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ORIGINAL ARTICLE
Year : 2020  |  Volume : 12  |  Issue : 2  |  Page : 113-118

Evaluation of Salivary 8-Isoprostane in Oral Lichen Planus: Case Control Study


Department of Oral & Maxillofacial Pathology, Azeezia College of Dental Science & Research, Meeyannoor, Kerala, India

Date of Submission18-Sep-2020
Date of Acceptance16-Nov-2020
Date of Web Publication16-Feb-2021

Correspondence Address:
Dr. Rathy Ravindran
Department of Oral & Maxillofacial Pathology, Azeezia College of Dental Science & Research, Meeyannoor, Kerala-691537
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jofs.jofs_220_20

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  Abstract 


Introduction: Lichen planus is a chronic inflammatory disease that affects the skin, nails, and the mucous membrane and oral mucosal involvement may frequently precedes skin manifestation. It is hypothesized that free radicals and reactive oxygen species has a strong relationship in the pathogenesis of oral lichen planus. F2-isoprostane are a group of bioactive prostaglandins and they are isomers of PGF2α is a most accurate approach to assess oxidant injury in vivo. The aim of the study was to evaluate the level of 8-isoprostane in saliva of patients with erosive and nonerosive oral lichen planus in comparison with healthy controls. Materials and Methods: The study participants were selected by convenience sampling from department of oral medicine and dermatology department of our institution. In this case control study, 36 patients with oral lichen planus and 36 control subjects were included. The lichen planus group is again subdivided into erosive and nonerosive lichen planus group. Unstimulated saliva was collected by passive drool method. The salivary 8–isoprostane level was measured by using 8-isoprostane ELISA kit. The statistical tests used were Chi square test, ANOVA and Pearson correlation. Results: The mean values of the salivary 8-isoprostane level an increase in the oral lichen planus group when compared with control group and is statistically significant (P ˂ 0.05). Conclusion: The present study showed that oxidative stress was increased in patients with oral lichen planus, reflected by higher concentration of salivary 8 isoprostane being higher in erosive lichen planus than nonerosive lichen planus.

Keywords: oral lichen planus


How to cite this article:
Dinesan AS, Ravindran R. Evaluation of Salivary 8-Isoprostane in Oral Lichen Planus: Case Control Study. J Orofac Sci 2020;12:113-8

How to cite this URL:
Dinesan AS, Ravindran R. Evaluation of Salivary 8-Isoprostane in Oral Lichen Planus: Case Control Study. J Orofac Sci [serial online] 2020 [cited 2021 May 8];12:113-8. Available from: https://www.jofs.in/text.asp?2020/12/2/113/309584



KEY MESSAGE: F2- isoprostanes provides a more unique tool to assess the role of oxidative stress in pathogenesis of lichen planus with more reliability.


  Introduction Top


Lichen planus is chronic inflammatory disease affecting skin, mucosa with prevalence of approximately 1% to 2% in general population. Oral lichen planus (OLP) frequently occurs in the fourth decade of life and affects women more than men in ratio of 1.4:1. Although the exact pathogenesis is not yet well-determined, in recent years studies on oxidant and antioxidant have shown to have role in pathogenesis. Oral lichen planus is a T cell-mediated autoimmune disease in which cytotoxic CD8+ T cells leads to apoptosis of oral epithelial cells.[1] Immune system impairment is proposed to be a main cause for development of this disease.[2] Histopathological changes such as development of band-like infiltration of lymphocytes supports this finding. Oral lichen planus has many etiological factors in which stress is considered to be a main cause. Free radicals and reactive oxygen species has a role in the pathogenesis of oral lichen planus. Free radicals may produce chemical modification and damage the proteins and nucleotides. Thus, they produce damage to basal keratinocytes. These free radicals degenerate the cell by means of lipid peroxidation. The inflammatory infiltrate of lichen planus contains mainly of CD4+ lymphocytes which is a source of reactive oxygen species. Free radical induced damage can lead to many alterations in the biological and physical properties of a cell, which can lead to impairment in normal cell function. So, the oxidative injury can be assessed well by measuring this lipid peroxidation products.[3]

F2 isoprostanes are a group of bioactive prostaglandins. They are formed by as a result of free radical mediated lipid peroxidation of arachidonic acid and cell membrane phospholipids. They are isomers of PGF2α (8-epi prostaglandin F2α). It can be used to assess oxidant injury in vivo. Other types of isoprostanes are isoprostanes D2- and E2, but they are less stable when compared to 8-isoprostane. F2 isoprostanes are present in all biological tissues and body fluids. Main advantages of isoprostanes are they are chemically stable, they are formed invitro, they cannot be altered by lipid contents in the diet. As they are formed by lipid peroxidation of arachidonic acid, they are independent of cycloxygenase action.[4],[5]

Gas chromatography–mass spectrometry (GC/MS) is considered to be the gold standard method for 8-iso PGF2α measurement. But they need solid-phase extraction (SPE) methods for sample preparation and they are laborious and it may lead to contamination and artefact generation. So in the present study 8-iso PGF2α can be measured by ELISA. It needs only less instrumentation and its result often correlate well with GC–MS, when measuring 8-iso PGF2α.[6]

Saliva is noninvasive and easy to collect. Its composition also changes according to various diseases.[7] Oxidative stress can also be measured in vivo in different types of samples like cells, solid tissues, urine, blood, and saliva. Several studies in the literature have shown the corresponding patterns of oxidative stress between serum and saliva, it suggest that saliva contains oxidation biomarkers similar to those in blood. So, it should be considered as a suitable media for diagnosing oxidative stress. On literature search, the role of oxidative stress in the pathogenesis has been studied using oxidant and antioxidant marker. Isoprostane, a more reliable marker has not been studied extensively in oral lichen planus. Hence, the present study was undertaken to evaluate the role of 8-isoprostane in the pathogenesis of lichen planus.[8]


  Materials and Methods Top


In this case control study, participants were selected by convenience sampling from department of oral medicine and dermatology department of our institution. Ethical approval for this study (protocol No. AEC/REV/2016/28) was provided by Azeezia Ethics Committee of Azeezia College of Dental Science & Research, Kerala, on 30 November 2016. The total sample size was 72 subjects. 36 patients with oral lichen planus satisfying the inclusion criteria were included in the study group and 36 healthy individuals constituted the control group. Oral lichen planus group was again sub grouped into erosive lichen planus and nonerosive lichen planus group. Erosive lichen planus group consists of erosive and atrophic lichen planus. Nonerosive group consists of reticular, papular and plaque-likes lichen planus lesions. All the participants were informed about the research study and agreed to participate by signing an informed consent form. The study was also approved by the Institutional Ethics Committee. The inclusion criteria were clinically and histopathologically diagnosed cases of oral lichen planus. Modified WHO diagnostic criteria of OLP was used. Oral lichen planus patients under treatment in last 2 months; Patients with lichenoid reaction and lichenoid dysplasia, oral mucosal disease other than lichen planus; individuals with systemic diseases such as diabetes mellitus, hypertension, Cardiovascular disease, lung diseases, renal diseases, neurodegenerative diseases, Chronic inflammatory diseases; Subjects with active caries (at least five clinical caries surface), smoking, periodontitis ;patients on vitamin supplementation, drugs, history of surgery last 2 months back were excluded from the study.[9],[10]

After obtaining an accurate medical history, completing a preliminary proforma, and lichen planus lesions were biopsied. Unstimulated saliva about 2 mL was collected from lichen planus patients and healthy controls by passive drooling method. [11]

Saliva collected before breakfast between 8 a.m and 10 a.m. Participants allow the saliva to passively drip into custom made saliva collecting funnel for 5 to 10 minutes till 2 mL saliva was collected. Saliva samples were stored in −80°C until analysis.

The salivary 8-isoprostane level was measured by using 8-isoprostane ELISA kit, (Item No.516351, Cayman, USA). This assay is based on the competition between 8-isoprostane and an 8 isoprostane acetylcholinesterase (AChE) conjugate (8-Isoprostane Tracer) for a limited number of 8-isoprostane-specific rabbit antiserum binding sites. Because the concentration of the 8-Isoprostane Tracer is held constant while the concentration of 8-isoprostane varies, the amount of 8-Isoprostane Tracer that is able to bind to the rabbit antiserum will be inversely proportional to the concentration of 8-isoprostane in the well. This rabbit antiserum-8-isoprostane complex binds to the rabbit IgG mouse monoclonal antibody that has been previously attached to the well. The plate is washed to remove any unbound reagents and then Ellman’s Reagent (which contains the substrate to AChE) is added to the well. The product of this enzymatic reaction has a distinct yellow color and absorbs strongly at 412 nm. The intensity of this color, determined spectrophotometrically, is proportional to the amount of 8-Isoprostane Tracer bound to the well, which is inversely proportional to the amount of free 8-isoprostane present in the well during the incubation. Concentration of 8 isoprostane were obtained in pg/mL.[12],[13]

The data collected was analyzed using by R software EZR version 1.32. The statistical tests used were Chi square, ANOVA, and Pearson correlation.


  Results Top


In the present study based on the age most of the patients (72.2%) were between 41 and 60 years of age in lichen planus group. Only 21% was between 30 and 40 years. (47 ± 8.6). In healthy controls most of them (58.3%) were between 30 and 40 years. Only 8.3% of the subjects were between 51 and 60 years [Table 1]
Table 1 Characteristics of study participants

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The mean age of the patients in oral lichen planus group were 47 ± 8.6 and healthy controls were 40.1 ± 6.8. Chi-square test was used to compare the age, between groups. It is found to be statistically significant (P < 0.01) [Table 2]. Based on gender most of the subjects in lichen planus group were female (75%). In healthy controls also females were more than males (75%) [Table 3].
Table 2 Comparison of age based on group

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Table 3 Comparison of gender based on group

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From the descriptive study of 8-isoprostane in saliva of lichen planus group and healthy controls, it was found that mean value of 8-isoprosatane in lichen planus group was 291.3±108.9 pg/mL and healthy controls were 49.3±28.1 pg/mL. Minimum value of 8-isoprostane in lichen planus group was 110.2 pg/mL and maximum value was 499.1 pg/mL. Minimum value of 8-isoprostane in healthy controls were 12.8 pg/mL and maximum value was 91.8 pg/mL. The mean value of 8-isoprostane in nonerosive lichen planus group was 235.5 pg/mL and erosive lichen planus group was 418.2 pg/mL [Table 4]. The lichen planus group showed higher concentration of 8-isoprostane (291.3 pg/mL) than healthy controls ([Figure 1]). In student t test, a significant difference in 8-isoprostane level of lichen planus group and healthy controls was observed (P < 0.01) [Table 5].
Table 4 Descriptive statistics for 8-isoprostane in saliva of nonerosive, erosive oral lichen planus and healthy controls

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Figure 1 Comparison of 8-isoprostane in saliva of nonerosive/erosive oral lichen planus patients and healthy controls

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Table 5 Comparison of 8-isoprostane in saliva of non-erosive/erosive oral lichen planus patients and healthy control

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The study variable 8-isoprosatne was compared between the groups by using ANOVA test. It was found to be statistically significant. Scheffe Multiple comparison analysis was used to compare 8-isoprostane level between each of these groups. Statistically significant difference was found in 8-isoprostane in saliva of nonerosive oral lichen planus group, erosive oral lichen planus group and healthy controls (P < 0.01) [Table 6].
Table 6 Comparison of 8-isoprostane in saliva of non-erosive, erosive oral lichen planus patients and healthy control

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The results showed that salivary 8-isoprostane is elevated in oral lichen planus group compared with that of the healthy individuals. Among the subgroups salivary 8- isoprostane is elevated in erosive lichen planus group than nonerosive group. The result of this study proves that oxidative stress is higher for oral lichen planus patients when compared with that of the healthy individuals. Among the subgroups oxidative stress is higher for erosive oral lichen planus group when compared with nonerosive lichen planus group ([Figure 2]).
Figure 2 Comparison of 8-isoprostane in saliva of nonerosive, erosive oral lichen planus patients and healthy controls

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Pearson correlation was used to check whether any association exist between level of 8-isoprostane and age of patients([Figure 3]). A negative correlation was obtained (r = −0.083). So there is no significant association exist between age and gender of the patients with level of 8-isoprostane in the present study (P ˃ 0.05) ([Figure 4]).
Figure 3 Scatter diagram for 8-isoprostane in saliva of nonerosive/erosive oral lichen planus patients with age

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Figure 4 Association of 8-isoprostane in saliva of nonerosive/erosive oral lichen planus with gender

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  Discussion Top


Saliva as diagnostic tool has many advantages being noninvasive, easy to collect, good patient compliance. Hence, in this study saliva was collected for assessing the oxidative marker. The use of F2 isoprostanes as markers of oxidative stress has several advantages over other oxidative stress markers such as they are chemically stable, they are formed in vivo and do not show diurnal variation and are present in detectable amounts in tissues and biological fluids. They are also unaffected by lipid contents in the diet. So, they can be used as a reliable marker for oxidative stress. In the present study a female predominance was noted which shows the higher prevalence of oral lichen planus in females than males. The studies by Shah et al. (2009)[14], Lopez-Jornet et al. (2014),[15] and Amirchaghmaghi et al. (2016)[1] found a female predominance. The mean age of the participants in both case and control group were 47 ± 8.6 and 40.1 ± 6.8, respectively, which is in accordance with the study of Scrobota et al. (2011).[16]

Among the 36 study subjects, 11(30.5%) participants were erosive lichen planus patients and 25(69.4%) were nonerosive lichen planus patients. This is in accordance with the study by Shah et al. (2009).[14]

The level of salivary 8-isoprostane is found to be higher in oral lichen planus group when compared with healthy individuals. The mean level of 8 isoprostane in lichen planus group is 291.3 ± 108.9 pg/mL and mean level of salivary 8–isoprostane in healthy controls is 49.3 ± 28.1 pg/mL. It is statistically significant P < 0.01. This shows that oxidative stress is higher for oral lichen planus group in comparison with the healthy individuals. Amirchaghmaghi et al. (2016)[1] in their study assessed the plasma level of 8 isoprostane in oral lichen planus patients and healthy individuals using sandwich enzyme linked immunosorbent assay (ELISA) and found a significant increase in the plasma level of 8-isoprostane in OLP group compared with the control group.

Panchal et al. (2015)[17] observed an increased level of oxidative stress in lichen planus patients in their study. The lipid peroxidation was measured in terms of MDA by using thiobarbituric acid (TBA) reactive substances method. They found that oxidative stress was higher in lichen planus patients and their study also highlighted the role of oxidative stress in pathogenesis of LP. Sander et al. (2005)[2] also observed a decreased antioxidant defence and increased oxidative damage to lipids, DNA and proteins in lichen planus in their study. Chalkoo et al. (2019)[18] also found that oxidative stress can be the possible etiologic factor for OLP and antioxidant may play a role in the treatment of the lesions. They evaluated the levels of nitric oxide (NO) in serum of patients with OLP and compared the levels with the controls. Their results showed that levels of NO were significantly higher in serum of cases with lichen planus than in normal.

Study by Jayasekharan et al. (2014)[19] showed that saliva can be used as an alternate and effective diagnostic tool in evaluating the oxidative stress status of an individual and antioxidants may be used to reverse the oxidative stress status in lichen planus.

Rekha et al. (2017)[20] observed that salivary antioxidant levels show a significant difference in response to oxidative stress in oral LP patients. They estimated the levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GPx) and uric acid (UA) in saliva of oral LP and compared with healthy controls. Their results showed that oxidative stress markers such as MDA and SOD are elevated, and GPx is decreased in the saliva of oral LP patients.

The mean level of salivary 8-isoprostane is higher in erosive lichen planus group(418.2±39.4) than nonerosive lichen planus group (235.5±77.5) in the present study which is in accordance with study by Amirchaghmaghi et al. (2016)[1]were higher 8–isoprostaneplasma level in erosive lichen planus group was found in comparison with nonerosive lichen planus. They concluded that free radicals and ROS play important roles in pathogenesis of lichen planus. In the present study there was no correlation exist between salivary 8-isoprostane (P ˃ 0.05) and age of patients. This is in accordance with the study by Montuschi et al. (1999)[21]. In which they found that there was no correlation exists between 8-isoprostane level and age in their study. Koregol et al. (2018)[22] found that salivary 8-isoprostane can be considered as a pathophysiological marker to measure oxidative stress. They determined the presence of salivary 8-isoprostane in healthy, chronic periodontitis and chronic periodontitis subjects with type II diabetes and they got a statistically significant difference in the levels of 8-isoprostane between healthy, chronic periodontitis and chronic periodontitis subjects with type II diabetes in their study.

Biomarkers of oxidative stress study by National Institute of Health found that F2- isoprostanes are the most accurate biomarker for assessing oxidant stress status in vivo when compared with other markers.[23] Montuschi et al. (2004)[21] reviewed about isoprostanes and they suggested that measurement of F2-isoprostanes has several advantages over other quantitative markers of oxidative stress and they also suggested that 8-isoprostane is considered as a more reliable marker for assessing oxidant injury when compared with other markers. The present study also shows that reactive oxygen species have a role in the pathogenesis of oral lichen planus.


  Conclusion Top


The salivary 8-isoprostane showed a significant increase in oral lichen planus patients compared with that of the healthy controls. The erosive lichen planus group showed a higher level of 8-isoprostane than nonerosive lichen planus group. The finding supports the hypothesis that oxidative stress plays a role in the pathogenesis of oral lichen planus. The measurement of F2-isoprostanes provides a more unique tool to assess the role of free radical in pathogenesis of lichen planus with more reliability. Further studies are suggested to assess the oxidative stress markers before and after treatment of oral lichen planus.

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Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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