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Increased level of plasma salusin-α and salusin-β in patients with multiple sclerosis
Multiple Sclerosis and Related Disorders 30 (2019) 76–80
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Multiple Sclerosis and Related Disorders journal homepage: www.elsevier.com/locate/msard
Increased level of plasma salusin-α and salusin-β in patients with multiple sclerosis
T
Murat Çakıra, , Seda Sabah-Özcanb, Hikmet Saçmacıc ⁎
a
Faculty of Medicine, Department of Physiology, University of Bozok, Yozgat, 66200, Turkey Faculty of Medicine, Department of Medical Biology, University of Bozok, Yozgat, 66200, Turkey c Faculty of Medicine, Department of Neurology, University of Bozok, Yozgat, 66200, Turkey b
ARTICLE INFO
ABSTRACT
Keywords: Salusin-α Salusin-β Multiple sclerosis Relapsing-Remitting Multiple Sclerosis
Background: Multiple Sclerosis (MS) is a potentially progressive autoimmune disorder of the central nervous system. The pathology of MS is characterized by inflammation, demyelination, reactive gliosis and neuronal damage. Salusin-α and salusin-β have been shown to be widely expressed in many tissues, including the central nervous system. In our study, we investigated whether salusin-α and salusin-β peptides had a relation with inflammation and whether it is related to Relapsing-Remitting Multiple Sclerosis (RRMS) disease. Methods: Forty healthy controls and forty patients with RRMS were included in the study. Salusin-α and Salusinβ levels were measured by Enzyme-linked Immuno-Sorbent Assay (ELISA). Results: Salusin-α and salusin-β levels were high at a significant level in RRMS patients compared to healthy controls (p < 0.0001). We found a strong positive correlation between salusin-α and salusin-β levels (p < 0.0001, r = 0,9925). Conclusion: In conclusion, we found that there was a relationship between salusin-α and salusin-β levels, and MS disease. Since RRMS is the first stage of MS and its most common type, it is important to perform biomarker studies in this period in terms of early planning of treatment. Although salusin-α and salusin-β levels increase in RRMS patients, further studies are needed to understand its relation with other neurological and inflammatory diseases to define it as a biomarker.
1. Introduction Multiple Sclerosis (MS) is a potentially progressive autoimmune disorder of the central nervous system. More than two million people worldwide suffer from MS. The pathology of MS is characterized by inflammation, demyelination, reactive gliosis and neuroaxonal damage (Zephir, 2018). MS disease occurs as a result of genetic predisposition and environmental triggers. Clinical symptoms vary depending on the lesion burden and localization and include muscle weakness, blurred vision, dizziness, fatigue and balance problems (Sawcer et al., 2011). Many studies of MS have identified various biomarkers for the diagnosis of the disease, the activity of the disease, and the prediction of the response of the patient to treatment (D'Ambrosio et al., 2015). Many of the biomarker examinations that can be used in the diagnosis of MS are performed in the cerebrospinal fluid (CSF) obtained by an invasive
procedure (Fitzner et al., 2015). Due to the difficulty of obtaining CSF, biomarkers that are examined in biomaterials which are acquired by non-invasive methods such as serum and plasma and which can be used in the diagnosis of the disease are needed. Salusin-α and salusin-β discovered in human embryo are composed of 28 and 20 amino acids, respectively. Salusins have been shown to be widely expressed in many tissues, including the central nervous system (Shichiri et al., 2003). Intravenous infusion of salusin-α and salusin-β to the rats have been reported to cause hypotension and bradycardia (Izumiyama et al., 2005). Salusins have also been shown to cause proliferation in vascular smooth muscle cells and fibroblasts (Shichiri et al., 2003). Salusins increase hypertrophy and growth in rat cardiomyocytes (Yu et al., 2004). It has also been reported that salusins have antiapoptotic effect in these cells (Xiao-Hong et al., 2006). Salusins have been shown to be associated with various diseases such as
Abbreviations: BMI, body mass index; CSF, cerebrospinal fluid; ELISA, Enzyme-linked Immuno-Sorbent Assay; EDSS, Expanded Disability Status Scale; HDL-c, highdensity lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; MS, Multiple sclerosis; NF-κB, Nuclear Factor-κB; NO, nitric oxide; ROS, reactive oxygen species; RRMS, Relapsing-Remitting Multiple Sclerosis ⁎ Corresponding author. E-mail address: [email protected] (M. Çakır). https://doi.org/10.1016/j.msard.2019.02.003 Received 20 November 2018; Received in revised form 28 January 2019; Accepted 3 February 2019 2211-0348/ © 2019 Published by Elsevier B.V.
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metabolic syndrome and acute renal failure (Cakir et al., 2017; Citil et al., 2014; Kimoto et al., 2010). Studies have shown that salusins are associated with inflammation and oxidative stress (Cakir et al., 2017; Xu et al., 2016; Zhao et al., 2017). Salusin-β has been reported to accelerate inflammation through the Nuclear Factor-κB (NF-κB) pathway (Koya et al., 2012). Although the physiological roles of salusins in the central nervous system are not clear; salusins have been shown to have neuroendocrine effects such as antidiuretic hormone release, blood pressure regulation and modulation of the functioning of the autonomic nervous system (Huang et al., 2015; Li et al., 2016; Li et al., 2015; Suzuki-Kemuriyama et al., 2016). In this study, we investigated whether salusin-α and salusin-β peptides, whose relation to inflammation were shown in the previous studies, is associated with Relapsing-Remitting Multiple Sclerosis (RRMS) disease.
2.3. Determination of plasma salusin-α and salusin-β levels Plasma salusin-α and salusin-β levels were measured using an Enzyme-linked Immuno Sorbent Assay (ELISA) kit with a minimum detectable concentration of 0.93 pg/mL and 1.75 pg/mL, respectively from Uscn Life Science Inc. (Wuhan, PR China). Optical density values for samples and standard samples were detected on a Spectramax ELISA reader (Molecular Devices) at 450 nm. The results are presented as pg/ mL. 2.4. Statistical analysis SPSS 20 package program was used for data analysis. It was determined whether the quantitative data showed normal distribution according to the Shapiro-Wilk normality test. Independent-samples ttest was used to compare normal distribution data between groups. The Mann-Whitney U test was used for the comparison of the groups that showed non-normally distributed data. Pearson correlation analysis was used for the correlation analysis of the data with normal distribution, and Spearman's correlation analysis was used for the correlation analysis of the data not showing normal distribution. Arithmetic mean ± standard deviation (SD) was used to define quantitative data. p < 0.05 was considered statistically significant.
2. Materials and methods 2.1. Subject All applications in this study were performed in accordance with the protocol approved by Bozok University Local Ethics Committee (2017KAEK-189_2018.05.30_06). In this prospective, cross-sectionally designed study, 40 RRMS patients (27 females, 13 males) and 40 healthy controls (26 females, 14 males) who applied to Neurology clinics between the ages range 21–60 years were included in the study. The following patient data were collected: age, gender, all symptoms, disease duration, the frequency of attacks, family history of MS, drug intake (related to MS) of patients were also recorded. Patients with another disease in the RRMS patient group were excluded from the study. While 10 of the patients were not receiving any prophylactic treatment, 5 were taking fingolimod, 2 were receiving natalizumab and 23 patients were taking first-line treatment (interferon, teriflunomide, dimethyl fumarate). The control group comprised healthy and age, body mass index (BMI) (kg/m2) and sex-matched volunteers. Leukocyte, neutrophil, lymphocyte, blood glucose, triglyceride, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), total cholesterol levels and BMI of all individuals, who were included in the study, was measured. A neurological examination of all the patients was carried out, and their disability was defined according to Expanded Disability Status Scale (EDSS) score and by using points for functional systems. Disability was measured with the EDSS, an ordinal scale of 0.0–10 (“no disability” to “death due to MS”), with higher scores reflecting the greater level of disability (Kurtzke, 1983). RRMS was defined according to the revised McDonald criteria 2010 and reporting neither attacks nor steroid use during the month before the interview was held. All patients who did not have concomitant neurological disorder were invited to participate. Other exclusion criteria were; congenital abnormalities of brain, recent head trauma, metabolic disease (diabetes mellitus, hypertension), acute-chronic infection, peripheral neuropathy, cardiac-thyroid-lung diseases, collagen tissue disease, renal failure, liver diseases, pregnant women and alcohol users.
3. Results Plasma salusin-α levels (Fig. 1A) were higher in the RRMS group (104 ± 192 pg/mL) compared to the control group (40 ± 136 pg/ mL) (p < 0.000). Plasma Salusin-β levels (Fig. 1B) were significantly higher in RRMS group (344 ± 640 pg / mL) than in the control group (128 ± 464 pg / mL) (p < 0.000). There was no significant difference between treated (n = 30) and untreated (n = 10) patients in the levels of salusins (p ˃ 0.05). When the lymphocyte levels between the groups were compared (Table 1), it was found to be lower in the RRMS group (0.4 ± 4.58) than in the control group (1.36 ± 4) (p < 0.025). When the correlation between the data was examined, a strong and positive correlation was detected between salusin-α and salusin-β levels (Fig. 2) (p < 0.000, r = 0,9925). When plasma salusin levels were compared with EDSS score and disease duration in terms of disease disability, no statistically significant correlation was found (p ˃ 0.05). Mean and standard deviation of EDSS and disease duration was 2.85 ± 1.18 and 8.3 ± 5.5 respectively. We found a weak negative correlation between salusin-α and lymphocyte levels (p < 0.040, r = −0,2307) when all individuals in the patient and control group are included. Similarly, we observed a weak and negative correlation between salusin-β level and lymphocyte level (p < 0.040, r = −0,2298). When the correlation between salusin-α and salusin-β levels and lymphocyte counts were examined separately in patient and control groups, we found no statistical significance (p ˃ 0.05). We found a negative correlation between age and levels of salusin-α (p < 0.025, r = −0,2503) and salusin-β (p < 0.025, r = −0,2497) (Fig. 3A and B). 4. Discussion Salusin-α and salusin-β was discovered in 2003 by Shichiri et al. in human embryo. Preprosalusin is cut from different amino acid regions, and salusin-α and salusin-β are formed. Preprosalusin is expressed in many parts of the central nervous system (Shichiri et al., 2003; Suzuki et al., 2011). Although salusins are commonly found in different regions of the brain, in the immune system and in many tissues in the body, the studies in the literature showed that the number of these peptides is limited. Salusin-α and salusin-β levels in human serum and urine were also determined (Sato et al., 2006). The studies on salusins have mainly focused on the relationship of these two peptides with the cardiovascular system. It has been shown that both peptides decrease heart rate
2.2. Biochemical analysis Blood was taken from patients and control group between 09.00 and 10.00 am. Baseline blood samples were collected from the subjects and patients into vacutainers containing Na2-EDTA (1.5 mg/mL), and blood samples were centrifuged for 10 min at 3000 rpm, after which the supernatant was quickly removed and kept frozen at −80 °C until the assays were performed by a specialist who was blind to patient status. The complete blood count, blood glucose level, blood lipids data of all the participants were obtained from the hospital registry system. 77
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Fig. 1. Comparison of plasma levels of salusin-α between control subjects and MS patients (A). Comparison of plasma levels of salusin-β between control subjects and MS patients (B).Data analyzed with independent sample t test. ***(p < 0.0001), (n = 80).
and blood pressure when administered intravenously in rats (Izumiyama et al., 2005). Salusin-α has been shown to decrease the formation of atherosclerosis by reducing foam cell formation, and salusin-β to increase atherosclerosis by increasing foam cell formation (Watanabe et al., 2008). In experimental gastric ulcer model, salusins have been reported to have protective effect (Tanyeli et al., 2017). In another study, the neuroprotective effect of salusin-β in the experimental Parkinson's disease model was shown. (Chang et al., 2018). In many studies, it was shown that there was no positive correlation between salusin-α and salusin-β levels, (Aydin, 2013; Sipahi et al., 2018; Watanabe et al., 2008); however, some studies showed a positive correlation (Celik et al., 2013). In our study, we found a positive correlation between salusin-α and salusin-β levels (P < 0,000, r:0,994). The correlation between salusin-α and salusin-β levels was found in both control and MS groups. MS is a serious health problem affecting the daily life of individuals due to the characteristic of its symptoms and progressive nature. The characteristic pathological sign of MS is that demyelinating plaques form perivenular inflammatory lesions (Karussis, 2014). Oligodendrocyte damage and demyelination occur as a result of inflammation
(Frischer et al., 2009). Infiltration of CNS immune cells causes more inflammation and damage. T-lymphocytes are the most important cells infiltrated into the tissue in MS. B-cells and plasma cells have tissue infiltration, albeit at a lower rate (Frischer et al., 2009). The release of proinflammatory cytokines induced by active lymphocytes promotes the activation of microglia and macrophages. It also leads to the development of phagocytic abilities of these cells and the production of various cytotoxic agents such as reactive oxygen species (ROS) and nitric oxide (NO) (Cunningham, 2013). Antibodies produced by active B Cells against the proteins and lipids of the myelin sheath are also included in this process (von Budingen et al., 2011). Studies have been conducted to investigate whether salusins might be associated with inflammation and inflammatory diseases. Serum salusin-α levels and salusin-β levels were found to be high in patients with Behçet's disease (Erden et al., 2014; Ozgen et al., 2011). In psoriasis patients, salusin-α levels were found to be low and salusin-β levels were high (Erden et al., 2015). In an in vitro study, it has been shown to induce the release of salusin-β from monocyte/macrophage cells as a result of stimulation with tumor necrosis factor-α (TNF-α) and lipopolysaccharide (Sato et al., 2010). In vitro studies, it has been reported 78
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and reactive oxygen species via the miR155/NOX2/NFκB signaling (Sun et al., 2016). In MS disease, proinflammatory cytokines, adhesion molecules and reactive oxygen species (ROS) production have been shown to increase in the neurodegenerative process (Cunningham, 2013). In this process, cytokine release is known to occur via the NFκB signaling pathway (Farh et al., 2015). In our study, the levels of salusins were higher in MS patients than in healthy subjects. The relationship between salusins and inflammation is likely to increase the level of salusins in RRMS patients. In our study, lymphocyte levels of MS group were significantly lower than the control group. This is due to the immunosuppressive drugs used by MS patients. There was no significant difference between treated and untreated patients in the levels of salusins. The absence of a difference in levels of salusins in treated and untreated patients suggests that salusins cannot be considered an inflammatory marker in RRMS patients. We consider this study as a pioneer that may guide the following researches on this issue. However, the role of salusins in the pathogenesis of the disease can be explained by supporting future large-scale longitudinal studies and different immunological and genetic parameters. There is a correlation between cortical lesions and cortical atrophy with increased EDSS score, indicating disability in MS (motor and ambulatory functions) (Geisseler et al., 2016). As the disease progresses to advanced stages, it is likely to worsen clinically. In our study, we did not find a correlation between salusin-α and salusin-β levels and the EDSS score in MS patients. In this group of patients that were not in the progressive phase, there may be no correlation with EDSS. Because, three months are needed to detect this situation in body fluids and to settle the ongoing inflammation process in the CSF although degeneration process is progressing (Walker et al., 2018). Our results could be expected to be more meaningful if we could evaluate myelin and axon damage with a marker that objectively shows the damage. In clinical practice, this necessitates the initiation of treatment in the early stage of the disease (Dobson and Giovannoni, 2018). This suggests that screening biomarkers are important in the diagnosis and follow-up phases of the disease.
Table 1 Baseline clinical and laboratory characteristics. Variables
Control (n = 40)
MS (n = 40)
P value
Male (n) Age BMI (kg/mm2) SBP (mm/Hg) DPB (mm/Hg) FBG (mg/dL) Leukocyte (mm3) Lymphocyte (mm3) Neutrophils (mm3) TG mmol/L LDL-c mmol/L HDL-c mmol/L TC mmol/L
(13 % 30.7) 38.5 ± 8 24.81 ± 2.64 100 ± 17.5 75.5 ± 7.3 85 ± 7.3 7.07 ± 1.49 2.28 ± 0.55 4.22 ± 1.31 122.3 ± 62.8 99.9 ± 25.3 56.1 ± 14 181.4 ± 36.5
(14 % 28.5) 36.5 ± 7.4 25.19 ± 3.66 115 ± 18.9 77.7 ± 18.9 90.1 ± 13.1 6.74 ± 1.86 2 ± 1.05* 3.95 ± 1.66 104.8 ± 36.7 104.8 ± 36.7 54.6 ± 16.4 188.7 ± 44.4
0.688 0.122 0.648 0.254 0.102 0.056 0.222 0.025 0.148 0.736 0.912 0.583 0.689
BMI, indicates body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting glucose; TG, triglyceride; LDL-c, low density lipoprotein cholesterol; HDL-c, high density lipoprotein cholesterol; TC, total cholesterol. All values are presented as mean ± SD. ⁎ P < 0.05 compared with control group.
5. Conclusion
Fig. 2. Correlation between serum levels of salusin-α and salusin beta. Data were evaluated with Pearson correlation analysis. Salusins levels are shown in pg/ml at study.
In conclusion, we found that there was a relationship between salusin-α and salusin-β levels, and MS. Since RRMS is the most common type of MS disease, our study was performed in this patient group. Since RRMS is the first stage of MS, it is important to conduct biomarker studies in this period in terms of early planning of treatment. The physiological role of salusins in the central nervous system and its relationship with neurodegenerative diseases has not been elucidated yet. Plasma salusin-α and salusin- β may be a biomarker for MS. However, further studies are needed to understand its relation with other
that salusin-β increases inflammation through activation of NF- κB, and that salusin-α has no effect (Koya et al., 2012; Zhou et al., 2014). In their study, Zhao et al. showed that salusin-β may increase the level of proinflammatory cytokine, adhesion molecules and oxidative stress via NOX2/ROS/ NFκB signaling (Zhao et al., 2017). In the study of Sun et al., salusin-β was shown to increase the level of monocyte adhesion
Fig. 3. Correlation between serum levels of salusin-α (A), salusin-β (B) and age. Data were evaluated with Spearman correlation analysis. Salusins levels are shown in pg/ml at study. 79
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neurological and inflammatory diseases to define it as a biomarker.
Sato, K., Koyama, T., Tateno, T., Hirata, Y., Shichiri, M., 2006. Presence of immunoreactive salusin-alpha in human serum and urine. Peptides 27 (11), 2561–2566. 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Conflict of interest Authors declare no conflict of interest in relation to the current work. Funding No sources of funding were received. References Aydin, S., 2013. Presence of adropin, nesfatin-1, apelin-12, ghrelins and salusins peptides in the milk, cheese whey and plasma of dairy cows. Peptides 43, 83–87. Cakir, M., Duzova, H., Taslidere, A., Orhan, G., Ozyalin, F., 2017. Protective effects of salusin-alpha and salusin-beta on renal ischemia/reperfusion damage and their levels in ischemic acute renal failure. Biotech. Histochem. 92 (2), 122–133. Celik, E., Celik, O., Yilmaz, E., Turkcuoglu, I., Karaer, A., Turhan, U., Aydin, S., 2013. Association of low maternal levels of salusins with gestational diabetes mellitus and with small-for-gestational-age fetuses. Eur. J. Obstet. Gynecol. Reprod. Biol. 167 (1), 29–33. Chang, Y., Yoo, J., Kim, H., Park, H.J., Jeon, S., Kim, J., 2018. Salusin-beta mediate neuroprotective effects for Parkinson's disease. Biochem. Biophys. Res. Commun. 503 (3), 1428–1433. Citil, C., Konar, V., Aydin, S., Yilmaz, M., Albayrak, S., Ozercan, I.H., Ozkan, Y., 2014. Brain, liver, and serum salusin-alpha and -beta alterations in Sprague-Dawley rats with or without metabolic syndrome. Med. Sci. Monit. 20, 1326–1333. Cunningham, C., 2013. Microglia and neurodegeneration: the role of systemic inflammation. Glia 61 (1), 71–90. D'Ambrosio, A., Pontecorvo, S., Colasanti, T., Zamboni, S., Francia, A., Margutti, P., 2015. Peripheral blood biomarkers in multiple sclerosis. Autoimmun. Rev. 14 (12), 1097–1110. Dobson, R., Giovannoni, G., 2018. Multiple Sclerosis - a review. Eur. J. Neurol. Erden, I., Demir, B., Ucak, H., Cicek, D., Dertlioglu, S.B., Aydin, S., 2014. Serum salusinalpha and salusin-beta levels in patients with Behcet's disease. Eur. J. Dermatol. 24 (5), 577–582. Erden, I., Ucak, H., Demir, B., Cicek, D., Bakar Dertlioglu, S., Ozturk, S., Aydin, S., 2015. Serum salusin-alpha and salusin-beta levels in patients with psoriasis. Eur. J. Dermatol. 25 (4), 352–353. Farh, K.K., Marson, A., Zhu, J., Kleinewietfeld, M., Housley, W.J., Beik, S., Shoresh, N., Whitton, H., Ryan, R.J., Shishkin, A.A., Hatan, M., Carrasco-Alfonso, M.J., Mayer, D., Luckey, C.J., Patsopoulos, N.A., De Jager, P.L., Kuchroo, V.K., Epstein, C.B., Daly, M.J., Hafler, D.A., Bernstein, B.E., 2015. Genetic and epigenetic fine mapping of causal autoimmune disease variants. Nature 518 (7539), 337–343. Fitzner, B., Hecker, M., Zettl, U.K., 2015. Molecular biomarkers in cerebrospinal fluid of multiple sclerosis patients. Autoimmun. Rev. 14 (10), 903–913. Frischer, J.M., Bramow, S., Dal-Bianco, A., Lucchinetti, C.F., Rauschka, H., Schmidbauer, M., Laursen, H., Sorensen, P.S., Lassmann, H., 2009. The relation between inflammation and neurodegeneration in multiple sclerosis brains. Brain 132 (Pt 5), 1175–1189. Geisseler, O., Pflugshaupt, T., Bezzola, L., Reuter, K., Weller, D., Schuknecht, B., Brugger, P., Linnebank, M., 2016. The relevance of cortical lesions in patients with multiple sclerosis. BMC Neurol. 16 (1), 204. Huang, X., Wang, Y., Ren, K., 2015. Deleterious effect of salusin-beta in paraventricular nucleus on sympathetic activity and blood pressure via NF-kappaB signaling in a rat model of obesity hypertension. Pharmazie 70 (8), 543–548. Izumiyama, H., Tanaka, H., Egi, K., Sunamori, M., Hirata, Y., Shichiri, M., 2005. Synthetic salusins as cardiac depressors in rat. Hypertension 45 (3), 419–425. Karussis, D., 2014. The diagnosis of multiple sclerosis and the various related demyelinating syndromes: a critical review. J. Autoimmun. 48-49, 134–142. Kimoto, S., Sato, K., Watanabe, T., Suguro, T., Koyama, T., Shichiri, M., 2010. Serum levels and urinary excretion of salusin-alpha in renal insufficiency. Regul. Pept. 162 (1-3), 129–132. Koya, T., Miyazaki, T., Watanabe, T., Shichiri, M., Atsumi, T., Kim-Kaneyama, J.R., Miyazaki, A., 2012. Salusin-beta accelerates inflammatory responses in vascular endothelial cells via NF-kappaB signaling in LDL receptor-deficient mice in vivo and HUVECs in vitro. Am. J. Physiol. Heart Circ. Physiol. 303 (1), H96–105. Kurtzke, J.F., 1983. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 33 (11), 1444–1452. Li, H.B., Lu, Y., Liu, J.J., Miao, Y.W., Zheng, T.Z., Su, Q., Qi, J., Tan, H., Yuan, Z.Y., Zhu, G.Q., Kang, Y.M., 2016. Salusin beta within the nucleus tractus solitarii suppresses blood pressure via inhibiting the activities of presympathetic neurons in the rostral ventrolateral medulla in spontaneously hypertensive rats. Cardiovasc. Toxicol. 16 (3), 223–234. Li, H.B., Qin, D.N., Suo, Y.P., Guo, J., Su, Q., Miao, Y.W., Sun, W.Y., Yi, Q.Y., Cui, W., Cheng, K., Zhu, G.Q., Kang, Y.M., 2015. Blockade of salusin-beta in hypothalamic paraventricular nucleus attenuates hypertension and cardiac hypertrophy in salt-induced hypertensive rats. J. Cardiovasc. Pharmacol. 66 (4), 323–331. Ozgen, M., Koca, S.S., Dagli, N., Balin, M., Ustundag, B., Isik, A., 2011. Serum salusinalpha level in rheumatoid arthritis. Regul. Pept. 167 (1), 125–128. Sato, K., Fujimoto, K., Koyama, T., Shichiri, M., 2010. Release of salusin-beta from human monocytes/macrophages. Regul. Pept. 162 (1-3), 68–72.
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Increased level of plasma salusin-α and salusin-β in patients with multiple sclerosis
T
Murat Çakıra, , Seda Sabah-Özcanb, Hikmet Saçmacıc ⁎
a
Faculty of Medicine, Department of Physiology, University of Bozok, Yozgat, 66200, Turkey Faculty of Medicine, Department of Medical Biology, University of Bozok, Yozgat, 66200, Turkey c Faculty of Medicine, Department of Neurology, University of Bozok, Yozgat, 66200, Turkey b
ARTICLE INFO
ABSTRACT
Keywords: Salusin-α Salusin-β Multiple sclerosis Relapsing-Remitting Multiple Sclerosis
Background: Multiple Sclerosis (MS) is a potentially progressive autoimmune disorder of the central nervous system. The pathology of MS is characterized by inflammation, demyelination, reactive gliosis and neuronal damage. Salusin-α and salusin-β have been shown to be widely expressed in many tissues, including the central nervous system. In our study, we investigated whether salusin-α and salusin-β peptides had a relation with inflammation and whether it is related to Relapsing-Remitting Multiple Sclerosis (RRMS) disease. Methods: Forty healthy controls and forty patients with RRMS were included in the study. Salusin-α and Salusinβ levels were measured by Enzyme-linked Immuno-Sorbent Assay (ELISA). Results: Salusin-α and salusin-β levels were high at a significant level in RRMS patients compared to healthy controls (p < 0.0001). We found a strong positive correlation between salusin-α and salusin-β levels (p < 0.0001, r = 0,9925). Conclusion: In conclusion, we found that there was a relationship between salusin-α and salusin-β levels, and MS disease. Since RRMS is the first stage of MS and its most common type, it is important to perform biomarker studies in this period in terms of early planning of treatment. Although salusin-α and salusin-β levels increase in RRMS patients, further studies are needed to understand its relation with other neurological and inflammatory diseases to define it as a biomarker.
1. Introduction Multiple Sclerosis (MS) is a potentially progressive autoimmune disorder of the central nervous system. More than two million people worldwide suffer from MS. The pathology of MS is characterized by inflammation, demyelination, reactive gliosis and neuroaxonal damage (Zephir, 2018). MS disease occurs as a result of genetic predisposition and environmental triggers. Clinical symptoms vary depending on the lesion burden and localization and include muscle weakness, blurred vision, dizziness, fatigue and balance problems (Sawcer et al., 2011). Many studies of MS have identified various biomarkers for the diagnosis of the disease, the activity of the disease, and the prediction of the response of the patient to treatment (D'Ambrosio et al., 2015). Many of the biomarker examinations that can be used in the diagnosis of MS are performed in the cerebrospinal fluid (CSF) obtained by an invasive
procedure (Fitzner et al., 2015). Due to the difficulty of obtaining CSF, biomarkers that are examined in biomaterials which are acquired by non-invasive methods such as serum and plasma and which can be used in the diagnosis of the disease are needed. Salusin-α and salusin-β discovered in human embryo are composed of 28 and 20 amino acids, respectively. Salusins have been shown to be widely expressed in many tissues, including the central nervous system (Shichiri et al., 2003). Intravenous infusion of salusin-α and salusin-β to the rats have been reported to cause hypotension and bradycardia (Izumiyama et al., 2005). Salusins have also been shown to cause proliferation in vascular smooth muscle cells and fibroblasts (Shichiri et al., 2003). Salusins increase hypertrophy and growth in rat cardiomyocytes (Yu et al., 2004). It has also been reported that salusins have antiapoptotic effect in these cells (Xiao-Hong et al., 2006). Salusins have been shown to be associated with various diseases such as
Abbreviations: BMI, body mass index; CSF, cerebrospinal fluid; ELISA, Enzyme-linked Immuno-Sorbent Assay; EDSS, Expanded Disability Status Scale; HDL-c, highdensity lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; MS, Multiple sclerosis; NF-κB, Nuclear Factor-κB; NO, nitric oxide; ROS, reactive oxygen species; RRMS, Relapsing-Remitting Multiple Sclerosis ⁎ Corresponding author. E-mail address: [email protected] (M. Çakır). https://doi.org/10.1016/j.msard.2019.02.003 Received 20 November 2018; Received in revised form 28 January 2019; Accepted 3 February 2019 2211-0348/ © 2019 Published by Elsevier B.V.
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metabolic syndrome and acute renal failure (Cakir et al., 2017; Citil et al., 2014; Kimoto et al., 2010). Studies have shown that salusins are associated with inflammation and oxidative stress (Cakir et al., 2017; Xu et al., 2016; Zhao et al., 2017). Salusin-β has been reported to accelerate inflammation through the Nuclear Factor-κB (NF-κB) pathway (Koya et al., 2012). Although the physiological roles of salusins in the central nervous system are not clear; salusins have been shown to have neuroendocrine effects such as antidiuretic hormone release, blood pressure regulation and modulation of the functioning of the autonomic nervous system (Huang et al., 2015; Li et al., 2016; Li et al., 2015; Suzuki-Kemuriyama et al., 2016). In this study, we investigated whether salusin-α and salusin-β peptides, whose relation to inflammation were shown in the previous studies, is associated with Relapsing-Remitting Multiple Sclerosis (RRMS) disease.
2.3. Determination of plasma salusin-α and salusin-β levels Plasma salusin-α and salusin-β levels were measured using an Enzyme-linked Immuno Sorbent Assay (ELISA) kit with a minimum detectable concentration of 0.93 pg/mL and 1.75 pg/mL, respectively from Uscn Life Science Inc. (Wuhan, PR China). Optical density values for samples and standard samples were detected on a Spectramax ELISA reader (Molecular Devices) at 450 nm. The results are presented as pg/ mL. 2.4. Statistical analysis SPSS 20 package program was used for data analysis. It was determined whether the quantitative data showed normal distribution according to the Shapiro-Wilk normality test. Independent-samples ttest was used to compare normal distribution data between groups. The Mann-Whitney U test was used for the comparison of the groups that showed non-normally distributed data. Pearson correlation analysis was used for the correlation analysis of the data with normal distribution, and Spearman's correlation analysis was used for the correlation analysis of the data not showing normal distribution. Arithmetic mean ± standard deviation (SD) was used to define quantitative data. p < 0.05 was considered statistically significant.
2. Materials and methods 2.1. Subject All applications in this study were performed in accordance with the protocol approved by Bozok University Local Ethics Committee (2017KAEK-189_2018.05.30_06). In this prospective, cross-sectionally designed study, 40 RRMS patients (27 females, 13 males) and 40 healthy controls (26 females, 14 males) who applied to Neurology clinics between the ages range 21–60 years were included in the study. The following patient data were collected: age, gender, all symptoms, disease duration, the frequency of attacks, family history of MS, drug intake (related to MS) of patients were also recorded. Patients with another disease in the RRMS patient group were excluded from the study. While 10 of the patients were not receiving any prophylactic treatment, 5 were taking fingolimod, 2 were receiving natalizumab and 23 patients were taking first-line treatment (interferon, teriflunomide, dimethyl fumarate). The control group comprised healthy and age, body mass index (BMI) (kg/m2) and sex-matched volunteers. Leukocyte, neutrophil, lymphocyte, blood glucose, triglyceride, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), total cholesterol levels and BMI of all individuals, who were included in the study, was measured. A neurological examination of all the patients was carried out, and their disability was defined according to Expanded Disability Status Scale (EDSS) score and by using points for functional systems. Disability was measured with the EDSS, an ordinal scale of 0.0–10 (“no disability” to “death due to MS”), with higher scores reflecting the greater level of disability (Kurtzke, 1983). RRMS was defined according to the revised McDonald criteria 2010 and reporting neither attacks nor steroid use during the month before the interview was held. All patients who did not have concomitant neurological disorder were invited to participate. Other exclusion criteria were; congenital abnormalities of brain, recent head trauma, metabolic disease (diabetes mellitus, hypertension), acute-chronic infection, peripheral neuropathy, cardiac-thyroid-lung diseases, collagen tissue disease, renal failure, liver diseases, pregnant women and alcohol users.
3. Results Plasma salusin-α levels (Fig. 1A) were higher in the RRMS group (104 ± 192 pg/mL) compared to the control group (40 ± 136 pg/ mL) (p < 0.000). Plasma Salusin-β levels (Fig. 1B) were significantly higher in RRMS group (344 ± 640 pg / mL) than in the control group (128 ± 464 pg / mL) (p < 0.000). There was no significant difference between treated (n = 30) and untreated (n = 10) patients in the levels of salusins (p ˃ 0.05). When the lymphocyte levels between the groups were compared (Table 1), it was found to be lower in the RRMS group (0.4 ± 4.58) than in the control group (1.36 ± 4) (p < 0.025). When the correlation between the data was examined, a strong and positive correlation was detected between salusin-α and salusin-β levels (Fig. 2) (p < 0.000, r = 0,9925). When plasma salusin levels were compared with EDSS score and disease duration in terms of disease disability, no statistically significant correlation was found (p ˃ 0.05). Mean and standard deviation of EDSS and disease duration was 2.85 ± 1.18 and 8.3 ± 5.5 respectively. We found a weak negative correlation between salusin-α and lymphocyte levels (p < 0.040, r = −0,2307) when all individuals in the patient and control group are included. Similarly, we observed a weak and negative correlation between salusin-β level and lymphocyte level (p < 0.040, r = −0,2298). When the correlation between salusin-α and salusin-β levels and lymphocyte counts were examined separately in patient and control groups, we found no statistical significance (p ˃ 0.05). We found a negative correlation between age and levels of salusin-α (p < 0.025, r = −0,2503) and salusin-β (p < 0.025, r = −0,2497) (Fig. 3A and B). 4. Discussion Salusin-α and salusin-β was discovered in 2003 by Shichiri et al. in human embryo. Preprosalusin is cut from different amino acid regions, and salusin-α and salusin-β are formed. Preprosalusin is expressed in many parts of the central nervous system (Shichiri et al., 2003; Suzuki et al., 2011). Although salusins are commonly found in different regions of the brain, in the immune system and in many tissues in the body, the studies in the literature showed that the number of these peptides is limited. Salusin-α and salusin-β levels in human serum and urine were also determined (Sato et al., 2006). The studies on salusins have mainly focused on the relationship of these two peptides with the cardiovascular system. It has been shown that both peptides decrease heart rate
2.2. Biochemical analysis Blood was taken from patients and control group between 09.00 and 10.00 am. Baseline blood samples were collected from the subjects and patients into vacutainers containing Na2-EDTA (1.5 mg/mL), and blood samples were centrifuged for 10 min at 3000 rpm, after which the supernatant was quickly removed and kept frozen at −80 °C until the assays were performed by a specialist who was blind to patient status. The complete blood count, blood glucose level, blood lipids data of all the participants were obtained from the hospital registry system. 77
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Fig. 1. Comparison of plasma levels of salusin-α between control subjects and MS patients (A). Comparison of plasma levels of salusin-β between control subjects and MS patients (B).Data analyzed with independent sample t test. ***(p < 0.0001), (n = 80).
and blood pressure when administered intravenously in rats (Izumiyama et al., 2005). Salusin-α has been shown to decrease the formation of atherosclerosis by reducing foam cell formation, and salusin-β to increase atherosclerosis by increasing foam cell formation (Watanabe et al., 2008). In experimental gastric ulcer model, salusins have been reported to have protective effect (Tanyeli et al., 2017). In another study, the neuroprotective effect of salusin-β in the experimental Parkinson's disease model was shown. (Chang et al., 2018). In many studies, it was shown that there was no positive correlation between salusin-α and salusin-β levels, (Aydin, 2013; Sipahi et al., 2018; Watanabe et al., 2008); however, some studies showed a positive correlation (Celik et al., 2013). In our study, we found a positive correlation between salusin-α and salusin-β levels (P < 0,000, r:0,994). The correlation between salusin-α and salusin-β levels was found in both control and MS groups. MS is a serious health problem affecting the daily life of individuals due to the characteristic of its symptoms and progressive nature. The characteristic pathological sign of MS is that demyelinating plaques form perivenular inflammatory lesions (Karussis, 2014). Oligodendrocyte damage and demyelination occur as a result of inflammation
(Frischer et al., 2009). Infiltration of CNS immune cells causes more inflammation and damage. T-lymphocytes are the most important cells infiltrated into the tissue in MS. B-cells and plasma cells have tissue infiltration, albeit at a lower rate (Frischer et al., 2009). The release of proinflammatory cytokines induced by active lymphocytes promotes the activation of microglia and macrophages. It also leads to the development of phagocytic abilities of these cells and the production of various cytotoxic agents such as reactive oxygen species (ROS) and nitric oxide (NO) (Cunningham, 2013). Antibodies produced by active B Cells against the proteins and lipids of the myelin sheath are also included in this process (von Budingen et al., 2011). Studies have been conducted to investigate whether salusins might be associated with inflammation and inflammatory diseases. Serum salusin-α levels and salusin-β levels were found to be high in patients with Behçet's disease (Erden et al., 2014; Ozgen et al., 2011). In psoriasis patients, salusin-α levels were found to be low and salusin-β levels were high (Erden et al., 2015). In an in vitro study, it has been shown to induce the release of salusin-β from monocyte/macrophage cells as a result of stimulation with tumor necrosis factor-α (TNF-α) and lipopolysaccharide (Sato et al., 2010). In vitro studies, it has been reported 78
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and reactive oxygen species via the miR155/NOX2/NFκB signaling (Sun et al., 2016). In MS disease, proinflammatory cytokines, adhesion molecules and reactive oxygen species (ROS) production have been shown to increase in the neurodegenerative process (Cunningham, 2013). In this process, cytokine release is known to occur via the NFκB signaling pathway (Farh et al., 2015). In our study, the levels of salusins were higher in MS patients than in healthy subjects. The relationship between salusins and inflammation is likely to increase the level of salusins in RRMS patients. In our study, lymphocyte levels of MS group were significantly lower than the control group. This is due to the immunosuppressive drugs used by MS patients. There was no significant difference between treated and untreated patients in the levels of salusins. The absence of a difference in levels of salusins in treated and untreated patients suggests that salusins cannot be considered an inflammatory marker in RRMS patients. We consider this study as a pioneer that may guide the following researches on this issue. However, the role of salusins in the pathogenesis of the disease can be explained by supporting future large-scale longitudinal studies and different immunological and genetic parameters. There is a correlation between cortical lesions and cortical atrophy with increased EDSS score, indicating disability in MS (motor and ambulatory functions) (Geisseler et al., 2016). As the disease progresses to advanced stages, it is likely to worsen clinically. In our study, we did not find a correlation between salusin-α and salusin-β levels and the EDSS score in MS patients. In this group of patients that were not in the progressive phase, there may be no correlation with EDSS. Because, three months are needed to detect this situation in body fluids and to settle the ongoing inflammation process in the CSF although degeneration process is progressing (Walker et al., 2018). Our results could be expected to be more meaningful if we could evaluate myelin and axon damage with a marker that objectively shows the damage. In clinical practice, this necessitates the initiation of treatment in the early stage of the disease (Dobson and Giovannoni, 2018). This suggests that screening biomarkers are important in the diagnosis and follow-up phases of the disease.
Table 1 Baseline clinical and laboratory characteristics. Variables
Control (n = 40)
MS (n = 40)
P value
Male (n) Age BMI (kg/mm2) SBP (mm/Hg) DPB (mm/Hg) FBG (mg/dL) Leukocyte (mm3) Lymphocyte (mm3) Neutrophils (mm3) TG mmol/L LDL-c mmol/L HDL-c mmol/L TC mmol/L
(13 % 30.7) 38.5 ± 8 24.81 ± 2.64 100 ± 17.5 75.5 ± 7.3 85 ± 7.3 7.07 ± 1.49 2.28 ± 0.55 4.22 ± 1.31 122.3 ± 62.8 99.9 ± 25.3 56.1 ± 14 181.4 ± 36.5
(14 % 28.5) 36.5 ± 7.4 25.19 ± 3.66 115 ± 18.9 77.7 ± 18.9 90.1 ± 13.1 6.74 ± 1.86 2 ± 1.05* 3.95 ± 1.66 104.8 ± 36.7 104.8 ± 36.7 54.6 ± 16.4 188.7 ± 44.4
0.688 0.122 0.648 0.254 0.102 0.056 0.222 0.025 0.148 0.736 0.912 0.583 0.689
BMI, indicates body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting glucose; TG, triglyceride; LDL-c, low density lipoprotein cholesterol; HDL-c, high density lipoprotein cholesterol; TC, total cholesterol. All values are presented as mean ± SD. ⁎ P < 0.05 compared with control group.
5. Conclusion
Fig. 2. Correlation between serum levels of salusin-α and salusin beta. Data were evaluated with Pearson correlation analysis. Salusins levels are shown in pg/ml at study.
In conclusion, we found that there was a relationship between salusin-α and salusin-β levels, and MS. Since RRMS is the most common type of MS disease, our study was performed in this patient group. Since RRMS is the first stage of MS, it is important to conduct biomarker studies in this period in terms of early planning of treatment. The physiological role of salusins in the central nervous system and its relationship with neurodegenerative diseases has not been elucidated yet. Plasma salusin-α and salusin- β may be a biomarker for MS. However, further studies are needed to understand its relation with other
that salusin-β increases inflammation through activation of NF- κB, and that salusin-α has no effect (Koya et al., 2012; Zhou et al., 2014). In their study, Zhao et al. showed that salusin-β may increase the level of proinflammatory cytokine, adhesion molecules and oxidative stress via NOX2/ROS/ NFκB signaling (Zhao et al., 2017). In the study of Sun et al., salusin-β was shown to increase the level of monocyte adhesion
Fig. 3. Correlation between serum levels of salusin-α (A), salusin-β (B) and age. Data were evaluated with Spearman correlation analysis. Salusins levels are shown in pg/ml at study. 79
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neurological and inflammatory diseases to define it as a biomarker.
Sato, K., Koyama, T., Tateno, T., Hirata, Y., Shichiri, M., 2006. Presence of immunoreactive salusin-alpha in human serum and urine. Peptides 27 (11), 2561–2566. 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