1 Volume 22 2023 e237270 Original Article Braz J Oral Sci. 2023;22:e237270http://dx.doi.org/10.20396/bjos.v22i00.8667270 1 Oral and Maxillofacial Diseases Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. 2 Surgical Oncology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.  3 Faculty of Health, Mashhad University of Medical Sciences, Mashhad, Iran. Corresponding author: Maryam Amirchaghmaghi, Ala Ghazi Contact information: Oral and Maxillofacial Diseases Research Center, Department of Oral Medicine of Mashhad Faculty of Dentistry, Vakilabad Blvd, Postal code:9177948959, Mashhad, Iran. amirchakhmaghim@mums.ac.ir, ghazial@mums.ac.ir Editor: Dr. Altair A. Del Bel Cury Received: October 14, 2021 Accepted: July 30, 2022 Assessment of salivary opiorphin in oral lichen planus Zohreh Dalirsani1 , Maryam Amirchaghmaghi1*≠ , Ala Ghazi1*≠ , Seyed Isaac Hashemy2 , Mahboobeh Taherizadeh3 Opiorphin is a pentapeptide, which could be isolated from human fluids and has a decreasing effect on pain. Aim: Since lichen planus is a chronic mucocutaneous disease, which causes pain or burning feeling in the oral mucosa, this study aimed to compare salivary opiorphin levels of oral lichen planus (OLP) patients with healthy subjects. Methods: This case-control study, was performed on 24 patients with OLP lesions and 21 healthy subjects. After collecting un- stimulated saliva, opiorphin levels were compared between two groups through statistical analyses. Results: There was not any significant difference between OLP patients and healthy subjects according to salivary opiorphin concentration (p=0.378). Also, in the OLP group, opiorphin concentration was not significantly different between males and females (p=0.601). Analytical analysis could not show any remarkable difference between various severity of OLP lesions regarding to salivary opiorphin levels (p=0.653). Conclusion: In this study, salivary opiorphin levels was not significantly different between patients with OLP and healthy subjects; however, more studies are suggested for better assessment of salivary opiorphin levels in various types of OLP lesions and its correlation with pain severity. Keywords: Oligopeptides. Lichen planus, oral. ≠ Maryam Amirchaghmaghi and Ala Ghazi contributed equally to this work. about:blank about:blank https://orcid.org/0000-0002-5898-3180 https://orcid.org/0000-0002-6504-2725 https://orcid.org/0000-0002-0064-1233 https://orcid.org/0000-0002-1323-5250 https://orcid.org/0000-0003-1061-3284 2 Dalirsani et al. Braz J Oral Sci. 2023;22:e237270 Introduction Opiorphin is a pentapeptide, which could be isolated from human fluids including saliva1. It seems that opiorphin inhibits encephalin- inactivating enzymes and increases half-life of encephalin2. Moreover, opiorphin could inhibit nociception due to chronic and acute stimulation, and perception of pain, this effect is compatible with morphine1. Since, this peptide has a preventive effect on pain, there is a hypothesis, which depict that every chronic disease imposes chronic pain or discomfort, might influence on opiorphin levels. Lichen planus is a chronic mucocutaneous disease, in which, the patients mostly tolerate a long time burning and pain in their oral mucosa. Despite huge progres- sion about etiology of OLP, the exact mechanism and definite treatment of this condition is unclear. The researchers have been trying to find some acceptable and noninvasive methods for diagnosis and assessment of efficacy of the treatments. Up to now, several stud- ies have been performed on some salivary biomarkers in OLP patients; however, none of them evaluate salivary opiorphin, as an index of chronic pain. This study was conducted to investigate salivary opiorphin concentrations of OLP patients and compare this marker with healthy subjects. Methods and Materials This case-control study was performed in the oral and maxillofacial medicine depart- ment of Mashhad Faculty of Dentistry from September 2018 until June 2019. Twenty-four patients with OLP (both keratotic and non-keratotic), and 21 healthy sub- jects, after signing a written informed consent form, enrolled in this study. Diagnosis of OLP was confirmed through clinical and histopathological examinations, according to WHO modified criteria3. The healthy individuals were selected from the patients, who were referred to Mash- had Faculty of Dentistry, who had no oral lesions or systemic diseases, or any pain; they had only been referred for common dentistry procedures. The inclusion criteria for the case group were: newly diagnosed oral lichen planus patients, as confirmed by clinical and histopathological examination; patients who had signed the informed consent form. The exclusion criteria for the case group were as bellow: the subjects, who had used any drugs interacting with opioids or analge- sics; the patients, whose histopathological examination was reported as lichenoid reactions. The exclusion criteria for the control group was the usage any medicine for relief of the pain, in recent days. Prepared checklists were completed for all subjects including demographic charac- teristics consisting of age, gender, and medical history; lesion properties including the site of lesion, clinical features (keratotic and non-keratotic), type and severity of 3 Dalirsani et al. Braz J Oral Sci. 2023;22:e237270 OLP lesion, based on Thongprasom criteria3. The patients were categorized into five categories 20-30, 30-40, 40-50, 50-60, and ≥60 years according to their age. The cor- relation of opiorphin levels, and sex and age groups was statistically analyzed. Assessment of opiorphin All subjects were asked not to drink, eat, or smoke for 90 minutes before collecting unstimulated saliva. Saliva samples were collected through spitting method between 9 to 12 a.m. The patients were asked to sit in a convenient position and after gather- ing the saliva in their mouth, spit into 50 ml falcon sterilized tube every 1 minute for 5-10 minutes4. The salivary samples were stored with ice and sent to the laboratory and kept at -20°C for 24 hours for mucolysis. In order to separate debris and mucous, the saliva samples were centrifuged for 15 minutes at 3000 rpm. Until collecting all samples and analysis, the samples were kept at -20°C. After that, the watery parts of the salivary samples were transferred to microtubes with a volume of 1.5 cc. Then opiorphin levels were measured using the kits of “Human Opiorphin ELISA Kit (Catalog # MBS760008)” according to the protocol of the company. The bioten double antibody sandwich was used for the assessment of opiorphin. First, 40 µL of the salivary sample and 10 µL of opiorphin antibody were added to 50 µL of standard solution and 50 µL of streptavidin-HPR; after that, the mixed solution was incubated at 37ºC for 60 min. Next, this solution was washed with 300 µL diluted buffer five times. Then, 50 µL of chromogen A and 50 µL of chromogen B solutions were added to prepare the sam- ples. After mixing them, the samples were incubated at 37ºC for 10 minutes. Finally, 50 µL of stop solution was added to them, which change the yellow color to blue color. After 10 minutes, the optical density was measured at the wavelength of 450 nm to assess the opiorphin concentration. The opiorphin levels were compared between healthy subjects and OLP patients through statistical analyses. Statistical analysis The data were entered into SPSS software (version 24) and statistical analysis was performed. Kolmogorov-Smirnov test was employed for quantitative data with normal distribution. In order to assess the qualitative variables between the groups, Chi-do and Fisher’s exact test were used. For quantitative variables with normal distribution, para- metric statistical methods including independent t-test and ANOVA, and for nonpara- metric methods in case of abnormal distribution, Mann-Whitney test and Kruskal-Wallis were employed. The significance level for the statistical tests was considered as 0.05. The protocol of this study was approved by ethical committee of Mashhad University of Medical Sciences (the ethical code: IR.MUMS.DENTISTRY.REC.1397.045). Results In this study, salivary opiorphin levels of 24 OLP patients and 21 healthy subjects were assessed. 4 Dalirsani et al. Braz J Oral Sci. 2023;22:e237270 Kolmogorov-Smirnov test showed that the variables of age and opiorphin have normal distribution. Therefore, independent t- test was used for assessment of these variables. The mean ages of OLP patients and healthy persons were 46.41±11.89 and 46.09±9.12 years, respectively. Statistical analysis revealed that there was no any significant vari- ations between the two groups regarding the mean age (p=0.920). Also, more number of OLP patients belonged to 50-60-year group; however, chi square test showed that there was no remarkable correlation between development of OLP and subjects’ age (p=0.389) (Table 1). The mean of salivary opiorphin levels were 2.76±0.66 and 2.93±0.55 for the case and control groups, respectively (Table 1 and Figure 1). Independent t- test did not show any significant difference between OLP patients and healthy subjects according to salivary opiorphin levels (p=0.378) (Table 1). Pearson’s Chi-Square test did not reveal any significant correlation between OLP patients’ age and salivary opiorphin levels (p=0.187). Furthermore, this statistical analysis did not show any remarkable relationship between healthy subjects’ age and salivary opiorphin concentration (p=0.597). In the case group, the mean of salivary opiorphin levels were 2.72±0.69 and 2.89±0.58 for the females and males, respectively. Statistical analysis did not show any remark- able difference between two genders in the OLP patients according to salivary opi- orphin levels (p=0.601) (Table 2). Also, in the control group, the mean of salivary opi- orphin levels were 2.79±0.40 and 3.07±0.67 for the females and males, respectively, which were not significantly different (p=0.257) (Table 2). Thus, we concluded that subjects’ gender has no effect on salivary opiorphin level. Table 1. demographic data and opiorphin levels in the case and control groups. Variables Number (%) Case group Control group p-value Number (%) Sex Male 6(62.1) 10(37.5) p=0.114*Female 18(37.9) 11(62.5) Total 24(100) 21(100) Age (years) 20-30 3(12.5) 0(0) p=0.389* 30-40 5(20.8) 8(38.1) 40-50 6(25) 6(28.6) 50-60 8(33.3) 6(28.6) ≥60 2(8.3) 1(4.8) Total 24(100) 21(100) Opiorphin 2.76±0.66 2.93±0.55 0.378** *Chi square test **Independent samples t-test 5 Dalirsani et al. Braz J Oral Sci. 2023;22:e237270 Moreover, according to opiorphin levels, there was no remarkable difference between OLP females and healthy females (p=0.758), as well as between OLP males and healthy males (p=0.587). Analytical analysis showed that opiorphin levels were not statistically different between various severity of OLP lesions, based on Tongprasom criteria (p=0.653); and there was no significant difference between keratotic, and non-keratotic types of OLP lesions (p=0.453). We concluded that there is no association between salivary opiorphin levels, and severity of OLP lesions. Discussion Some biomarkers, which are secreted into saliva, could indicate some physiologic and pathologic conditions. Opiorphin is a pentapeptide, which might be isolated from human saliva1 and could alleviate pain due to inflammation and physical damage. Also, this biomarker is a natural suppressor of enzymes, which protects enkepha- lins and endorphin from degradation. Since enkephalin and endorphin are the native painkillers; this process could reduce the pain sensation. Moreover, opiorphin has anti-depressant5, anti-inflammatory and anti-tumoral effects1,6. Sobocińska et al.7 (2020) studied the effects of sialorphin and spinorphin on a mouse model of colitis and suggested these peptides as anti-inflammatory elements. Nejad et al.6 (2020) study revealed that salivary opiorphin levels in the patients, who had painful oral soft tissue lesions including traumatic and inflammatory Table 2. opiorphin levels based on the sex in the case and control groups. Variables Sex Case group Control group p-value* Opiorphin Female 2.72±0.69 2.79±0.4 0.758 Male 2.89±0.58 3.07±0.67 0.587 p-value* 0.601 0.257 *Independent samples t-test Figure 1. box plot of opiorphin levels in the case and control groups. 5.00 * 18 4.00 3.00 2.00 1.00 OLP Healthy Group O pi or ph in 6 Dalirsani et al. Braz J Oral Sci. 2023;22:e237270 conditions, as well as, in patients with oral pre-malignant or malignant lesions, were higher than in healthy subjects; their study confirmed that the severity and type of pain could effect on opiorphin levels. Therefore, assessment of patients’ salivary opiorphin could be an indicator of the severity of pain. Furthermore, these rising opiorphin levels in oral cancer patients could suggest an anti-tumoral role of this marker. An in-vivo study on a mouse model of melanoma showed that opiorphin and sialor- phin conjugate to a proapoptotic and antimicrobial peptide (called klak) leading to the formation of compounds, sialo-klak and opio-klak. These compounds have positive cytotoxic effects on cancer cells8. In the present study, the opiorphin levels were assessed in the patients involved with OLP, as potentially malignant lesions. Although opiorphin levels of OLP patients were higher than in healthy persons, the difference was not significant. Concurrently, similar study on painful oral lesions, showed that the opiorphin levels in these patients were higher than the controls6. Furthermore, Ozdogan et al.9 (2019) study showed that rate of salivary opiorphin concentration has a positive correlation with the severity of tooth pain. They concluded that inflammation due to the pulp or periodontal diseases could raise opiorphin levels. In the present study, which was performed on 24 OLP patients including 7 keratotic and 17 non-keratotic OLP; there was not statistically significant difference between salivary opiorphin levels of patients with keratotic and non-keratotic lesions. Also, this biomarker level did not noticeably rise in severe lesions based on Tongprasom crite- ria. Therefore, this study could not prove that inflammation due to an erosive form of OLP increase opiorphin levels in these patients. The results of this study were contradictory with Ozdogan et al.9 (2019) study, which observed that there was a direct correlation between opiorphin concentration and severity of inflammation. With respect to the fact that another study did not perform on various types of OLP lesions and opiorphin levels, a comprehensive study with a greater sample size on different severity of OLP is suggested for better judgment about the relationship of salivary opiorphin levels and severity of OLP lesions. In this study, there was no significant difference between the salivary opiorphin con- centration of OLP patients and healthy subjects; as well as, various types of OLP lesions. Further studies are recommended to evaluate salivary opiorphin levels in dif- ferent scores of OLP lesions and its correlation with pain severity. Author Contribution Zohreh Dalirsani, Maryam Amirchaghmaghi, Ala Ghazi and Seyed Isaac Hashemy contributed in designing and performing the project. Mahboobeh Taherizadeh con- tributed in analyzing the data. All authors actively participated in the manuscript’s findings and have revised and approved the final version of the manuscript. 7 Dalirsani et al. Braz J Oral Sci. 2023;22:e237270 Conflict of interest The authors declare no conflict of interest in this study. References 1. Wisner A, Dufour E, Messaoudi M, Nejdi A, Marcel A, Ungeheuer MN, et al. Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways. Proc Natl Acad Sci U S A. 2006 Nov;103(47):17979-84. doi: 10.1073/pnas.0605865103. 2. Sujatha S, Priyadharshini R, Niloofar Khansari N, Yashoda Devi BK, Shwetha V, Pavan Kumar T, et al. An analgesic to bridge the gap between Narcotics and NSAIDs: opiorphin. Int J Basic Clin Pharmacol. 2018;7(7):1432-6. doi: 10.18203/2319-2003.ijbcp20182695. 3. Thongprasom K, Luangjarmekorn L, Sererat T, Taweesap W. Relative efficacy of fluocinolone acetonide compared with triamcinolone acetonide in treatment of oral lichen planus. J Oral Pathol Med. 1992 Nov;21(10):456-8. doi: 10.1111/j.1600-0714.1992.tb00974.x. 4. Shirzaiy M, Dalirsani Z. The effect of glycemic control on salivary lipid peroxidation in type II diabetic patients. Diabetes Metab Syndr. 2019 May-Jun;13(3):1991-4. doi: 10.1016/j.dsx.2019.04.004. 5. Javelot H, Messaoudi M, Garnier S, Rougeot C. Human opiorphin is a naturally occurring antidepressant acting selectively on enkephalin-dependent delta-opioid pathways. J Physiol Pharmacol. 2010 Jun;61(3):355-62. 6. Nejad NK, Ramakrishna P, Kar A, Sujatha S. Quantitative analysis and expression of salivary opiorphin in painful oral soft-tissue conditions: A descriptive study. J Global Oral Health. 2020;3(2):123-7. doi: 10.25259/JGOH_41_2020. 7. Sobocińska M, Salaga M, Fichna J, Kamysz E. Anti-inflammatory effect of homo- and heterodimers of natural enkephalinase inhibitors in experimental colitis in mice. Molecules. 2020 Dec;25(24):5820. doi: 10.3390/molecules25245820. 8. Kamysz E, Smolarczyk R, Cichoń T, Jarosz-Biej M, Sikorska E, Sobocińska M, et al. Antitumor activity of opiorphin, sialorphin and their conjugates with a peptide klaklakklaklak. J Pept Sci. 2016 Nov;22(11-12):723-30. doi: 10.1002/psc.2936. 9. Ozdogan MS, Gungormus M, Ince Yusufoglu S, Ertem SY, Sonmez C, Orhan M. Salivary opiorphin in dental pain: A potential biomarker for dental disease. Arch Oral Biol. 2019 Mar;99:15-21. doi: 10.1016/j.archoralbio.2018.12.006.