Cardioprotective molecular mechanism of syringic acid against isoproterenol induced post- myocardial toxicity in male albino wistar rats

2.1 Animals

Male albino wistar rats were allowed to acclimatization for one week in animal house. 25 °C temperature, 12/12 light and dark cycles and ventilation were subjected to better management. Rats were provided with sufficient food and water. Sri Krishnadevaraya University, ananthapuramu, India got ethical clearance for conducting experiments on animals from Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Regd. 1889/GO/Re/S/16/CPCSEA), and this study was accepted by the Indian Animal Ethics Committee (IAEC) protocol No. SKU / Biochem / 08 / 2017.

2.2 Experimental protocols

Totally 24 rats were divided into 4 groups, each group with 6 rats.

Group I: Control group: rats received Dimethyl sulfoxide (DMSO) alone for 7 days

Group II: ISO + SA group: on 1st and 2nd days (with a gap of 24 h), rats received ISO (30 mg/kg bw) and dialy intake of SA (50 mg/kg bw) orally for 7 days

Group III: ISO group: on 1st and 2nd days (an interval of 24 h was maintained), rats received ISO (30 mg/kg bw)

Group IV: ISO + MP group: on 1st and 2nd days (with a 24 h interval), rats received ISO (30 mg/kg bw) and rats dialy intake with metaprolol (MP) (10 mg/kg bw) orally for 7 days.

DMSO was used to dissolveSA, water was used to soluble MP and ISO. Rats of group II and IV were subjected to oral post-treatment with SA and MP respectively through intragastric tube for 7 days. On 1st and 2nd days of experiment period (with a gap of 24 h), ISO was injected sub-cutaneously to rats of group II, III and IV and rats of group II and IV were treated with SA and MP the respectively, immediately after the second dose of ISO and continued post-treatment for upto 7 days.

2.3 Sample collection

All rats were sacrificed using cervical decapitation method following anaesthetization with sodium pentobarbital (35 mg/kg I.P.) at the end of study period. Serum and hearts were gathered right away and finally homogenate of heart tissue was prepared after washing with physiological saline (ice-cold) and subjected to centrifugation for the collection of supernatant to carry out different biochemical examinations. At −80 °C, serum and heart tissues were conserved for further analysis.

2.4 Biochemical measurements

All marker enzymes for heart injury such as creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were estimated by diagnostic kits except gamma glutamyl transferase (GGT) which was analysed by the method of Young and Stirling Donald, 1995. Anti-oxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were measured by the methods of Marklund and Marklund, 1974; BEERS and SIZER, 1952 respectively. High sensitivity-C reactive protein (hs-CRP) was measured with diagnostic kit. Body weights and heart weights of rats were also measured.

2.5 Western blot

Left ventricles of myocardium were taken to separate proteins. Cardiac tissues were homogenized, lysed and 10% (w/v) Polyacrylamide gel and 0.1% (w/v) SDS were used to resolve tissue homogenate by the technique of electrophoresis. A film of nitrocellulose was utilized for the transfer of proteins electrophoretically. Incubation of nitrocellulose layer was done with primary and secondary antibodies that are linked with horse-radish peroxidase enzyme. The formation of antigen-antibody complexes were recognized with the enhanced chemiluminiscence detection reagent kit (Biorad). 1:1000, 1:10,000 dilutions were used for anti-Nuclear factor-kappa B (NF-kB) and anti-Tumor necrosis factor-α (TNF-α) antibodies and secondary antibodies respectively.

Quantification of western blot was done with image J software (version 1.49) and expressed as percentage of control group.

2.6 Ultra structural studies of heart tissues by transmission electron microscopy (TEM)

Tiny pieces of heart tissues were rinsed by 0.1 M phosphate buffer (pH 7.2). After the cutting of 1mm thick pieces of heart tissues, fixation was done into 2.5% ice chilled glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and maintained for 12 h at 4 °C, finally post fixed with the help of osmium tetroxide (buffered). TEM examination was performed by the heart tissue processing. Staining of grids containing sections was done with the help of uranyl acetate (2%) and lead acetate (0.2%). TEM (4000×) was utilized for analyzing these segments of heart.

2.7 Statistical study

To perform the statistical study, results were subjected to Duncan’s Multiple Range (DMR) test by considering p value (p < 0.05). Data was expressed as means ± SD.

3.1 Effect of SA on cardiac marker enzymes

SA effect on Cardiac marker enzymes such as CK-MB, LDH and GGT in heart tissue and serum are shown in table 1. A significant (p < 0.05) change of CK-MB, LDH and GGT levels in heart and serum were found to be decreased and augmented respectively in ISO managed rats, compared to control rats. When ISO managed rats were post-treated with SA and MP, these marker enzyme levels were significantly (p < 0.05) brought up and down in heart and serum respectively, compared to ISO alone operated rats.

3.2 Effect of SA on anti-oxidant enzymes

SOD and CAT are affected by SA post-treatment in heart tissues (Table 2). A significant (p < 0.05) reduced activities of SOD and CAT were detected by ISO administration in rats, while comparing with control rats. SA and MP dramatically increased the activities of these enzymes significantly (p < 0.05) after post-treatment, compared to ISO injected rats.

3.3 Effect SA on expression levels of NF-kB and TNF-α

Fig. 1 demonstrate the effect of SA on the expressed protein levels of NF-kB and TNF-α in heart tissues. There was a significant (p < 0.05) increase in protein expression of NF-kB and TNF- α in ISO treated rats, when compared with control rats. Decreased levels of NF-kB and TNF- α were found significantly (p < 0.05) in SA and MP post-treated rats, compared to ISO administered rats.

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Fig. 1

Effect of post-treatment of SA on the protein levels of NF-kB and TNF-α levels in heart tissues of control and isoproterenol (ISO) treated rats. Results are mean ± SD values with superscript letters (a, b, c and d) significantly differ from one another [p<0.05, Duncan’s multiple range test (DMRT)].

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3.4 Effect of SA on hs-CRP

SA effect on inflammatory markers is exhibited in Fig. 2. A significant (p < 0.05) increase in the serum hs-CRP levels were detected in ISO injected rats in contrasting with control group of rats. Levels of hs-CRP in the serum revealed a significant (p < 0.05) decrease in SA and MP post-treated rats, when compared to ISO administered rats.

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Fig. 2

Effect of post-treatment of SA on high sensitivity c - reactive protein (hs-CRP) levels in serum samples of control and isoproterenol (ISO) treated rats. Results are mean ± SD values with superscript letters (a, b, c and d) significantly differ from one another [p<0.05, Duncan’s multiple range test (DMRT)].

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3.5 Effect of SA on body weights and heart weights

Impact of SA on weights of body and heart are expressed in Figs. 3A and B. After the completion of experimental period, in ISO group we found that body weights were decreased significantly (p < 0.05) and heart weights were increased significantly (p < 0.05). Interestingly we observed that a significant (p < 0.05) increase in body weights and a significant (p < 0.05) decrease in heart weights in rats, which were post-treated with SA and MP for 7 days.

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Fig. 3A

Effect of post-treatment of SA on body weights of control and isoproterenol (ISO) treated rats. Results are mean ± SD values with superscript letters (a, b and c) significantly differ from one another [p<0.05, Duncan’s multiple range test (DMRT)].

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Fig. 3B

Effect of post-treatment of SA on heart weights of control and isoproterenol (ISO) treated rats. Results are mean ± SD values with superscript letters (a, b and c) significantly differ from one another [p<0.05, Duncan’s multiple range test (DMRT)]. * Group is significantly not different with ISO+SA group.

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3.6 Effect of SA on ultra structural changes of myocardial tissues

Post-treatment effect of SA on heart tissues in control and ISO treated groups are shown in Fig. 4. Irregular ‘z’ lines, swollen mitochondria and fragmented myofibrils were found in the ISO administered heart tissues, when compared to control rats. SA post-treated heart tissues exhibited ‘z’ lines with regular arrangement, decreased content of fragmented myofibrils and decrease in the swelling of mitochondria.

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Fig. 4

Electron photomicrographs (TEM 4000×) of the heart tissues of normal, treated and isoproterenol (ISO) treated experimental rats.

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4.1 Effect of SA on cardiac marker enzymes

Cardiac marker enzymes such as CK-MB levels demonstrated that a marked increment in serum and reduction in heart tissue levels were detected in rats, which were induced by ISO. The reason may reflect that ISO administration leads to the necrosis of heart tissue, cellular damage with loss of permeability and functional integrity of the cell membrane (IR et al., 2008; Alam et al., 2018). Further increment in serum LDH and GGT, while decrement in heart LDH and GGT supported the cardiomyocyte membrane damage in ISO administered rats. SA post-administration exhibited a marked reduction in ISO induced elevated serum marker enzymes. Serum marker enzyme levels are reduced due to action of SA on maintaining integrity of the membrane, thereby preventing these enzymes leakage. Myocardial injury, which is caused by ISO administration, is mediated through the β1-adrenergic receptor.

Acute stimulation of β-adrenergic receptor not only produces reactive oxygen species (ROS) but also depresses total cellular anti-oxidant capability, down regulates the catalytic nature of copper-zinc SOD enzyme, m-RNA and protein and level of glutathione, leads to the damage to the integrity of the membrane and reducing the contractile capability of the heart and toxicity of myocytes, finally leads to the myocardial necrosis (Rathore et al., 1998; Srivastava et al., 2007). In our current approach, we detected that SA conserved the myocardium from ISO-caused myocardial functional and structural damage via decreasing the levels of serum diagnostic marker enzymes. Our results are in agreement with the previous results (Jia et al., 2017; Mnafgui et al., 2016; Ulla et al., 2017).

4.2 Effect of SA on anti-oxidant enzymes

SOD and CAT which are endogenous anti-oxidant enzymes are the first-line defense system against oxidative stress, destroying oxygen (O₂) and hydrogen peroxide (H₂O₂) before they interact to produce highly reactive hydroxyl radical. For the effective removal of oxygen stress in intracellular organelles, an effective equilibrium must be established between these anti-oxidant enzymes. Free radical induced cell injury is prevented efficiently with the help of SOD and CAT. SOD is the only enzyme, which uses a free radical as a substrate. Enhancement in the activity of SOD is a chief requirement to scavenge free radicals. CAT and/or GSH are in inactivated state even though SOD is in activated state; this situation is detrimental because SOD forms more cytotoxic product, hydrogen peroxide, which is not removed by the CAT and/or GSH when they are in inactivated state. So increase in SOD must sync with the increase in the activities of CAT and GSH for an overall beneficial effect (Syeda Nishat Fathima, 2019).

In ISO treated rats, we found a drastic decrease in SOD and CAT content in heart tissues. This may be due to ISO causes an imbalance between anti-oxidative defense system and production of ROS leads to the occurrence of MI (Zhou et al., 2008) through the generation of adrenochromes such as quinines via ISO auto-oxidation, finally leads to the synthesis of highly toxic O₂ free radicals such as superoxide (O₂⁻) and H₂O₂. Moreover Ca²⁺ overload and their consequences are crucial in ISO-caused injury and synthesis of ROS (Díaz-Muñoz et al., 2006). Cell membrane phospholipids and fatty acids are the key targets for the attack by ROS forming lipid peroxides, which survive for longer periods than ROS and can begin the chain reactions that enrich the oxidative damage to cells (Sharma et al., 2012). However the depletion of the anti-oxidant systems such as SOD occurs when the oxidative pressure increases. Reduction in the activity of SOD in ISO induced rats might be due to the enhanced formation of ROS, such as O₂⁻ and H₂O₂, which could reduce the activity of SOD. Post-treatment with SA to ISO administered rats increased these anti-oxidants due to possessing free radical scavenging, anti-oxidant activities and by increasing the availability of GSH levels. Earlier report supports the results of our research (Ashokkumar et al., 2018).

4.3 Effect SA on expression levels of NF-kB and TNF-α

Regulation of oxidative pressure, expression of inducible nitric oxide synthase, apoptosis and NF-kB activation will be carried out by the binding of TNF-α with TNF- α receptor (TNFR1) and TNFR2 (Ozsoy et al., 2008; Varfolomeev and Vucic, 2008). Nuclear factor-kappa B (NF-kB) is a complex of proteins, which participates in the transcription of DNA, production of cytokines and survival of cells. NF-kB is actually activated by the central receptor namely receptor activator of NF-kB (RANK), a type of TNFR. In normal conditions, NF-kB receptor is under inactivation by exhibiting interaction with Inhibitory kappa-B (Ik-B) in the cytoplasm. Whenever NF-kB is to be stimulated, Ik-B will get phospharylated and degraded, which results in the release of NF-kB from Ik-B and enters the nucleus, where we can observe binding with target gene promoter. An inflammatory response was shown generally in CVD. In myocardial infarctions, studies have found increased levels of pro-inflammatory cytokines such as TNF-α and NF-kB, which depend on the NF-kB activation (Karin and Ben-Neriah, 2000). In our research, we detected enhanced levels of NF-kB and TNF-α expression in the heart tissues of ISO operated rats, when compared with control rats, which might be due to ISO induced inflammation. Upon post-treating the rats with SA, we got a result that showing the drastic decrease in the NF-kB and TNF-α expression in the heart tissues, when compared to ISO rats. This result was due to the anti-inflammatory nature of SA, which may be brought by preventing the Ik-B degradation or by directly blocking the RANK. Our results show agreement with the past studies (Dai et al., 2019; Jia et al., 2017; Guo et al., 2011).

4.4 Effect of SA on hs-CRP

A major sensitive marker of inflammation, tissue damage and infection is indirectly participate in the coagulation process is a classic acute-phase protein, called hs-CRP. The lifespan of the hs-CRP is nearly 19 h in the plasma. There was a best correlation between total infarct size in AMI and circulating hs-CRP levels in clinical studies. Thus we can conclude that hs-CRP is a reliable diagnostic indicator for underlying coronary inflammation along with extent of myocardial necrosis. However previous research clearly exhibited the correlation between hs-CRP and AMI in human beings (Badiger et al., 2014; Carrero et al., 2019). Co-deposition of CRP was seen with activated complement in AMI (Lagrand et al., 1997) and research outcomes have clearly demonstrated that the response of CRP not only reflect the damage to tissues in this context but also show a marked role in the development of myocardial injury (Griselli et al., 1999). A recognizable role of CRP was noticed in the pathogenesis of CVD and as a predictor of CVD contain many effects of CVD such as clotting, oxygen radical synthesis, enhancing the adhesion molecule expression and plasminogen activator inhibitor1 and destabilization of plaque, which could lead to CVD.

A drastic surge in the hs-CRP levels was detected in the rats of ISO group, when compared to the normal group of rats. This may be occurred due to necrosis of myocardial cells, caused by ISO. CRP attaches to the phosphocholine, which is expressed on the surface of dead and dying cell. This results in the complement system activation, enhancing phagocytosis by macrophages (Bray et al., 2016). Levels of hs-CRP were decreased significantly in the serum of SA post-treated rats, upon comparing with control rats. This may be due to anti-oxidant and anti-inflammatory activity of SA.

4.5 Effect of SA on body weights and heart weights

In the present study, ISO injection was associated with decrease in body weights and marked increase in heart weights. Decrease in the body weights were linked to consumption of low diet by ISO administration (Patel et al., 2010). However post-treatment with SA exhibited a marked increase in body weights, which could be attributed to the capacity of SA to increase the diet intake in the rats of SA group. Increase in heart weight is might be due to increase in water content, increase in protein content and edematous intravascular space (Patel et al., 2010). When post-treated with SA, a recognizable decrement in heart weights was noticed. This effect will come due to SA post-treatment showed a marked decrease in the water content and protein content in heart tissues. Previous research studies are in good support with our research output (Wang et al., 2019).

4.6 Effect of SA on ultrastructural studies of myocardial tissues-TEM

To evaluate the cardioprotection of SA in post-myocardial infarction, TEM study was conducted to support the biochemical and molecular studies. Our results confirmed the cardioprotection of SA by exhibiting regeneration of heart tissues, which was previously damaged by ISO. Thus in our research, we found that all biochemical, molecular studies were further supported by TEM observations. Due to the generation of ISO induced more number of free radicals, mitochondrial damage was observed along with the dysfunction of mitochondria, which are the main characteristics of heart diseases. Heart tissues of ISO administered rats showed fragmentation of myofibrils as well as detectable alterations in size, shape and swelling of mitochondria. Deposition of products of lipid peroxidation occurs due to the GSH depletion, which is the major cause for the mitochondrial swelling. A significant regular arrangement of ‘Z’ lines, myofibrils as well as reduced swelling of mitochondria was detected in ISO administered rats, which were post-treated with SA. This outcome could be due to the free radical scavenging property and anti-oxidant activity of SA. Therefore mitochondrial swelling reduces by decreasing accumulation of lipid peroxidation products in mitochondria by increasing the GSH synthesis.