Cardiovascular disorders is a significant public health issue worldwide. Globally, heart failure is becoming a serious and expanding public health concern [1]. According to data from the American Heart Association, 6.2 million Americans over 20 years have heart failure. According to predictions, there will be a 46% increase in the prevalence of heart failure between 2012 and 2030, affecting nearly 8 million people over the age of 18, with a total percentage increase from 2.42% in 2012 to 2.97% in 2030 [2]. Coronary artery disease is the most common underlying cause of heart failure in high-income nations. According to Yuyun et al. [3], hypertensive heart disease, cardiomyopathy, and rheumatic heart disease have historically been identified as the main causes of heart disease in sub-Saharan Africa (SSA). An increase in the frequency of cardiovascular disease risk factors like diabetes and hypertension, heart failure is becoming more common in Sub-Saharan Africa as a result of an epidemiological transition. According to Agbor et al. [4], it represents 9.4%–42.5% of total hospital admissions in Africa. Owing to the significant cost that cardiovascular disease (CVD) places on public health systems, prevention is now a crucial aspect of clinical practice and research. It is focused on identifying and managing a number of risk factors, both modifiable and non-modifiable [5]. Scientists and the general public are becoming more aware of the detrimental effects of a diet high in fat and its tendency to promote atherosclerosis, cardiovascular illnesses, fatty liver disease, and stroke [6]. For instance, CAD is essentially non-existent in traditional African civilizations, yet rates among African Americans are comparable to those among Caucasian Americans. Many dramatic variations in rates over time within nations and across populations that migrate suggest that environmental factors, such as diet and lifestyle, rather than genetics, are the main determinants of many diseases.

The original purpose of consuming fish oil was to ensure enough intake of vitamin A and D, as they have long been recognized as critical micronutrients for the body's regular processes (homeostasis). The interest and understanding of the importance of marine omega-3 polyunsaturated fatty acids (PUFAs) in human health did not grow until the well-known publication of Dyerberg and Bang on the Inuit diet in the mid-1970s [7].

PUFAs have contributed to a breakthrough in the understanding of the health benefits of omega-3 fatty acids obtained from fish oils, which are primarily blends of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In order to support adequate neuronal functioning (learning capacity, mental development, and visual acuity) during the early stages of life, DHA is physiologically essential and must be present in high amounts in the brain and retina [8]. On the other hand, EPA is thought to be helpful in preventing adult cardiovascular disorders in adults. The proportions of EPA and DHA that offer the greatest benefit are also unknown, as is the quantity of n-3 PUFA required to produce health advantages [9].

Cameroon, like other low- and middle-income countries, is currently undergoing a socio-economic transition characterized by improving standards of living, rapid urbanization, and westernization of lifestyles, including an increased level of unhealthy diet, insufficient physical activity, tobacco use, and the harmful use of alcohol [10], thus increasing the prevalence of cardiovascular diseases. Existing drugs such as doxorubicin used in the treatment of heart failure showed some side effects that negatively affect health. More still, drugs available in pharmacies for cardiovascular diseases like Doxorubicin (approximately 320,000 FCFA) and Gemfibrozil (approximately 50,000 FCFA) are very expensive and not easily affordable by some of the population. Treatment of these cardiovascular diseases using food supplements is less expensive than getting drugs from the pharmacy. Sea foods are rich in PUFAs; these include crustacean fishes because algae are their main source of feeding. Sea fish consumed in Cameroon are rich in PUFAs are mostly consumed, and that could possess beneficial effects on health [11].  So, oil from raw Sardinella maderensis PUFA-rich fish could be a promising solution for decreasing the rate of cardiovascular diseases in Cameroon [12]. The current work will look at the potential of sardine fish oil to protect against doxorubicin-induced cardiotoxicity in an experimental rat model. It will also assess the effects of fish oil on oxidative stress, lipid profile, and serum enzyme activity.

Sample Collection and Preparation

Freshly caught Atlantic sardine fish (Sardinella maderensis) were purchased at the cold store, CONGELCAM, in Buea, Southwest region, Cameroon. As soon as they were bought, they were transported to the University of Buea's Life Science Teaching Laboratory and stored in a freezer for later use. The fish/ice ratio was 1:2 (w/w). The fish's internal organs were removed, and it was thawed with tap water before usage. The fins, scales, and bones were removed, and the fish was cut lengthwise along the backbone to yield the maximum amount of flesh, which was used to make fish fillet.

Oil Extraction and Quality Assessment

Oil was extracted using the Bligh and Dyer method (AOAC, 1995). The physicochemical properties of the oils were determined following the AOCS method (1992) to assess their quality and purity. These included free fatty acid content, saponification value, iodine value, peroxide value, p-anisidine value, and TOTOX value.

Animal Bioassay

Ethical Clearance

An ethical clearance for animal handling and care was obtained from the University of Buea-Institutional Animal Care and Use Committee (UB-IACUC) with Ref (2023/03/UB/IACUC/BTU/FS) of March 10, 2023, in order to carry out this study.

Animal Distribution and Grouping

A total of 36 male Wistar rats (180–200 g) aged 3–4 months were purchased at the animal house of the Department of Zoology and Animal Physiology, Faculty of Science, University of Buea, and were divided into six groups each consisting six animals. Each group was kept in well ventilated cages in a well-ventilated lab where they were raised and were allowed to acclimatize to the laboratory conditions (temperature of 24-27°C and 12-hour light-dark cycle) for one week prior to the commencement of the experiment. The animals were fed ad libitum (a solid pellet diet) and allowed free access to water throughout the experiment.

The rats were randomly divided into a control group, a negative control group, and a positive control group, and the remaining groups were the test groups (Table 1). Sardinella maderensis oil was given by oral gavaging according to Hocking et al. [13] using a curved 18 gauge, 2.25 mm ball diameter, and 2 inches in length. After the last intraperitoneal injection of doxorubicin (day 28), 24 hours later,       all the experimental animals were anesthetized by intraperitoneal administration of both ketamine (60 mg/kg) and xylazine (10 mg/kg) [14] and were sacrificed for serum preparation and organ collection.

Table 1: Treatment Administered to the Rats during the 28 Days’ Study.

Assessment of Body Weight, Food Intake, and Water Intake

Body weight, food intake, and water intake were recorded every day for 28 days using a weighing balance [19].

Collection of Blood and Serum Preparation

Blood was collected from each anesthetized rat by using capillary tubes. The collected blood was stored in microcentrifuge tubes, and following clot formation, serum was obtained by centrifuging the whole blood at 3,000 rpm for 20 minutes. Serum was stored for further biochemical analyses (serum enzyme activity, lipid profile, and oxidative stress).

Measurement of Serum Enzymes and Lipid Profile

Lipid profile: Triglycerides, total cholesterol, and HDL cholesterol were evaluated, and LDL cholesterol was calculated from measured values of total cholesterol, triglycerides, and HDL cholesterol according to the relationship: [LDL-chol] = [total chol] - [HDL-chol] [TG]/5 and [TG]/5 were estimates of VLDL-cholesterol [53]. Serum markers (aspartate transferase, alanine transferase, lactate dehydrogenase, and creatine kinase) were measured with commercial kits (Chronolab) according to the methods described by [20-24]. Measurement of Nitric Oxide (NO)/Nitrite

The dosage of NO was done using liver homogenate of β-GaIN/LPS mice, indirectly through the determination of nitrite by Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylene diamine in 2.5% phosphoric acid) [25]. The absorbance of the preparation was measured at 570 nm with the spectrophotometer (Genesis 20), and the nitrite level was determined by using the sodium nitrite standard curve.

Oxidative Stress

Malondialdehyde (MDA) Determination

MDA as an indicator of lipid peroxidation in tissues was determined in the heart, lungs, kidneys, brain, liver, and serum of rats by the method described by [26]. Tissue homogenate (1 ml) was added to 0.5 ml of trichloroacetic acid (20%) and 1 ml of thiobarbituric acid (0.67%). The mixture was allowed to react for 10 minutes at a high temperature (90 ?C, water bath). The mixture was centrifuged, and the absorbance of the supernatant was measured at 530 nm. The concentration of MDA was quantified with an extinction coefficient of 1.56 × 105 M-1?cm-1 and expressed as µM of MDA per g of protein.

Reduced Glutathione Level

The assay of reduced glutathione was performed following the protocol described by Kumar et al. [27]. Liver tissue homogenates (10 µL) were added to Ellman’s reagent (1500 μL). The mixture was then incubated for 1 hour at room temperature, and the absorbance was determined at 412 nm. The amount of reduced glutathione (mol/mg of protein) was calculated with the molar extinction coefficient (13,600/M × cm).

Catalase Activity

Catalase activity was evaluated according to the protocol of [25]. Briefly, 25 µL of homogenate and 375 µL of phosphate buffer (0.1 M, pH 7.5) were mixed. Hydrogen peroxide solution (100 μL, 50 mM) was introduced in the mixture, and the reaction was stopped one minute later by adding 1 mL of dichromate/pure glacial acetic acid. All tubes were heated (100 ?C) for 10 minutes. After cooling, the absorbance was read at 620 nm, and catalase activity was determined using the calibration curve and expressed as mmol of H2O2/min/mg of protein.

Superoxyde Dismutase (SOD) Activity

The activity of superoxyde dismutase was evaluated according to the protocol established by Temdie et al. [25]. Liver homogenate (67 µL) and carbonate buffer (833 µL, 50 mM, pH 10.2) were added to the adrenaline solution (100 μL, 0.3 mM). The absorbance of the preparation was read at 480 nm, 20 s, and 80 s after the introduction of adrenaline in the medium. The specific activity of SOD was expressed as SOD units per mg of protein.

Data Analysis

GraphPad software data analysis program version 3 was used for statistical analysis (GraphPad Software, USA). The data were analyzed by one-way analysis of variance (ANOVA) and presented as means ± standard deviation (SD). Individual differences were determined by the Bonferroni post-test. A value of less than 0.05 was considered statistically significant.

Oil Quality Indices of Sardinella maderensis Fish Oil 

The physicochemical properties of Sardinella maderensis fish oil evaluated are presented in Table 2. The free fatty acid value was evaluated in terms of acid value and converted to percentage as shown in the table. The table revealed that the physicochemical properties were within the accepted range by Codex Alimentarius (2017), except for the free fatty acid value with a value of 0.98, which is slightly lower than the standard value.

Table 2: Characterization of Sardinella maderensis Fish Oils Physicochemical Properties.

Invivo Studies

Changes in Body Weight 

The change in weight for all the groups of rats is summarized in Figure 1. The weekly change in weight for all the groups was low as compared to the normal groups from the second week. However, across all the groups, there was an increase in weight from the third week.

Figure 1: Weekly Change in Weight.

Effect of Sardine Fish Oil Treatment on Serum Markers

Table 3 summarizes the serum marker concentrations in the serum of the rats. Heart failure induced by doxorubicin caused a significant increase in serum marker concentrations compared to the other groups. The effect of treatment with sardine fish oil showed a significant reduction in the serum cardiac markers for the test groups compared to the negative control group. Treatment with sardine fish oil showed a significant reduction in AST, ALT, ALP, and LDH levels compared to the Gemfibrozil-treated group (positive control). Also, the activity of fish oil ALT in Test Group I was lower than that of the Positive Control Group, however, having a greater value than the latter (p<0.001, p<0.01, p<0.05).

Table 3: Serum Marker Concentrations of the Different Groups.

Values are mean ± SD (n = 6). Data were analyzed by one-way ANOVA followed by a Bonferroni post-test for multiple comparisons shown on the table with letters (a, b, c, d, and e). The same letters show the p-values are not significant. *Significant change with respect to Normal Group, Negative Control group, the Positive Control group, Test group I, II and III with (250, 500, and 1000 mg/kg, respectively) (*: p<0.05, **: p< 0.01, and ***: p < 0.001).

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