion, chief cell degeneration, and RBC congestion. Diclofenac treatment caused significant up-regulation of all the parameters (p < 0.001) compared to the control group. Concomitant rutin treatment at 10 mg/kg with diclofenac showed significant improvement of only inflammatory cell infiltration (p < 0.001) compared to only diclofenac treatment. Still rutin at 20 mg/kg significantly down-regulated all the parameters as mentioned above (p < 0.001) (Figure 7). SEM analysis of the gastric tissue indicates the protective effect of rutin against diclofenac-induced gastric damage based on the intactness of the ridge to pit pattern in the gastric tissue surface architecture ( Figure S5, Supplementary Material). The observations are in line with histopathological findings.

Rutin shielded hepatic tissue injury
The level of MDA and GSH in hepatic tissue was significantly increased and decreased, respectively, upon treatment with diclofenac alone compared to the control group (p < 0.001) ( Figure S6 and Figure S7, Supplementary Material). Although rutin at 10 mg/kg did not significantly affect GSH levels, it displayed a marked effect on MDA content (p < 0.05). Rutin at 20 mg/kg with diclofenac showed marked improvement in the MDA and GSH content compared to only diclofenac treatment (p < 0.001 and p < 0.01, respectively). Histopathological examination of hepatic tissue showed that rutin has the ability to prevent diclofenac-mediated hepatic tissue injury when given in combination with diclofenac. These assessments are based on the following standard features: inflammatory cell infiltration, RBC congestion, hepatocyte ballooning, and hepatocyte necrosis (Figure 8). Diclofenac treatment caused significant upregulation of all the parameters compared to the control (p < 0.001). Rutin (10 and 20 mg/kg) significantly down-regulated all the parameters like inflammatory cell infiltration (p < 0.001 and p < 0.01, respectively), RBC congestion (p < 0.05 and p < 0.01, respectively), hepatocyte ballooning (p < 0.01), and hepatocyte necrosis (p < 0.01) in comparison to the diclofenac alone (Figure 9). Based on the intactness of ridge to pit

Discussion
Diclofenac is one of the extensively used NSAIDs to treat various inflammatory disorders. Nowadays, its use is prescribed at lower dose levels and/or for shorter duration (Spitz 2013, Ghosh et al. 2015. The reasons behind the restricted use of this effective drug are mainly cardiovascular and gastrointestinal toxicities. In the present study, we also observed the induction of cardiac injury under the experimental regimen. It is reported that diclofenac causes the generation of ROS, leading to apoptotic cell death in the cardiac tissue (Abdulmajeed et al. 2015, Abdel-Daim et al. 2018. In the quest to explore any role of rutin for diclofenac-induced cardiac injury in the rat model, we gratifyingly observed the protective action of rutin mainly at 20 mg/kg dose level after oral administration in the presence of diclofenac. Injury to any particular organ is associated with releasing specific and/ or nonspecific markers. As evident in the literature, CK-MB and cTnT are the two specific biomarkers for cardiac injury (Abdel-Daim et al. 2018, Oda and Derbalah 2018). On the other hand, LDH and SGOT are the two nonspecific markers of cardiac injury (Gupta et al. 2021). All these marker levels are elevated during degenerative/necrotic changes in the cardiomyocyte membrane leading to cardiotoxic situations (Latimer 2011, Oda andDerbalah 2018). The present study results suggest that rutin can restrict the elevation of these marker enzymes during diclofenac-induced cardiac injury. Injury to any organ mainly happens due to oxidative stress, linked to higher ROS generation. Thus, anti-oxidant defense phenomena activate upon any oxidative stress-mediated organ damage (Maity et al. 2012, Arafa et al. 2020. GSH helps to neutralize ROS and protect cells from oxidative stress-mediated cell damage (Chow et al. 2003). MDA, a by-product of lipid peroxidation, is served as a marker for oxidative stress. Our experimental results reveal that rutin can restrict exhaustion of cellular anti-oxidant defense and exert preventive action against oxidative stress due to diclofenacinduced cardiac injury.
Damage to any organ is associated with the release of inflammatory cytokines. In this direction, it is reported that TNF-a and IL-6 levels are enhanced in the serum during diclofenac-mediated cardiotoxic conditions (Zhang et al. 2019). The present experimental results indicate that rutin can inhibit these pro-inflammatory cytokines mainly TNF-a during diclofenac-mediated cardiotoxic situations.
ANP, a cardiac hormone, helps in maintaining the balance of body fluids and blood pressure. It is produced mainly by the atrial myocytes during cardiac injury (Kim and Kass 2016). ANP is reported to induce apoptosis in cardiac myocytes Figure 9. The bar-graph for scoring histopathological parameters of hepatic tissue among the different study groups: (A) inflammatory infiltration, (B) RBC congestion, (C) hepatocyte ballooning, and (D) hepatocyte necrosis. Data were compared between the following groups: control vs. rutin alone or diclofenac alone; diclofenac alone vs. combination of diclofenac and rutin. NS represents statistically insignificant. Significance level: Ã p < 0.01, ÃÃ p < 0.01, and ÃÃÃ p < 0.001. (Najenson et al. 2018). So elevated level of ANP contributes to ROS generation and subsequently induces oxidative stress leading to myocardial injury (Shah et al. 2011). On the other hand, FABP3 is mostly present in the heart muscle and rapidly releases during cell injury. It also promotes oxidative stress and apoptosis (Song et al. 2012, Shingu et al. 2018. The present study results suggest that rutin has a pronounced effect on down-regulating ANP and FABP3 that can minimize oxidative stress and apoptosis leading to protection of cardiac damage. The anti-apoptotic effect of rutin can also be explained by the specific marker for apoptosis. It is linked to mitochondrial impairment and Bcl-2 is considered as a gatekeeper for the apoptotic response (Takahashi et al. 2004). Procaspse-3, an apoptotic marker that upon activation resulting in the proteolytic cleavage leading to cell death (Zhang et al. 2005). The above-mentioned effect of rutin on various enzymes/proteins, along with histopathological observations and SEM findings, illustrates that rutin is efficient in conserving the heart against diclofenac-induced cardiac injury. Therefore, rutin possibly acts by minimizing the following events toward its cardioprotective effect against diclofenacinduced cardiac injury.
The present experimental results suggest that rutin can be additionally beneficial to prevent diclofenac-mediated gastric damage via normalizing the PGE 2 and TXB 2 levels as well as minimizing the oxidative stress in the liver tissue.

Conclusion
Based on the present investigational results, rutin is found to be an effective candidate against diclofenac-induced cardiac injury. Lowering in oxidative stress, inhibition of inflammation, and reduction of apoptosis by rutin possibly contributed to restrict the diclofenac-mediated cardiac injury. The present research work is the first-time report regarding the involvement of ANP, FABP3, and MYL3 in diclofenac-induced myocardial injury leading to cardiac damage. Moreover, rutin can be additionally beneficial to protect against diclofenacinduced gastric damage and hepatic injury. Further research work should be focused on developing phytotherapeutics that can counteract the dose-dependent side effects of diclofenac.