Investigating the Association of IL-1beta IL-8 & IL 11 with Commonly used Cardiovascular Biomarkers (CK-MB & Troponin) in Patients with Myocardial Infarction (MI)

Background: Myocardial infarction is a major cause of death and disability worldwide. The term myocardial infarction should be used when there is an evidence of myocardial necrosis in a clinical setting consistent with myocardial ischemia. Cardiac troponins (cTn) have emerged as the preferred diagnostic biomarkers. If troponin assays are not available, the best alternative is creatine kinase (CK-MB). Numerous studies have reported the ability of various inammatory biomarkers to predict future cardiovascular events. Therefore, we have decided to investigate inammatory cytokines (IL-1β, IL-8 & IL-11) to compare with routine biomarkers. Methods: 40 patients who were diagnosed with myocardial Infarction (at the rst 24 hours), have voluntarily participated in this research project. 5 cc of whole blood was collected, both myocardial routine biomarkers (Tropinin & CK-MB), serum lipid proles and blood sugar were evaluated after diagnosis of MI. Meanwhile, we have assessed the serum levels of three Interleukins (IL-1β, IL-8 & IL-11) by ELISA method. Results: First we have assessed blood sugar and all lipid proles. B.S and HDL were higher in males, while TG, Cholesterol and LDL were higher in females. Our data demonstrated that Troponin continued to increase signicantly throughout the 72 hours, while CK-MB fell dramatically over the period in question. Our result also revealed the rise in IL-1β serum levels (after 72 hours of MI diagnosis). However, this was not a signicant increment. On the other hand, both IL-8 and IL-11 have followed the same decreasing pattern till the end of experiment. It is interesting to note that IL-8 showed a dramatic fall during the experimental period. Conclusion: We concluded that inammatory cytokines biomarkers MI cases. A noticeable changes of plasma levels MI and follow-up.

IL-8 and IL-11 have followed the same decreasing pattern till the end of experiment. It is interesting to note that IL-8 showed a dramatic fall during the experimental period.

Conclusion:
We concluded that in ammatory cytokines would be a promising diagnostic biomarkers for MI cases. A noticeable changes of IL-8 plasma levels made it a decent candidate for MI diagnosis and follow-up.

Background
Myocardial infarction (MI) known as heart attack caused by the deprivation of adequate blood ow in part of the heart muscle which brings about some di culties such as the lack of oxygen supply and hurting heart muscles. MI is unfortunately highly associated with death and related di culties around the world. The main common symptoms are: chest pain, shortness of breath, sweating, nausea, vomiting, abnormal heart beating and some other factors. However, anyone who meets one of the following criteria would be diagnosed for MI: detecting any changes in cardiac biomarkers (speci cally Troponin), ischemia, ECG changes associated with new myocardial ischemia and last but not least is the pathological ndings of MI (1,2). Lots of myocardial infarction cases cannot be diagnosed due to their asymptomatic feature as well as the minor and not speci c ones. These are often recognized via the electrocardiogram (ECG) examination that represent the abnormal Q wave. We have to take into account that some biomarkers have been released into the blood circulation as a result of heart injury. Researches revealed that these biomarkers might be able to detect ischemia faster and even with higher sensitivity than ECG results. Detectable cardiac biomarkers which are found in the circulation as a consequence of cardiac injury include: myoglobin, cardiac troponin, creatinine kinase (CK), LDH and some other factors.
Both troponin and creatinine kinase (speci cally CK-MB) have the higher sensitivity and speci city than the other factors mentioned above (3,4).
In terms of sensitivity and tissue-speci city, cardiac troponin is the better match to recognize the myocardial injury rather than some other mentioned biomarkers. Cardiac troponin isoforms (cTnT and cTnI) are measured for myocardial infarction because they can be detected approximately 2-4 h after the onset of MI, reach their peak after 24-48 h and remain in the blood circulation for the next 1-2 days on average (5,6). In times of no availability for troponin assessment, CK-MB could be the great alternative. It depicts the same pattern and kinetics as troponin, with slight differences in details, although it is not comparatively sensitive to detect small MI. The serum levels of CK-MB start to increase 4-9 h after the beginning of MI symptoms (like troponin), peaks at around 24h and nally decreases to reach its baseline within the next 48-72 h. Since it clears from the circulation faster than troponin, it makes this a better biomarker to detect reinfarctions in patients (7)(8)(9)).
An array of researches depict the association between in ammation and MI as well as some other cardiovascular events. Apart from the probable e ciency of routine cardiac biomarkers for diagnosis of myocardial infarction, it has been reported that some in ammation biomarkers would play an important role in either future cardiovascular di culties' anticipation or diagnosis such as: C-reactive protein (CRP), growth differentiation factor-15 (GDF-15), e interleukin-1 receptor family member called ST2 (ST2), IL-6 and some other interleukins (10,11).
As far as classifying the interleukins concerned, interleukin 1 (IL-1) is the rst recognized amongst the other cytokines. IL-1 cDNAs' cloning resulted in two linked isoforms (IL-1α and IL-1β) with complete different functions. Despite the two isoforms, IL-1α and IL-1β, introduced as IL-1 family, there are 11other members in this family. As for IL-1β, unlike IL-1α, a myriad levels of regulation need for its biological activation. Pro-IL-1β is converted into a mature form as a result of proteolytic process caused by IL-1β converting enzyme named caspase-1 (12,13).
Interleukin-8 (IL-8), also known as chemokine C-X-C motif ligand 8 (CXCL8), is typical pro-in ammatory chemokine associated with the recruitment and activation of neutrophil and monocytes. IL-8 gene encodes a protein with 99 amino acids which is nally processed into either a 77 amino acid's protein in nonimmune cells or 72 amino acid's protein in monocytes and macrophages. Although there is an argument over the probable role of IL-8 in ischemia/reperfusion injury, there is still a lack of human researches to con rm this assumption (14,15).
Interleukin-11 (IL-11) is a multifunctional cytokine and member of IL-6 family along with some other cytokines such as: IL-6, IL-31, Cardiotrophin-2 (CT-2), Cardiotrophin-like cytokine (CLC), etc. Although it was discovered just the past quarter century, the minority of investigation has been done on its functions and activities. Its multifunctional property recently attracts researchers' attention. There have been some arguments over its effect on various di culties like in ammations and cancers. There is also the variety of upregulation and downregulation in ammatory responses have been represented as a result of its in ammatory microenvironment. Studies showed that IL-11 might take part in cardiovascular diseases via targeting cardio myocytes in order to reduce the cardiovascular injuries(16, 17).
In spite of several studies on the effectiveness of main cardiac biomarkers (Troponin and CK-MB) on MI prognosis and follow up, there are still some doubt over their complete sensitivity and speci city. Thus, the present study was designed to recognize the probable role of in ammatory biomarkers (IL-1β -IL-8 -IL-11) to facilitate the MI diagnosis and also their follow up advantages. We assume that the simultaneous measurements of both routine biomarkers and in ammatory ones would make it easier to diagnose MI as well as the probability of reinfarction in these patients.

Material And Methods
Study population 40 patients, who were diagnosed with Myocardial Infarction (at the rst 24 hours of stroke), have participated voluntarily in this research project. We have to take into account that all the patients with the history of in ammation and cancer diseases or whoever diagnosed with any type of diseases resulted in increment in the amount of in ammatory factors, have been omitted from the project. All participants were thoroughly informed prior to the study and also before taking part in the research. Their informed consents were also obtained before sampling. This study was approved and carried out by following the ethical guidelines and regulations which were con rmed by ethics committee of the Fasa University of Medical Sciences.
Blood Sample Collection and Interleukin measurements 5 cc of whole blood were collected by seasoned nurses. Then all blood samples were centrifuged to obtain their serum. Both routine myocardial biomarkers (Tropinin & CK-MB) were evaluated as the rst steps of MI diagnosis. All serum lipid pro les and their blood sugar were evaluated as well. Subsequently, we have assessed the serum levels of three Interleukins (IL-1β, IL-8 & IL-11) using the enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer's instructions (Bioassay Technology Laboratory-China).

Follow-up
After the rst round of sampling we would like to follow up the patient's condition, via the measurements of both biomarkers and mentioned interleukins, 72 hours after the onset of MI. Therefore, the same amount of blood samples has been collected for the assessment of three interleukins' (IL-1β -IL-8 -IL-11) serum levels as well as the main biomarkers (Troponin & CK-MB), in order to follow-up the patients' conditions. This could make it easier to compare the probable effects of cardiac biomarkers and in ammatory biomarkers for the MI cases follow-up.

Statistical analyses
All the data was represented as mean ± standard error (SEM). SPSS statistical software (SPSS, Chicago, IL, USA, version 21) was applied to analyze the data. The Wilcoxon test was used to compare data obtained from the variables (Troponin, CK-MB, IL-1β, IL-8 and IL-11). Spearman's correlation coe cient was employed to establish the relationship between changes in the values of parameters. Continuous variables were compared using the Wilcoxon signed-rank test. Normal distribution of data was checked using the Shapiro-Wilk test (P < 0.05). P ≤ 0.05 was considered to be statistically signi cant.

Results
In this study we have investigated cardiac biomarkers (troponin and CK-MB) in 40 MI patients. We also have evaluated three different interleukins in order to compare with routine biomarkers. First their blood sugar and lipid pro les were measured in both sexes. It is interesting to note that B.S and HDL were higher in males, while TG, Cholesterol and LDL were higher in females (Table 1).

In ammatory Biomarkers
Our result revealed the increasing pattern for IL-1β over the time past. However, the measurements showed no signi cant rise within the second sampling time (time point 2) compared with the time 1 (Fig. 3). On the other hand, both IL-8 and IL-11 have followed the same pattern of decreasing throughout the experimental time, which means they both decreased after 72 hours. It is interesting to note that just IL-8 illustrated a dramatic decrement during the whole experimental time (Fig. 4

Discussion
In the present study we investigate three interleukins (IL-1β -IL-8 and IL-11) as probable and practical biomarkers along with cardiac biomarkers (troponin and CK-MB) in order to have e cient and timely diagnosis of MI. Since the sensitivity and speci city of biomarkers for diagnosing myocardial injuries is of high importance, we could take the advantage of in ammatory biomarkers assessments as a complementary way of diagnosis as well as follow-up procedure, together with the routine biomarkers. These would be bene cial to follow-up patients in terms of treatment steps and anticipating eventual reinfarction.
In the current research, blood sugar and lipid pro les of each patient (both sexes) were measured at the beginning of the experiment. A higher amounts of blood sugar and HDL have been reported in males, while TG, cholesterol and LDL were higher in females. Haseeb A. Khan et al noticed that total cholesterol (TC), LDL and HDL decreased signi cantly in acute myocardial infarction patients (AMI). They mentioned that although TC reduction did not prevent the AMI risk, a decrement in HDL would make these patients vulnerable to AMI incidence(18). Also, Amit Kumar Shrivastava and his colleagues, who evaluated 400 AMI patients, illustrated a signi cant decrease in TC, LDL and HDL and a remarkable elevation in TG and in ammatory markers such as : IL-6 and IL-10 within the rst 2 days of sampling. They represented the serum level's correction of mentioned factors at 7 days of AMI onset. Thus, the assessments of serum lipid pro les are highly recommended after the MI diagnosis in order to reduce the risks (19). There are also more researches which highlights the importance of lipids and lipoprotein measurements in cardiovascular diseases like Bahattin Balci who underlined An increase in TG and low density lipoproteins and decrease in LDL, HDL and TC in patients with Acute Coronary Syndrome (ACS). He suggested that fasting lipid pro le assessment and statin therapy for these patients, as long as they admit to the hospital, would probably reduce morbidity and mortality (20).
Studies have claimed that both gender and age differences have in uenced the amounts of serum lipid pro les in patients with MI. In 2017 Zhixiong Zhong et al showed the higher TC, LDL, HDL and TG levels in females than males in elderly Hakka patients with acute myocardial infarction in southern China. They also mentioned a higher chance of dyslipidemia in non-elderly male patients than elderly one. Therefore, they had a great suggestion of lipid-lowering therapy for their elderly patients in order to decrease the cardiovascular di culties' risks (21). Our study con rms that the amount of serum lipid pro le is sexdependent and any contradiction between these studies would stem from a population differences.
In some recent studies many researches have introduced how serum lipid pro le evaluation could be essential within the 24 hours of admission with acute coronary syndrome symptoms. In 2018 William T. In the current study the amount of Troponin and CK-MB were changed with the same pattern similar to the nding of other researches. TnC blood levels increased within the rst hours of patient's admission, continuing its rise to reach its peak and stay high for the next 2-3 weeks. Although CK-MB demonstrated the same increasing trend as TnC, it has decreased to reach its normal levels with the next 48-72 hours.
Various researches depicted that CK-MB is no longer evaluated as an emergency biomarker and could be used to estimate infarct size and prognosis processes (24,25).
Due to the importance of having biomarkers with high sensitivity and speci city for MI diagnosis along with patient's follow-up, we decided to investigate some in ammatory cytokines (1L-1β, IL-8 & IL-11) and compare them with the routine biomarkers.
Regarding the IL-1β blood levels, the increasing pattern has been depicted not only in the rst sampling but after 72 hours. This showed a marked similarity with the troponin changes throughout the experimental period. In a recent study Johanne Silvain et al. found that high IL-1β levels were closely associated with cardiovascular mortality, major cardiovascular events (MACE) and as a whole all-cause mortality. These ndings were represented as a result of 90-days and one-year follow up of patients with acute MI. They have also support the necessity of using IL-1β inhibitors in the acute MI patients as a treatment and risk reductions(26). Paul M Ridker et al. reported that the anti-in ammatory therapy approach toward IL-1β with Canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, without any changes of LDL cholesterol levels could substantially reduce the cardiovascular diseases' incidents in the patients with the MI history (27). According to the results of another study, it has been demonstrated that IL-1β levels were in connection with repairing process of patients with the MI history(STEMI). They indeed mentioned the importance of this in ammatory cytokine and dysfunctional myocardial remodeling after the reperfused MI that would bring about myocardial function impairment and heart failure(28).
In the recent letter published in 2019 Hirotaka Mor et al. illustrated that that nolotinib, prescribing for chronic myeloid leukemia (CML) patients, could increase the IL-1β serum levels and cardiovascular events. Despite the effectiveness of nilotinib for CML, it should not be prescribed for patients with the cardiovascular history and risk factors. They have also showed that IL-1β might contribute to apoptosis through releasing from injured cardio myocytes in the AMI patients. Apoptosis was induced by IL-1β in cardio myocytes of murine MI models. Taken together, IL-1β would elevate the risk of apoptosis and should be controlled speci cally in high risk patients (29). Recently Mona Panahi et al. were represent the adverse effects of post-MI in ammation which led to heart failure (HF). A timely decision like blockade of in ammatory cytokines (IL-1) could prevent the incidence of post-MI excessive in ammation which curb the repairing processes by immune system (30).
Moreover, myriad of researches have demonstrated the plausible association between IL-1β + 3954C/T polymorphism (rs1143634) and MI incidence. This has been proved via a meta-analysis by Yizhen Fang et al., speci cally amongst Caucasian populations (31). We have to take into account that although the circulation levels of IL-1β is very low to investigate, this in ammatory biomarkers' assessment would be bene cial in order to eliminate its probable adverse impact on cardiovascular remodeling in reperfused MI patients.
We also studied IL-8 and IL-11 in MI cases. Both in ammatory biomarkers followed the same falling pattern in an experimental period. IL-8 has decreased dramatically after the 2nd time point, while IL-11 changes was not statistically signi cant. Kathrin Schömig et al. showed the association between IL-8 levels and progenitor cells in circulation of AMI patients. Progenitor cells are released during in ammation, giving rise to neovascularization. This would clarify the possibility of bene cial impacts induced by in ammatory biomarkers instead of their mere adverse effects (32). On the contrary Christian Shetelig et al. ( 2018) reported the link between IL-8 high levels and some cardiovascular di culties in STEMI patients such as: microvascular obstruction, large infarct size, LV remodeling and further clinical post-MI incidences. They have supposed the possible IL-8's therapeutic approach toward identifying high risk STEMI patients as well as post-infarct in ammation reduction (14). Our result con rms the increase in the IL-8 serum levels with the onset of MI. Owing to the probable effects of this in ammatory biomarker to deteriorate the post-MI conditions, it could have been used as an in ammatory regulation target in post-infarct patients in order to curb new events.
In another recent review in 2018 researches have provided the results related to the bene ts of cytokines' (IL-1, -6, -8, MCP-1, CC chemokines, CXC chemokines and TNF-α) inhibition on cardiac function. However, adverse or even neutral results was indeed represented for some of these cytokines (IL-1, IL-6, IL-8, and MCP-1). There were still some con icting animal model results. They have showed the use of monoclonal antibody against IL-8 for ischemic reperfusion rabbit model and its association with infarct size reduction, while in another study the IL-8 receptor overexpression in a chronic MI rat model caused a decrease in infarct size and in ammatory cells and nally LEVF improvement (33). In a case-control study by S Zarrouk-Mahjoub et al., 30  This inequality between pro and anti-in ammatory cytokines would lead to heart failure (34). Our results reconcile with the previous studies regarding the increased IL-8 levels just after MI onset.
Since there was a belief about the link between in ammation and coronary heart disease (CHD), Zhengxia Liu et al. have evaluated the in ammatory cytokines levels (CD121a, interleukin [IL]-1β, IL-8, and IL-11) in CHD and CHD-free patients. Although the CD121a levels were high in MI patients, IL-1β levels were unchanged throughout the experiment. Like CD121a, IL-8 levels were also high in AMI patients. Meanwhile they have mentioned the highest levels of IL-11 in CHD patients rather than non-CHD ones (35). In a case report in 2016, 4 different cases have been treated with IL-11, known as a cardio protective cytokine, in ST elevation myocardial infarction (STEMI) patients. There was not any adverse drug reaction throughout the treatment with recombinant human IL-11 (rhIL-1) and ultimately all cases left the hospital without any HF symptoms(36).
According to the IL-11 treatment, Masanori Obana et al. have illustrated that IL-11 treatment for coronary artery ligation animal model could alleviate post MI cardiac brosis via STAT3. They have proved a notable upregulation of IL-11 mRNA in cardiac myocytes of MI cases. Therefore, intravenous administration of IL-11, almost after ligation, could decrease the brosis area. These all gave a new perspective for IL-11 treatment against post-MI complications (37). In a recent study in 2019 researchers have investigated the association between IL-11 levels and cardiac prognosis of chronic HF patients. Although the speci c mechanisms of IL-11 remained unknown, higher levels of IL-11 have been reported throughout the cardiac events which might have severe outcomes(38). Our result con rmed the IL-11 increment that has been reported throughout the cardiac evets in mentioned studies. Moreover, due to the con icting evidences from different researches we suggest a thorough evaluation of pro and antiin ammatory cytokines as well as a long term follow-up till the patients' stable conditions. Some limitations can be enumerated for present study. The sample size was relatively small owing to the lack of enough volunteers to join the research study. Furthermore, we recommend the evaluation of various related pro and anti-in ammatory cytokines simultaneously along with in depth clinical MI and HF investigations in the hospital to conduct a concrete conclusion.

Conclusion
In conclusion, lipid pro le variations should be taken into consideration as crucial factors which curb the incidence of reinfarction and cardiac deterioration. Moreover, in ammatory cytokines, both proin ammatory and anti-in ammatory ones, would be bene cial to apply as routine biomarkers for MI diagnosis and follow-up. Signi cant changes in IL-8 would show the importance of this cytokine as a diagnostic factor within the onset of the MI.

Declarations
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funds
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors for the research, authorship, and/or publication of this article.    P≤0.05*.