Abstract: Background and Objective: Earthworms are oligochaete soil macroinvertebrates that play an essential role in soil fertility and production throughout time. In the present study, an attempt has been made to assess the therapeutic effects of earthworm extract against myocardial infarction induced by epinephrine injection in rats. Materials and Methods: Myocardial infarction was induced in rats by injecting 2 mg kg–1 epinephrine subcutaneously daily for 2 days. The rats were split into three groups: Control, MI and MI were given earthworm extract (60 mg kg–1, orally) for 7 days. Results: The treatment with earthworm extract restored ST-segment near normal, improved cardiac troponin T, creatine kinase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total proteins, albumin, creatinine, uric acid, urea, malondialdehyde, nitric oxide, reduced glutathione, catalase, glutathione-S-transferase. According to the histological study, the earthworm extract-treated group exhibited remarkable improvement in the studied heart muscles, liver and kidneys in various areas. Conclusion: Earthworm extract possessed a therapeutic effect against epinephrine-induced myocardial infarction. Earthworm extract improves cardiac, liver and renal biomarkers and restores the EEG pattern to its original state. The therapeutic effect of earthworm extract results from its antioxidant and anti-inflammatory activities.
INTRODUCTION
Cardiovascular disease (CVD) is the primary cause of mortality worldwide1. The CVD encompasses a wide range of ailments, including heart and blood vessel abnormalities, Angina, MI and heart failure2,3.
Myocardial infarction (MI) is an abrupt and prolonged decrease in myocardial blood flow that results in myocardial necrosis. It has marked by chest aches or discomfort that might spread to the shoulder, arm, back, neck or jaw4,5. For many years, MI has been one of the most frequent disorders seen in clinical practice and its prevalence has risen dramatically in recent years6.
The pathogenic mechanism of myocardial injury is still not completely understood, however, the significance of oxygen-derived free radicals in myocardial injury has been demonstrated7. Overproduction of reactive oxygen species (ROS) leads to cell membrane damage in the ischemic myocardium, resulting in contractile failure and finally acute MI8,9. As a result, treatment therapies with antioxidant or free radical scavenging action may help to reduce oxidative stress linked to IHD.
Epinephrine, a catecholamine secreted by the adrenal medulla, is one of the neurohormones implicated with cardiac stress, responsible for the "fight or flight" response10. It is also utilized in the emergency room to treat acute respiratory distress in patients with croup syndrome or bronchiolitis11,12. On the other hand, in high doses, Epinephrine was found to play a vital role in generating the highly cytotoxic reactive oxygen species (ROS) and depletion of the antioxidant enzyme associated with MI8. The ROS act directly on cells, causing lipid, protein and DNA damage with overproduction of NO2 and nitrosative derivatives and eventually cardiomyocytes damage12.
Many current medications, such as organic nitrates, calcium channel antagonists and blockers, help avoid cardiovascular illnesses, but their usage is generally restricted due to side effects and negative responses13. This is one of the reasons why many people all around the world, especially those in wealthy nations are turning to complementary and alternative medicine, which includes natural products. Plants and animals can provide natural products for use as natural medicines. The earthworm is one of the natural medications available14. The earthworm is an oligochaete soil macro invertebrate that serves a critical role in soils’ long-term fertility and productivity15. It has been widely used in traditional medicine around the world, especially in Asia, including India, Myanmar, China, Korea and Vietnam to treat fever, stomach discomfort, neck pain, brain diseases and digestive problems due to its multiple therapeutic characteristics16,17. Earthworm extract displays unusual antioxidant properties due to containing polyphenols18.
In the present study, an attempt has been made to assess the therapeutic effects of earthworm extract against myocardial infarction induced by epinephrine injection in rats.
MATERIALS AND METHODS
Study area: The experiments were performed at Cairo University between January and March, 2022.
Chemicals and reagents: The MISR Company provided Epinephrine for pharmaceuticals. All the other chemicals utilized were obtained from the Biodiagnostic Company (El Motor St, Dokki, EGY).
Collection of earthworms: Earthworms were gathered from commercial vermiculture in the Giza Governorate and kept in plastic tubs with decomposed organic debris until they were employed for research.
Earthworm extract preparation: According to Dewi et al.19, the whole extracted collected earthworm (1 kg) was rinsed with running water to remove the mucous from the surface Earthworms were immersed in distilled water for 6 hrs to remove dirt from their bodies with a continuous water exchange. After being cleaned, worms are then mashed into small pieces, smoothed and moved into a glass tube. About 80% of ethanol was added for 2 days and then evaporated in a water bath. The extract was then centrifuged at 3000×g for 10 min. The supernatant is filtered and then lyophilized (Christ freeze dryer) to get the extract.
Experimental animals: Adult male Wistar rats (Rattus norvegicus), weighing 190-200 g, were selected for the experiments. Ten rats were used to determine LD50, while eighteen rats were used for the main experiment. Rats were purchased from the National Research Center (NRC, Dokki, Giza). Animals were kept in hygienic polycarbonate boxes (6 rats per box), in the well-ventilated animal house of the Department of Zoology, Faculty of Science, Cairo University, Egypt. They were supplied with a standard laboratory diet and water ad libitum. The animals were housed under suitable conditions and handled a 12/12 hrs light-dark cycle at (22-25 ) room temperature. Before the experiment began, the animals were housed in the laboratory for 7 days to acclimate to laboratory conditions.
Ethical consideration: The Cairo University, Faculty of Science, Institutional Animal Care and Use Committee (IACUC) (Egypt) (CU/I/F/64/20) approved the study’s experimental protocols and procedures. All the experiments were carried out under international guidelines for the care and use of laboratory animals.
Acute toxicity study (LD50): The LD50 of AcCF was determined according to the method described by Chinedu et al.20.
Myocardial infarction induction: Myocardial infarction was induced in rats by injecting 2 mg kg–1 epinephrine subcutaneously once daily for 2 successive days21.
Experimental design: Eighteen rats were divided into three groups (6/group) as the following:
| • | Group 1 (control): Rats were injected with physiological saline on the 1st and 2nd day at an interval of 24 hrs and then received distilled water orally for 7 days |
| • | Group 2 (MI): Rats were injected with epinephrine (2 mg kg–1, S.C.) on the 1st and 2nd day at an interval of 24 hrs then received distilled water orally for 7 days |
| • | Group 3 (E. worm extract): Rats were injected with Epinephrine (2 mg kg–1, S.C.) on the 1st and 2nd day at an interval of 24 hrs, then received earthworm extract (45 mg kg–1, orally) for 7 days |
Measurement of ECG: One day after the final dose of Epinephrine the rats were kept overnight fasting and then they were anaesthetized with light anaesthetic 3% sodium pentobarbital. Rats were placed supine and leads of the conventional electrocardiograph (ECG 100 G single-channel Electrocardiograph-china) were connected to the dermal layer of both front paws and hind legs for the measurement of ECG.
Animal handling and collection of the samples: The blood samples of the animals were immediately collected in sterile centrifuge tubes by the exsanguination method. The heart, kidney and liver were enucleated and transferred to a filter paper for removing blood traces. Pieces of the tissues were stored at -80 for biochemical analyses. In 10% formal saline, another piece of them was suspended for preparative fixation for Histopathological fixation.
Biochemical estimation: The accessible kits were used for the estimation of cardiac troponin T, creatine kinase, lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), albumin, total protein, cholesterol, triglyceride (TG), creatine, uric acid and urea using biodiagnostic kits (Giza, Egypt).
Determination of oxidative stress parameters: In ice-cold 0.1 mol L–1 Tris-HCl buffers (pH 7.4), the heart, liver and kidney tissues were homogenized (10% w/v). The homogenate was centrifuged at 3000 g for 15 min at 40°C and the resultant supernatant was utilized to evaluate oxidative stress markers. Biodiagnostic kits were used to measure malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), glutathione-s-transferase (GST) and nitric oxide (NO) according to the manufacturer’s instructions (Giza, Egypt).
Histological examination: The heart, liver and kidney tissues were rapidly removed and embedded in paraffin, cut into 5 m sections and stained using hematoxylin and eosin (H&E). Photomicrographs were taken of the sections after they were examined under a light microscope.
Statistical analysis: Results were expressed as Means±SE. The comparisons within groups were carried out utilizing One-Way Analysis of Variance (ANOVA) with the Duncan post hoc test to compare the group means and p<0.05 was regarded statistically significant. The statistical analysis was performed using SPSS for Windows.
RESULTS
ECG patterns: The control rats showed a regular ECG pattern, whereas Epinephrine treated rats showed a marked ST-segment elevation. These changes were restored to near normal in earthworm extract treated rats (Fig. 1).
Cardiac function markers: There was a significant elevation (p<0.05) in the cardiac troponin T, creatine kinase, LDH and AST levels of the MI group compared to the control group. While significant decrease (p<0.05) was observed in the aforementioned parameters after treatment with earthworm extract compared with the MI group (Table 1).
Liver function markers: Compared with the control group, epinephrine-treated rats resulted in a significant increase (p<0.05) in levels of ALT, ALP and a decrease in TP. However, the treatment with earthworm extract ameliorates these changes in the ALT, ALP and TP (Table 2).
| Fig. 1(a-c): | ECG of the (a) Control group, (b) MI group and (c) Earthworm extract group |
| Table 1: | Effect of E. worm on cardiac biomarkers of myocardial infarction rats |
|
Experimental groups |
|||
| Parameters |
Control |
MI |
E. worm |
| cTn1 (ng mL–1) |
0.12±0.00a |
1.11±0.03c |
0.51±0.05b |
| LDH (U L–1) |
1415.99±28.27a |
2703.39±92.92b |
1566.57±135.70a |
| AST (U mL–1) |
127.25±3.95a |
169.66±5.65b |
139.01±4.58a |
| Creatine kinase-MB (ng mL–1) |
665.97±29.80a |
1382.80±24.62b |
742.88± 33.65a |
| Values are Mean±SE (n = 6 per group), each value not sharing a common letter superscript is significantly different (p<0.05) | |||
| Table 2: | Effect of E. worm on liver functions of myocardial infarction rats |
|
Experimental groups |
|||
| Parameters |
Control |
MI |
E. worm |
| ALT (U mL–1) |
42.89±1.04a |
67.95±2.38b |
46.49±1.55a |
| ALP (U L–1) |
220.55±8.12a |
425.60±16.22c |
299.90±9.01b |
| Albumin (g dL–1) |
4.82±0.08b |
4.52±0.10c |
4.69±0.03a,b |
| Total Protein (g dL–1) |
6.91±0.12b |
6.26±0.16a |
6.45±0.25a,b |
| Values are Mean±SE (n = 6 per group), each value not sharing a common letter superscript is significantly different (p<0.05) | |||
| Table 3: | Effect of E. worm on lipid profile of myocardial infarction rats |
Experimental groups |
|||
| Parameters | Control |
MI |
E. worm |
| Cholesterol (mg dL–1) |
72.85±3.49a |
77.28±3.39a |
74.49±3.38a |
| TG (mg dL–1) |
76.15±8.11a |
86.31±6.63a |
66.80±5.20a |
| Values are Mean±SE (n = 6 per group), each value not sharing a common letter superscript is significantly different (p<0.05) | |||
| Table 4: | Effect of E. worm on kidney functions of myocardial infarction rats |
Experimental groups |
|||
| Parameters | Control |
MI |
E. worm |
| Creatinine (mg dL–1) |
0.75±0. 01a |
1.14±0.03c |
0.83±0.02b |
| Urea (g dL–1) |
55.74±1.99a |
76.40±1.32b |
60.55±2.55a |
| Uric acid (mg dL–1) |
1.55±0.04a |
2.18±0.14b |
1.59±0.05a |
| Values are Mean±SE (n = 6 per group), each value not sharing a common letter superscript is significantly different (p<0.05) | |||
Lipid profile: Table 3 showed a non-significant change (p>0.05) in cholesterol and TG concentration of the MI group compared to the control group. Similarly, cholesterol and TG concentration showed non-significant change (p>0.05) after earthworm extract administration compared with the MI group.
Kidney function markers: A significant increase (p<0.05) in the concentration of creatinine, uric acid and urea of the MI group compared to the control group was shown in Table 4. On the other hand, a significant decrease (p<0.05) was noticed in these parameters after the earthworm extract treatment compared with the MI group.
| Fig. 2(a-f): | Photomicrograph of the heart, (a-b) Microscopic observation of heart sections in the control group, (c-d) Microscopic observation of heart sections in MI group and (e-f) Microscopic observation of heart sections in earthworm extract group (H&E) Infiltration of mononuclear inflammatory cells (arrow), Congested blood arteries (arrowhead) |
Oxidative stress markers: The MDA and NO levels in the MI group showed a significant increase (p<0.05), while GSH, CAT and GST decreased compared with the control group. The values of the aforementioned parameters returned to near-normal levels after treatment with earthworm extract (Table 5).
Histopathology of the heart: The endocardium, myocardium and epicardium had a normal histological structure in the control group. The myocardial had cardiomyocytes that were cylindrical and branched, with acidophilic sarcoplasm and centrally placed oval nuclei (Fig. 2a, b). Several histological abnormalities were seen in the cardiac muscles of the MI group. The cardiac myocytes revealed a multifocal region of fibrosis associated with myocarditis, which was characterised by the infiltration of mononuclear inflammatory cells (arrow). Cardiomyocyte dispersion was seen in several of the studied areas due to inflammatory oedema. Degenerated cardiomyocytes that were vacuolated and profoundly eosinophilic were commonly detected. Severely congested blood arteries (arrowhead) were observed (Fig. 2c, d). The Earthworm group showed normal histological structure in most examined sections except for a few sections that displayed edematous exudates with a fewer number of infiltrated inflammatory cells (Fig. 2e, f).
Histopathology of the liver: The livers from the control group had normal histologic structure, with hepatocytes grouped in typical lobular architecture with central veins (CV) surrounded by radiating hepatic cords. The portal triads had a typical histological structure, including hepatic artery, portal vein and bile duct branches (Fig. 3a, b).
| Table 5: | Effect of E. worm on oxidative stress biomarkers of myocardial infarction rats |
Experimental groups |
||||
| Parameters |
Organs |
Control |
MI |
E. worm |
| MDA (nmol g–1 tissue) |
Kidney |
2.94±0.19a |
5.02±0.16b |
3.35±0.21a |
|
Liver |
3.16±0.13b |
4.19±0.13c |
2.15±0.10a |
|
|
Heart |
2.06±0.19a |
3.41±0.22b |
1.55±0.10a |
|
| NO (μmol g–1 tissue) |
Kidney |
4519.64±301a |
6968.47±308c |
5434.30±300.76b |
|
Liver |
1944.98±59.01a |
2895.79±113.27c |
2615.19±31.21b |
|
|
Heart |
1615.20±106.95a |
2497.16±102.20c |
2225.55±12.30b |
|
| GSH (mg g–1 tissue) |
Kidney |
0.54±0.03c |
0.32±0.02a |
0.44±0.01b |
|
Liver |
0.61±0.01b |
0.36±0.03a |
0.65±0.07b |
|
|
Heart |
1.36±0.08c |
0.91±0.01a |
1.19±0.05b |
|
| GST (U g–1 tissue) |
Kidney |
0.64±0.08b |
0.28±0.02a |
0.40±0.02a |
|
Liver |
2.09±0.15c |
1.40±0.01a |
1.74±0.06b |
|
|
Heart |
0.32±0.01c |
0.10±0.00a |
0.22±0.04b |
|
| CAT (U g–1 tissue) |
Kidney |
181.30±5.82c |
106.33±16.30a |
147.44±3.88b |
|
Liver |
183.45±2.48b |
128.19±5.26a |
168.69±8.18b |
|
|
Heart |
137.14±1.37c |
39.83±2.77a |
52.98±1.07b |
|
| Values are means±SE (n = 6 per group), each value not sharing a common letter superscript is significantly different (p<0.05) | ||||
| Fig. 3(a-f): | Photomicrograph of the liver, (a-b) Microscopic observation of heart sections in the control group, (c-d) Microscopic observation of heart sections in MI group and (e-f) Microscopic observation of heart sections in earthworm extract group (H&E) Central veins (CV), mononuclear inflammatory cells (arrowhead) |
| Fig. 4(a-f): | Photomicrograph of the kidney, (a-b) Microscopic observation of heart sections in the control group, (c-d) Microscopic observation of heart sections in MI group and (e-f) Microscopic observation of heart sections in earthworm extract group (H&E) Inflammatory cells infiltration (arrow), hyperemia (arrowhead) |
The MI group revealed many histological changes in the investigated hepatic tissue. Random hepatic necrosis was observed in the hepatic lobules, which was coupled with necrobiotic alterations in the hepatocytes. In several of the investigated sections, the portal region revealed oedema associated with fibroplasia. In the hepatic parenchyma, multifocal aggregations of mononuclear inflammatory cells (arrowhead) were seen (Fig. 3c, d). Improvement of hepatic tissue was detected in rats who received earthworm.
However, few sections showed portal oedema associated with few inflammatory foci in the hepatic lobules (Fig. 3e, f).
Histopathology of the kidney: Microscopic investigation of kidney tissue from the control group (Fig. 4a) indicated normal histology of both the renal cortex and the medulla, the renal cortex included both glomeruli and renal tubules. The renal medulla had both kinds of tubules and collecting ducts. Examining the MI group (Fig. 4b, c) exhibited significantly congested renal parenchyma that was visible in the renal cortex, followed by dilated renal blood vessels and the formation of eosinophilic casts in the tubular lumen. Some examined medullary regions showed numerous inflammatory cells infiltration between renal tubules (arrow). Concerning the earthworm group (Fig. 4d, e) apparently normal kidney cytoarchitecture in numerous examined sections. Few sections showed mild hyperemia in the renal cortex (arrowhead) with few detectable renal casts and mildly dilated tubules in the medullary tubules.
DISCUSSION
Cardiovascular disorders are linked to high rates of morbidity and death worldwide22. Myocardial infarction (MI) is caused by an epinephrine overdose, which produces coronary vasoconstriction, raises myocardial oxygen demand and reduces myocardial blood flow23.
In the current study, myocardial infarcted rats showed elevation in ST-segment in EEG and increases in the levels of cardiac troponin T (cTnT), creatinine kinase (CK) and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST). Abnormalities on an ECG are the most common criterion for a definitive diagnosis of myocardial infarction24. These changes might be caused by oxidative stress that results in loss of cell membrane in the damaged heart25,26. Furthermore, myocardial creatine kinase leakage has been linked to ST elevation27. Epinephrine can also cause cardiac necrosis and expedite myocardial cell death28, as seen in the current study’s heart histology of MI- mice. These ECG changes are substantiated by the fact that epinephrine injection resulted in a significant increase in cardiac troponin T (cTnT), creatinine kinase (CK) and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST), which are markers of cardiac necrosis29.
The cTnT, a well-established cardiomyocyte damage biomarker, is the chosen biomarker for acute MI diagnosis30. Although the mechanism of troponin release is unknown, it has been proposed that catecholamine-induced oxidative stress causes beta-adrenoreceptor hyperactivation, which leads to complicated functional, structural myocardial injury and permanent cellular damage and results in a release of cell proteins into the bloodstream8,31.
The LDH, AST and CK, which are diagnostic indicators of cardiac tissue damage, leak out of injured tissues into the bloodstream when the cell membrane becomes permeable or ruptures. Changes in plasma membrane integrity and/or permeability are reflected in the quantity of these cellular enzymes present in plasma26,32,33.
The current study showed that earthworm extract had cardioprotective properties, confirming normal ST-segment, restoring heart function to normal levels and protecting cardiac tissue from harm caused by a high dosage of Epinephrine.
The current investigation found a relation between MI and the liver by the significant increase in alanine amino transaminases (ALT), alkaline phosphates (ALP) and a reduction in total protein (TP) and albumin levels. In addition, histopathology analysis of liver tissue in the MI group demonstrated histological damage. The liver is a vital organ implicated in cellular redox equilibrium due to the synthesis of different intracellular antioxidant enzymes responsible for ROS clearance34. Clinical studies have demonstrated that hepatic necrosis can occur following an acute myocardial infarction35,36. Previous research has indicated that epinephrine-induced MI rats had reduced enzymatic free radical defence mechanisms, leading to increased oxidative stress that can contribute to pathological liver damage37.
The earthworm extract treated rats showed improvement in liver function markers and histology. Suppresses the creation of reactive oxygen groups or scavenges them while concurrently modulating the genes responsible for antioxidant enzyme synthesis16, inflammation suppression38, cell regeneration acceleration and hepatic cell secretory processes enhancement are the hepatoprotective mechanisms of earthworm extract39.
Current findings suggested that the kidney's capacity to operate had deteriorated, as demonstrated by the significant increase in creatinine, urea and uric acid and histopathological investigation. One of the fundamental MI caused by i.p., epinephrine injection is renal complication40. The pathogenetic mechanism of epinephrine-induced acute renal failure has been linked to ischemia insult, which contributes to the maintenance of renal dysfunction40,41.
The renoprotective action of earthworm extract was discovered for the first time in this study, which was validated by restoring kidney function markers to normal levels and histological examination of renal tissue.
A high dosage of epinephrine produced oxidative stress in MI rats’ hearts, livers and kidneys, as evidenced by large increases in MDA and NO levels and significant decreases in GSH, CAT and GST levels. The administration of high doses of epinephrine has been shown to cause significant oxidative stress and necrotic lesions in various organs42. Quinone metabolites of epinephrine can cause oxidative stress by reacting with oxygen to form ROS and interfering with antioxidant enzymes43,44.
One of the most significant oxidative mechanisms that lead to cardiac cell damage is the conversion of Epinephrine to adrenochrome by superoxide anions45,46. Adrenochrome (C9H9NO3) is a poisonous ortho-quinone molecule that binds with the sulphydryl groups of numerous proteins, including enzymes and reacts with oxygen to produce O2– and H2O2 are part of its essential characteristics47.
Earthworm extract showed potent antioxidant activity against MI-induced oxidative stress. It is a phenolic compound with anti-inflammatory48 and antioxidant properties49. Because phenols contain hydroxyl groups, they are particularly effective for scavenging free radicals50. Since the earthworm’s polyphenol content is high, the earthworm’s anti-inflammatory and antioxidative properties may be linked to it41.
These results showed the therapeutic ability of earthworm extract in the treatment of myocardial infarction, which opens the area to use this extract in clinical studies. More advanced studies on earthworm extract could be needed using many doses and periods before the clinical applications.
CONCLUSION
It may be concluded from the findings of this study that earthworm extract has therapeutic for myocardial infarction. Earthworm extract improves cardiac, liver and renal biomarkers and restores the EEG pattern to its original state. The therapeutic effect of earthworm extract results from its antioxidant and anti-inflammatory activities.
SIGNIFICANCE STATEMENT
This study discovers the therapeutic effect of earthworm extract for the treatment of myocardial infarction can be beneficial for the treatment of heart diseases. This study will help the researcher to uncover the critical areas of using natural products isolated from earthworms in medicine that many researchers were not able to explore. Thus a new theory on the uses of earthworm natural products in medicine may be arrived at.