Abstract
Background
Vascular inflammation has an important role in the development and progression of coronavirus disease 2019 (COVID-19). Also, a high cytokine level predicts a poor prognosis for COVID-19. In this study, we aimed to investigate the effect of salusin-α and salusin-β peptides in determining the severity of the disease in the acute period of COVID-19.
Method
The investigation involved studying a group of 74 hospitalized individuals who had tested positive for SARS-CoV-2 through polymerase chain reaction (PCR) tests. The patients were divided into two groups: those who did not reach the primary endpoint and were discharged without complications and those who reached the primary endpoint (a composite of ICU admission and/or mortality). Salusin-α and salusin-β levels of serum samples taken at the time of application were statistically compared.
Outcome
There was no statistically significant difference in salusin-α levels between the groups (p=0.279). However, salusin-β levels were found to be significantly higher in patients who reached the primary endpoint compared to those who did not reach the primary endpoint and the healthy control group.
Conclusion
The results of the analysis showed that a salusin-β level of 12.45 ng/mL predicts the risk of complications such as intensive care unit admission or mortality, with a sensitivity of 83.8% and a specificity of 40.5% for the estimation of the primary endpoint. The obtained data support our hypothesis, but observational studies with larger sample sizes are needed to evaluate salusins’ determination of COVID-19 prognosis.
Introduction
Novel coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), leading to mortality and morbidity. 1 Clinical manifestations and cardiovascular complications associated with vasculopathy have been frequently reported in these patients.2-4 Vascular endothelium is involved in the development of complications. The expression of Angiotensin-converting enzyme-2 (ACE-2), the main entry receptor of the virus, in vascular cells and the presence of SARS-CoV-2 in endothelial cells in fatal COVID-19 patients have also been demonstrated. 5 Endothelial cell dysfunction has been suggested to contribute to the poor prognosis of COVID-19. 6
In previous years, Shichiri et al. 7 discovered the multifunctional endogenous bioactive peptides salusin-alpha (α) and salusin-beta (β) synthesized from preprosalusin. These peptides are expressed in many organs and tissues, including the heart, blood vessels, brain, and kidneys. In blood, urine, and tissues, they are found in two forms: salusin-α (28 amino acids) and salusin-β (20 amino acids).8,9 While salusin-α shows mild to moderate mitogenic and hemodynamic activity, salusin-β has been shown to induce bradycardia and hypotension through parasympathetic stimulation, as well as to promote atherosclerotic effects by enhancing macrophage foam cell formation in rats. 10 Salusin-β accelerates the inflammatory response in vascular endothelial cells and increases oxidative stress in vascular smooth muscle cells. 11
In assessing the severity and complications of the disease during the acute period in the follow-up of COVID-19 patients, it is crucial to consider evaluating endothelial pathophysiology and employing endothelial function tests for follow-up assessments. Given the central role of endothelial inflammation in COVID-19, biomarkers that regulate vascular function, such as salusin, have emerged as potential candidates for prognostic evaluation. The existing literature does not uncover any analogous studies examining salusin-α and salusin-β biomarkers in predicting the progression of COVID-19 disease. In this study, we aimed to investigate the effect of salusin-α and salusin-β peptides in determining the severity of the disease in the acute period of COVID-19.
Methods
Study Site and Population
This prospective study was conducted in Canakkale Onsekiz Mart University Hospital, a tertiary pandemic hospital, between 01.04.2022-10.07.2022. The study enrolled patients hospitalized in the inpatient ward and adult intensive care unit, and whose nasopharyngeal swab samples were positive for the SARS-CoV-2 PCR test. Hospitalization and admission to intensive care were determined according to the COVID-19 Diagnosis and Treatment Guidelines of the Ministry of Health. 12
Patients with active infectious/inflammatory disease, malignancy, diabetes mellitus, decompensated chronic liver disease; history of myocardial infarction, percutaneous coronary intervention or coronary artery surgery, heart failure or valvular heart disease, cerebrovascular disease; patients with estimated glomerular filtration rate (eGFR) <30 mL/min/1.73 m2 calculated by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula, patients on dialysis, pregnant and breastfeeding patients were excluded from the study.
Information on demographics, comorbidities, duration of hospitalization, disease severity, complications and mortality, peripheral blood hemoglobin, leukocytes, lymphocytes, platelets, CRP (C-reactive protein), levels of procalcitonin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), albumin, D-Dimer and clinical information of the patients were obtained from the hospital automation system.
Study Outcomes
Patients were divided into two groups based on their clinical course: those discharged without complications (Group 1) and those requiring intensive care or experiencing mortality (Group 2 - Primary Endpoint).
The primary composite endpoint of this study was defined as the occurrence of serious clinical outcomes, namely admission to the ICU and/or in-hospital death. Patients were divided into two groups based on whether or not they reached this primary endpoint during their clinical course.
Power analysis was performed using G*Power (Version 3.1). With a small effect size (f=0.3), an alpha level of 0.05, and a power of 80%, the minimum total sample size was 111, with 37 individuals in each of the three subgroups. Accordingly, the study enrolled a total of 111 participants divided into three distinct groups: Group 1 (n=37); Group 2 (n=37); and Group 3 (n=37) served as the healthy control group.
Collection and Storage of Blood Samples
Venous blood samples of the patients enrolled in the study were collected with a vacuum blood collection system into BD Vacutainer™ SST™ II Advance Tubes—Blood serum tubes (Becton Dickinson, Franklin Lakes, NJ). After 30 minutes, blood samples were centrifuged at 1500 g for 15 minutes to separate the serum. They were stored refrigerated at -80°C until the day of the study.
Measurement of Biochemical Parameters
On the study day, serum samples were gradually allowed to reach room temperature and analyzed for salusin-α (Catalog no: 710620; AFG Bioscience, Northbrook, IL, USA) and salusin-β (Catalog no: 710621; AFG Bioscience, Northbrook, IL, USA) by ELISA.
The hemogram analyses were conducted using the Mindray BC 6200, procalcitonin and ferritin analyses were performed on the Cobas e601, and other tests were analyzed on the Cobas c702 devices.
Statistical Analysis
Data were analyzed using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp. Armonk, NY: USA. Released 2018). Mean ± standard deviation, median (min-max), and number (percentage) were used for descriptive analysis. Statistical assessments comparing the parameters to be analyzed in the patient groups were conducted using either parametric or non-parametric significance tests, depending on whether the assumptions for parametric tests were met. For group comparisons by patients’ sociodemographic and clinical characteristics, the Mann-Whitney U, Kruskal-Wallis, and Chi-Square tests were used. Logistic regression analysis was used to determine the relationship between clinical characteristics and achievement of the primary endpoint. The predictive value of salusin-β for the clinical course was evaluated by Receiver-operator characteristic (ROC) curve analysis. P<0.05 was accepted as statistical significance, and exact p values were given.
Ethical Considerations
The necessary permissions were obtained from the Scientific Research Platform of the Turkish Ministry of Health (decision dated 25.08.2021 and numbered 2021-08-23T21_41_02) and the Canakkale Onsekiz Mart University Medical Faculty Clinical Research Ethics Committee (decision dated 22.10.2021 and numbered 07-23).
Written and verbal consents were obtained from the participants and first-degree relatives of the unconscious participants. 2013 Revised Helsinki Criteria were followed.
Results
The study included 111 participants, including 74 patients diagnosed with COVID-19 [Group 1 (patients without the endpoint) (n=37), Group 2 (Patients with the endpoint, defined as ICU admission and/or mortality) (n=37)] and 37 in the control group. Only salusin-α and salusin-β levels were considered in the control group. The patient cohort was classified into two groups based on their clinical outcomes. Group 1 encompassed individuals discharged without complications, regardless of whether they had reached the primary endpoint. Conversely, Group 2 comprised patients who had successfully achieved the primary endpoint. Of the patients, 42 (56.8%) were men, and the median age was 68 years.
General Characteristics of Patients Diagnosed With COVID-19
p<0.05 was considered statistically significant. Continuous data were expressed as median (minimum-maximum) and categorical data as number (percentage). COPD, chronic obstructive pulmonary disease; CVD, cardiovascular disease; SD: standard deviation
Comparison of Biochemical Values by Groups
p<0.05 was considered statistically significant. Data were presented as median (minimum-maximum). ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; GGT: gamma glutamyl transferase; CRP: C-reactive protein; LDH: lactate dehydrogenase.
Comparison of Salusin-α and Salusin-β Values Between Groups
*p<0.05 was considered statistically significant. Data are expressed as median (min-max).
Pairwise comparisons revealed no statistically significant difference in salusin-β levels between Group 1 patients and the healthy control group (p=1.00). Similarly, no statistically significant difference was found between Group 1 and Group 2 (p=0.08). Likewise, no statistically significant difference was found between Group 2 and the healthy control group (p=0.08).
Logistic Regression Analysis of Independent Variables for the Primary Endpoint
*CRP: C-reactive protein; LDH: lactate dehydrogenase; SO2: oxygen saturation.
ROC analysis of serum salusin-β to predict the primary composite endpoint showed an AUC of 0.701 (95% CI: 0.581-0.821); p=0.003), with an optimal threshold at 12.45 ng/mL, 83.8% for sensitivity, 40.5% for specificity, 58.7% for positive predictive value and 71.4 for negative predictive value (Figure 1). ROC curve analysis of Salusin-β
Discussion
In this study, we aimed to evaluate the performance of salusin-α and salusin-β peptides in determining the severity of the disease in the acute phase of COVID-19. Our study compared patients who reached the primary endpoint with those who did not, alongside healthy controls, revealed no significant difference in salusin-α levels. In contrast, salusin-β levels were significantly higher in patients who achieved the primary endpoint compared to healthy controls and patients who did not achieve the primary endpoint.
Vascular inflammation and endothelial dysfunction play a central role in how COVID-19 damages the body, often triggering a wide range of clotting complications — from blockages in tiny blood vessels to clots in major arteries.13-15 Different clinical presentations due to COVID-19 include pulmonary embolism, venous thromboembolism, myocardial infarction, cerebral infarction. D-dimer level is one of the criteria used to detect thrombosis in patients. Measuring D-dimer level and coagulation parameters from the early stage of the disease has been reported to be useful in controlling and managing COVID-19 disease.13-15 In our study, patients who experienced the worst outcomes — those admitted to the ICU or who did not survive — showed markedly higher levels of both salusin-β and D-dimer, pointing to these two markers working together to drive disease severity. The logical link between these two markers can be explained via the inflammatory-thrombotic cascade: Impaired endothelial cells can express various adhesion molecules (VCAM-1, ICAM-1, and selectins) and chemokines (MCP-1) that promote monocyte recruitment. Furthermore, impaired endothelial cells can release proinflammatory cytokines such as IL-6 and TNF-α, increasing vascular inflammation. 16 In a study with experimental mice to demonstrate the effect of salusins on atherosclerosis and vascular inflammation, Zhou et al. 16 showed that salusin-β increased IL-6 and TNF-α levels through I-κBα/NF-κB pathway, and in another study in human umbilical artery endothelial cells, they showed that this was achieved by activation of p38 MAPK/NF-κB and JNK/NF-κB pathways and upregulation of VCAM-1 and MCP-1 expressions. 17 The importance of endogenous salusin-β in promoting monocyte and macrophage adhesion to aortic endothelial cells was first shown in an experimental mouse study by Koya et al. 18 Additionally, it was discovered that it activates the redox-sensitive transcription factor NF-B, which results in an increase in the number of VCAM-1 receptors in endothelial cells, a proinflammatory adhesion molecule. Therefore, the sharp rise in salusin-β seen in our severe COVID-19 patients reflects just how intense the ‘cytokine storm’ and widespread vascular damage can become — and helps explain why D-dimer levels climb so high, and outcomes turn so poor. This suggests that salusin-β and D-dimer are not simply two separate warning signs appearing side by side, but rather two interconnected signals that tell the same story: the body has transitioned from widespread inflammation into clinically significant clotting. The salusin-β levels in our study were found to be significantly higher than those reported in previous studies related to different diseases. This marked elevation likely reflects the ‘cytokine storm’ and widespread endothelial dysfunction specifically triggered by severe SARS-CoV-2 infection. This may be due to the excessive release of biologically active peptides from vascular tissues during SARS-CoV-2 infection compared to other inflammatory conditions. When groups were compared based on pairwise comparisons, those meeting the primary endpoint had significantly higher salusin-β levels than both the healthy control group and those who did not. As a result, salusin-β appears as a potential prognostic marker for forecasting the course of COVID-19, demonstrating its value in prognostic evaluations.
An in vitro study by Esfahani et al. 19 showed that salusin-α had no inhibitory effect on proinflammatory cytokines via the NF-κB pathway in human umbilical vascular endothelial cells. In an in vitro study in human umbilical vascular endothelial cells, Zhou et al. 17 showed that salusin-α had no effect on NF-κB activation; it selectively decreased VCAM-1 protein, but not VCAM-1 mRNA, TNF-α, IL-6 or MCP-1. This suggested that salusin-α had little effect on the inflammatory response in human umbilical vascular endothelial cells. Again, in an in-vivo study in ApoE-deficient experimental mice, Zhou et al. 16 showed that salusin-α had no effect on the NF-κB pathway. The absence of a difference between COVID-19 disease severity and salusin-α levels in our study could be attributed to the purported lack of influence of salusin-α on the NF-κB pathway and inflammatory response.
It has been reported that many factors, including genetic factors, influence the course of the disease in COVID-19 patients.20,21 A recent study in the literature specifically supports the idea that HPA-3 polymorphisms and related antibodies (anti-HPA-3a) trigger platelet consumption in COVID-19, predisposing to the coagulation process and consequently to elevated D-dimer levels. 21 Current data in the literature also emphasize that this inflammatory environment in COVID-19 is closely related to coagulation disorders. Talebzadeh et al. 22 demonstrated that inflammatory processes (via Vitamin D deficiency) in COVID-19 patients increase thrombotic risk by affecting platelet parameters (MPV and platelet count). Although our study did not evaluate platelet parameters in measuring the severity of COVID-19, current literature suggests that such salusin-induced inflammatory environments may be linked to activation of the coagulation system. A recent study in Iran investigated whether specific genetic variations in mannan-binding lectin serine protease 2 (MASP2) predispose patients to COVID-19, and how the lectin pathway and endothelial activation contribute to the course of COVID-19. The study found that higher levels of MASP2 were closely associated with higher D-dimer levels and worse patient outcomes. This finding is consistent with our study’s findings. While MASP2 reflects vascular damage triggered by complement activation, salusin-β influences the pro-inflammatory side of the process – fueling endothelial dysfunction and the coagulation cascade via the NF-κB pathway – suggesting that these two processes are not independent but mutually influential. 23
Other hematological and biochemical findings in our study are consistent with the literature. Mobinikhaledi et al. 24 showed a strong association between high neutrophil counts and LDH levels and the need for intensive care, and low lymphocyte counts and mortality in COVID-19 patients. Similarly, in our study, significantly higher leukocyte, neutrophil, LDH, and D-dimer levels and marked lymphopenia were detected in patients who reached a poor clinical outcome (Group 2). 24 Similarly, the fact that elevated CRP and prolonged Prothrombin Time (PT) levels at admission are early prognostic indicators for severe COVID-19 pneumonia and the need for intensive care supports our findings in our study, which show a poor prognosis associated with elevated CRP and D-dimer levels. 25
Limitations
The study’s limitation is that it was conducted in a single center with a limited number of patients.
Furthermore, according to our study findings, the diagnostic performance of salusin-β has some limitations. At the optimal threshold, this marker showed relatively low sensitivity (83.8%) and significantly low specificity (40.5%). These values suggest a high probability of false positive results, which at this stage may potentially limit its clinical use as a standalone diagnostic tool. Consequently, studies with larger sample sizes are needed to improve these threshold values and increase overall diagnostic accuracy through the integration of additional biomarkers or clinical parameters.
Conclusion
We suggest that salusin-β can be used as a prognostic marker in COVID-19 once it is validated in multicenter studies with larger sample sizes. If validated, serum salusin-β could serve as a complementary biomarker in risk stratification models, potentially identifying high-risk COVID-19 patients early in the disease course.
Footnotes
Authors’ Note
We confirm that neither the results of this study nor any part thereof have been published previously in any peer-reviewed journal between 2022 and 2025.
Acknowledgements
We express our sincere gratitude to our colleagues who contributed to this study and to the patients who participated. This work was derived from the first author’s medical residency thesis. The authors acknowledge the financial support provided by the Canakkale Onsekiz Mart University Scientific Research Projects Coordination Unit (BAP) under project number TTU-2022-3850. An abstract of this study was presented as an oral presentation at the 26th Turkish Congress of Clinical Microbiology and Infectious Diseases (KLİMİK 2026), held in Antalya, Turkey, from 29 April to 3 May 2026.
ORCID iDs
Ethical Considerations
The necessary permissions were obtained from the Scientific Research Platform of the Turkish Ministry of Health (decision dated 25.08.2021 and numbered 2021-08-23T21_41_02) and the Canakkale Onsekiz Mart University Medical Faculty Clinical Research Ethics Committee (decision dated 22.10.2021 and numbered 07-23).
Consent to Participate
Written and verbal consents were obtained from the participants and first-degree relatives of the unconscious participants. 2013 Revised Helsinki Criteria were followed.
Funding
This work was produced from the first author’s medical residency thesis. The authors would like to acknowledge the financial support provided by the Çanakkale Onsekiz Mart University Scientific Research Projects Coordination Unit (BAP) through project No. TTU-2022-3850.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Data Availability Statement
The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary material. Raw data that support the findings of this study are available from the corresponding author, upon reasonable request. This work was produced from the first author’s medical residency thesis.