Therapeutic activity of sarpogrelate, and dopamine D2 receptor agonists on cardiovascular and renal systems in alloxan-induced diabetic rats

Background Dopamine D2 agonists are notable medications in the treatment of Parkinsonism, hyperprolactinemia, and hyperglycemia. An aliation showed between the enlistment of myocardial injury ailment and long haul treatment with dopamine D 2 agonist drugs identied with the partial initiation of 5-HT 2a receptors. Methods The investigation aims to represent the activity of sarpogrelate, a particular 5-HT (2A) receptor blocker, in diminishing myocardial injury prompted by extended haul utilization of D 2 agonist drugs in diabetic rodents. Both bromocriptine and cabergoline managed independently and combined with sarpogelate for about a month to diabetic nephropathy rats. Results Bromocriptine and cabergoline created a signicant reduction in BGL, BP, and kidney hypertrophy index in diabetic nephropathy rats. Administration of bromocriptine, cabergoline, alone, or in combination with sarbogrelate fundamentally diminished blood concentrations of ALP, AST, urea, and creatinine. Bromocriptine and cabergoline alone showed noteworthy ascending of LDH-1, Troponin I, and TNFα1 levels in the serum (p < 0.05). Paradoxically, utilizing bromocriptine and cabergoline with sarpogrelate treatment altogether diminished the degree of the myocardial biomarkers in the serum. A mix of bromocriptine or cabergoline with sarpogrelate diminished the level of the myocardial infarct size in the heart assessed utilizing the TTC staining method. Conclusions The examination exhibited that both bromocriptine and cabergoline could be utilized securely in blend with sarpogrelate for a long duration of treatment for diseases like hypertension and diabetes.

sympathetic nerve activity inhibition. Bromocriptine may secure against I/R injury of the kidney utilizing p44/42 mitogen-initiated protein kinase actuation [8] and preventing chronic nephropathy [9].
Conversely, the bromocriptine-actuated hypotension was wholly nulli ed by pretreatment with metoclopramide, a dopamine D2 receptor antagonist that crosses the barrier [10,11]. However, the use of D2 agonists is often related to cardiovascular complications, including hypotension and myocardial disorder. There is an on the spot relationship between the utilization of D2-like R agonist in patients with Parkinson's disease and cardiopathy, especially in the early phase of therapy [12]. D2 receptors are pleiotropic receptors having multiple effects in that activating them inhibits adenylyl cyclase, resulting in inhibition of voltage-gated calcium ions and activation of potassium conductance [13]. Early studies suggested that anterior pituitary hyperplasia correlated with hypersecretion of prolactin [14]. The use of D2 receptor agonists resulted in a reduction in tumor mass.
It demonstrated that long term use of bromocriptine therapy was associated with an increased risk of developing heart disease, which occurred in a cumulative dose-dependent treatment [15]. Dopamine D2 receptor agonists classi ed as ergot dopamine D2 agonists and non-ergot D2 agonists. Bromocriptine and cabergoline (ergot derivative) are related to valvular cardiopathy since the two drugs have both dopamine D2 and serotonin 5-HT (2A) receptors agonistic activities. Pramipexole (non-ergot derivative) shows few incidences of heart disease onset since it is no effect on 5-HT (2B) receptors [16,17,18]. Both sarpogrelate and ketanserin (selective 5HT2a/2b antagonists) constrict cardiac brokenness, infarct size, and changes within the ECG because of MI. Likewise, these outcomes bolster the view that serotonin and 5-HT (2A) may augment the harmful impacts of ischemic injury within the heart [19,20]. Sarpogrelate found to possess bene cial impacts in peripheral vascular sickness, restenosis after coronary stenting, pneumonic hypertension, intense and relentless myocardial localized necrosis [21]. The current study aims to identify the possible protective effect of sarpogrelate, a 5-HT (2A) receptor blocker, associated with long term use of D2 agonist drugs therapy on cardiac and renal functions in diabetic nephropathy model rats.

Animals
Wister albino male rats 5-7 weeks of age weighing 150-200g obtained from the animal house of Batterjee medical college. The animals were housed three per cage under controlled standard laboratory conditions in monitored ventilated cages and spontaneously given food and water. The steps of the research were approved by the ethical committee of research of Batterjee medical college. Tutelage was taken, particularly with relevant housing conditions, to avoid or minimize the animals' discomfort. The animals kept on solid oored cages with a deep layer of sawdust, and cages changed daily. Three laboratory technicians administered the treatment, and two investigators collected the data randomly. Data was coded prior to analysis so that the treatment group cannot be identi ed before analysis is completed. At the end of the study, all animals were euthanized by thiopental (intravenous injection, 150 mg/kg) for tissue collection.

Induction of diabetes by alloxan
Alloxan monohydrate dissolved in sterile normal saline. Diabetes was induced in 30 rats (150-200 g) by a single intraperitoneal injection of alloxan (5%) 150 mg/kg. The rats kept fasting for 12 h before the injection of alloxan. Fasting plasma glucose measured by obtaining tail blood samples. The rats which showed a plasma glucose level of 200 mg/dl or more were considered diabetic and taken in the study [22].

Experimental design
Tests took place four weeks after the induction of diabetes. The rats divided randomly into six diabetic groups and one healthy control group, each group of 6 rats. The sampling calculation is done based on resource equation method [23]. Once there was a stable rising in the urea and creatinine levels in the blood, drugs injected. 7. Diabetic group treated with a combination between cabergoline and sarpogrelate by the same doses.

Determination of blood glucose level
Blood glucose levels tested on the 0 day, 1st, 7th, 14th, and 21st days from the start of the experiment.
Blood samples collected from the tail of the fasting animals. One millimeter of its end cut and a drop of blood used for blood glucose tests using an advanced glucometer (Roche, USA). The accuracy of glucometer checked with the O-toluidine method [24].

Blood Pressure Recording
Basal blood pressure was measured employing a non-invasive blood pressure recorder apparatus (Ugobasile instruments, Italy).
Each rat placed in an exceeding restrainer and appropriate cuff sensor mounted on its tail and warmed to about 33-35 °C. The tail cuff was in ated to a pressure above 200 mmHg, systolic blood pressure; diastolic blood pressure was measured directly by the tail-cuff and pulse sensor two hours after treatment of drugs [25].

Estimation of liver & kidney functions
Alkaline phosphatase and aspartate aminotransferase activities determined using the method described by King and King (1954) [26]. The procedure of Tietz et al. (1994) [27] used to determine serum creatinine concentration, while the serum urea concentration determined by the method of Kaplan (1965) [28]. After the end of the study, the kidneys washed with normal saline and weighed. The kidney hypertrophy index calculated by determining the kidney weight and body weight ratio (g/g) x10 3 .

Estimation of serum biomarkers of myocardial injury
The blood was withdrawn through the retro-orbital venous plexus method, kept at 37°C for 30 mins, and centrifuged at 4°C, 3000 rpm for 10 mins. Then the separated serum stored at -20°C for various biochemical analyses. The severity of the cardiac injury assessed by the estimation of lactate dehydrogenase (LDH-1) and cardiac troponin I (cTnI) in serum. LDH-1 and troponin levels analyzed by spectrophotometric methods using commercially available diagnostic kits consistent with the methods of Nieland, 1955 [29].
Serum TNF-α1 concentrations Serum TNF-alpha cytokines level was identi ed by the ELISA technique using a quantitative sandwich enzyme immunoassay technique (Abcam Company). The test was done according to the company's instructions. The ELISA reader (optical density at 405nm immediately) calculated and applied on a standard curve to sort out the concentration of the cytokines.

Evaluation of Myocardial Infarct Size by TTC
For evaluation of the myocardial infarcted area, the hearts were removed, washed in phosphate-buffered saline, frozen and stored at −20°C, the frozen hearts sliced into 1 mm sections along the long axis from apex to base. Triphenyl tetrazolium chloride (TTC) staining accustomed to assess myocardial tissue viability and determine myocardial infarction size. The tissue slices incubated in 1% TTC PBS solution, pH 7.4, at 37°C for 20 min. Tissues xed in 10% PBS-buffered formalin overnight at 2°C-8°C. Either side of every TTC-stained tissue slice was photographed with the photographic camera to differentiate the red-stained viable and also the white-unstained necrotic tissues [30]. Digital photographs downloaded to a computer. Areas stained in white and red were measured using SigmaScan software (SPSS Science) in trace-measurement mode. That mode was wont to measure either the ischemic or the infarcted areas, which may be a sum of calibrated pixels during a de ned region. This was done manually by drawing an image layer on the photograph [31]. The following equation calculated the infarction size percentage: The percentage of Infarct volume = Infarct volume / Total volume of slice X 100

Statistical analysis
The data and statistical analysis done comply with the recommendations on experimental design and analysis in pharmacology [32]. The results expressed as mean± SE. The signi cance of the differences between the values performed by a one-way ANOVA test and Dunnett's Multiple Comparison Test using GraphPad Prism software. P < 0.05 was considered to be a signi cant difference.

Results
Estimation of blood glucose levels The diabetic control group showed a signi cant increase in BGL compared to the healthy control group. On repeated administration of the bromocriptine (4 mg/kg), cabergoline (0.6 mg/kg) individually or in combination with sarbogrelate a signi cant (p < 0.05) decrease in blood glucose by time compared to the diabetic control group (Table 1).

Kidney hypertrophy index
The results showed that the kidney hypertrophy index signi cantly increased in the diabetic group of rats compared with the normal control rats. However, the index markedly reduced by both bromocriptine and cabergoline treated groups, even individually or mixed with sarpogrelate (as seen in Table 2). There is no effect of using sarpogrelate on the diabetic kidney index.

Hemodynamic Parameter (Antihypertensive activity)
Alloxan-induced diabetes in rats caused a signi cant rise in blood pressure after three weeks from the induction of diabetes. Daily oral administration of bromocriptine and cabergoline individually or in combination with sarpogrelate showed a signi cant decrease in blood pressure (Table 3). There is no effect of sarpogrelate on BP in diabetic rats.

Estimation of Liver and kidney Functions:
The serum concentrations of AST, ALP, urea, and creatinine as an indicator of liver and kidney functions recorded in table 4. Data revealed that the diabetic control group had a signi cant increase in the previous biomarkers' serum concentrations compared to the healthy control rats. Daily administration of bromocriptine, cabergoline even alone, or in combination with sarbogrelate caused signi cantly decreased serum concentrations of the previous biomarkers, comparing to the positive control of diabetic rats. There is no marked effect of sarpogrelate treatment on the previous biochemical indicators in the diabetic rats.

Myocardial biomarkers
Bromocriptine and cabergoline treated animals for one month in doses 10 mg/kg, and 0.6 mg/kg respectively displayed signi cant elevation of LDH-1 level in the serum. By contrast, using a combination of bromocriptine and cabergoline with sarpogrelate treatment signi cantly decreases the level of the biomarkers in the serum. The results in table 5 indicated that the qualitative test of troponin I reagent kit showed only positive results with bromocriptine and cabergoline treated groups. The results of the ELISA test indicate that the expression levels of TNF-alpha 1 in the diabetic rats' groups treated with bromocriptine or cabergoline individually were signi cantly higher comparing to the diabetic control rats. The groups of combination between bromocriptine and cabergoline with sarbogrelate showed lower TNFalpha 1 expression levels comparing to the groups treated with bromocriptine or cabergoline individually ( Table 5).

Evaluation of myocardial injury
Myocardial infarction size was used as an indicator of the progress of the myocardial injury. The hearts of bromocriptine 4 mg/kg, and cabergoline 0.6 mg/kg treated groups showed a signi cant increase in risk area infarction. By contrast, the combination of bromocriptine or cabergoline with sarpogrelate (50 mg/kg) reduced the percentage of the myocardial infarct size (Figures 1A, 1B).

Discussion
Both bromocriptine and cabergoline induced hypoglycemic activity may be due to enhanced suppressive hepatic glucose production [33]. The exact mechanism of action as antidiabetic is not entirely identi ed; bromocriptine decrease hepatic production of glucose, increase glucose transporter production, or increase or mimic glucagon-like peptide-1 activity [34]. Its contribution to hypoglycemia may be due to adjustments in the neural control of seasonal and patterns of food intake and nutrient storage [35]. Bromocriptine was approved by the Food and Drug Administration (FDA) in May 2009 for the treatment of type 2 diabetes.
In the present study, diabetic nephropathy is the most common cause of renal complication and the leading of hypertension. Both bromocriptine and cabergoline showed a marked antihypertensive activity, which agrees with some previous reports [36,37]. This action related to dopamine D2 receptors acting drugs, which can lower the blood pressure by inhibition of Na/k ATPase activity, vasodilation, and inhibition of sympathetic nerve activity. Both bromocriptine and cabergoline induced a marked improvement in the kidney function by decreasing urea and creatinine serum levels. The pharmacological pathways that explain this effect have not been su ciently determined. The result agrees with some of the previous studies suggest that the development of therapies directed to increase renal D 2 receptor expression/function may provide novel and practical approaches in the treatment of renal injury [38].
The high noted adverse effect of bromocriptine and cabergoline on heart represented in myocardial injury and infarction approved in the present study by using relative overdoses for a month of treatment manner. The action may be related to dopamine D2 and serotonin 5-HT (2A) receptors agonistic properties, increasing the heart pumping rate. It has been reported that both bromocriptine and cabergoline (ergot derivative) have been associated with heart disease since the two drugs have both dopamine D2 and serotonin 5-HT (2A) receptors agonistic properties [17,18]. It is hypothesized that one component of  [40].
According to the current observed data, the combination of bromocriptine and cabergoline with the new chemical agent sarpogrelate (selective 5HT2a/2b antagonists) decreases the adverse effects of these two drugs on the heart. The protective effects of sarpogrelate on myocardial tissue were approved by its ability to decrease the secretion of myocardial biomarkers. As shown by decreasing LDH-1, Troponin I, and TNF alpha 1 during the treatment of bromocriptine and cabergoline. Sarpogrelate attenuates cardiac dysfunction, infarct size; these results support the view said that serotonin and 5-HT (2A) might contribute to the harmful effects of ischemic injury in the heart. Regarding the biochemical study, sarpogrelate drug can be considered a safe drug on liver and kidney functions.

Conclusions
According to the given study, both bromocriptine and cabergoline can be used safely in combination with sarpogrelate for the treatment of many diseases like hypertension, diabetes, and Parkinsonism. Thus, we hypothesize that sarpogrelate deserves additional experimental and clinical research related to cardiovascular diseases. Ethics approval and consent to participate:

Abbreviations
The study was approved in December 2019 by the ethical committee of research of Batterjee medical college. The committee consists of four people. The research protocol and the study plan were submitted.
The committee held a comprehensive discussion of risks and bene ts before reaching the approval conclusion. During the study, the research coordinator of the ethics committee updated the progress of the research. In addition, the research experiments on animals followed the guidelines of the experimental animals' research and ethics committee "EAREC", King Abdulaziz University.

Consent for publication
Not applicable Availability of data and materials Not applicable

Competing interests
No competing interests Funding There was no funding available for the current manuscript submitted.

Authors' contributions
All authors conceived and planned the experiments. MF carried out the implementation of the experiments. RM planned and carried out the statistical analyses and contributed to sample preparation.
MF took the lead in writing the manuscript. All authors contributed to the interpretation of the results, provided critical feedback and helped shape the research, analysis, and manuscript.   Values shown are means ± SEM; n = 6 rats per group. * P < 0.05, signi cantly different from normal control group; # P < 0.05, signi cantly different from diabetic control group. Values shown are means ± SEM; n = 6 rats per group. # P < 0.05, signi cantly different from diabetic control group. a P < 0.05, signi cantly different from diabetic group treated with bromocriptine. b P < 0.05, signi cantly different from diabetic group treated with cabergoline. Sarbogrelate treated group, (E) Cabergoline+ Sarbogrelate treated group, (F) Cabergoline+ domperidone treated group. B: Effect of the tested drugs on percentage of myocardial infarct size in alloxan-induced diabetic rats. Values shown are means ± SEM; n = 6 rats per group. # P < 0.05, signi cantly different from diabetic control group. a P < 0.05, signi cantly different from diabetic group treated with bromocriptine. b